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Novel Approach to Activity Evaluation for Release-Active Forms of Anti-Interferon-Gamma Antibodies Based on Enzyme-Linked Immunoassay
Elena S. Gavrilova ,
* E-mail: GavrilovaES@materiamedica.ru
Affiliation OOO "NPF "MATERIA MEDICA HOLDING", Moscow, Russian Federation
Sergey A. Bobrovnik,
Affiliation Department of Molecular Immunology, Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, Kiev, Ukraine
Gordon Sherriff,
Affiliation Ab Biotechnology Limited, Penicuik, United Kingdom
Andrey A. Myslivets,
Sergey A. Tarasov,
Oleg I. Epstein
Elena S. Gavrilova,
Elena S. Gavrilova Sergey A. Bobrovnik ... Oleg I. Epstein
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Following publication of this article [1], concerns were raised about the scientific validity of the study as well as a potential competing interest that was not declared. The PLOS ONE Editors discussed the concerns with the authors and consulted external experts. In light of our editorial assessment and advice received in the expert consultations, we are retracting this article due to concerns about the scientific validity of the research question, study design, and conclusions.
Specifically, we are concerned about the overall design of the study, which aims to detect effects of a reagent diluted to such a degree that the solution is not expected to contain biochemically relevant levels of antibody. The consulted experts also raised concerns about the validity and rigor of the immunoassay system used in the study. The enzyme-linked immunosorbent assay (ELISA) used was adjusted to give barely detectable signals, which renders the assay particularly susceptible to interference. In light of these issues, we consider that the article does not present sufficient or reliable evidence to support the conclusions.
In addition to these scientific concerns, the Competing Interests statement was incomplete on the published article. The Competing Interest statement read: "GS is an employee of Ab Biotechnology Limited. ESG, AAM, SAT and OIE are employees of MATERIA MEDICA HOLDING, whose company funded this study. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials." The third sentence of this disclosure is inaccurate, and ought to have disclosed that OOO "NPF "MATERIA MEDICA HOLDING" markets the release-active antibody solution used in the study.
During the journal's post-publication assessment of this work, concerns were also raised about insufficient methodological reporting in the published article that obfuscate the assessment of the study design. The authors provided additional details about methods and reagents used in response to the journal's queries, but even in consideration of these the fundamental concerns remain about the premise of the study, the assay used, and the overall reliability of the conclusions. In light of these issues, the PLOS ONE Editors retract this article.
ESG, SAB, GS, SAT, and OIG do not agree with retraction and stand by the results in the article. AAM did not respond.
3 May 2018: The PLOS ONE Editors (2018) Retraction: Novel Approach to Activity Evaluation for Release-Active Forms of Anti-Interferon-Gamma Antibodies Based on Enzyme-Linked Immunoassay. PLOS ONE 13(5): e0197086. https://doi.org/10.1371/journal.pone.0197086 View retraction
Selection of a suitable assay to measure the activity of drug agents based on release-active forms of anti-interferon-gamma antibodies (RA forms of Abs) is an important step forward in the investigation of such agents. In this study, the enzyme-linked immunosorbent assay was utilized to examine the effect of RA forms of Abs specific for human interferon gamma on the interaction between monoclonal anti-interferon gamma antibodies and recombinant human interferon gamma. The experimental data and the results obtained by using relevant mathematical analysis showed that such RA forms of Abs are able to modulate the monoclonal antibody interaction with both soluble and immobilized (to the assay plate well) interferon gamma. These data demonstrated the importance of using relatively low concentrations of both soluble and plate-immobilized interferon gamma to detect the effects of RA forms of Abs to interferon gamma on the binding of monoclonal antibodies to interferon gamma. It has been suggested that the observed influence of RA forms of Abs on 'antibody-antigen' interaction could be used to detect and analyze the activity of drugs containing RA forms of Abs.
Citation: Gavrilova ES, Bobrovnik SA, Sherriff G, Myslivets AA, Tarasov SA, Epstein OI (2014) Novel Approach to Activity Evaluation for Release-Active Forms of Anti-Interferon-Gamma Antibodies Based on Enzyme-Linked Immunoassay. PLoS ONE 9(5): e97017. https://doi.org/10.1371/journal.pone.0097017
Editor: Salvatore V. Pizzo, Duke University Medical Center, United States of America
Received: January 6, 2014; Accepted: April 14, 2014; Published: May 9, 2014
Copyright: © 2014 Gavrilova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The work was sponsored by OOO "NPF "MATERIA MEDICA HOLDING". The funder & Ab Biotechnology Limited provided support in the form of salaries for authors ESG, AAM, SAT, OIE and GS but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the author contributions section.
Competing interests: GS is an employee of Ab Biotechnology Limited. ESG, AAM, SAT and OIE are employees of MATERIA MEDICA HOLDING, whose company funded this study. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
Antibody-based drugs are widely available among marketed medicinal products. To date, approximately 30 commercial therapeutic monoclonal antibodies (mAbs) are available for sale in the USA and Europe [1]. However, despite the success of these therapeutics, the use of antibody-based agents remains challenging [2], [3] and considerable efforts at designing and modifying antibody-containing pharmaceuticals or antibody derivatives are ongoing. An example of this is the using of antibody fragments or the production of fusion proteins by coupling antibody fragments to toxins or cytokines [4]–[6]. Another approach to overcome the obstacles associated with the use of mAbs are the attempts to facilitate qualitative penetration of antibodies into the cell by means of microinjections, electroporation etc. [7], [8].
In the last decade, a number of publications devoted to the so-called "release-active forms of drugs" have appeared [9]–[18]. It was observed that during the process of decreasing the initial concentration of a drug substance by multiple consecutive dilution or grinding (trituration) with lactose that the end products of such a process have physicochemical and biological properties which are different from the initial substance properties [11]–[14]. The main feature of these release-active forms is their ability to exert a modifying influence on the starting material. Several drugs based on release-active antibodies have already been introduced into clinical practice and have a pro-antigen nature of action, caused by a direct modifying effect of these drugs on the appropriate antigen.
One of the most studied substances used for the preparation of antibody-containing RA drugs is the anti-IFN-gamma antibody. The efficacy and safety of drugs containing RA forms of Abs to interferon gamma have been extensively studied in various experimental models as well as in clinical studies [17]–[23]. It was shown during these studies that RA forms of Abs change the conformation and binding affinity of interferon gamma (IFN-gamma), demonstrated by changes in antigen-antibody interaction. Therefore, an enzyme-linked immunosorbent assay (ELISA) seems to be one of the most appropriate techniques for quality control of RA-based medicines. The purpose of the present study was to develop an ELISA that could permit detection of RA forms of Abs to IFN-gamma. The study involved a number of experiments to evaluate the applicability of the ELISA assay and determine the optimal conditions for the detection of the modulatory effect produced by RA forms of Abs to IFN-gamma, based on their ability to impact the specific binding of antibodies to interferon gamma.
Preparation of anti-IFN-gamma release-active dilutions
RA forms of Abs to IFN-gamma were supplied as ready-to-use solutions by OOO "NPF "MATERIA MEDICA HOLDING" (Russia, Moscow). Affinity-purified rabbit polyclonal antibodies to recombinant human interferon gamma were manufactured in accordance with current European Union requirements for Good Manufacturing Practice for starting materials (EU Directive 2001/83/EC as amended by Directive 2004/27/EC) by Angel Biotechnology Holdings plc (UK, Edinburgh) as a starting material for commercial production of Anaferon for Children for therapeutic oral application. RA forms of antibodies to IFN-gamma were obtained using routine methods described in the European Pharmacopoeia (7th Edition, 2011). All dilutions were prepared in glass vials. Antibodies to IFN-gamma (2.5 mg/ml) were mixed with a solvent (ethanol–water solution) and shaken for 1 min to produce the C1 dilution. All subsequent dilutions consisted of one part of the previous dilution to 99 parts of solvent (ethanol–water solution for intermediate dilutions and distilled water for the final dilution), with succussion between each dilution. Thus, RA forms of Abs to IFN-gamma contain release-active dilutions of antibodies to IFN-gamma consisting of a mixture of C12+C30+C50 final dilutions. Solutions were prepared in sterile conditions, avoiding direct intense light, and were stored at room temperature.
PBS solution was utilized as a placebo (control) instead of antibodies to IFN-gamma to prepare RA forms using the above described method. RA form of buffer (solvent for antibodies) was chosen as a control in order to meet appropriate ICH guideline, where residual solvent is referred to as one of the applicable controls for demonstration of analytical assay specificity [24]. Generally, it might be also reasonable to use RA forms of different rabbit polyclonal antibodies as the control. Nevertheless, it was not the subject of this study as such specificity has been proven previously for this type of drugs [15], [25].
Drugs and chemicals
Mouse monoclonal antibodies to human interferon gamma (1 mg/ml) were purchased from Mabtech AB, Germany. Recombinant human interferon gamma (1 mg/ml) was obtained from Dialab, Russia, and was used in both a soluble form and as a solid phase coated on the microplate.
Standard ELISA protocol
Microtest Plate 96Well (SARSTEDT AG & Co., Germany, Cat No 82.1582.001) plates were coated with 0.1 ml/well of interferon-gamma at 0.25–10.0 µg/ml in phosphate buffered saline (PBS) (pH 9.2, lab-made) for 18 hours at 4°C. The next day, the coated microplates were washed four times using Tris-buffered saline (TBS) and blocked with 0.1% Tween and 1% bovine serum albumin in PBS for 1 h at room temperature. The blocking solution was removed and the wells were washed four times with TBS.
RA forms of Abs to IFN-gamma or control, mAbs and IFN-gamma (where applicable) were prepared as described below for each individual experiment. 100 µL of each sample was transferred to the wells and assayed in triplicate. TBS alone was used as a blank. The samples were incubated for 45 minutes at room temperature while shaking (shaker-incubator Dynatech, USA) and anti-IFN-gamma antibodies were captured on the plate surface. Unbound material was removed by washing four times with TBS, followed by addition of 100 µL per well of goat anti-mouse IgG antibodies-horse radish peroxidase (HRP) conjugate (Sigma, USA, Cat A4416), added at a dilution of 1∶1000 and incubated for 1 h at room temperature. The microplate was then washed four times with TBS. Color development was performed using 100 µL per well of mixture of 10 mg/ml orthophenylenediamine (Sigma, USA, Cat P1526) and 0.03% hydrogen peroxide (PE "Yan", Ukraine) in PBS (pH 13.5) adjusted to pH 5.0 for 10 min at room temperature. The reaction was stopped by adding 60 µL/well of 2 M sulfuric acid and the absorbance at 490 nm was measured using an ELx800 Absorbance Microplate Reader (BIO-TEK Instruments, Inc., USA). The assay conditions for the ELISA were optimized under previous pilot experiments (data not shown).
