Comparative characterization of antigenic epitopes in the immunodominant region of the protein encoded by open reading frame 3 in Burmese and Mexican strains of hepatitis E virus.

To analyse the effect of strain-specific sequence variation on the antigenic properties of the protein encoded by the open reading frame 3 (ORF 3) of hepatitis E virus (HEV), two sets of short overlapping peptides spanning amino acids 91 to 123 of this protein from Burmese and Mexican strains were synthesized and tested with sera obtained from outbreaks of enterically transmitted non-A, non-B hepatitis in three different regions of the world (Mexico, Turkmenistan and Kenya). The data suggest strain-specific variation in the antigenic reactivity of the ORF 3 protein. The C-terminal region of this protein contains several antigenic epitopes located in the most variable positions. Individual sera were found to interact with different groups of epitopes from each set of peptides. The antigenic epitopes of the Mexican strain appear to be less conformation-dependent than those of the Burmese strain. The most immunoreactive epitope of the ORF 3 protein from the Mexican strain was localized at amino acid positions 95 to 101. The ORF 3 protein of the Burmese strain contains an immunodominant epitope at amino acid positions 112 to 117. Some of these short peptides may be useful for the development of a diagnostic assay to discriminate between the Burmese and Mexican strains.

Specificity and association constants of 33 monoclonal antibodies to human IgA epitopes.

We used an immunofluorescent sequential-saturation-of-antibody assay and an interactive computer program for Scatchard analysis to determine association constants (Ka) of 33 murine monoclonal antibodies (Mabs) specific for human IgA epitopes. Ka ranged from 0.37 to 690 x 10(7) liters per mole (an approximate 1900-fold difference). Specificity was validated with a panel of 18 highly purified IgA1 and IgA2 myeloma proteins and secretory IgA using an immunofluorometric assay. Western blots of bacterial IgA protease digests were used to locate the epitopes of IgA specific Mabs in either the Fab, Fc, or hinge region. Mabs specific for unique epitopes on secretory IgA or free secretory component (FSC) were produced and evaluated.

Estimation of association constants of 42 monoclonal antibodies to human IgG epitopes using a fluorescent sequential-saturation assay.

We measured the association constant (Ka) for 42 murine monoclonal antibodies (MAbs) to human IgG epitopes. Included are antibodies, previously evaluated in an IUIS/WHO collaborative study, to various epitopes on the four subclasses of human IgG - IgG Fc, IgG Fab, kappa, and lambda - and to selected IgG allotopes. We used a sequential-saturation immunofluorescent assay and interactive computer program to determine the Ka by Scatchard analysis. Kas ranged from unmeasurably low by this method (approximately 10(6) L/M) to 3.8 X 10(9) L/M. Some class specific MAbs had large Ka differences for different subclasses and some subclass specific MAbs had large Ka differences for molecules of the same subclass but of different light-chain types.

Two-site immunoenzymometric assays for serum IgG subclass infant/maternal ratios at full-term.

Improvements were made in the range, precision, convenience, automation, and reliability of our previously published two-site immunoenzymometric assays using mouse monoclonal antibodies specific for human IgG and its subclasses. The serum concentration ratios for these immunoglobulin isotypes were measured in neonatal/maternal paired sera from 119 normal full-term deliveries. These ratios are significantly different than 1.0 (P = 0.001) for total IgG, IgG1, and IgG2 (show non-equality of paired neonatal/maternal sera concentrations) but are not significantly different than 1.0 for IgG3 and IgG4. On average, IgG1 is elevated 61%, and IgG2 is depressed 11% in the full-term neonate with respect to its own mother. In some pregnancies, active transport of IgG1 may be selectively enhanced by low material IgG1 concentration and selectively inhibited by high levels.

Hypergammaglobulinemia associated with human immunodeficiency virus infection.

The serum concentrations of 11 Ig isotypes (IgG, IgG1, IgG2, IgG3, IgG4, IgA, IgA1, IgA2, IgM, IgD, and IgE) were measured in four relatively small groups of homosexual (or bisexual) males. All these patients were seropositive for HIV. Two of the groups (nonprogressors) were clinically stable for approximately 2 years and were characterized either as asymptomatic or with PGL. The third group (progressors) developed AIDS 2-38 months after blood specimens were taken. The fourth group had AIDS. A fifth group of anti-HIV-seronegative heterosexual males completed the study. The geometric mean IgA serum concentration was more markedly elevated over normal control sera than any of the other study groups and was the only Ig isotype that was significantly higher in the progressor than in the nonprogressor group. The geometric IgG1 serum concentration was significantly higher in asymptomatic nonprogressors, PGL-nonprogressors, progressors, and AIDS patient groups than that in HIV-seronegative normals. In contrast, the geometric mean IgG2 serum concentration is depressed in all the anti-HIV-seropositive patients (but not significantly with the AIDS group). Multivariate analysis showed the Ig-isotype assays to have much less predictive power for progression to AIDS than the T-helper cell assays.

