Gordon R. Dreesman

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.

Herpesvirus-induced antigens in squamous-cell carcinoma in situ of the vulva.

Antigens induced by herpes simplex virus Type 2 (HSV2) were found to be associated with squamous-cell carcinoma in situ of the vulva in nine of 10 patients. The HSV2-induced antigens are DNA-binding proteins that are normally present in the nuclei of infected cells, but in the cells of the carcinomas in situ they were found in the cytoplasm. Whole-virion structural antigens were not present, although there was serologic evidence of previous HSV2 infection in patients tested for the presence of antibodies. The observations reported here and the recent parallel rise in the prevalence of both HSV2 infections and vulvar carcinoma in situ, particularly in women under 40 years of age, suggest an association of HSV2 infection with this type of neoplasia, the nature of which remains to be determined.

Defective Virions of Herpes Simplex Viruses

Serial undiluted passage of clonally purified herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in 4 different types of cells resulted in (i) partial loss (84–98%) of infectivity and (ii) the app

Cellular and humoral immunity in guinea pigs to two major polypeptides derived from hepatitis B surface antigen.

Guinea pigs immunized with hepatitis B surface antigen (HBsAg), types adw, adr and ayw, and with two major polypeptides derived from HBsAg/adw developed cell-mediated immunity as determined by the macrophage migration inhibition assay. Peritoneal exudate cells from animals immunized with a 22000- or a 25000-mol. wt. polypeptide derived from HBsAg/adw showed significant migration inhibition after challenge with either polypeptide or with purified HBsAg. Significant inhibition of macrophage migration was not observed when polypeptide-sensitized cells were challenged with normal human serum or with normal human liver extract. Similarly, a cell-mediated immune response was not observed in peritoneal exudate cells from animals sensitized to normal human serum or normal human liver extract which were challenged with either of the polypeptides. The humoral immune response to either of the polypeptides, as measured by radioimmunoassay, was substantially lower than that observed in animals immunized with intact particles. This apparent difference between cellular and humoral responses suggests that the macrophage migration assay is a sensitive indicator of the immunogenicity of the smaller mol. wt. HBsAg-derived polypeptides in guinea pigs.

Application of a modified computer algorithm in determining potential antigenic determinants associated with the AIDS virus glycoprotein

A computer program was developed for use on an Apple IIe that utilized the parameters developed by Hopp and Woods (T. P. Hopp and K. R. Woods, 1983, Mol. Immunol. 20, 483-489) for predicting the hydrophilic regions of a given protein. This program will produce a listing of the hydrophilic sequence averages and graphically illustrates the peak areas. The hydrophilic averages over a hexapeptide length can be used to predict protein structure. In conjunction with the Chou-Fasman predictive scheme for protein secondary structure determination, the possible antigenic determinants for the envelope glycoprotein of three viruses isolated from patients with acquired immunodeficiency syndrome (AIDS) were predicted. These predicted determinants could be used to generate synthetic peptides that represent a potential vaccine preparation or in developing a diagnostic assay that specifically detects the agent.

Coxsackievirus aggregates in muscle cells of a polymyositis patient.

Muscle biopsy specimens of a patient with polymyositis showed crystalline structures resembling picornavirus aggregates within muscle cells. The patients serum reacted in an indirect immunofluorescence assay with autologous muscle cells. A strongly positive immunofluorescence staining was also noted when a section of muscle tissue was reacted with coxsackievirus A9 antiserum, and a weakly positive reaction was noted with coxsackievirus B2 antiserum. No staining was observed after treatment with antiserum to poliovirus type 1 or echovirus types 11 and 22.

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.

Alteration of Hepatitis B Antigen (HB Ag) Determinants by Reduction and Alkylation

Previous studies have demonstrated that sulphydryl groups and/or disulphide bonds play an important role in the tertiary structure of a number of animal viruses since biological activities such as infectivity and haemagglutination are destroyed by treatment with either alkylating (sulphydryl binding) or reducing reagents (Allison, Buckland & Andrewes, 1962; Carver & Seto, 1968; Hare & Chan, 1968; Neurath & Rubin, 1968) The differential effect of these different reagents is illustrated by the fact that a reducing agent such as dithiothreitol (DTT) destroys the infectivity of many enteroviruses, but these same viruses are unaffected by treatment with sulphydryl binding reagents (Allison et al. 1962; Carver & Seto, 1968). Amino acid analyses of highly purified preparations of hepatitis B antigen (HB Ag) have shown that the 20 to 25 nm particles contain increased levels of cysteine (or cystine) amino acid residues (4.8% molar concentration) when compared to other animal viruses (0.3 to 2.0%) (Dreesman et al. 1972a).

Glycoproteins Associated with Hepatitis B Antigen

The total carbohydrate content of purified hepatitis B antigen (HB Ag) ranged between 3.6 and 6.5% as determined by the phenol-sulfuric acid method. Disc-sodium dodecyl sulfate PAGE of purified HB Ag followed by periodate-Schiff staining revealed one major and two minor glycoproteins with molecular weights of 22,000,27,000 and 32,000 daltons, respectively. Both ad and ay subtypes of HB Ag contained these glycoproteins.