The work included 4 different groups of experiments.
Experiment 1. Study of the relationship between antibody concentration and color development for different mAbs dilutions.
The experiment was performed in accordance with the standard ELISA protocol. For this experiment only mAbs without RA forms of Abs to IFN-gamma or control were used as test samples. Dilutions of mAbs were 1∶80000 (0,0000125), 1∶40000 (0,000025) and 1∶20000 (0,00005). The concentration of IFN-gamma adsorbed onto the plate was 1 µg/ml. An example of concentration/color relationship is presented in Fig. 1.
PowerPoint slide
Figure 1. Linear relationship between mAbs to IFN-gamma dilutions and optical density.
The concentration of IFN-gamma absorbed on the plate is 1 µg/ml. Dilutions of mAbs are on X axis (where 0.00001 means dilution of starting antibodies to 1∶100000 times, 0.00002 means dilution of starting antibodies to 1∶50000 times etc.). Optical densities are average values of three measurements. The error bars are the standard deviations.
https://doi.org/10.1371/journal.pone.0097017.g001
Experiment 2. Study of the influence of adsorbed IFN-gamma concentration on detection of RA forms of anti-IFN-gamma antibodies.
During these experiments plates were coated with interferon-gamma at 0.25–6.0 µg/ml in order to determine the influence of the IFN-gamma concentration. Test samples were prepared from mAbs pre-diluted in the range 1∶5000–1∶135000 in TBS and RA forms of Abs to IFN-gamma or control, mixed at a ratio 1∶4 v/v respectively. 100 µL of each sample was transferred into the wells and incubated for 45 minutes at room temperature while shaking. After shaking the standard ELISA protocol was followed.
Experiment 3. Competitive ELISA. Study of the effect of RA forms of Abs to IFN-gamma on the interaction between mouse monoclonal antibodies to IFN-gamma and interferon gamma.
The effect of RA forms of Abs to IFN-gamma on the interaction between monoclonal anti-IFN-gamma antibodies and IFN-gamma was evaluated using the method of Friguet et al. [26] modified by Stevens [27]. The samples were prepared following two different procedures: In the first, 0.3 mL of the RA forms of Abs to IFN-gamma were added to mAbs samples (pre-diluted to 1∶10000, 0.1 mL); the resulting mixtures were incubated for 45 min at room temperature and each sample was then supplemented with 0.1 mL of an appropriate concentration of IFN-gamma solution to give a 1∶50000 antibody final dilution. The prepared mixtures were incubated for 18–20 h at room temperature to reach antigen-antibody equilibrium. During the second procedure, RA forms of Abs to IFN-gamma (0.3 mL) were added to different concentrations of interferon gamma. The mixture were incubated for 45 min at room temperature and then supplemented with 0.1 mL of mAbs solution and incubated for 18–20 h at room temperature. Phosphate buffered saline (PBS) treated in the same fashion as RA forms of Abs was used as the control.
After incubation for 18–20 h, 0.1 mL of each test preparation was added to the wells of the plate pre-coated with IFN-gamma and the plate incubated for 45 min under continuous shaking at room temperature. During this time, the portion of mAbs not bound to the interferon gamma in the test samples bound to the interferon gamma coated on the plate, and this amount of antibody was then determined by the remainder of the standard ELISA protocol.
Experiment 4. Indirect ELISA. Study of the dynamics of binding between mAbs and immobilized interferon gamma.
The effect of RA forms of Abs to IFN-gamma on binding of mAbs to the immobilized IFN-gamma with time was evaluated using the following technique: samples of mAbs were mixed with RA forms of Abs to IFN-gamma (or controls) at a ratio of 1∶4 v/v. The resulting sample preparations were transferred at fixed time intervals into the wells containing immobilized interferon gamma, and the microplate was incubated at room temperature under continuous shaking. The samples were added to the test plate so that at the time of simultaneous removal of all the samples from the plate the duration of incubation was 10 min, 15 min, 20 min, 30 min etc., and the amount of bound antibody was measured by the standard ELISA protocol.
Competitive ELISA.
Mean optical density (OD) values for each test sample were plotted on a graph presenting unbound antibody concentrations against the concentrations of IFN-gamma in the samples of antigen-antibody mixtures, and these values were also used to generate linear functions using the Stevens equation [27]:
(1)where li – concentration of IFN-gamma in the sample of mixture of mAbs and IFN-gamma; Ai – optical density (OD) measured for samples with IFN-gamma at concentration li; A0 – OD measured for samples without IFN-gamma; Kd – dissociation constant for antigen-antibody reaction.
According to the equation (1), the slope in the above described plots with against 1/li is equal to Kd, i.e. the value inversely proportional to the affinity constant of antibody-antigen binding. Thus, this approach can be used to determine how the test sample (here RA forms of Abs to IFN-gamma) influences the affinity of interaction between mAbs and IFN-gamma interaction reaction, provided that this factor does not affect the antigen concentration li.
Indirect ELISA.
Since it is known that binding of bivalent antibodies to immobilized antigen is practically irreversible [28]–[30], this reaction is described by the following differential equation:
(2)where t – reaction time; k – binding constant of the reaction between antibody and immobilized antigen; A – concentration of antibody in the solution.
Solution of equation (2) with the initial conditions t = 0, A = A0 gives the following equation: (to define A values related to t)
It is much more convenient to measure the dynamics of antibody binding to the immobilized antigen rather than the dynamics of reduction of antibody concentration in the solution with ELISA assays. Therefore equation (3) can be rewritten to obtain an equation defining changes in the amount of antibody bound to the immobilized antigen instead of the variables relating to the antibody concentration in the solution. This transformation could be done in the following way: it is clear that at any time point t the amount of antibody bound to the plate P is equal to the amount of antibody leaving the solution, i.e. the A0–A value. As the reaction is almost irreversible, the whole amount of antibody in the solution should become bound to the immobilized antigen as time approaches infinity (i.e. at t→∞, P∞ = A0). Hence, A and A0 values from equation (3) could be substituted for equivalent P∞-P and P∞ values, respectively:
Thus, equation (4) describes the time-dependent changes in the amount of product in the first-order irreversible reaction (here the level of antibody binding to plate with time). In terms of our study, this equation is very convenient, as instead of the amount of bound antibody used in equation (4) it is possible to employ a value proportional to this amount - OD in the plate wells as determined by ELISA. However, because equation (4) is transcendental, it unfortunately suggests no analytical solution, which explains the current reason for using the Guggenheim graphical method [31] or its modification - Kezdy-Swinbourne method [32], [33].
Nevertheless, an analytical solution to the equation (4) for a special case can be found i.e. when the assay measures the amount of reaction product Pi and P2i (color development in the wells measured by ELISA is proportional to the amount of antibody bound to the plate) at such time points ti and t2i when t2i = 2ti. We have shown previously [34] that these conditions may allow for the following analytical solution to the equation (4):
First, having defined in assay Pi and P2i values at time points ti and t2i, it is possible to calculate P∞ following the formula:
Then, with P∞ value known, it is also possible to calculate k as: (6)
For improved assay precision in calculating P∞ and k values, a number of Pi and P2i measurements should be performed at time points ti and t2i, respectively. Further, according to equations (5) and (6), it is possible to first generate a linear plot with the assay Pi2 values against 2Pi-P2i to define P∞, and then, knowing P∞, to generate a linear plot with ln(P∞/(P∞-Pi)) against ti to define k. According to equations (5) and (6), the slopes of the described linear plots will be equivalent to P∞ and k, respectively. By measuring these slopes improved precision can be achieved in defining the values of interest (P∞ and k), than by using individual measurements of P1 and P2.
Thus, assays to evaluate the effect that RA forms of Abs to IFN-gamma have on the time-dependent changes in the binding of mAbs to plate-immobilized IFN-gamma can be performed by using the above method for calculating P∞ and k values of both mAbs samples (in the presence of RA forms of Abs to IFN-gamma) and controls.
Data processing and all statistical calculations used in this protocol were performed using SAS-9.3 statistical software. Analyses on continuous variables were carried out using a three-way analysis of variance (ANOVA) for experiment 2 and two-way ANOVA for experiments 3 and 4. Experiment 1 did not require any statistical analysis. If the overall ANOVA results were significant, multiple comparisons were done using the Bonferroni post-hoc test. The data in the Figures 1, 2A-D, 3A and 4A are presented as mean optical density per group ± standard deviation.
Figure 2. The influence of absorbed IFN-gamma concentration on detection of RA forms of anti-IFN-gamma antibodies.
The figure shows the influence of the concentration of IFN-gamma adsorbed onto microtiter plates on the mAbs dilution curves observed in the presence of control or RA-antibodies to IFN-gamma (RA Abs to IFNg). Test samples prepared of mAbs, pre-diluted up to 1∶5000–1∶135000 in TBS, and either RA forms of Abs to IFN-gamma (RA Abs to IFNg) or control, mixed at a ratio 1∶4 v/v respectively. 100 µL of each sample was transferred into the wells (3 wells for each sample) and was incubated for 45 minutes at room temperature while shaking (shaker-incubator Dynatech, USA). The remainder of the experiment was conducted in accordance with the standard ELISA protocol. Dilutions of mAbs on X axis are those observed after mixing with RA forms of Abs to IFN-gamma or control (where 0.00001 means dilution of starting antibodies to 1∶100000 times, 0.00002 or 2E-05 means dilution of starting antibodies to 1∶50000 times etc.). The Y-axis displays 490 nm optical densities. The error bars represent the standard deviations of the measurements. The concentrations of IFN-gamma adsorbed on the plate are: A 6 µg/ml B - 2 µg/ml C - 0.7 µg/ml; D - 0.25 µg/ml. RA samples are significantly different from the control (F31/60 = 93.4; p<0.0001) in the case of the C and D curves.
Figure 3. Effect of RA forms of anti-IFN-gamma antibodies on antigen (soluble form)-antibody interactions.
The figure displays the effect of RA forms of Abs to IFN-gamma (RA Abs to IFNg) on the ability of soluble IFN-gamma to bind anti-IFN-gamma mAbs. The amount of mAbs which have not been bound by the respective antigen after reaching the dynamic equilibrium condition was measured by adding mAbs samples with different concentrations of antigen (shown on X axis of A where 0.00025 means dilution of starting interferon-gamma to 1∶4000 times, 0.0005 means dilution of starting interferon-gamma to 1∶2000 etc.) into antigen-coated wells of the assay plate for 45 min. (A): the influence of mAbs (1∶50000 dilution) in the mixture with IFN-gamma in different concentrations in presence of RA forms of Abs to IFN-gamma or control after equilibration. The error bars represent the standard deviations of the measurements. The sample RA Abs to IFNg prepared by procedure 1 (RA Abs to IFNg -1) was mixed with mAbs initially. IFN-gamma in different concentrations was added 45 minutes later. RA Abs to IFNg prepared by procedure 2 (RA Abs to IFNg-2) was mixed with IFN-gamma at different concentrations and mAbs was added 45 min later. It was shown that the order of reagent addition during pre-incubation is not important. RA samples are significantly different from control (p<0.0001) for each RA sample. RA Abs to IFNg-1 and RA Abs to IFNg-2 do not differ significantly from each other (p = 0.2). (B): there are linear relationships from 1/li calculated from "A" curves and the equations for these linear functions where the slopes are equal to the dissociation constants for antigen-antibody reaction.
