Rapid assay for simultaneous detection and differentiation of immunoglobulin G antibodies to human immunodeficiency virus type 1 (HIV-1) group M, HIV-1 group O, and HIV-2.

A rapid immunodiagnostic test that detects and discriminates human immunodeficiency virus (HIV) infections on the basis of viral type, HIV type 1 (HIV-1) group M, HIV-1 group O, or HIV-2, was developed. The rapid assay for the detection of HIV (HIV rapid assay) was designed as an instrument-free chromatographic immunoassay that detects immunoglobulin G (IgG) antibodies to HIV. To assess the performance of the HIV rapid assay, 470 HIV-positive plasma samples were tested by PCR and/or Western blotting to confirm the genotype of the infecting virus. These samples were infected with strains that represented a wide variety of HIV strains including HIV-1 group M (subtypes A through G), HIV-1 group O, and HIV-2 (subtypes A and B). The results showed that the HIV genotype identity established by the rapid assay reliably (469 of 470 samples) correlates with the HIV genotype identity established by PCR or Western blotting. A total of 879 plasma samples were tested for IgG to HIV by a licensed enzyme immunoassay (EIA) (470 HIV-positive samples and 409 HIV-negative samples). When they were tested by the rapid assay, 469 samples were positive and 410 were negative (99.88% agreement). Twelve seroconversion panels were tested by both the rapid assay and a licensed EIA. For nine panels identical results were obtained by the two assays. For the remaining three panels, the rapid assay was positive one bleed later in comparison to the bleed at which the EIA was positive. One hundred three urine samples, including 93 urine samples from HIV-seropositive individuals and 10 urine samples from seronegative individuals, were tested by the rapid assay. Ninety-one of the ninety-three urine samples from HIV-seropositive individuals were found to be positive by the rapid assay. There were no false-positive results (98.05% agreement). Virus in all urine samples tested were typed as HIV-1 group M. These results suggest that a rapid assay based on the detection of IgG specific for selected transmembrane HIV antigens provides a simple and reliable test that is capable of distinguishing HIV infections on the basis of viral type.

A rabbit B cell determinant for a conserved portion of myelin basic protein, rabbit encephalitogenic sequence 65-74.

Synthetic peptide S24 (TTHYGSLPQKG) represents residues 65-74 of myelin basic protein (MBP) and contains the major determinant involved in the development of experimental allergic encephalomyelitis (EAE) in rabbits. This peptide is completely conserved in all nonprimate mammals for which sequence information is available. Although it is clear that peptides containing the S24 region are capable of inducing EAE, previous serologic studies have resulted in the conclusion that the determinant is "buried" or sequestered in intact MBP. Employing a liquid phase radioimmunoassay, we studied Ab responses to the S24 determinant in six rabbits injected with rat myelin. Two of the six animals developed small but measurable responses to the S24 determinant. In one of these rabbits, the response was boosted with a covalent conjugate of S82 and methylated BSA (MBSA). We also measured antibodies to the S24 determinant in rabbit antisera to human, monkey, dog, bovine, and the large and small forms of rat MBP. By nonequilibrium inhibition analysis, we determined that the antibody responses to these antigens were all directed to a determinant composed of residues 66-71 of MBP, and that intact MBP inhibits the binding of these antibodies to radiolabeled S24. The results demonstrate that the rabbit encephalitogenic region of myelin basic protein is exposed in the intact molecule both as an immunogen and as a reactant in liquid-phase assays; furthermore, they demonstrate that MBP antigenicity leading to B cell responses does not necessarily depend on sequence differences between the injected protein and its counterpart in the host species. The latter finding reinforces the contention of Atassi that autoantibody responses are not exclusive to "evolutionary hypervariable locations."

Comparative levels of endogenous myelin basic protein-serum factors (MBP-SFs) in adult and suckling mice (B6CBAF1 and B6C3HF1, strains) and in neurologically mutant mice of the same genetic background.

Normal adult B6C3HF1 and B6CBAF1 mice contained at least 10 times higher levels (1.17 microM) of endogenous myelin basic protein-serum factors (MBP-SFs) than previously found in adult Lewis rats. In rat MBP-SF levels in the adult (0.03 microM) were much less than in the suckling animals (0.74 microM). In mice, by contrast, the adult (1.17 microM) and suckling (0.75 microM) levels were similar. Suckling mice from 9 different neurologically mutant strains and their clinically normal littermates had MBP-SF levels (0.5 microM) slightly below that of normal suckling mice of the same genetic background (0.75 microM).

Affinity purification of an acylated and radiolabelled synthetic derivative of residues 75-83 of bovine myelin basic protein, [125I]S79. A model for the purification of picomole quantities of specific peptide fragments of myelin basic protein and antibodies against them.

