Studies on Hepatitis a Virus: I. Expression of Viral Capsid Peptides in Escherichia Coli and Their Application for an Immunoassay. II. Development of an Assay for Viral Detection by Anti-Hav Antibodies.
Date of Award
Doctor of Philosophy (PhD)
Ronald J. Siebeling
A panel of eleven monoclonal antibodies of the IgG and IgM class were developed against the HM 175 strain of the Hepatitis A virus. Immunoglobulin specificity for the Hepatitis A virus was determined and the antibodies were applied toward the development of an antigen capture assay linked to a nucleic acid amplification step (RT-PCR), for detection of the Hepatitis A virus. The neutralizing mouse monoclonal antibody and a rabbit polyvalent serum were assessed as capture antibodies for the immunoaffinity stage of the assay. Fifty microliter microfuge tubes were coated with the capture antibody. Formalin inactivated HAV was diluted to concentrations ranging from 10$\sp8$ to 10$\sp2$ virions per aliquot. After incubation the virion capsids were disrupted by heating and the viral genome was reverse transcribed. A conserved diagnostic sequence of 191 base pairs was selected for amplification and detection of the HA virus. After the RT step, the reaction was supplemented for the PCR step. After amplification, the amplified products were resolved by agarose gel electrophoresis. The genome regions of capsid proteins VP1 and VP3 of the Hepatitis A virus (HAV) were individually amplified by PCR. Oligonucleotide primers containing flanking restriction endonuclease sequences were designed for amplification of the capsid genes. The expression vectors pTTQ18 and pAX4b+ were selected for generation of $\beta$-galactosidase HAV fusion peptides. The fusion products contained either the alpha ($\alpha$) fragment (pTTQ18) or the entire $\beta$-galactosidase peptide (pAX4b+). The recombinant peptides were expressed in Escherichia coli. Rabbit anti-HAV sera raised against the intact HA virion reacted with the recombinant peptides by immunoblot and enzyme immunoassay. A neutralizing mouse monoclonal antibody generated against the intact HAV reacted only against the VP1 recombinant peptide by immunoblot and enzyme immunoassay. Affinity purification of the recombinant HAV peptides resulted in higher recovery of the fusion peptide containing the entire $\beta$-galactosidase molecule. Cleavage of the VP1 recombinant peptide generated in the pAX expression vector system was assessed. The cleaved peptides reacted with both the rabbit polyvalent and the monoclonal antibody. A peptide corresponding to the expected size of VP1 (33 kDa) was demonstrated by immunoblot.
Baez, Luis Antonio, "Studies on Hepatitis a Virus: I. Expression of Viral Capsid Peptides in Escherichia Coli and Their Application for an Immunoassay. II. Development of an Assay for Viral Detection by Anti-Hav Antibodies." (1993). LSU Historical Dissertations and Theses. 5482.