Figure 4. Effect of RA forms of anti-IFN-gamma antibodies on antigen (absorbed form)-antibody interactions.
The figure shows changes in presence of control or RA forms of Abs to IFN-gamma in mAbs binding with interferon gamma adsorbed onto the plate (0,5 ug/ml); the error bars are the standard deviations for the groups. (A). Using the curves presented in Figure (A) and the method described in Materials and Methods, P∞ and k were determined for mAbs in presence of control (B and C) or RA forms of Abs to IFN-gamma (RA Abs to IFNg) (D and E). RA sample is statistically different from control (F15/31 = 72.6; p<0.0001). (B), (C), (D) and (E) show equations of linear relationships obtained using MS Excel.
Overall ANOVA results and all post-hoc analyses results were accessed using the GLM (general linear models) procedure.
It was established in previous experiments, that the optimal dilution for the mAbs we used in ELISA measurements are dilutions of 1∶20000 or higher. This dilution is derived from the observed, directly proportional, relationship between antibody concentrations and color development in the plate wells at this or higher dilutions (Fig. 1, Table S1). Therefore the 1∶20000 dilution of mAbs (or higher dilutions) was chosen as the starting point for the preparation of test samples in the majority of subsequent assays.
It was also determined that the results of examining the effect of RA forms of Abs to IFN-gamma on the interaction between mAbs and IFN-gamma were influenced by the concentration of IFN-gamma adsorbed onto the plate surface. The number of experiments performed as described at Experiment 2 of Experimental design section demonstrated that the binding between mAbs and immobilized IFN-gamma was inhibited by RA forms of Abs to IFN-gamma to a greater or lesser extent depending on the chosen conditions (the example of the results of such experiments is shown in Fig. 2, Table S2). Moreover, the inhibition of mAbs binding to the respective antigen was higher when the concentration of IFN-gamma immobilized on the plate was minimal (F31/60 = 93.4; p<0.0001 for curves C and D, Fig. 2). This relationship leads to an assumption that to successfully detect release-activity of RA forms of Abs by using ELISA, it is crucial that the amount of antigen immobilized on the plate be extremely low to ensure more effective competition of RA forms of Abs to IFN-gamma with mAbs for binding to the antigen. With increasing amount of plate-immobilized antigen, the competition becomes insignificant and the effect of RA forms of Abs to IFN-gamma decreases or even becomes undetectable in some experiments (Fig. 2).
Thus, the results showed that it is possible to detect release-activity of RA forms of Abs that is associated with their effect on Ab-Ag interaction by using the described technique, provided however the amount of antigen adsorbed on the plate surface is minimal. It should be noted that these amounts of antigen are so low that they fall close to the sensitivity limit of the ELISA. It seems that only these conditions allow the detection of the competition between RA forms of Abs to IFN-gamma and mAbs in the ELISA.
Furthermore, it cannot be ruled out that the effect of RA forms of Abs on the reaction between antibodies and immobilized IFN-gamma is associated not only with the competition of RA forms of Abs to IFN-gamma with mAbs for binding to interferon gamma, but also with the immediate impact of RA forms of Abs to IFN-gamma on the properties of antigen and mAbs, which also could have influenced the binding capacity of mAbs. The data obtained previously point to the existence of such a possibility [14]. In order to detect the activity of RA forms of Abs to IFN-gamma, we investigated the effect of RA forms of Abs on the interaction of mAbs with soluble interferon gamma, using a technique frequently employed for measuring antibody affinity for a respective antigen by ELISA [26].
Fig. 3a (Table S3) displays the results of one of the experiment series carried out to study the effect of RA forms of Abs to IFN-gamma on the ability of soluble IFN-gamma to bind monoclonal anti-IFN-gamma Ab. In these experiments, the amount of mAbs which have not been bound by the respective antigen after reaching the dynamic equilibrium condition was measured by adding antibody samples with different concentrations of antigen into the antigen-coated wells of the assay plate for 30–60 min [26]. During this time, an amount of antibody proportional to the concentration of free antibody in the antigen-containing preparations is bound by the antigen immobilized on the plates, thereby permitting quantification of both free antibody in each of the test samples and antigen-bound antibody. Further, by plotting values against 1/li (Fig. 3b) according to equation (1) it is possible to calculate the affinity of the antigen-antibody reaction.
The experiment presented in Fig. 3 was performed by the two different methods as described in Experiment 3 of Experimental design section. Firstly, the RA samples were mixed with solutions containing different concentrations of interferon gamma; these were incubated for forty five minutes, then monoclonal antibodies were added. The second technique involved mixing the RA samples first with monoclonal antibody followed forty five minutes, by the addition of different concentrations of IFN-gamma solution. As seen in Fig. 3a, the order of addition of the RA samples being tested into mixtures with either IFN-gamma or monoclonal antibody had no effect on the experimental outcome. In both cases, the concentration of free monoclonal anti-IFN-gamma in the sample preparations with interferon gamma, i.e. the concentration of antibody not bound by interferon gamma, was somewhat decreased in the presence of anti-IFN-gamma in comparison with the control (F23/56 = 28.1; p<0.0001 for each of comparison), whereas there were no differences between the two types of target samples (Post-hoc comparison with the Bonferroni adjustment p = 0.2).
The affinity constants of the mAbs reaction with interferon-gamma calculated as described above were 3.86×108 M−1 (control) vs 5.56×108 M−1 and 5.92×108 M−1 (two types of mAbs samples with RA forms of Abs to IFN-gamma). Hence, assuming that RA forms of Abs to IFN-gamma exert an effect only on the properties of mAbs and have no effect on IFN-gamma and with respect to the obtained results, it is possible to conclude that the affinity of mAbs to interferon-gamma increases to a certain degree (approximately by 1.5 times) in the presence of RA forms of Abs to IFN-gamma.
However, RA forms of Abs to IFN-gamma may be producing a more potent effect on the conformation of IFN-gamma molecules rather than on mAbs affinity. These rabbit antibodies are specific for human interferon gamma, which means they exhibit specificity for its recombinant analogue. Hypothesizing that IFN-gamma molecules in solution, due to molecular dynamics, are present in a range of conformational states (where only in some of these states are the antigenic determinants of IFN-gamma complementary to mAbs molecules), we can infer that any influence inducing a shift towards augmentation or reduction of complementary IFN-gamma molecules will lead respectively to enhancement or inhibition of their interaction with antibodies. It is reasonable to assume that interferon-complementary molecules, represented by anti-IFN antibodies, can induce a shift towards augmentation in a fraction of the molecules capable of binding mAbs. Therefore, the influence of RA forms of Abs to IFN-gamma is likely to alter the conformation of IFN-gamma molecules rather than altering mAbs conformation and this kind of influence may be the main reason for changes seen in mAbs-IFN-gamma interactions.
If the above inferences are true, the observed leftward shift in the binding curves obtained for mAbs-antigen reaction (Fig. 3a) may also be associated with the apparently increased concentration of IFN-gamma in the samples, where it is represented as a mixture with RA forms of Abs to IFN-gamma. This is probably because the increase in the concentration of IFN-gamma molecules prone to conformational changes (due to which these molecules exhibit complementarity to mAbs) is caused by release-activity of RA forms of Abs to IFN-gamma.
It should be noted that if this apparent increase of IFN-gamma concentration (which is actually an increase of the concentration of molecules with conformations that account for the complementary activity of IFN-gamma toward mAbs) is close to a constant value for this particular type of sample containing RA forms of Abs to IFN-gamma and is minimally dependent on the concentration of IFN-gamma in the sample preparations, then the observed increase will be far more pronounced at low concentrations of IFN-gamma than when using high IFN-gamma concentrations. This observation should imply that notable differences between control samples and samples containing RA forms of Abs to IFN-gamma are likely to occur with low antigen concentrations, whereas with high concentrations of IFN-gamma these differences will be almost undetectable. Our results support this observation.
Another approach in the evaluation of the effects of RA forms of Abs on the interaction between mAbs and IFN-gamma is the study of the kinetics of antibody binding to the antigen immobilized on the plate. Examples of curves obtained for time-dependent changes in the level of binding between mAbs samples (with or without RA forms of Abs to IFN-gamma) and IFN-gamma immobilized on the plate surface (as described in Experiment 4 of Experimental design section), are presented in Fig. 4 (Table S4). As shown, there are evident differences between the curves as was observed in the previous series of experiments, with suppression of mAbs binding to the immobilized IFN-gamma in the presence of RA forms of Abs to IFN-gamma, as compared to controls. What is evident in the comparison of these curves is that there are more pronounced differences at shorter incubations, while the curves tend to become more similar with increasing time of incubation.
We believe that the trends observed in the experiments may be interpreted as evidence to suggest the following: after supplementing IFN-gamma with the mAbs mixture containing RA forms of Abs to IFN-gamma, they begin to compete for binding to the available antigenic determinants of the solid-phase IFN-gamma. At the same time, RA forms of Abs to IFN-gamma apparently exert an influence on the molecular dynamics of the immobilized IFN-gamma so that the fraction of IFN-gamma molecules with the conformation complementary to mAbs paratopes gradually increases with time. Owing to this effect, the rate of increase in the amount of mAbs bound to the immobilized IFN-gamma in the presence of RA forms of Abs to IFN-gamma is higher than in the control wells, where the amount of epitopes for mAbs remains constant (F15/31 = 72.6; p<0.0001). Therefore, the binding curve of mAbs assayed in the presence of RA forms of Abs to IFN-gamma has a steeper slope than that generated for the controls.
It should be noted that in some of our initial experiments (after 10- to 30-min incubation of the plates with sample preparations) the level of antibody binding to IFN-gamma present in samples with RA forms of Abs to IFN-gamma was markedly lower in comparison to the controls. However, after more prolonged time (60 min or more), the amount of bound antibody was nearly identical in the control and experimental wells; in some instances, the amount of bound antibody in the controls was even lower than in assay variants with RA forms of Abs to IFN-gamma. As a result, the antibody binding curves obtained for controls and experimental wells could intersect at 80–120 min after the onset of the experiment.
