Early procedures for the assay of vitamin B.sub.12 (also referred to as cobalamin or cyanocobalamin) employed bacterial species, such as Euglena gracius or lactobacillus leichmannie which lacked the intrinsic ability to synthesize vitamin B.sub.12. Such microbiological assays determined vitamin B.sub.12 levels in an extract of a plasma sample from a human subject by measuring the growth of the vitamin B.sub.12 sensitive organism in a vitamin B.sub.12 free medium to which a known amount of the plasma sample extract had been added. Since the growth of the sensitive organism was, within certain limits, proportional to the vitamin B.sub.12 concentration in the medium, it was possible by measuring a suitable growth parameter, such as optical density of the test solution after incubation for a selected period of time and at a certain selected temperature, and comparing this parameter to values observed by running the same test with samples containing different, known concentrations of vitamin B.sub.12 to determine the concentration of vitamin B.sub.12 in the plasma sample extract.
The microbiological assay, while being a selective and sensitive assay, is difficult to perform requiring sterile techniques and thus requires highly skilled technicians. Moreover, it is difficult to maintain colonies of the required vitamin B.sub.12 sensitive microorganisms. Additionally, results are not available for several days, and the tests cannot be carried out on patients who are taking antibiotics or other drugs. Thus when radioreceptor assays were subsequently developed, the art rapidly dropped the microbiological methodology and switched to the more convenient assay.
The radioreceptor assay utilized competitive binding inhibition by vitamin B.sub.12 contained in a patient's plasma sample to the binding of known concentrations of a radiolabeled vitamin B.sub.12 derivative, i.e., .sup.57 Co-vitamin B.sub.12 to receptor sites on an intrinsic factor (IF) preparation. The intrinsic factor preparations were derived from the stomachs of higher mammals, primarily hogs. These preparations were impure in the sense that they were contaminated with substantial, even major amounts of R-proteins. Unlike intrinsic factor, which binds vitamin B.sub.12 in a highly selective manner, the R proteins have non-specific binding characteristics and will bind to the natural analogs of vitamin B.sub.12 which are closely related structurally to vitamin B.sub.12 but which are biologically inactive.
This contamination of intrinsic factor with R-protein was not considered by the art to affect the assay procedure, since the natural analogs of vitamin B.sub.12 were known to be present normally in the human gut but were not believed to be absorbed and present in the blood stream and tissue. However, in a paper by Kolhouse, et al., in The New England Journal of Medicine, Vol. 299, No. 15, pp. 785-789 (Oct. 12, 1978), it was demonstrated that such biologically inactive analogs do, in fact, naturally exist in the human blood stream. These analogs were measured as true cobalamin by the commercially available radioassay for vitamin B.sub.12, and this was believed to be the reason for observed discrepancies wherein serum B.sub.12 values obtained by radioassays were consistently higher than those obtained with microbiologic assays.
The clinical implications of these discrepancies were noted by Cooper and Whitehead in the same issue of The New England Journal of Medicine at pp. 816-818 in a paper entitled, "Evidence That Some Patients With Pernicious Anemia Are Not Recognized By Radiodilution Assay For Cobalamin In Serum". These authors in their discussion of the situation make the following statement:
"This reliability [of the microbiological assay] appears not be be true of the radiodilution assays tested. The data show that whereas these assays give quantitative and reproducible results, they did not detect all patients with clinically proved deficiency of cobalamin . . . .
These data are of concern because of the widespread use of radiodilution assay and the use of assay for cobalamin in serum to screen for deficiency of the vitamin . . . . It appears that analogues of cobalamin are present in human serum and plasma and that these analogues bind effectively to non-intrinsic-factor cobalamin binders in most commercial preparations of `intrinsic factor. ` . . . The well documented specificity of authentic intrinsic factor for cobalamin itself indicates that radiodilution assays using pure authentic intrinsic factor would provide a reliable method for determining cobalamin in serum."
After these two papers were published there has been intensive activity by commercial vitamin B.sub.12 radioassay manufacturers to modify their kits to try to overcome these serious problems. As described by Kubasik, et al., Clin. Chem. 26/5, 598(1980), there have been two basic approaches to achieve a solution:
"1. Negate the R-protein binding sites: This can be accomplished by flooding the IF-R protein combination binder with an analog such as cobinamide. Cobinamide will not bind the IT, but will bind to R-proteins. If added in great excess (&gt;100-fold), it will quench all the non-specific cobalamin binding sites
2. Purify the IF: IF concentrate can be purified by prolonged treatment with proteolytic enzymes, such as trypsine (E.C. 3.4.21.4) or chymotrypsin (E.C.-3.4.21.1), or both, or by affinity chromatography."
Evaluation of these approaches by the authors resulted in the following conclusion:
"From our data presented here, it appears that although the `cobinamide`-blocked binder can be used, we agree . . . that pure or `purified` IF would be the better choice of a binder, for analytical reasons. Any possible non-specific effects due to other binders would be eliminated."
Criteria which were employed in established purity of the intrinsic factor preparations were as follows:
1. Binding of bioactive cabalamin was completely inhibited when active IF was first incubated with antibody to IF.
2. Comparing binders in assays performed at acidic and basic pH, and at basic pH in the presence of cobalamin analogs. A purified IF preparation exhibits very low cobalamin binding ability at acidic pH, and the binding at basic pH will be little affected by the presence of cobalamin analogs.
3. Purified IF does not measure "cobinamide" (cyanohydroxycobinamide) even at extremely high concentrations.