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Biomedical subjects

G R Dreesman

Publications and source records attributed to G R Dreesman.

At least 109 records · Page 6Linked to original sources
PubMed1975

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.

Chloramines↗
PubMed1975

Cell-mediated immunity in guinea pigs to subunits derived from hepatitis B surface antigen.

Guinea pigs immunized wit- hepatitis B surface antigen (HBSAg), types adw and ayw, and with two polypeptides (400,000 and 24,000 molecular weight) developed cell-mediated immunity (CMI), as determined by radioimmunoassay. Peritoneal exudate (PE) cells from guinea pigs immunized with the 40,000-molecular-weight polypeptide migrated poorly (30 to 88% inhibition) after challenge with the immunizing subunit or with purified HBSAg, type gdw or ayw. Response of the same PE cells to the 24,000-molecular-weight subunit was significantly reduced. Similar but less striking evidence for CMI was observed was PE cells derived from guinea pigs inoculated with purified type-specific HBSAg or with the 24,000-molecular-weight polypeptide. Humoral responses were predictable and showed a reasonable degree of correlation with the CMI response. PE cells from control animals inoculated with normal human serum or polyacrylamide gel were not inhibited after challenge with purified preparations of HBSAg or with the 400,000-molecular-weight polypeptide, but did show CMI with their respective immunogens. In addition, PE cells from guinea pigs inoculated with normal human serum were inhibited from migrating after challenge with the 24,000-molecular-weight subunit, suggesting that the latter may contain an antigenic determinant related to a human serum protein.

Animals↗
PubMed1975

Comparative biophysical studies of hepatitis B antigen, subtypes adw and ayw.

Comparative biophysical and biochemical analyses were performed on purified preparations of hepatitis B antigen (HBs Ag) subtypes adw and ayw, including isoelectric pH evaluations, analysis of the different morphological forms, molecular weight determinations, and analysis of the polypeptides by polyacrylamide gel electrophoresis, Both HBs Ag-positive plasma and purified HBs Ag were analyzed by electrofocusing in a sucrose ampholyte gradient. Four distinct populations of HBs Ag with a pH range of 4.5 plus or minus 0.1 to 5.4 plus or minus 0.1 for unfractionated plasma samples and 3.9 plus or minus 0.05 to 4.9 plus or minus 0.05 for purified samples were detected in both adw and ayw preparations. Electron microscopic studies of each population of purified HBs Ag revealed 19- to 27-nm spheres in each fraction. Purified material labeled with 125I by the chloramine-T method behaved as one major population with an isoelectric pH value of 3.9 plus or minus 0.1. Purified adw preparations revealed a major population with a molecular weight of 3.7 times 10-6 and a second one of 4.6 times 10-6. Purified preparations of ayw contained one population with a molecular weight of 4.6 times 10-6. Polyacrylamide gel electrophoretic analysis of purified HBs Ag revealed nine polypeptides for ayw and seven for adw particles. These studies indicate that purified preparations of HBs Ag are heterogeneous and that distinct differences can be detected between the two subtypes.

Centrifugation, Density Gradient↗
PubMed1975

Production of antibody to individual polypeptides derived from purified hepatitis B surface antigen.

Purified preparations of hepatitis B surface antigen (HBsAg) were solubilized with sodium dodecyl sulfate and urea under reducing conditions and subsequently fractionated by preparative sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis (PAGE). Pools of the individual fractions eluted from the preparative PAGE were concentrated and purified further by analytical PAGE. Five purified polypeptides were isolated from HBsAg, types adw and ayw, with molecular weights of 19,000, 24,000, 27,000, 35,000, and 40,000. Each preparations was emulsified in Freund complete adjuvant and injected into guinea pigs. Antibody to each HBsAg type was measured by radioimmunoassay. The 19,000 molecular weight polypeptide derived from ayw particles and the 27,000 molecular weight subunit obtained from both types failed to elicit an antibody response. The other three polypeptides derived from the ayw particles elicited group-specific antibody responses. Similar group-specific reactivities were observed in the testing of anti-adw 35,000 and anti-adw 40,000 molecular weight polypeptide sera. However, guinea pigs immunized with the 19,000 and the 24,000 molecular weight polypeptides of the adw type produced antibody that reacted preferentially with adw particles. This indicates that either these subunits carry predominately d determinants or that, because of the low levels of material used for inoculation, no immune response or an undetectable one was elicited to the a or w components.

