In general, immunoassays are produced by first determining epitopes that are specifically associated with a virus and then determining which of the epitopes is preferred for the assay being developed. When the immunodominant epitopes are isolated, their sequences are determined, and genetic material for producing the immunodominant epitopes is produced. Methods of producing proteins by either chemical or biological means are known, as are assays used to detect the presence of antibodies to particular epitopes.
In producing immunoassays the overall object is to obtain an immunoassay which is both highly sensitive and highly selective. More specifically, the immunoassay must be designed such that it can detect even very low levels of the material it is designed to detect, i.e., it is highly sensitive. An assay having a high degree of sensitivity ensures that a sample, which has been tested, is not contaminated with the material the assay is designed to detect. For example, a highly sensitive assay that detects even the slightest presence of antibodies for a given virus is desirable in that it makes it possible to detect and thus discard samples that contain any amount of the antibody indicating that the samples contain the virus.
Although a high degree of sensitivity is desirable in an assay, it is not desirable if the assay is falsely indicating the presence of the material, i.e. the assay is providing a false positive result. Such false positive results can occur when the analyte has a high degree of similarity with another material present in the sample. The ability on an assay to differentiate between two similar but different materials relates to its selectivity.
An immunoassay with a high degree of selectivity will detect the presence of a material being assayed for even when that material is present in the sample in combination with other materials having a similar structure. Thus, a highly selective immunoassay will eliminate most false positive results. In general, as selectivity increases sensitivity decreases. This occurs, in part, due to the high degree of variability in viruses. Assays which are designed to be highly sensitive must take into account the high degree of variability between different viruses. As virus variability is accommodated to improve sensitivity, the selectivity decreases. Alternatively, as one produces an immunoassay that is more and more selective with respect to a particular virus, the likelihood of the assay becoming so selective as to have decreased sensitivity increases.
To a large extent the problem of providing for improved selectivity (less false positives) is dealt with by searching for and finding the most immunodominant epitopes. The problem of sensitivity (low concentration detection) is dealt with by providing immunodominant epitopes from a variety of different regions of the virus.
Current assays are designed to utilize relatively few peptides selected as “major epitopes” or highly immunodominant epitopes. The assay sensitivity is dependent on the number of major epitopes available on the solid support. If the availability of epitopes is limited by the number of peptides that can be coated on the solid phase, then that assay will have reduced sensitivity. These results can be demonstrated as poor assay dilution sensitivity and poor seroconversion sensitivities and/or false negative determinations (Chien, D. Y. et al. (1993) J. Gastroenterology and Hepatology 8:S33–39).
There is currently a need to improve the sensitivity and selectivity of assays for antibodies to pathogens in biological fluids and thereby improve diagnosis of pathogen infection resulting in improved screening of blood supplies.