M.Sc. / It is the widely accepted view that the human immunodeficiency virus (HIV) is the causative agent of acquired immunodeficiency syndrome (AIDS) and that South Africa harbors mainly HIV type 1 subtype C (HIV-1 C). Extensively characterized biological isolates (especially of HIV-1 subtype C) for use in HIV/AIDS vaccine and drug development are not readily available. This study evaluated three different protocols for the expansion of virus from infected PBMC's of 68 HIV/AIDS patients (designated HJ1 — 22 and INN1 — 97). Factors influencing the success of a protocol for the expansion of HIV-1 were 1) the amount and time of addition of IL-2 and PHA to the culture media; 2) the fact that freshly isolated clean PBMC's (treated with PHA prior to co-cultivation) was necessary while infected PBMC's could be used fresh or frozen; 3) whether the absence or presence of polybrene as a tissue culture additive had any effect. The I-11V-status of patients could be confirmed with rapid tests and/or NASBA assays, while successful expansion of the virus could be confirmed or refuted by determining p24 levels of sera or culture supematant (with values ranging from <7.8pg/ml to about 280pg/ml). Less sensitive assays like the reverse transcriptase (RT) and gpl 20 ELISA's give much lower absorbance values when compared to the p24 ELISA. Using expanded virus to infect PBMC's and T-cell lines (PM1, U87.CD4-CCR5, U87.CD4-CXCR4 and CEM.NKR-CCR5) and then measuring p24 levels showed that the final protocol chosen was capable of producing high titre, biologically active virus. To further test the biological activity of the isolates, the virus was used in assays evaluating the potential inhibitory ability of natural products and neutralizing antibodies. PCR using universal primers (SK22/SK38/SK39) was not consistently successful in amplifying out the correct sized region of gag (for SK22/SK39 a fragment of 600bp and a 115bp fragment for SK38/SK39 was obtained but not for all the samples). Primers (Cgag189(+/-)) were designed during this project to specifically amplify an 189bp region of gag from subtype C, which proved (in some instances) to be more successful. PCR amplification of the proviral DNA fragments was confirmed by sequencing of selected PCR products. Virus titre was determined by calculating TCID50 (login: 1.054). By modifying expansion and detection protocols it is possible to standardize the process to suit a particular isolate and/or circumstance. This production of large volumes of high titre, biologically active isolates has filled a desperate need for reagents to aid HIV researchers in the development of an effective vaccine or other drug therapy.

Immunochemical reagents were characterized under carefully controlled laboratory conditions using conventional high performance liquid chromatography instrumentation. The stationary phase was prepared by attaching antigen molecules to an insoluble support through a covalent linkage. Experiments were carried out by introducing antibody molecules into the mobile phase and monitoring their interaction with the stationary phase. Monoclonal antibodies were employed because of their more homogeneous properties compared to polyclonal antisera. Radioisotopes were employed to study low level adsorption on the stationary phase. Recovery experiments were carried out in which it was possible to account for all of the material introduced into the mobile phase. Antibodies were purified over a preparative scale antigen affinity column following labeling to insure high immunoreactivity. Studied under normally dissociating conditions, irreversible adsorption of picomole amounts of protein on the antigen stationary phase was greater than on other ligand modified stationary phases. This accumulation decreased with repeated use of the affinity column. The present study provides a framework for evaluation of other immunoaffinity systems and demonstrates that reproducible recovery of immunologically active material in high yield is possible. Monoclonal antibodies labeled with fluorescein were different from unlabeled molecules in binding and physical characteristics. Computer simulations were used to describe binding behavior. Although fluorescein labels improve detection sensitivity over native protein absorbance, their use in this case decreased binding affinity significantly. Heterogeneity of affinity purified fluorescein labeled and unlabeled monoclonal antibodies was examined with two dimensional gel electrophoresis. In addition to increased charge heterogeneity in the labeled antibody fragments, both light and heavy chains possessed more negative character. These results agree with each other. Fluorescein contains a carboxylic acid group, and modification of antibody light chains may interfere with binding affinity. The number and location of labels covalently attached to antibodies must be carefully controlled to obtain maximum detection sensitivity and preserve immunoreactivity.

There is no abstract available for this thesis. / Department of Chemistry

Thesis (Ph. D.)--Ohio University, March, 2004. / Includes bibliographical references (leaves 158-181).

Call number: LD2668 .T4 1985 M33 / Master of Science

Thesis (Ph. D.)--State University of New York at Binghamton, Department of Chemistry, 2007. / Includes bibliographical references.