Evaluation of monoclonal antibodies having specificity for human IgG sub-classes: results of an IUIS/WHO collaborative study.

Seventy-four monoclonal antibodies (McAb) of putative specificity for human IgG (11), the IgG sub-classes (59) or Gm allotypes (4) have been evaluated for reactivity and specificity in eight laboratories employing different assay techniques or protocols. For the IgG, IgG3, IgG4, G1m(f) and G3m(u) specificities McAb have been produced that can be satisfactorily applied in most methodologies employed and have potential as reference reagents. The IgG1 and particularly IgG2 specificities proved problematical with all McAb evaluated demonstrating apparent assay restriction and whilst performing well in some assays proved to be poor or inactive reagents in others. However, the study identifies McAb individually suited to application within most commonly employed methodologies. Epitope display is the probable variability rather than capricious behaviour by the McAb. IgG1 and IgG2 were the least immunogenic of the sub-class proteins and there is evidence that epitope display is influenced by the physical and chemical procedures used to immobilize or fix antigen - a common requirement in the assay systems studied.

Evaluation of thirty-one mouse monoclonal antibodies to human IgG epitopes.

Stable clones of 31 mouse hybridomas that produce monoclonal antibodies (MAbs) against human IgG antigenic determinants were obtained. The number of hybridomas of different specificity described are: 2 anti-IgG1 Fc, 1 anti-IgG2 Fc, 1 anti-IgG2 Fd, 2 anti-IgG3 Fc, 2 anti-IgG3 hinge, 3 anti-IgG4 Fc, 3 anti-IgG4 Fd, 2 anti-nG4m(b), 4 anti-IgGFc, 2 anti-IgGFd, 1 anti-kappa, 1 anti-lambda, 1 anti-non IgG1, 2 anti-non IgG2, 2 anti-non-IgG3, 2 anti-non-IgG4. Evidence is presented validating their specificity. Some MAbs demonstrated to be avid, potent, and specific for well defined IgG-subclass epitopes may be partially or completely inactive in other assay systems, presumably because of different presentations of antigen epitopes. In general, this problem requires careful writing of protocols describing the use of MAbs.

Characterization of antibody responses in legionellosis with an immunofluorometric assay.

A solid-phase immunofluorometric assay was used to qualitatively characterize and precisely measure human immunoglobulin class-specific antibody responses in legionellosis. Stable antigen preparations consisted of cells grown at 25 degrees C that were killed, fixed with Formalin vapors, washed, and lyophilized. Working-curve material consisted of dilutions of selected convalescent sera. Linear regressions of logit transformations of relative fluorescence intensities versus the logarithm of the relative concentrations of sera were determined to give immunoglobulin class-specific antibody levels from uninfected and infected individuals. Each fluorescence intensity obtained with immunoglobulin class-specific antibody was converted to a multiple of the median fluorescence intensity obtained with sera from uninfected individuals. A presumptive-positive acute-phase legionellosis serum was defined for each immunoglobulin class by a multiple of the normal median fluorescence intensity that was greater than the multiple of the normal median from approximately 97% of the uninfected population.

Quantitative characterization of specificity and potency of conjugated antibody with solid-phase, antigen bead standards.

An immunofluorescent assay was used to characterize precisely the potency and specificity of fluorescein-conjugated immunoglobulin class-specific and polyvalent anti-sera. Stable antigen standards consisted of highly purified immunoglobulin antigens covalently bound to polyaminostyrene beads. Linear regressions of weighted logit transformations of relative fluorescent intensities versus the logarithm of relative conjugate concentrations were determined. The potency of conjugates was compared using two different methods derived from the logit transformation. Inappropriate specificities were measured in some conjugates described as immunoglobulin 'class-specific'.

Aplasia of the optic nerve.

A case of unilateral true aplasia of the optic nerve in an otherwise normal and healthy child is presented. Twenty-eight previously reported cases of aplasia have been reviewed and classified according to criteria which we have presented. Only six of these cases, including the present case, represent true aplasia, manifested by total blindness and complete absence of the optic disc and retinal vessels.