Animals↗
PubMed1975

Humoral and cellular immunity in guinea pigs to polypeptides derived from HBsAg-containing particles.

Recent studies in this laboratory have shown that purified HBsAg contains at least 7-9 polypeptide subunits ranging in molecular weight (MW) from 18,000 to 120,000; at least three of these are associated with carbohydrates. In addition, two glycosphingolipids have been extracted which are structurally similar to the blood group antigenic substance. The glycoproteins and glycolipids are of particular interest since it has been reported that carbohydrate is associated with the antigenic activity of HBsAg. Antisera to these subunits have been produced in guinea pigs. These results stimulated further 'in vivo' investigations into the possible development of a subunit vaccine which would exclude genes of a viral or cellular nature and would not be infectious. Guinea pigs were immunized with HBsAg, subtypes adw and ayw, and with two polypeptides (40,000 MW and 24,000 MW) in complete Freund's adjuvant. Control animals were inoculated with normal human serum or with polyacrylamide gel. Animals were boostered on day 14 and tested on day 30 for the development of cell-mediated immunity (CMI) by the macrophage inhibition assay, and for humoral immunity by PHA and RIA. Migration of peritoneal exudate (PE) cells was inhibited 30-88% in guinea pigs immunized with the 40,000-MW polypeptide when challenged with purified HGsAg, subtype adw or ayw, or with the 40,000-MW preparation, but the results were inconclusive with the 24,000-MW subunit. Similar evidence of CMI was observed with PE cells derived from guinea pigs inoculated with purified subtype-specific HBsAg or with the 24,000-MW polypeptide. Humoral responses were predictable, and showed a reasonable degree of correlation with the CMI response. Control animals failed to respond to challenge with purified preparations of HBsAg or the 40,000-MW polypeptide, but did show CMI with their respective immunogens and - in the case of guinea pigs inoculated with normal human serum - with the 24,000-MW subunit, suggesting that the latter may contain an antigenic determinant related to a human serum protein.

Animals↗
PubMed1975

Antibody to herpes simplex virus type 2-induced nonstructural proteins in women with cervical cancer and in control groups.

Sera obtained from 15 patients with cervical cancer, 10 patients with breast cancer, and 15 control women, individually matched with the cervical cancer patients, were examined for antibodies to early proteins synthesized in herpes simplex virus type 2 (HSV-2)-infected cells. The method used was an indirect radioimmune precipitation test followed by polyacrylamide gel electrophoretic analysis of immune precipitates. The relative reactivity to a major early nonstructural protein (VP134) was used to compare these selected sera. The results obtained suggest that cervical cancer patients possess sera with a higher reactivity to VP134 than breast cancer patients or matched healthy women,and that serum reactivity is independent of the level of neutralizing antibodies to HSV-2.

Antibodies, Viral↗
PubMed1975

Radioimmunoassay of human serum antibody specific for adenovirus type 5-purified fiber.

A radioimmunoassay (RIA), utilizing a second antibody to separate immune complexes, was developed to provide a sensitive and specific measure of serum antibody to adenovirus type 5 (Ad 5) fiber. Purity of fiber antigen was ascertained by sodium dodecyl sulfate urea-polyacrylamide gel electrophoresis and isoelectric focusing in ampholyte pH gradients. After labeling with 125I to high specific activity, the iodinated fiber did not exhibit loss of antigenic reactivity and remained stable for 3 weeks when stored at minus 20 degrees C with supplemental protein. Rabbit anti-Ad 5 serum with a neutralization titer of 1:320 precipitated 50% of the labeled fiber at a serum dilution of 1:50,000 when tested by the RIA. In competition assays as little as 0.5 ng of unlabeled fiber per millimeter was sufficient to inhibit the 125I fiber-antibody reaction. Serum specimens from 20 volunteers, obtained before and after vaccination with purified Ad 5 fiber or hexon subunit vaccine, were tested by RIA, hemagglutination-inhibition (HI), and neutralization tests. A comparison of mean antibody titers of post-inoculation sera showed that the RIA was 300 and 1000 times more sensitive than the HI and neutralization tests, respectively. Moreover, 19 of the men who were negative by the standard serologic tests before vaccination were shown to have anti-fiber antibody, with a mean RIA titer of 1:1028. Specificity of the RIA was demonstrated by the lack of an increase in antibody to Ad 5 fiber among those individuals vaccinated with the hexon subunit. Thus, the development of a highly sensitive and reproducible RIA allows for the detection of antibody specific for the Ad 5 fiber in serum which contains antibodies to the different virion antigenic determinants associated with Ad 5.

