Clinical correlation of the immunological markers of HIV infection in individuals from Thailand.

OBJECTIVE: To evaluate the usefulness of T-cell subsets, beta-microglobulin (B2M), p24 antigen and anti-p24 antibodies as differentiating and prognostic markers in HIV-infected Thai patients. DESIGN: Sixty-one HIV-infected patients in various stages of disease (six AIDS, three AIDS-related complex, 34 persistent generalized lymphadenopathy and 18 HIV-asymptomatic) were followed prospectively for 2 years. Patients were examined and immunological markers assessed every 6 months at least. Any HIV-related complications were treated symptomatically and clinical staging was re-evaluated at each visit. Due to financial constraints, none of the patients were given antiretroviral drugs. METHODS: T-cell subsets were enumerated by indirect immunofluorescence using OKT4 or OKT8 for T-helper and T-suppressor cells, respectively. beta 2M and p24 antigen were quantified by enzyme-linked immunosorbent assay and anti-p24 antibodies were by immunoblot assay. RESULTS: Our preliminary study revealed that the decrease in CD4+ T-cells or anti-p24 titre and the increase in p24 antigen or beta 2M correlated well with disease staging, as defined by the Centers for Disease Control. Absolute number and percentage of CD4+ T-cells, absolute number of CD8+ T-cells, beta 2M level and p24 antigen and anti-p24 antibody levels at entry could be used as reliable prognostic markers for HIV progression. The combination of p24 antigen with the number of CD4+ T-cells substantially increased the prognostic value, compared with either used alone. CONCLUSIONS: The annual rate of clinical progression from asymptomatic to symptomatic HIV infection in our study was 6.8%. The results we obtained in this preliminary study may be used as baseline data for planning future therapeutic interventions in Asian patients.

Comparison of the sensitivity of various anti-HIV tests in early seroconversion sera.

Paired sera from 4 patients with proven HIV infection whose initial specimens obtained 14-51 days earlier were indeterminate were simultaneously retested with 7 screening anti-HIV test kits and the immunoblot assay. The study aimed to evaluate the sensitivity of various new and old anti-HIV screening tests. The test kits evaluated were 4 ELISA test kits from Wellcome (Wellcozyme), Organon (Vironostika anti-HTLV-III), Pasteur (Rapid Elavia) and Diagnostic Biotechnology (DB, HIV-1 ELISA), 2 rapid tests based on microfiltration enzyme immunoassay procedure from Rapport (SUDS) and Disease Detection International (SeroCard), and 1 particle agglutination (PA) test (Serodia-HIV). Immunoblot strips from Diagnostic Biotechnology (HIV-1 Western blot) were used to confirm the HIV infection in these serum specimens. Out of the 4 initial serum specimens tested, all were positive by PA, 2 by SUDS, Wellcome and Pasteur, 1 by SeroCard and DB, and none by Organon. When tested by immunoblot, 1 was negative (i.e., completely without any bands) whereas 3 were indeterminate (i.e., 1 with very weak band for p18, 1 with weak band for p24, 1 with very weak band for gp160. All repeat specimens obtained 14-51 days later (mean 32.5 +/- 16 days) were positive by all screening tests as well as immunoblot. Therefore, with these 4 early seroconversion sera, the sensitivity of the PA was 100%, that of SUDS, Wellcome and pasteur was 50%, of that SeroCard and DB was 25%, and Organon, 0%. None of these sera was considered positive by immunoblot.

[Monoclonal antibodies to non-assembled alpha and beta chain of human chorionic gonadotropin (hCG)]. / Monoklonale Antikörper gegen die nichtassemblierte alpha- und beta-kette des humanen Choriongonadotropins (hCG).

Using a panel of monoclonal antibodies (MCA) to human chorionic gonadotropin (hCG) and its alpha and beta subunits and additional MCA to the non-assembled, free alpha and beta chains which were produced in the course of the present experiments, it was possible to extend the previously established epitope map of hCG. Two MCAs turned out to be specific for the free alpha chain of the glycoprotein hormones (GPH) and, thus, did not react with holo hCG. Two other MCA recognized two epitopes (beta 6 and beta 7) on the free form of the hCG beta only. Again, no holo hormone cross-reaction was observed. Whereas previously only nine antigenic hCG determinants (3 alpha, 4 beta, 2c) had been demonstrated, it was now possible to distinguish fourteen epitopes (5 alpha, 5 beta, 4c). In addition, epitope maps were established for the non-assembled, free subunits of hCG. These comprise six epitopes on the alpha chain (alpha 1- alpha 6) and seven on the beta chain (beta 1- beta 7). Both so far generally accepted premises of Judith Vaitukaitis, claiming the beta chain of the GPH to be immunologically species-specific, the beta chain to be immunologically hormone-specific, were clearly disproven by demonstrating inter- and intra-species cross-reaction of some MCA. Based on the immunological topography of the holo hCG molecule and its free subunits, highly specific and sensitive immuno-enzymometric assays (IEMA) with predictable specificities were designed, measuring either non-assembled subunits alone or in combination with the intact hormones.

Monoclonal antibodies against the free subunits of human chorionic gonadotrophin.