Using the hypothesis described in Materials and Methods we can successfully evaluate quantitative characteristics of the binding process of mAbs to the immobilized IFN-gamma as observed in the presence or absence of RA forms of Abs to IFN-gamma. By plotting Pi2 values against 2×Pi-P2i it is possible to define P∞ for controls and test samples, which is calculated as the slope of the obtained linear function (Fig. 4b). The calculations yield the control P∞ value of 1.14 and the mAbs P∞ value in the presence of RA forms of Abs to IFN-gamma of 1.53. Given that P∞ indicates the Pi value that could be obtained if the experiment lasted indefinitely and the reaction was completed, we see from the data derived that the mAbs sample appeared to have 1.53/1.14 = 1.34 fold higher antibody concentration in the presence of RA forms of Abs to IFN-gamma. This implies that, owing to their release-activity, RA forms of Abs to IFN-gamma produced in this experiment a 1.34-fold increase in the amount of mAbs with paratopes possessing complementary conformations toward IFN-gamma epitopes. These data support the previous results pointing to the ability of RA forms of Abs to enhance antibody activity [13], with the mechanism of this process, however, still remaining unknown.
By calculating the P∞ value according to equation (6) it is also possible to define the binding rate constants of mAbs both in the presence of RA forms of Abs to IFN-gamma and in the control wells. As predicted by the hypothesis, ln(P∞/(P∞-Pi)) values related to time ti produced linearity, which enabled calculation of the slopes of the curve (Fig. 4d and 4e) that were equivalent to binding rate constants k. The rate constants appeared to be identical (approximately 8.4×10−5 sec−1) for both types of experiments (i.e. in the presence or without RA forms of Abs to IFN-gamma), indicating that antibody affinity for interferon-gamma was not altered by the release-activity of RA forms of Abs to IFN-gamma. Hence, the release activity is likely to induce an increase in the amount of mAbs with the conformation complementary to IFN-gamma epitope, whereas they have no effect on other antibody-antigen interaction parameters.
Summarizing the data obtained, we can conclude: RA forms of Abs to IFN-gamma are able under certain conditions to influence the interaction of mAbs to IFN-gamma with the respective antigen, e.g. interferon gamma. Detection of this effect caused by the release-activity of RA forms of Abs on antibody reaction with the antigen provides an opportunity to measure this activity, and to make further attempts at establishing the relationship between the therapeutic effect of RA forms of Abs and such activity. Achieving this goal would provide new options for studying physicochemical and biological properties of RA forms of Abs, which in turn would enable investigations into the biological effects of RA forms of Abs as observed in humans and animals.
The occupancy of microtiter plates (A) and optical density values (B) in Experiment 1.
https://doi.org/10.1371/journal.pone.0097017.s001
(DOCX)
We thank Alison Catalano for critical discussions and providing language help.
Conceived and designed the experiments: ESG SAB GS SAT OIE. Performed the experiments: SAB. Analyzed the data: SAB AAM. Contributed reagents/materials/analysis tools: SAB. Wrote the paper: ESG SAB GS SAT. Contributed to the writing of the manuscript: OIE.
1. Buss NAPS, Henderson SJ, McFarlane M, Shenton JM, de Haan L (2012) Monoclonal antibody therapeutics: history and future. Curr. Opin. Pharmacol, 12(5), 615. doi: 10.1016/j.coph.2012.08.001.
2. Frenzel A, Fröde D, Meyer T, Schirrmann T, Hust M (2012) Generating Recombinant Antibodies for Research, Diagnostics and Therapy Using Phage Display. Curr. Biotechnol. 1, 33–41. doi:10.2174/22111550111201010033
3. Lipman NS, Jackson LR, Trudel LJ, Weis-Garcia F (2005) Monoclonal Versus Polyclonal Antibodies: Distinguishing Characteristics, Applications, and Information Resources. ILAR J. 46(3), 258–268. doi:10.1093/ilar.46.3.258
4. Boado RJ, Zhang Y, Zhang Y, Xia CF, Pardridge WM (2007) Fusion antibody for Alzheimer's disease with bidirectional transport across the blood-brain barrier and abeta fibril disaggregation. Bioconjugate Chem. 18(2): , 447–55. doi: 10.1021/bc060349x.
5. Cao M, Cao P, Yan H, Lu W, Ren F, et al.. (2009) Construction, purification, and characterization of anti-BAFF scFv-Fc fusion antibody expressed in CHO/dhfr- cells. Appl Biochem Biotechnol. 157(3): , 562–74. doi: 10.1007/s12010-008-8434-6.
6. Vanhove B, Laflamme G, Coulon F, Mougin M, Vusio P, et al.. (2003) Selective blockade of CD28 and not CTLA-4 with a single-chain Fv–α1-antitrypsin fusion antibody. Blood, 102(2): , 564–570. doi:10.1182/blood-2002-08-2480.
7. Chames P, Van Regenmortel M, Weiss E, Baty D (2009) Therapeutic antibodies: successes, limitations and hopes for the future. Br J Pharmacol, 157(2): , 220–233. doi: 10.1111/j.1476-5381.2009.00190.x.
8. Hansel TT, Kropshofer H, Singer T, Mitchell JA, George AJT (2010) The safety and side effects of monoclonal antibodies. Nat Rev Drug Discov, 9: , 325–338 doi:10.1038/nrd3003
9. Bailbé D, Philippe E, Gorbunov E, Tarasov S, Epstein O, et al.. (2013) The novel oral drug Subetta exerts an antidiabetic effect in the diabetic Goto-Kakizaki rat: comparison with rosiglitazone. J Diabetes Res. 2013, 763125. doi: 10.1155/2013/763125.
10. Dugina JL, Petrov VI, Babayeva AR, Martyushev-Poklad AV, Tcherevkova EV, et al.. (2005) A randomized, open-label, comparative, 6-month trial of oral ultra-low doses of antibodies to tumor necrosis factor-alpha and diclofenac in rheumatoid arthritis. Int J Tissue React. 27(1): , 15–21.
11. Epstein OI, Vorobeva T, Berchenko O, Garbuzova S, Geiko V, et al.. (1996) Information and ontological models of adaptation. IMPE, Moscow.
12. Epstein OI (2009) Ultra-Low Doses (History of one research). Publishing Office of the Russian Academy of Medical Sciences, Moscow.
13. Epstein OI (2012) Release-activity: a long way from the phenomenon to new drugs. Bull Exp Biol Med. 154(1): , 54–58. doi: 10.1007/s10517-012-1874-6.
14. Epstein OI (2013) Release activity phenomenon and spatial homeostasis hypothesis. Usp. Fiziol. Nauk. 44(3): , 54–76 (in Russian).
15. Nicoll J, Gorbunov EA, Tarasov SA, Epstein OI (2013) Subetta treatment increases adiponectin secretion by mature human adipocytes in vitro. Int J Endocrinol. 2013, 925874. doi:10.1155/2013/925874
16. Sakat SS, Mani K, Demidchenko YO, Gorbunov EA, Tarasov SA, et al.. (2014) Release-active dilutions of diclofenac enhance anti-inflammatory effect of diclofenac in carrageenan-induced rat paw edema model. Inflammation,37(1): , 1–9. doi: 10.1007/s10753-013-9705-0.
17. Tarasov SA, Zarubaev VV, Gorbunov EA (2012) Activity of ultra-low doses of antibodies to gamma-interferon against lethal influenza A (H1N1) 2009 virus infection in mice. Antiviral Res. 93: , 219–224. doi:10.1016/j.antiviral.2011.11.018.
18. Vasil'ev AN, Sergeeva SA, Kachanova MV, Tarasov SA, Elfimova UV, et al.. (2008) Use of ultralow doses of antibodies to gamma-interferon in the treatment and prophylaxis of viral infections. Antibiot Khimioter. 53: , 32–35.
19. Aver'ianov AV, Babkin AP, Bart B, Volchetski??? AL, Minina ES, et al.. (2012) Ergoferon and oseltamivir in treatment of influenza: results of multicentre randomized comparative clinical trial. Antibiot Khimioter. 57(7–8): , 23-30 (in Russian).
20. Erman ES, Osidak LV, Sukhovetskaya VF, Drinevskii VP (2009) Efficiency of interferon inductor anaferon (pediatric formulation) in prophylaxis of acute respiratory infections in sickly children. Bull Exp Biol Med. 148(2): , 270–4.
21. Shishkina LN, Sergeev AN, Kabanov AS, Skarnovich MO, Evtin NK, et al.. (2008) Study of efficiency of therapeutic and preventive anaferon (pediatric formulation) in mice with influenza infection. Bull. Exp. Biol. Med. 146: , 763–765. doi:10.1007/s10517-009-0378-5.
22. Shishkina LN, Skarnovich MO, Kabanov AS, Sergeev AA, Olkin SE, et al.. (2010) Antiviral Activity of Anaferon (Pediatric Formulation) in Mice Infected with Pandemic Influenza Virus A (H1N1/09). Bull. Exp. Biol. Med. 149(5): , 612–614. doi: 10.1007/s10517-010-1006-0.
23. Verevshchikov VK, Borzunov VM, Shemiakina EK (2011) Ergoferon and improvement of etiopathogenetic therapy of influenza and acute respiratore viral infection in adults. Antibiot Khimioter. 56(9–10): , 23–6 (in Russian).
24. ICH harmonized tripartite guideline (2005) Validation of analytical procedures: text and methodology Q 2 (R1). Available: http://www.ich.org/. Accessed 2014 Apr 10.
25. Epstein OI, Sherstoboev EY, Martyushev-Poklad AV, Dugina YL, Sergeeva SA, et al.. (2004) Dose-dependent effects and specificity of action of antibodies to endogenous regulators in ultralow doses. Bull Exp Biol Med. 137(5): , 460–2.
26. Friguet B, Chaffotte AF, Djavadi-Ohaniance L, Goldberg ME (1985). Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J. Immunol. Methods. 77: , 305–319. doi: 10.1016/0022-1759(85)90044-4.
27. Stevens FJ (1987) A modification of an ELISA-based procedure for affinity determination: correction necessary for use with bivalent antibody. Molec. Immunol 24: 1055–1060
28. Bobrovnik SA (1998) Dynamics of the interaction of monoclonal antibodies with antigens immobilized on plates. Ukr. Biokhim. Zh. 70(3): , 118–128 (in Russian).
29. Nygren H, Werthen M, Stenberg M (1987) Kinetics of antibody binding to solid-phase-immobilised antigen. Effect of diffusion rate limitation and steric interaction. J. Immunol. Methods. 101: , 63–71. doi: 10.1016/0022-1759(87)90217-1.
30. Werthén M, Nygren H (1993) Cooperativity in the antibody binding to surface-adsorbed antigen. Biochim. Biophys. Acta 1162: 326–32
31. Guggenheim EA (1926) XLVI. On the determination of the velocity constant of a unimolecular reaction. Philos Mag J Sci. 2(9): , 538–543. doi:10.1080/14786442608564083.
32. Kezdy FJ, Jaz J, Bruylants A (1958) Cinétique de L'action de L'acide Nitreux sur les Amides I. Méthode Générale. Bull. Soc. Chem. Belg. 67(11–12) , P.687–706. doi: 10.1002/bscb.19580671104.
33. Swinbourne ES (1960) Method for obtaining the rate coefficient and final concentration of a first-order reaction. J. Chem. Soc. 473: , 2371–2372. doi: 10.1039/JR9600002366.
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Disabled Vehicle, Accident Procedures, And Truck Fires - This short program is simply a guide to help drivers avoid excessive down time, properly report disabled vehicles, and basic accident reporting procedures. We'll discuss the basics, but follow your company's procedures. If you have a question about the procedures, ask your supervisor for some answers.
Topics included in this safety video are: stop the vehicle if any unusual noise, inspect (vehicle, fire extinguisher, First Aid kit, and emergency reflectorized triangles), flares, stopping and safeguarding a disabled vehicle, stay inside the vehicle, if you're involved in an accident (notify your dispatcher immediately, brief description of the accident, exact location of the accident, whether there are any known injuries, if vehicle is disabled then let dispatcher know the details so emergency crews can be dispatched, get the names, addresses, telephone numbers, and insurance information from the people involved, ask to see their driver's license and insurance papers if at all possible, don't say anything except get the basic information and wait for the police, don't move the vehicle until instructed to by the police or qualified authorities), eliminating evidence, driver's written reports, special notes, and vehicle fires.
WITH PURCHASE OF ANY ENGLISH VHS OR DVD YOU WILL RECEIVE A FREE WRITTEN MATERIAL CD-Rom.
The CD-ROM of written materials may include: Leaders Guides, Questions & Answers, Manuals, and Power Points.