Among the antibodies contained in a rabbit antiserum to synthetic peptide sequence TTHYGSLPQKAQGHRPQDEG (S82) of bovine myelin basic protein (residues 65-83 plus glycine), was a population reactive with a C-terminal determinant of S82 and cross-reactive with S79 (AQGHRPQDEG) but not S6 (AQGHRPQDENG). This antibody population was purified 153-fold by affinity chromatography from a minicolumn containing S79 coupled to CH-Sepharose 4B(TM) and eluted with 3 M MgCl2. The purified antibodies were then coupled to CNBr-activated Sepharose 4B(TM) and used to purify 125I-labelled, acylated S79 ([125I]S79), 3 M MgCl2 once again having been used to elute the labelled ligand. Sips distribution studies revealed appreciable heterogeneity of binding affinities of unpurified antibodies in their reaction with affinity-purified [125I]S79 or of purified antibodies in their reaction with unpurified [125I]S79 (heterogeneity constant a = 0.34 and 0.36, respectively). In contrast Sips distribution data indicated considerable restriction of binding of the purified antibodies in their reaction with purified labelled ligand (a = 0.92) with an average affinity constant of K0 = 1.56 X 10(8) M-1. The results indicate that the heterogeneous spectrum of binding affinities originally displayed by the unpurified S79-reactive antibodies in their reaction with unpurified labelled S79 was due both to the presence of some antibodies characterized by high affinity binding (K0 greater than 10(9) M-1) and of some labelled ligand with low binding affinity. The affinity chromatographic method as here described should prove advantageous in purifying and eventually characterizing picomolar amounts of serum factors, previously postulated to be fragments of myelin basic protein, that are reactive with reagent antibodies up to an affinity level of 10(8) M-1.

Immunogenicity of synthetic peptide sequences S81 and S82 (residues 68-83 and 65-83) of bovine myelin basic protein. Time-course of antibody responses in rats and rabbits.

The timing and intensity of the antibody responses to the marker determinants of synthetic peptide S81 and S82 sequences of bovine myelin basic protein (MBP) (residues 68-83 and 65-83, respectively) were studied in 20 Lewis rats and 6 rabbits. All rats immunized with either peptide in CFA responded with antibody development. All rabbits immunized with S82 and CFA developed both antibodies and experimental allergic encephalomyelitis. In contrast only one rabbit developed antibodies against S81 and none of the S81-challenged rabbits developed disease. On the basis of extrapolation of linear time-response curves to zero activity, the time of appearance of anti-peptide antibody activity in the Lewis rats was 15.1 +/- 1.7 days after a single immunization, a week longer than the normal latent period before appearance of anti-MBP antibodies. The time of appearance of anti-S82 antibody activity in rabbits exhibiting linear response curves was 18 days, 4 days after a booster immunization with S82 in incomplete Freund's adjuvant. The development of clinical signs of experimental allergic encephalomyelitis occurred within 4 weeks after initial challenge (a few days after boosting) and continued for 8--13 days in all S82-immunized rabbits.

Equilibrium competitive inhibition analysis of synthetic peptide antigens from myelin basic protein as affected by the dual-dilution phenomenon.

It was shown that 125I-labelled and unlabelled forms of synthetic encephalitogenic peptide S82 (residues 65-83 plus glycine) of bovine myelin basic protein (MBP-Bov) were equally competitive in dual-dilution radioimmunoassays with rat- and rabbit-anti-S82 antisera without causing much deviation even at the extremes of the dual-dilution binding curves (solved in terms of total S82). With other antisera the deviations caused by the addition of unlabelled S82 were much greater than would be expected among repetitive assays with labelled antigen alone, and the excessive deviations were usually more prominent in one region of the dual-dilution binding curve than in another. Thus, establishing equivalence between labelled and unlabelled antigen with respect to one antiserum even at several dilutions does not establish proportionate sharing with respect to all antisera at all antigen concentrations. A method of dual-dilution equilibrium competitive inhibition analysis was devised that took this precaution into account. By means of the method, synthetic MBP-Bov peptides representing different parts of the S82 sequence were compared with homologous S82 peptide for their inhibitory effects upon dually diluted [125I]S82-anti-S82 systems. By this process several different S82 determinants were pinpointed, some with high affinity antibodies, others with low affinity antibodies, yet others equally well at high or low affinity.

Equilibrium and nonequilibrium competitive inhibitions of antipeptide antibody binding by parent myelin basic protein and 18 related peptide sequences.

Equilibrium and nonequilibrium competitive inhibition analyses of a number of antisera to peptide S81 and S82 sequences were carried out through the use of inhibition radioimmunoassays with [125I]S81, [125I]S82, and [125I]S79 and a panel containing 18 related peptides and five myelin basic protein preparations. Two principal determinants were identified, one of them sequential, the other nonsequential. The sequential determinant involved a peptide at or near the C-terminal end of S82 that could be blocked by an interchange of asparagine for glycine at the C terminus. The nonsequential determinant was dominant for a number of rabbit and rat antisera, both anti-S82 and anti-S81, and was shared not only by S81 and S82 but also by S8 and S80, i.e., the family of residues of bovine MBP sequence 69-83. Neither determinant was expressed in any of the myelin basic protein preparations, and the nonsequential determinant was not expressed in peptide sequences smaller than S8.

