Use of normal serum as a satisfactory binding protein in radioassay of Vitamin B12

Abstract

Recent investigations of radioassay procedures for serum Vitamin B12 have shown a disturbing discrepancy between the clinical state of Vitamin B12 deficiency and the normal result produced for serum Vitamin B12 in a number of patients. It was estimated that this discrepancy occurred in more than 10% of Vitamin B12 deficient patients, a frequency which is unacceptably high. Investigation of the problem by Kolhouse <i>et al</i>. revealed that serum contained Vitamin B12 analogues which are not biologically active in man but are measured as Vitamin B12 in many commercial assays because of the contamination of the intrinsic factor binder with ‘R' protein. The ‘R' protein can bind the analogues whereas the intrinsic factor is specific for Vitamin B12. Kolhouse recommended that clinical laboratories and commercial manufacturers change their assays and use only purified intrinsic factor as binder, and this applied to laboratories which used human serum as the source of Vitamin B12 binding protein. The purpose of this report is to present evidence that normal human serum is a satisfactory source of Vitamin B12 binding protein and that assays using serum did not suffer the same problems that occur with commercial kits. As in other laboratories, the awareness of the authors of the problem started with a clear discrepancy between serum Vitamin B12 results using a commercial kit (Quanta-Count B12 Radioassay, Bio Rad Laboratories) and the clinical diagnosis of 5 patients. However, instead of repeating the Vitamin B12 analyses by a microbiological method, the analyses were repeated by a radioassay using normal human serum as binder. Results consistent with the patient's diagnoses were obtained in all cases. Subsequently 49 specimens with mainly low Vitamin B12 levels were assayed by the commercial radioassay kit, radioassay using serum binder and microbiological assay. Six samples gave normal levels by commercial kit but low levels by the serum binder radioassay and microbiological assay. The agreement between the serum binder radioassay and microbiological assay was good overall. Over a 12 mth period all serum radioassay results for Vitamin B12 below 350 pg/ml were checked by microbiological assay. A total of 264 samples were assayed in this way. The results showed good agreement between the 2 methods, with no major difference in classification evident. Thus it is concluded that normal human serum is a satisfactory binder for radioassay of Vitamin B12.