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redpajama_set_name: RedPajamaGithub


text:
BATTLE AT BRISTOL: TENNESSEE VOLUNTEERS VS…
Home » The Art of Living » Tailgating & Homegating » BATTLE AT BRISTOL: TENNESSEE VOLUNTEERS VS VIRGINIA TECH HOKIES
BATTLE AT BRISTOL: TENNESSEE VOLUNTEERS VS VIRGINIA TECH HOKIES
The Art of: Tailgating & Homegating
Tags: ACC, Battle At Bristol, SEC, tailgating, Tennessee, Virginia Tech,
Football time is here and what a weekend we just had! Some close games, some upsets, some come from behind wins! Making for my favorite time of the year…Fall…football…family…friends…food!
This Saturday, Sept. 10, 8 p.m.The Bristol Motor Speedway in Bristol, Tennessee will be the site of the largest football game ever. Over 150,000 fans will be on hand to root for their favorite team, the Tennessee Volunteers or the Virginia Tech Hokies.
What makes this game so intriguing…Bristol sits on the state border of Tennessee and Virginia. There's the Bristol, Tennessee side and the Bristol, Virginia side. The schools are approximately the same distance from the Speedway.
ESPN COLLEGEGAME DAY WILL BE BROADCASTING THERE Click here for video
On our trip back from Vermont and Maine, Dave and I decided to stop over in Blacksburg to visit the Virginia Tech campus. We loved the campus and its beautiful buildings.
Burress Hall
War Memorial Chapel on the Drill Field
Lane Stadium
And of course I had to include a few from the campus of the University of Tennessee.
Ayres Hall on The Hill
The Knoxville Skyline overlooking The Tennessee River and The University of Tennessee
Ready for The Vol Navy
Neyland Stadium and running through the T
Smokey, the Tennessee Vols mascot.
As we enjoy the college rivalries, game day spectacles, marching bands, food, tailgating and friendships, I would like to remember those 32 students and faculty members at Virginia Tech who lost their lives on that terrible day, April 16, 2007.
Dave and I visited the April 16 Memorial and were moved by the 32 engraved Hokie stones. Two had flowers on them in rememberance of their birthdays.
Nicole Peterson, 19 at the time would have been 28.
Dr. Liviu Librescu, 76 years old on that terrible day would have been 86.
"The revelation that Dr. Liviu Librescu blocked the door of his classroom in Norris Hall on the morning of April 16 so that his students could escape through the windows came as no surprise to his family, friends, and colleagues. The renowned aeronautical engineering educator and researcher had demonstrated profound courage throughout the 76 years of his life…" (from Virginia Tech We Remember site…click here to read his life story.)
What tears at my heartstrings, he was a Holocaust survivor.
As we get ready to enjoy the big event in Bristol, I would like fans to remember that football is a game. Even though it brings so much enjoyment to our Saturdays, in the big scheme of things, life is about sportsmanship, courage, respect for others, love for family, country and friends.
And for all of us who have a House Divided…
the center beauty, Darcy will be sporting her VT attire instead of UT Vols. Her son, Sean, played baseball at VT. Husband, John will be really divided as his daughter, Amanda and soon to be husband, Trevor are avid UT newly graduated alums.
Amanda, Kathy, John, Darcy, Trevor and Cindy
Watch for upcoming stories from my tailgating travels.
Come celebrate with me. I'll save you a seat at the table.
3 COMFORT FOODS FOR NFL PLAYOFF GAMES
The Art of: Food, Tailgating & Homegating
I realized something the other day when thinking of chili recipes for the... continue reading
BIG COLLEGE FOOTBALL GAMES – LABOR DAY WEEKEND
Are you ready for some football? Game Day Recipes for 4 of the big games... continue reading
Michael Redding says:
Thanks Kathy for another great post and reminding us what it is all about. You have a such a special way with words and pictures that touched my heart. I will be pulling for our Vols on Sat. at Bristol. What a great venue and atmosphere that will be for a football game. Wish I was there to experience it live.
KathyMillerTime says:
Thank you Michael for your special comment. I felt very emotional writing this story because of the bigger picture. I am a true believer in good sportsmanship and look forward to both the Vol fans and Hokie fans enjoying the moment with class.
The largest game ever this weekend? 150,000 is a lot of mouths to feed. Not sure how you're going to do that.
HO HO Bob! Leave it to you for a laugh! Think I will have a lot of cooking to do!
Cindy Dickinson says:
What a great story Kathy! You have a fantastic way with words and I love the way you put everything in prospective, especially when it comes to families. Thanks for sharing.
Thank you for your comment Cindy! As you know families are important especially when they are on opposite sides in a football match. I try not to forget my manners when rooting for my team lol. Ask my kids if I do a good job .

