Immunological and biophysical properties of hepatitis B antigen labeled by the chloramine-T and by the lactoperoxidase methods.
Optimal conditions were sought for the radiolabeling of microgram quantities of hepatitis B surface antigen (HBs Ag) employing the chloramine-T or lactoperoxidase iodination procedures. Preparations of HBsAg labeled by these procedures are referred to as chloramine-T preparations and lactoperoxidase preparations, respectively. Labeled HBsAg having specific activities between 10-20 muCi/mug were found to display the greatest degree of sensitivity for unlabeled HBsAg and for anti-HBs using a double-antibody radioimmunoassay (RIA-DA). Increasing the specific activity above this level redulted in a decreased affinity of labeled 1251-HBs Ag for anti-HBs, indicating that soluble antigenic alterations had developed. At equivalent specific activities, chloramine-T preparations competed less effectively for unlabeled HBs Ag than lactoperoxidase preparations, and anti-HBs endpoint titers were slightly reduced, especially among preparations of high specific activity (greater than or equal to 65 muCi/mug). Chloramine-T preparations of HBs Ag (sp. act. 15--30 muCi/mug) showed essentially no antigenic deterioration over a 2-month period at minus 196 degrees C or minus 70 degrees C. Utilization of optimally labeled 1251-HBs Ag has increased the sensitivity of the RIA-DA for unlabeled HBs Ag 30-fold to a level below 1 ng/ml and enhanced antiamine-T method revealed that only the most acidic population was labeled (pH 3.75+/-0.5). In contrast, six antigenic components with distinct pI values ranging from 3.7 to 5.2 were detected by RIA-DA in both unlabeled HBs ag and in the chloramine-T preparation. This indicated that the chloramine-T method did not radically change the relative number or charge of each of the pI populations present in purified preparations of HBs Ag. Analysis of HBs Ag iodinated by the lactoperoxidase procedure revealed the presence of three of four populations of particles with pI values ranging from 3.9 to 4.5, suggesting that this procedure labels HBs Ag more uniformly.
D002700:Chloramines; D000939:Epitopes; D006509:Hepatitis B / Q000276:immunology; D006510:Hepatitis B Antibodies; D006511:Hepatitis B Antigens; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D007525:Isoelectric Focusing; D007553:Isotope Labeling / Q000379:methods*; D007784:Lactoperoxidase; D011485:Protein Binding; D011863:Radioimmunoassay; D012964:Sodium / Q000378:metabolism
D002700:Chloramines; D000939:Epitopes; D006510:Hepatitis B Antibodies; D006511:Hepatitis B Antigens; D007457:Iodine Radioisotopes; D012964:Sodium; D007784:Lactoperoxidase