Adenoviridae↗
PubMed1974

Modified radioimmunoassay for murine sarcoma-leukemia virus group-specific antigen.

Iodination of disrupted Moloney strain murine sarcoma-leukemia virus resulted in labeled group-specific (gs) protein which was subsequently purified on an isoelectrofocusing column. This iodinated purified gs antigen, prepared from a relatively small quantity of purified virus, was used in a radioimmunoassay. A radioimmunoassay inhibition method was developed so that antibody specific for mammalian C-type gs antigen could be measured in undiluted or low dilutions of test serum without altering the known reagents of the test. The gs antigen isolated from purified Moloney strain murine sarcoma-leukemia virus has an isoelectric point (pH 5.95) which is significantly lower than that reported for other murine leukemia viruses.

Antibodies, Viral↗
PubMed1974

Major polar lipids of hepatitis B antigen preparations: evidence for the presence of a glycosphingolipid.

The major phospholipids of hepatitis B antigen, subtype adw, were characterized as phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine. In addition, two carbohydrate-containing lipids were observed, one of which was characterized as a non-sialic acid-containing, water-soluble glycosphingolipid. Upon hydrolysis the glycosphingolipid yielded both hydroxy and non-hydroxy fatty acid as well as a long-chain base which was putatively identified as dihydrosphingosine.

Chromatography, Paper↗
PubMed1974

Expression of the major internal viral polypeptide in cells transformed by wild-type and temperature-sensitive murine sarcoma virus.

Phenotypic expression of the murine intraspecies and interspecies antigenic determinants of the major type C viral structural 30,000-dalton polypeptide, p30, was measured by radioimmunoassay inhibition in cell lines from different species. Uninfected normal rat kidney (NRK) cells did not contain detectable levels of murine intraspecies and interspecies p30 antigen, whereas rat cells transformed by and producing murine sarcoma virus (MSV)-Moloney leukemia virus (M-MSV-MuLV) contained high levels of both murine intraspecies and interspecies p30 antigen. Significant amounts of murine intraspecies and interspecies p30 antigen were detected in wild-type MSV-transformed nonproducer NRK cells. The control of p30 antigen expression was examined in temperature-sensitive MSV-transformed nonproducer cells [NRK(MSV-1b)] which are cold sensitive for maintenance of the transformed phenotype. Both murine intraspecies and interspecies p30 antigens were detected in NRK(MSV-1b) cells when grown at the permissive (39 C) or nonpermissive (33 C) temperature, suggesting that p30 antigen expression is not correlated with maintenance of the transformed phenotype. The results demonstrate that previously undetectable p30 antigens are expressed in MSV-transformed nonproducer NRK cells, and suggest that the expression of p30 antigen may be a useful marker for viral gene expression in mammalian cells.

Animals↗
PubMed1974

Papovavirus structural polypeptides: comparison of human and rabbit papilloma viruses with simian virus 40.

A comparative study of the structural polypeptides of purified human papilloma virus, rabbit papilloma virus, and simian virus 40 (SV40) was performed by electrophoretic analysis on 10% polyacrylamide gels containing sodium dodecyl sulfate either by Coomassie blue staining or by electrophoresing purified virus preparations labeled with radioactive iodine. Analysis of human papilloma virus on stained gels revealed 6 bands with a major polypeptide of molecular weight 63,000 daltons comprising 60% of the virion protein. Analysis of rabbit papilloma virus on stained gels revealed 5 bands with a major polypeptide of molecular weight 60,000 daltons comprising 48% of the virion protein. Under conditions which yielded 6 polypeptides for SV40, in agreement with published data, 4 and 7 polypeptides were regularly detected in iodinated preparations of human and rabbit papilloma viruses, respectively. Isoelectrofocusing of iodinated virions revealed isoelectric points of pH 3.7 for SV40 and pH 4.0 for both rabbit and human papilloma viruses.

Animals↗