PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 50382

Immunological and biophysical properties of hepatitis B antigen labeled by the chloramine-T and by the lactoperoxidase methods.

F B Hollinger·M Morrison·R Chairez·G R Dreesman
View original source ↗Export metadata ↓

Abstract

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.

Explore related subjects

ChloraminesEpitopesHepatitis BHepatitis B AntibodiesHepatitis B AntigensHydrogen-Ion ConcentrationIodine RadioisotopesIsoelectric FocusingIsotope LabelingLactoperoxidaseProtein BindingRadioimmunoassaySodiumJournal ArticleResearch Support, U.S. Gov't, P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

F B Hollinger, M Morrison, R Chairez, G R Dreesman. 1975. Immunological and biophysical properties of hepatitis B antigen labeled by the chloramine-T and by the lactoperoxidase methods.. https://doi.org/10.1016/0022-1759(75)90083-6

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed1979

13C NMR analysis of methionine sulfoxide in protein.

The 13C epsilon NMR signal of methionine sulfoxide is 22.6 ppm downfield from that of methionine. This affords a method by which the extent of methionine oxidation can be determined in intact protein. We demonstrate the utility of this approach with beta-galactosidase enriched with 13C in its methionine methyls.

Chloramines↗
PubMed1979

Myeloperoxidase, hydrogen peroxide, chloride antimicrobial system: nitrogen-chlorine derivatives of bacterial components in bactericidal action against Escherichia coli.

In the presence of Escherichia coli, myeloperoxidase-catalyzed oxidation of chloride ion resulted in formation of long-lived chloramine and/or chloramide derivatives of bacterial components. The same amount of these nitrogen-chlorine (N-Cl) derivatives was obtained with either hypochlorous acid (HOCl) or the myeloperoxidase system, indicating that myeloperoxidase catalyzed the oxidation of chloride to HOCl. Identical killing was obtained with HOCl or the myeloperoxidase system. About 30 to 50% of the oxidizing equivalents of HOCl were detected as N-Cl derivatives of peptides or peptide fragments that were released from the bacteria. The apparent molecular weight distribution of the peptides decreased with increasing amounts of HOCl, suggesting that peptides were fragmented by oxidative cleavage of chloramide derivatives of peptide bonds. The remaining 50 to 70% of the oxidizing equivalents of HOCl were rapidly consumed in peptide bond cleavage or the oxidation of other bacterial components. There was a close correspondence between the oxidation of bacterial sulfhydryls and bactericidal action. The N-Cl derivatives were lost and the oxidation of bacterial sulfhydryls increased over a period of several h at 37 degrees C. These changes were accompanied by increased killing. The increase in sulfhydryl oxidation and killing could be prevented by washing the bacteria to remove the N-Cl derivatives. Therefore, the N-Cl derivatives could oxidize bacterial components long after the myeloperoxidase-catalyzed oxidation of chloride was complete.

Chloramines↗