Discordant results on body fluid levels of human chorionic gonadotrophin (hCG) free alpha- and beta-subunits under physiological and pathophysiological conditions, prompted us to raise a total of 260 monoclonal antibodies (MCA) against free hCG-alpha, free hCG-beta, holo-hCG, human follicle-stimulating hormone and bovine luteinizing hormone; 153 MCA recognizing the human alpha-subunit and 28 reacting with hCG-beta were extensively analysed for their intra- and inter-species cross-reactivity with homologous hormones, and for the compatibility of epitopes recognized by them. The immunological topography of free hCG-alpha and free hCG-beta was resolved by these MCA, and epitope maps were designed. Six antigenic determinants on the free alpha-chain (alpha 1-alpha 6), clustered in three spatially distinct domains, and seven epitopes on the surface of free hCG-beta (beta 1-beta 7), could be distinguished. Strikingly, three alpha-chain epitopes (alpha 4, alpha 5 and alpha 6) were shared between various species, which is in contradiction to the concept of immunological species-specificity of alpha-subunits. Three determinants were found to be present only on the free subunits but not on holo-hCG (alpha 6, beta 6 and beta 7), and only two determinants (beta 1 and beta 7) were hormone-specific for hCG. Based on this information, an immunoenzymometric assay for the free alpha-subunit of human glycoprotein hormones was established, with a sensitivity of 1.3 pg/well and a cross-reactivity with holo-hCG of less than 0.005%.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigenic features of human follicle stimulating hormone delineated by monoclonal antibodies and construction of an immunoradiomometric assay.

The characterization of human (h) FSH with 181 monoclonal antibodies (MCA) allowed the elucidation of its antigenic topography. One- and two-site, limited as well as excess reagent type radioimmuno- and enzymoimmunoassays revealed three main categories of MCA molecular binding specificities; two thirds of all antibodies were directed against the alpha-subunit and one fourth toward the beta-chain, and less than one tenth recognized the conformationally (c) intact holohormone. With high frequency immunization schedules these specificities were shifted toward a higher proportion of beta-MCA. On the basis of intra- and interspecies cross-reaction studies as well as epitope contiguity analyses by sandwich assays, the three main categories could be further subdivided into nine epitopes: 1) five epitopes associated with the alpha-subunit, two of which were suprisingly shared by other species, and two being iodination sensitive, 2) two evolutionary conserved structures on the beta-subunit, adjacent to each other, and 3) two c-determinants, one of these present also on hTSH. The epitopes were arranged in three major antigenic domains, which seems to be a common homologous construction principle of the four human glycoprotein hormones: a central domain, consisting of three identically arranged alpha- and similarly located c-epitopes, is flanked by a single spatially distinct domain on each subunit. The establishment of an epitope map was followed by the construction of an immunoradiometric assay with a sensitivity of 0.25 ng hFSH/ml and an apparent cross-reactivity vs. hLH, hTSH, and hCG of less than 1%.

Probing the receptor-interaction of glycoprotein hormones with monoclonal antibodies.

Having recently analyzed with monoclonal antibodies (MCA) the immunologic surface of human chorionic gonadotropin (hCG) as consisting of 9 distinct epitopes exposed on the molecule in a characteristic topographical manner (Schwarz, S., Berger, P., and Wick, G., Endocrinology 118, 189-197, 1986) we now attempted to confirm this result on a more general basis, e.g. by incorporating MCA that were just recently obtained and previously not included. What we were, however, interested most was the question whether the tentative model of the epitope map of hCG could represent a "target" with which hCG-related hormones such as LH, FSH, and TSH (the family of glycoprotein hormones, GPH) would (partially) match. Indeed, repeating various immunizations with GPH of human as well as animal origin revealed a remarkable reproducibility in terms of several anticipated epitope specificities of MCA. This indicates that MCA can be regarded as reliable probes for mapping epitopes and, as we have presumed, of receptor interaction domains of GPH as well. Extending the originally used 2-site MCA binding exclusion approach by an interspecies crossreactivity (Xr) analysis we now are able to refine our epitope model of hCG such that 2 additional epitopes were found which were not previously resolvable. Most surprisingly, two of 5 epitopes on the alpha subunit were now also detected on various non-human GPH, which is in striking contrast to a seemingly well established dogma. Yet all five alpha epitopes of hCG are present on hLH, hFSH, and hTSH as well and arranged in the same spatial relationship to each other as on hCG. Even the 2 conformational epitopes and their close topographical relationship to the alpha-epitopes appear to be remarkably conserved on all human GPH. Among the beta-epitopes we have found one that is not shared by hLH and that - surprisingly - is not the C-terminal peptide (CTP) by which hLH differs from hCG. On the basis of this refined epitope map a way was paved along which it should be feasible to elucidate the sterical relationship of the epitopes to the receptor interaction domain(s) of hCG. To this end the MCA were tested in principally two ways: first, as to which of the 11 MCA with different epitope specificities would be able (or not) to inhibit by preincubation the binding of radiolabeled hCG (or hLH, respectively) to rat testis LH/hCG receptors? Secondly (and inversely) which of the 11 epitopes of hCG would still be accessible to binding by radiolabeled MCA when the (unlabeled) hormone is bound to the receptor?(ABSTRACT TRUNCATED AT 400 WORDS)