Irina Ionescu-Matiu

Utilization of the biotin/avidin system to amplify the sensitivity of the enzyme-linked immunosorbent assay (ELISA)

The biotin/avidin system was incorporated into the enzyme-linked immunosorbent assay (ELISA) technique to increase the sensitivity of the standard ELISA for the detection of mouse antibody to hepatitis B surface antigen ((anti-HBs and HBsAg, respectively). Two biotin/avidin ELISA designs were studied. In both assays, 96-well polystyrene plates were coated with HBsAg, post-coated with 0.5% gelatin and incubated with dilutions of mouse anti-HBs. In the biotin/avidin (BA) ELISA, reagents were added to antibody reacted wells in the following sequence: biotinylated goat anti-mouse IgG (b-GAMG), avidin-alkaline phosphatase (Av-AP) and substrate. The order of reactants after mouse antibody in the biotin/avidin/biotin (BAB) ELISA was b-GAMG, avidin, biotinylated alkaline phosphatase (b-AP) and substrate. The sensitivities of BA ELISA, BAB ELISA and a standard ELISA using a glutaraldehyde conjugated goat anti-mouse enzyme were compared to AUSAB (a commercial radioimmunoassay) using a panel of 23 mouse anti-HBs sera. All 3 ELISAs were more sensitive than AUSAB; the standard ELISA, BAB ELISA and BA ELISA were respectively 50, 1173 and 4134 times more sensitive than AUSAB for detection of mouse anti-HBs activity.

Characterization of anti-hepatitis B surface antigen monoclonal antibodies.

17 monoclonal antibodies generated against purified hepatitis B surface antigen (HBsAg), subtype ayw, were characterized by solid-phase radioimmunoassays. Eleven of these antibodies had specificity against the group-specific alpha determinant of HBsAg, two demonstrated antibody activity against the w HBsAg subtype, one against human serum albumin, and three against human IgG. All monoclonal antibodies were of the IgG class.

A common human anti-hepatitis B surface antigen idiotype is associated with the group a conformation-dependent antigenic determinant.

Abstract Human antibodies that express a common anti-hepatitis B surface antigen idiotype were directed against the group-specific a determinant. The ability of hepatitis B surface antigen (HBsAg)-derived polypeptides to inhibit the idiotype-anti-idiotype reaction was dependent on conformation, since denatured HBsAg viral polypeptides virtually lost their inhibitor capacity when compared to native polypeptides. This report further characterizes a common idiotype associated with human antibodies to HBsAg.

Antigenic relationship of a hepatitis B surface antigen-derived polypeptide and human serum albumin.

Four major polypeptides with mol. wt. of 22000, 25000, 52000 and 68000 were isolated from solubilized preparations of hepatitis type B surface antigen (HBsAg). These four populations, referred to as P22, P25, P52 and P68, respectively, were used to immunize guinea-pigs. Guinea-pigs were also inoculated with HBsAg and with purified human serum albumin (HuSA). These antisera were utilized to establish that intact HBsAg particles are associated with HuSA antigenic reactivity. HuSA antigenic determinants were associated with purified preparations of P68. HuSA antigenic activity was not detected with purified preparations of P22, P25 and P52 or with respective specific antisera to each of the above. However, purified P68 contained the antigenic determinants of both host protein and hepatitis B virus-specified protein origin.

Immunogenicity of Conjugates and Micelles of Synthetic Hepatitis B Surface Antigen Peptides

A cyclic peptide containing the amino acid sequence 122 through 137 of the major hepatitis B surface antigen (HBsAg) polypeptide was synthesized. The immunogenicity of this synthetic peptide, aggregated in micelles or covalently coupled to tetanus toxoid, was assessed in mice. Antibodies against HBsAg (anti-HBs) were obtained with both preparations, administered either in saline suspension or adsorbed on aluminum gel. The peptide-tetanus toxoid conjugate was more immunogenic than the peptide micelles, producing high levels of specific anti-HBs.

Evidence for the presence of repeating antigenic determinants in the major and minor polypeptides derived from hepatitis B surface antigen

Abstract The immunogenicity of the four major and seven minor hepatitis B surface antigen (HBsAg)-derived polypeptides was evaluated in guinea pigs and compared to that of 22-nm HBsAg particles. Both humoral and cell-mediated immune responses were determined. All antisera reacted with homologous HBsAg/ ayw and P22/ ayw . However, the glycosylated polypeptides (P25, P27, P31, and P52) generally elicited the most intense immune responses. At least nine polypeptides induced antibodies which also reacted with heterologous HBsAg/ adw particles and P22/ adw. Whole HBsAg/ ayw particles induced transformation in lymphocytes obtained from guinea pigs immunized with the major or minor polypeptides. These data tend to demonstrate that all HBsAg-derived polypeptides contain in their structure the group-specific and at least one of the subtype-specific antigenic determinants, and these determinants are also exposed on the surface of the HBsAg particle. It seems possible that a limited nucleotide sequence confers the same virus specificities to most if not all of the constitutive proteins of HBsAg.

Hepatitis B Surface Antigen Polypeptide and Synthetic Peptide Vaccines

The hepatitis B vaccine recently licensed for use in the United States has undergone several field trials in high-risk populations and has proven to be immunogenic, efficient and safe. The hepatitis vaccine is unique in that the hepatitis B virus (HBV), the ultimate source of the vaccine, cannot be grown in tissue culture. The immunizing antigen, purified from plasma of healthy human carriers of HBV, consists of noninfectious 22 nm particles of hepatitis B surface antigen (HBsAg), produced as excess viral surface protein and released in the bloodstream. Although the HBsAg particles are highly purified and inactivated, there is some concern that the vaccine may contain traces of host material or infective adventitious agents, which may trigger undesirable effects in the recipients. Ideally, a hepatitis B vaccine candidate should have the following properties: 1) be free of normal human serum protein contaminants, 2) be free of potential residual infectious HBV, 3) be free of other viruses and nucleic acid, 4) have a high degree of immunogenicity when administered in conjunction with an adjuvant suitable for use in humans, 5) be independent of human HBV carriers as a source of immunogenic material, 6) have a reproducible composition, 7) cost less than the present vaccine, and 8) be available in unlimited supplies.

Comparison of Three Immunoassays for Screening Anti-Hepatitis B Hybridomas

Three micro solid phase immunoassays were developed for screening secreting anti-hepatitis B hybridoma clones: a micro-solid phase radioimmunoassay (micro-SPRIA)1 and two enzyme-linked immunosorbent assays (ELISA). In all three procedures, aliquots of hybridoma culture fluid were first added to antigen-coated wells. Goat anti-mouse IgG (GtaM) was used as second antibody. For micro-SPRIA, Gt α m was iodinated by the chloramine T method. For the ELISAs, we compared two methods of coupling the antibody to alkaline phosphatase (AP): with glutaraldehyde (ELISA-glutaraldehyde) and with N-succinimidyl 3-(2-pyridyldi-thio) propinate (SPDP) (ELISA-SPDP). All three tests were more sensitive than the commercially available AUSAB (Abbott), which could not detect low enough levels of antibody to be used for hybridoma screening. ELISA-glutaraldehyde was the least sensitive of the three and produced a number of false positive results. Micro-SPRIA was 5 times more sensitive than ELISA-glutaraldehyde, and ELISA-SPDP was 2.5 times more sensitive than micro-SPRIA. This higher sensitivity of ELISA-SPDP allowed us to detect a greater number of secreting clones at an earlier time after fusion.