redpajama_set_name: RedPajamaCommonCrawl


text:
ICRC launches video on the human cost of ignoring the Geneva Conventions
The International Committee of the Red Cross (ICRC) has launched an ad-style video that illustrates the terrifying human cost of ignoring the Geneva Conventions and aims to raise awareness of international humanitarian law. The 60-second film, Victory by Any Means, which uses real clips from conflicts around the world, takes viewers on a journey from utopian dream to humanitarian nightmare. It is the hardest-hitting campaign ad of its kind from the ICRC.
"The bombing of hospitals, the massive displacement of civilians and the scourge of sexual violence in war have almost become commonplace," said ICRC President, Peter Maurer. "Human suffering in conflict is nothing new. But collectively, we're failing in our response to it. Not enough countries, not enough armies, not enough armed groups, are abiding by the fundamental human values enshrined in the Geneva Conventions. When international humanitarian law is violated we all, ultimately, pay the price."
Starting with an upbeat voiceover and stirring music the ad initially depicts a utopian dream, using imagery of what seems to be a safe and prosperous nation. As it continues, the voiceover becomes more threatening and the mood darkens. Viewers begin to see footage of violations of international humanitarian law – with schools and hospitals being bombed, degrading treatment inflicted on prisoners and the use of chemical weapons. The film ends on scenes of destruction and desolation – the result of a "victory by any means" approach to war.
"The Geneva Conventions are based on the principle that wars have limits, and these limits are as necessary today as they ever were," said the Director of international law and policy at the ICRC, Helen Durham.
"Every state around the world has signed up to the Geneva Conventions, which prohibit attacks on medical facilities, torture and the mistreatment of prisoners. Those with the power to make such practices stop have a legal duty to do so. The Geneva Conventions help preserve our humanity during times of conflict. If we lose that, what are any of us left with?"
Viewers of the film are invited to visit the therulesofwar.org website to learn more on the Conventions. The ad will be distributed on Facebook and other social channels. It was created in collaboration with global marketing and technology agency DigitasLBi and customer engagement agency Kitcatt Nohr.
https://youtu.be/hokUQ7pQmoc


Tim is a corporate international tax partner, based in BDO's London office. He has over 25 years of wide-ranging tax experience. Before joining BDO LLP in 2008, Tim worked for other major accountancy firms as well as spending several years working in industry as the head of tax at a global technology company. Relevant experience; Tim enjoys helping clients to devise and implement pragmatic, commercial tax strategies which fit their business needs as they expand internationally. He has significant experience of international mergers and acquisitions, tax compliance and dealing with tax authority challenges. Tim has a particular interest in taxation of software and other intellectual property. He specialises in working with online gaming, technology and similar international businesses.


Here you can view recent pictures of Holiday Inn Express Burnley and the local attractions of Lancashire. Whether you're wondering just how scrummy your breakfast will be, or how clean and modern your room will be – you can find out here! Simply click on a thumbnail to see the full size image.
If you would like know more about anything you see, just get in touch!


text:
Q: Is it possible to clear/remove TapGestureRecognizer after tapped event in Xamarin Forms? I tried to apply this Image to be like ImageButton. Then I want to disable it after Tapped/Clicked event. By the way, I got some problems that I cannot Clear/Remove GestureRecognizers in Tapped event.
This is my code:
Image img = new Image { HorizontalOptions = LayoutOptions.End, VerticalOptions = LayoutOptions.End, Source = ImageSource.FromResource("Ns.ico.png") };

var tapImg = new TapGestureRecognizer();

tapImg.Tapped += (object sender, EventArgs e) =>
{
     img.Source = ImageSource.FromResource("Ns.ico_disable.png");
     img.GestureRecognizers.Clear; //<--- this line is problem
};

img.GestureRecognizers.Add(tapImg);

How should I solve this problem but still the same purpose?

A: Clear is a method call so your code should be img.GestureRecognizers.Clear();
Or you could make a Tapped method and unsubscribe from it when tapped
tapImg.Tapped += Gesture_Tapped;

void Gesture_Tapped(object sender, EventArgs e)
{
    tapImg.Tapped -= Gesture_Tapped;
}

redpajama_set_name: RedPajamaStackExchange


text:
JCILPS calls for action against violators of ILPS rules
IMPHAL: The Joint Committee on Inner Line Permit System (JCILPS) has urged the state government to take up necessary action to repatriate persons belonging to outside states who had entered the state in violation of the rules and regulations of ILPS.
While briefing the media persons JCILPS convener Y.K. Diren told the government that the unauthorized non-locals should all be sent back to their own states at the earliest without favoring any person and also urged local people with resources to avoid bringing in non-locals to the state.
It remarked that a total restriction on entry of outsiders into the state have been enforced since the issuance of a notification served by the state government on March 18, 2020 in connection with Covid-19 pandemic in order to prevent the spread of the disease from incoming arrivals from the red Zone areas.
However, many outsiders were found travelling freely towards the state during this pandemic with the opening of airways which was kept under quarantine in the different state CQC in which the people of the state were born panicked, he added.
While the JCILPS members expressed shock at the free movement non-locals at the Impha International Airport, it also queried on what terms the state security force and the concerned state authority they were allowed to enter the state amidst the travelling restrictions into the state.
It further mentioned that the average people are feeling unhappy and aggrieved at the free movement of the outsiders, and a question has been raised whether the situation had happened due to the state authority being unaware of such an occurrence or it had intentionally given them a free hand.
Hence, JCILPS task force have been formed and deployed at different places in order to control the free movement of the outsiders and to pick out those in staying in violation of the rules at keys entry points of the state. Mentioning the issuance of temporary cards with validity of 6 months, the committee appealed to the state administration to form a specific set of guidelines to be followed by non-Manipuris who are living in rental accommodations.


Set of three rare candleholders by Gunnar Ander for Ystad Metall. Made from glass in saturated red, green and petrol blue, with brass bases and handles wreathed with cane. Make a very warm, colorful group, spread light in a beautiful way.


My two all time favorite TV characters. Buffy supplanted Star Trek as my favorite series (though Game of Thrones may well take the Seven Kingdoms of TV in my mind).
Lack of good remasters on blu-ray and overall 20th Anniversary treatment says otherwise... unfortunately.
Wouldn't there have to be more BtVS and/or Angel spin-off series and movies across decades for the franchise to achieve that level of pop culture phenomenon to people coming of age and yet to be born over the next 30 years ? (going by Star Trek's endurance across 6 decades and Buffy's across 3 decades, so far?) Buffy has two shows and one film. Trek's got 6 (soon to be 7) TV shows and 13 films.
Also, Trek debuted when there were a lot less options on TV. I think more people in the world have Trek as a pop culture touchstone than they do the Buffyverse.
Just curious, which genre brings in more dollars -- sci-fi or fantasy ? (tough to categorize Buffy, but overall I'd put it in the fantasy camp) Which genre has more TV and/or film titles made within its category ?
Kris Rodenberry was already an experienced veteran when he began Star Trek and could devote much of his second half to iterating the 'verse. Joss is by today's standards relatively young as powerhouse project runners go and is still marking out a space for himself. but I agree, Buffyverse projects which are in no sense direct continuations of either show (for example, a Cordelia comics mini-series showing in detail what a Higher Being in the Buffyverse might get up to) would be a good idea for him at some point.


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text:
Poiana Câmpina (în trecut, Poiana, Poiana Sărată și Poiana de Prahova) este o comună în județul Prahova, Muntenia, România, formată din satele Bobolia, Pietrișu, Poiana Câmpina (reședința) și Răgman.

Așezare
Comuna se află pe malul drept al râului Prahova, în dreptul municipiului Câmpina de pe malul celălalt. Este traversată de șoseaua județeană DJ100E care spre est duce la Câmpina (DN1) și mai departe spre Telega; și spre est duce către Provița de Jos și Adunați. La Poiana Câmpina, din acest drum se ramifică șoseaua județeană DJ101P, care duce spre sud de-a lungul Prahovei la Florești și Filipeștii de Târg. Prin comună trece și calea ferată Ploiești-Brașov, pe care este deservită de gara Câmpina și de halta Bobolia.

Demografie

Conform recensământului efectuat în 2011, populația comunei Poiana Câmpina se ridică la  de locuitori, în scădere față de recensământul anterior din 2002, când se înregistraseră  de locuitori. Majoritatea locuitorilor sunt români (97,05%). Pentru 2,4% din populație, apartenența etnică nu este cunoscută.
Din punct de vedere confesional, majoritatea locuitorilor sunt ortodocși (94,84%). Pentru 2,42% din populație, nu este cunoscută apartenența confesională.

Politică și administrație
Comuna Poiana Câmpina este administrată de un primar și un consiliu local compus din 13 consilieri. Primarul, , de la , este în funcție din . Începând cu alegerile locale din 2020, consiliul local are următoarea componență pe partide politice:

Istorie
La sfârșitul secolului al XIX-lea, comuna Poiana cuprindea satele Poiana, Bobolia, Slobozia, Vrăjitoarea și Piatra de Sus, făcând parte din plaiul Prahova al județului Prahova. Pe atunci, în comună exista o școală mixtă cu 68 de elevi (dintre care 24 de fete), 5 mori și o pivă pe râul Prahova, o moară pe râul Doftana și 3 biserici — una aparținând schitului Poiana construit de spătarul Toma Cantacuzino în 1688, una în Bobolia, înființată la 1838 și una în Slobozia înființată de Mihai Cantacuzino, metoh al mănăstirii Sinaia.

Anuarul Socec din 1925 consemnează comuna Poiana în aceeași plasă, cu satele Bobolia, Piatra, Poiana, Slobozia și Vrăjitoarea, cu 2200 de locuitori. În 1931, comuna primește pentru prima oară numele de Poiana de Câmpina și este comună suburbană a comunei urbane Câmpina. În preajma lui 1938, comuna a fost inclusă în plasa Câmpina a aceluiași județ.