Studies of rat antibodies specific for myelin basic protein (MBP). Antibody-dependent cell-mediated lysis of MBP-sensitized targets in vitro.

Rats immunized with rat myelin basic protein (MBP) in an encephalitogenic regimen produce antibodies which cooperate with spleen cells from unimmunized rats in the specific lysis of MBP-sensitized chicken erythrocytes (MBP-CRBC) in vitro. Antibodies against MBP capable of participating in antibody-dependent cell-mediated cytotoxicity (ADCC) were detected in serum 9 days post immunization, and prior to the onset of experimental allergic encephalomyelitis (EAE). Measurements based on a sensitive radioimmunoassay technique had previously established that anti-MBP activity emerges by day 9, early enough to participate in events leading up to EAE. The ADCC assay was designed to further characterize functions of immunoglobulins associated with the humoral response to MBP, with emphasis on the cytophilic properties of early antibodies. The assay described was found to be nearly as sensitive as the RIA and could detect specific syngeneic rat anti-MBP at concentrations of about 0.03 pmoles/ml serum. Whether ADCC-type reactions take place in the central nervous system during acute EAE is not known; however, the presence of early cytophilic antibody in serum suggests that humoral immunity may play an ancillary role to that demonstrated for T-cell immunity in EAE.

Endogenous myelin basic protein-serum factors (MBP-SFs) in Lewis rats. Evidence for their heterogeneity and reactivity with anti-MBP antibodies of different affinities.

MBP-SF, previously described as an endogenous myelin basic protein-serum factor in Lewis rats with a suggested function as a neuroautotolerogen, appears not to be a single factor but a heterogeneous collection of serum factors (MBP-SFs), most probably small fragments of MBP, each cross-reactive with a different region of the multideterminant parent molecule. The heterogeneity of the MBP-SFs in any serum sample is defined and limited by the spectrum of binding affinities of the antibody populations represented in a given reagent anti-MBP antiserum. Some samples of normal Lewis rat serum have been found to contain high affinity MBP-SFs which coexist with low affinity anti-MBP antibodies whereas other sera have shown the reversed pattern, viz. low affinity MBP-SFs and high affinity antibodies. Additional sera have been found to contain MBP-SFs of several different affinities. In time-course studies of rats sensitized to neuroantigen-adjuvant a variety of MBP-SFs and anti-MBP antibodies of different affinities may be observed in sequentially collected sera from a given animal. In no animal has any serum sample been found to contain the full spectrum of MBP-SFs. Although some MBP-SFs have been found to increase temporarily during the 2nd week after neuroantigen/CFA sensitization, all MBP-SFs tend to disappear in the 2nd week and to be replaced by anti-MBP antibodies of differing affinities 3-4 weeks following sensitization.

Myelin basic protein serum factor. An endogenous neuroantigen influencing development of experimental allergic encephalomyelitis in Lewis rats.

Age-related concentrations of myelin basic protein serum factor (MBP-SF), an endogenous neuroantigen detected and quantitated by inhibition of binding of rat myelin basic protein (RMBP) antibody with 125I-RMBP reagent antigen and immunochemically indistinguishable from native RMBP in this respect, reach peak levels as high as 21 ng/microliter among 2-3-wk-old normal suckling Lewis rats. Levels then progressively decline to low, usually undetectable levels of less than or equal to 0.6 ng/microliter MBP-equivalents in adult animals by 7 wk of age. MBP-SF levels are inversely related to the age-related increasing capacity of maturing Lewis rats to develop experimental allergic encephalomyelitis (EAE) after sensitization to MBP of syngeneic, but not xenogeneic, origin. MBP-SF appears to be an endogenous neuroimmunoregulatory product of potential importance for immunologic tolerance to autologous RMBP in Lewis rats.

Development of a localized hemolysis-in-gel assay for Vi antigen: characterization of the Vi-specific PFC response of nude and normal mice.

An assay to detect specific plaque-forming cells (PFC) to Vi antigen (Vi) was developed and the optimal conditions for sensitization of sheep erythrocytes (SE) and plaque development were determined. Using PFC and passive hemagglutination (PHA) assays, Vi-specific immune responses of athymic (nude) and normal mice were characterized. Vi was found to elicit only IgM PFC. No discernable secondary response was detected following a second injection of antigen. Nude and normal mice responded in a quantitatively similar manner to all doses of Vi tested and responded similarly on varying days following immunization. Also, both nude and normal mice produced the greatest number of Vi-specific PFC 4 days following immunization with an optimally immunogenic dose of Vi (1.0 microng/mouse). These results indicate that functional thymus-derived cells are not necessary to elicit an immune response against Vi antigen.