În 1950, comuna a fost arondată orașului regional Câmpina din regiunea Prahova și apoi (din 1952) din regiunea Ploiești. Satul Vrăjitoarea a primit în 1964 numele de Pietrișu. În 1968 comuna Poiana Câmpina a redevenit parte a județului Prahova, devenind din nou comună suburbană a orașului Câmpina. În 1989, s-a renunțat la conceptul de comună suburbană, iar Poiana Câmpina a fost subordonată direct județului Prahova.

Monumente istorice
Singurul obiectiv din comuna Poiana Câmpina inclus în lista monumentelor istorice din județul Prahova ca monument de interes local, clasificat ca monument de arhitectură, este fostul schit "Poiana" (secolele al XVII-lea–al XVIII-lea) din satul Poiana Câmpina.

Personalități născute aici
 Ion Bălu (1933 - 2021), critic, istoric literar.

Note

Poiana Câmpina

redpajama_set_name: RedPajamaWikipedia


text:
Peter Carroll
Professor Peter Carroll
House: Clive
Memories of School:
Very happy ones, particularly as a member of Clive House (I was prefected, then 'de-prefected', then re-prefected!) and the School Rugby 1st XV. Persons I remember include Derek Milton and Les Duxbury.
Since School:
Back to the UK, to Leicester University (BA), then Southampton University (MSc), then lectured at Newcastle upon Tyne, then to Fiji (1979-1982) as a lecturer at the University of the South Pacific, followed by 14 years in Brisbane, Australia.
In-between married Sue, had two children, Dan and Sam! In 2001 moved to Hobart, Tasmania, as Dean of the Faculty of Commerce at the University of Tasmania.
I live in Hobart and would love to hear from any Old Cambrians, but particularly those in the 1959-1964 era. Just click on my e-mail link below.
(Registered - 27th Jan 2005)
If anyone wishes to contact Peter, please Click Here to e-mail him


Fantastic home on Eastman Lake. Completely re-modeled with granite counters, knotty pine vaulted ceilings, sun room, office, open floor plan, great room opens out to lakefront. New roof in 2013, greenhouse, landscaped, fenced, private dock, custom kitchen, outdoor cooking area on patio, all furnishings and boats negotiable. Move-in ready.


text:
Albert Guigui (syndicaliste) - (1903-1982)
Albert Guigui (Grand-Rabbin de Belgique) - (1944-..)


text:
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Join A-List directors in helping make Orson Welles final film + interview with producer Filip Jan Rymsza
July 1, 2015 April 28, 2017 filmdoctor 0 Comment Andy Wooding, Filip Jan Rymsza, Filip Jan Rymsza interview Film Doctor, film doctor, Orson Welles, Orson Welles The Other Side of the Wind, The Other side of the Wind movie
Hi Film Folk!
We have a very special post today regarding Citizen Kane actor, writer and director Orson Welles's last film which needs YOUR help to finish.
Join Clint Eastwood, Edgar Wright, J.J. Abrams, Wes Anderson, Peter Bogdanovich, Steven Soderbergh, Brett Ratner, Sofia Coppola and Jason Reitman in making Orson's very last film, The Other Side of the Wind reach screens.
Full campaign details are here but SCROLL DOWN for a chat with producer Filip Jan Rymsza about working with Jurassic World, Back to the Future and Indiana Jones producer Frank Marshall on the project and what he's learned about Mr. Welles himself!
Orson Welles in 1937 – Photograph by Carl Van Vechten
Producer Filip Jan Rymsza said:
"This project came through a mutual friend who told me at Cannes that the rights to the last Orson Welles film was available. I like projects that are challenging by nature. This is unique, it's a total one of a kind. There's never been anything like it and there won't be another project like this again.
It's the centenary of Orson's birth so it's time. This is an event. Let's do this. If not now, then when?
It's been expensive to do from a filmmaking process and it's been hard from a rights stand point, from the way Orson was shooting and with the material being scattered. It's one thing to see it through but it's another, necessary thing, to make sure it's right."
You can own a DIGITAL COPY of the film once completed, along with all sorts of Orson goodies.
There are TICKETS to the LONDON, LA, PARIS and NYC PREMIERES available – so think about foregoing a couple of weekends of drinking or a few cinema trips and be a part of film history!
Filip also described how wonderful it was to see Orson's process close up:
"Going through the process of seeing how he worked as a director and how a film looks when the production was spread out of 6 years was amazing [Welles shot the film from 1970-76). How do you shoot different actors at different times, years apart, within a specific scene? You have to shoot around certain people and that informed his style.
Having big obstructive frames with foreground characters with their backs to the camera and having to shoot that way because you either don't have that actor available or you don't even know who that actor is because you haven't cast him yet. A lot of scenes were done like that with John Huston. He wasn't on it at the time of shooting, so the style was borne from that necessity. It became a signature Welles style – he used it in Othello and Touch of Evil. Being able to keep that in your head when many times you don't have a script supervisor on set. To carry that in your head for over 6 years and have it come together so seamlessly is amazing."
From left: Peter Bogdanovich, Bill Weaver,
(seated), Orson Welles and Oja Kodar on location during the filming of Orson Welles' last unfinished film "The Other Side of the Wind." Photo by José María Castellví
When asked what the legendary director's final film will be like Filip responded:
"My impression of it still lives with the script. It's fascinating as it's Orson Welles making a film about directing and a certain type of director – the swashbuckling, adventurous, macho type. John Huston was that type of director in a way and, of course, John Ford. He influenced Orson more than any other types of director. There's a bit of autobiography in it, a bit of Hemingway in it – it's about the notion of an artist. He's got a lifetime's worth of experience. He said he could have written a Tolstoy type of book on the character because he knew it so well!"
Super Producer
And Filip had an amazing time working with Jurassic World and Back to the Future producer, Frank Marshall:
"He's humble and hands on and he's so dedicated to this film. Disproportionately so! We've been collaborating for about 3 years now. He's been on the set of Jurassic World and whenever there's something that needs to be dealt with he calls immediately and is very much on it. This was a very important part of his career, working with Orson. For everything that I've been told and have read and heard, he (Orson) was not an easy person to work with – he challenged people in many different ways and Frank was no exception. Frank was fired multiple times and never left and stuck around at the hotel and proved himself very necessary."
HELP make Orson's last film a reality, and be a part of history, with a small or large contribution. Spreading the word is HUGELY appreciated by everybody involved, so please do SHARE and RT.
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Any questions/thoughts/experiences of your own??? Leave a comment below!
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New Temporary Hospital To Treat Coronavirus Patients
Military to provide support to temporary hospital with 4,000 beds for coronavirus patients.
Ministry of Defence vehicles deliver protective equipment to hospitals in London (MoD/Crown Copyright)
MILITARY TO SUPPORT NHS ENGLAND
New 4,000 bed hospital in East London will be built with military support
NHS England, with the help of UK Armed Forces, will open a new temporary hospital for treating several thousand more patients with coronavirus.
The Nightingale Hospital (named after Florence Nightingale), which will be run by NHS England and based at East London's Excel Conference centre in London, is expected to admit its first patients early next week. The hospital will comprise two wards able to accommodate 4,000 people in total. Initially, the hospital will provide up to 500 beds equipped with ventilators and oxygen.
Earlier media reports stated the new hospital will be staffed by medics from the Army, Royal Air Force and the Royal Navy. However, the MoD stated that the Nightingale is not a military run field hospital, but an "NHS facility with the military providing support." It is believed the military will provide mostly logistics and infrastructure support to help transform the Excel centre into a temporary hospital. It is unclear whether military medics will actually work alongside NHS staff in the new hospital.
"Our military planners and engineers are working hand-in-hand with the NHS to support their development of the Nightingale Hospital," said Defence Secretary Ben Wallace. "The Armed Forces have already been distributing personal protective equipment to meet the increased demand and we stand ready to assist further in any capacity needed."
Royal Navy surgeons, GPs, nurses and medical specialists are already working alongside their NHS colleagues in hospitals across the country, particularly Derriford in Plymouth and Queen Alexandra in Portsmouth – to treat and look after those affected by the virus.
There were calls by naval charities for RFA Argus, a casualty receiving ship which contains 100 hospital beds, to be relocated from Plymouth to London to help alleviate pressure on the NHS. But unlike USNS Comfort and Mercy, two US naval hospital ships which have been deployed to New York and LA to help alleviate pressure on medical resources in both cities, the Argus, which is operated by the Royal Fleet Auxiliary, is not designated a hospital ship under the Geneva Convention and therefore does not contain the typical markings ( a red cross). Most of the hospital beds on board the Argus are low dependency, with a small number of high dependency beds.
What the UK military lacks in terms of hospital ships, it more than makes up for in terms of providing the highest levels of combat care and combating infectious diseases. In Afghanistan, the British Army's 22 Field Hospital, based in Aldershot,created a world-class trauma hospital. In 2014, more than 100 members of the field hospital were deployed to Sierra Leone to work in a treatment facility for local and international healthcare workers that may have contracted the Ebola virus. Their work in Sierra Leone gave NGOs the confidence and quality of care they needed to remain on the frontline and to successfully defeat the outbreak in West Africa, which was one of the largest outbreaks of Ebola in recent years.
In addition to the new Nightingale hospital, on 18 March, the Defence Secretary announced a COVID Support Force to assist public services with their response to the coronavirus outbreak. As of 23 March, 250 military personnel were deployed to assist civil authorities, and 20,000 armed forces personnel remain on standby to assist.
UK Armed Forces are assisting the government in a multitude of ways. Around 150 personnel from all three services will be trained to drive supplies of oxygen to NHS facilities if required.
The military is also assisting with the distribution and delivery of Personal Protective Equipment to NHS staff. Military aircraft are being used to distribute aid to UK territories and to transfer critically ill COVID-19 patients to hospitals where they can receive better treatment. Joint Helicopter Command is on standby to provide aviation capabilities to access isolated communities that may not have access to urgent medical care during the coronavirus crisis.
"You can expect to see your military across the United Kingdom assisting further over the coming weeks and months," said Lt. Gen. Tyrone Urch, Commander Standing Joint Command.
The head of the Royal Navy, First Sea Lord Admiral Tony Radakin, said: "In times of crisis, we are ready to support and assist the government wherever needed. But our duties remain unchanged, and we continue to deliver on operations around the world."
Despite the global coronavirus threat, Royal Navy operations in the Middle East are continuing as normal with the frigate HMS Argyll traversing the Mediterranean to take over from destroyer HMS Defender patrolling the Gulf and Indian Ocean. The Argyll is due to remain in the Gulf for the next seven months alongside Royal Navy warships – frigate HMS Montrose and minehunters Ledbury, Blyth, Shoreham and Brocklesby.
This story is adpated from announcements made by the UK Ministry of Defence.
US European Command Remains Ready, Despite COVID-19 Threat


We are a family owned plant Nursery offering an extraordinary selection of native plants, flowering and fruit trees and other landscape material. We have a large selection of cacti and other unusual indoor plants. Our experienced and knowledgeable staff is available for advice and consultations both at the Nursery and at your home or business. We also offer container planting service and beautiful pottery.
We hold educational sessions and other seasonal community events to engage people of all ages. Conveniently located in Smarr, Georgia we serve garden enthusiasts in the Middle Georgia area. We specialize in locating specific plants desired by our customers. We plan to expand our business soon to also provide locally farmed products, gardening workshops/crafts and garden themed giftshop.


In an display of excellent (read: unconscionable) communication skills, RadioShack recently followed through with its plans to dismiss 400 people at their Fort Worth headquarters WITH AN EMAIL.
No doubt internal communicator pundits (Shel, Ron, others) are having a stroke about this, and well they should be. HR people, PR people, and any other people with souls should be shaking their heads at this. I'm not sure what to say about this, but…wow.
There must be a logical reason why calling those employees (albeit, not a fun job) into a meeting (one-on-one, 100 at a time, all at once) and have the decency to tell them to their faces that they were being let go was considered an unreasonable concept.
I knew there was a reason besides crappy products, deplorable service, and hapless employees that I didn't like RadioShack.


According to the recent report by IMARC Group, entitled "Precision Agriculture Market: Global Industry Trends, Share, Size, Growth, Opportunity and Forecast 2018-2023", the global precision agriculture market reached a value of around US$ 5 Billion in 2017. Precision agriculture, also known as satellite agriculture, refers to one of the most popular innovations in modern farming. It is the concept of managing accurate variabilities in the farm which helps in producing more output with less agricultural inputs, thereby minimizing the production cost. Precision agriculture relies on specialized equipment, software and IT services so as to provide a real-time data about the condition of soil, ambient air and crops, as well as other important information like local weather predictions and labour costs. This data is used by the farmers to know about the precise planting times, crop rotation and harvesting time.
Nowadays, farmers are increasingly depending on smartphones, internet, WiFi, Zigbe and low-power wide-area (LPWA) for their day-to-day farming activities. These technologies assist in efficiently planning and executing several operations, such as planting, purchasing, harvesting and inventory control, with zero negligence and errors. Moreover, these help in spreading awareness amongst small as well as large farmers about the latest developments in the industry. This acts as one of the major factors which has led to a rise in the demand for precision agriculture across the globe. Further, agriculture development authorities as well as state-owned enterprises are cooperating together with agro-processing organizations, and food and beverage manufacturers in order to promote the growth of sustainable agricultural activities. This aims at enhancing the productivity, food security and efficiency of agricultural process, in turn, proliferating the demand for precision agriculture. Looking forward, the market value is projected to reach US$ 10 Billion by 2023, expanding at a CAGR of more than 13% during 2018-2023.
Based on technology, the market has been segregated as GNSS/GPS systems, GIS, remote sensing and variable rate technology (VRT). Currently, GNSS/GPS systems represent the largest technology segment of precision agriculture as the accuracy of these technologies have improved, which in turn, is enabling a large-scale adoption of precision agriculture for geo-positioning and field-mapping.
On the basis of type, the market has been segmented as automation and control systems, sensing and monitoring devices, and farm management systems. At present, automation and control systems are mostly preferred in precision agriculture as they aid in minimizing the labour costs as well as electricity costs.
The market is also bifurcated on the basis of component which include hardware and software components. Amongst these, hardware components account for the majority of the market share as their consistent evolution has resulted in the introduction of new devices with enhanced location and data processing capabilities.
Based on application, yield monitoring represents the biggest segment, followed by crop scouting, mapping, soil monitoring and precision irrigation. Yield monitoring systems are used for measuring and recording information based on the grain flow, grain moisture and farm area covered. Additionally, they help the farmer in maximizing the crop yield and understanding the field variability.
Region-wise, the market has been classified into North America, Europe, Asia Pacific, Middle East and Africa, and Latin America. Amongst these, North America is the leading market due to increasing adoption of precision agriculture in various countries like the United States and Canada.
The competitive landscape of the market has also been examined with some of the key players being Agribotix, Bayer CropScience AG, Case IH Agriculture, ClearAg Operations, Conservis Corporation, Deere & Company, Farmers Edge Inc., Fairport Farm Software, AGCO Corporation, Granular AG, Grownetics Inc., IBM, Mapshots Inc., Raven Industries Inc. and Relx Group.


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Why Philanthropy Needs Reinventing
by michaeld | Jul 1, 2013 | Columns | 0 comments
By Eric Friedman
We have an unhealthy relationship with charitable giving. Giving USA reports that Americans donate about $300 billion annually to nonprofits-averaging about $1000 per person. We love the charities we give to and feel good when we give to them. This is a nation of do-gooders.
At the same time, philanthropy is broken and almost everyone knows it. The causes that receive the most donations are not necessarily the ones that make the greatest impact. Instead, the personal whims and preferences of donors determine where dollars flow regardless of need or impact. Donors typically have confidence, which is largely based on reputation and trust, in the organizations to which they give. Many donors do not know how to define a "high performing" nonprofit, let alone how to identify one or assess whether there are more worthy charities. The donor community has a reputation for responding to inspirational anecdotes in professionally-written fundraising material rather than asking for meaningful evidence of performance; simultaneously, they express concerns about high overhead costs and program effectiveness. Philanthropy is broken, and it needs to be reinvented.
But how? There are three key areas that can create a domino effect of improving charitable giving.
1. There must be greater honesty about charitable giving. Although the media often reports on fraud and waste in the charitable world, such instances are not the most problematic lack of honesty. The larger issue is more common and very subtle. For example, today's dominant paradigm of giving is that donors give to the causes they care about and have personal ties to–the university they attended, the illness that touched their lives, or the nonprofits promoting the type of art or music they enjoy. This type of giving itself is not dishonest, but the fact that so few people acknowledge that giving based on the personal preferences of the donor dramatically reduces the impact of the donations. While these are "good" causes, this type of giving is very different from trying to make the biggest dent in the causes that are most effective at helping others.
Is philanthropy about helping those in need or pursuing the personal passions of the donor? If it is the former, then shouldn't a central tenet be to try to provide the most help possible? In this case, such donor-driven strategies should be widely regarded as failed philanthropy.
Being honest is not just about not lying, but also requires a certain level of frankness. Not every good cause is equally good, and not every donor is equally deserving of praise. As long as the philanthropic community views those who donate millions to their favorite opera houses to be as generous as those who help the poorest people in the world, there is a lack of honesty. We should reserve the highest public praise for donors with the most altruistic motives and better understand our own reasons for giving. This type of honesty would lead donors to be less whimsical and more thoughtful about their giving choices.
2. There must be better ways for donors to evaluate nonprofits. Although there are several charity rating agencies, their weaknesses are often so glaring as to make them virtually useless. Most focus almost exclusively on financial metrics such as the percentage of donations going to fundraising and overhead costs. This is far from a good measure of performance–just because a charity only spends 10% on fundraising and overhead costs doesn't mean the programs funded by the remaining 90% are effective. Even among the charity rating agencies that have advanced beyond pure financial measures of performance, most give their top ratings to hundreds of extremely different charities, effectively watering down the value of their ratings and expressing little conviction about what really works best. These rating agencies can be counterproductive to their own missions, as they give donors a false sense of confidence when providing high ratings for charities that may not be very effective.
There are only a small number of charity evaluators that present their views of the best of the best charities; the most notable are GiveWell and Giving What We Can. More of these evaluators, as well as a higher profile for the existing ones, would go a long way to make charitable giving more effective. Such charity evaluators will develop and thrive as donors express more interest in them.
3. Donors must take more responsibility for the impact of their giving. According to a study by Hope Consulting, only 35% of donors do any research before giving, and only 9% do more than two hours of research. Instead of giving with all "heart" and no "head," a better balance is needed. Donors must think about not just what causes tug at their hearts, but also what are the greatest problems and where are charities most likely to make an impact. The irony is that as donors make their giving decisions based less on emotional appeals and more on evidence about what works best, the increased conviction they have in their giving will ultimately provide even greater emotional satisfaction.
A reinvented, better world of philanthropy will not happen overnight. But it can happen gradually, as one donor at a time takes responsibility for their own giving.
Eric Friedman is an individual donor who has spent several years trying to understand how to maximize the impact of his giving, including traveling to Africa to see his giving in action. He is also an actuary. He graduated from Stanford University with majors in mathematics and economics. He lives in Oak Park, Illinois.
Website: http://www.reinventingphilanthropy.com
Reinventing Philanthropy: A Framework for More Effective Giving will be available for purchase in September 2013 from www.amazon.com, www.barnesandnoble.com and through all major booksellers