Ab Avidity Research Articles

Albumin nitrosylated by activated macrophages possesses antiparasitic effects neutralized by anti-NO-acetylated-cysteine antibodies.

Activated macrophages exert an L-arginine-dependent cytostatic effect on the extracellular parasite, Trypanosoma musculi. This effect is not observed in the absence of albumin in the culture medium but is restored by the addition of albumin, indicating the presence of an albumin-nitric oxide (NO) adduct acting as an effector molecule. Since L-cysteine represents a privileged target for NO, an immunochemical approach was performed using an acetylated-cysteine-BSA conjugate. This conjugate was nitrosylated using sodium nitrite as a NO donor. Binding of NO to the conjugated haptens was assayed using spectrophotometry. It was completely abolished by mercuric chloride, confirming the presence of an S-NO bond. Polyclonal Abs were obtained after immunizing rabbits with S-nitroso-acetylated-cysteine (NO-ac-Cys) conjugates. Using the enzyme-linked immunosorbent assay method, Ab avidity and specificity were determined by competition experiments between NO-ac-Cys-conjugated compounds and other nitrosylated or non-nitrosylated compounds. The resulting cross-reactivity ratios showed that conjugated NO-ac-Cys-BSA was the best recognized compound. These Ab were used for an in vitro study of the kinetics of NO-derived compounds from activated murine macrophages. Anti-NO-ac-Cys Ab inhibited the antimicrobial effect of activated macrophages on the extracellular parasite, T. musculi. Moreover, the L-arginine-dependent antiparasitic activity of supernatants from Calmette-Guerin bacillus-activated macrophages required the presence of albumin and was also inhibited by anti-NO-ac-Cys Ab, showing the effector role of S-nitroso-albumin.

The V-region repertoire of Haemophilus influenzae type b polysaccharide antibodies induced by immunization of infants.

Haemophilus influenzae type b (Hib) is a significant pathogen for young children, and three Hib vaccines (named PRP-OMPC, HbOC, and PRP-T) are currently available for young children. Extensive studies of anti-Hib polysaccharide (PS) antibodies (Abs) have shown that the V regions of Abs against the Hib PS comprise a VH gene in the VH3 gene family and a VL gene from various K kappa and V lambda subgroups. To study immunogenic properties of the three vaccines in young children, we determined the VL subgroups and avidities of anti-Hib-PS Abs induced by the three clinically available conjugate vaccines. Ab avidity was measured by determining the concentration of a Hib-PS oligomer that abrogates half of the binding of immunoglobulin G anti-Hib-PS Abs to microwells. The PRP-OMPC vaccine induced lower-avidity Abs than the prelicensure HbOC vaccine (P = 0.05). When we compared anti-Hib-PS Abs expressing V kappa Ia, V kappa II, and V lambda subgroups, a greater Ab response was induced by the prelicensure HbOC vaccine than other vaccines (P < 0.05). When anti-Hib-PS Abs with the V kappa III subgroup were compared, however, both PRP-T and prelicensure HbOC vaccines induced a comparable response, which in turn was greater than those induced by the PRP-OMPC or the postlicensure HbOC vaccine (P < 0.001). The VL repertoire of Abs induced with the prelicensure HbOC or PRP-T vaccine in young children is dominated (about 80%) by anti-Hib-PS Abs using subgroup V kappa II. However, anti-Hib-PS using V kappa II VL accounts for only about 40% of the total anti-Hib-PS Abs induced with the PRP-OMPC vaccine or the postlicensure HbOC. Our data suggest that immunogenic properties of Hib vaccines in young children vary depending on the vaccine preparations as well as the vaccine types.

Protection from lethal coronavirus infection by immunoglobulin fragments.

Molecular mechanisms of in vitro and in vivo virus neutralization by specific Ab remain largely undefined. Murine coronaviruses provide an excellent animal model for such studies. To determine the role of Ab bivalency and the contribution of its Fc portion in the neutralization of viral infectivity and passive protection of mice by an in vitro neutralizing and in vivo protective mAb (7-10A), F(ab')2 and Fab fragments were generated and their biologic properties were examined. The two fragments reacted in ELISA like the whole Ab against viral Ag or specific anti-idiotypic Abs. The affinity constants of the different Ab preparations were determined by surface plasmon resonance using immobilized anti-idiotypic Abs. The apparent affinity constant of the whole Ab molecule was 7.0 x 10(9) M-1 and was reduced 2-fold for F(ab')2 fragments and 14-fold for Fab molecules. Like whole Ab, both F(ab')2 and Fab fragments could neutralize virus in vitro and passively protect mice in vivo. However, the efficiency of in vivo neutralization by Fab fragments was reduced compared with the bivalent molecules, despite almost identical half-lives of both types of Ab fragments. These results demonstrate that in vitro and in vivo virus neutralization mechanisms by this Ab are independent of Fc-mediated functions and bivalency, but are probably influenced by Ab avidity. Also, this is the first report of in vivo protection against a viral infection by Fab fragments of antiviral Ab.

Functional differences in idiotypically defined IgG1 anti-polysaccharide antibodies elicited by vaccination with Haemophilus influenzae type B polysaccharide-protein conjugates.

Abstract We investigated the relationship between the form of the Haemophilus influenzae type B (Hib) polysaccharide (PS)-protein conjugate vaccine, Id expression, and Ab quality. Two post-vaccination pools were prepared from sera of infants vaccinated with either Hib PS oligomers coupled to CRM197, a mutant diphtheria toxin (HbOC), or with higher m.w. Hib PS coupled to an outer membrane protein complex of Neisseria meningitidis group B (Hib-OMP). The mean anti-Hib PS Ab avidity of the serum pool from the infants vaccinated with HbOC was threefold higher than that of the pool from infants vaccinated with Hib-OMP. Using sequential immunoabsorption, three IgG1 idiotypically-defined anti-Hib PS fractions were isolated from each of the serum pools: Hibld-1, Hibld-2, and a Hibld-1/-2-depleted population, designated Hibld-0. Hibld-1 and Hibld-2 are idiotypic markers for anti-Hib PS Abs expressing kappa II-A2 and lambda VII V regions, respectively. Hibld-1 anti-Hib PS Abs had significantly higher avidity, 2- to 19-fold higher in vitro bactericidal activity, and were more protective against Hib bacteremia in infant rats, than the respective Hibld-2 Abs isolated from each of the pools. Comparing the two vaccines, Hibld-1 anti-Hib PS Abs elicited by HbOC had significantly higher avidity and 10-fold higher bactericidal and rat protective activity than the Hibld-1 Abs elicited by Hib-OMP. These findings demonstrate that the molecular form of the Hib PS immunogen dictates both V region usage and quality of Ab function.

Increase of auto anti-phosphatidylinositol antibodies in plasma of female rats during the appearance of DMBA-induced malignant mammary tumors

Using a lipid-adapted ELISA, antiphosphatidylinositol (PtdIns) antibodies (Ab) have been found in sera of cancer patients. With the same procedure, we have checked their possible raising in plasma of female rats during the appearance of 7,12-dimethylbenz[ a]anthracene (DMBA)-induced mammary tumors. Twenty days after DMBA administration, the mean anti-PtdIns Ab level was already higher than that of control rats (oil). Immunochemical analysis of the anti-PtdIns Ab site showed a rather high Ab avidity (8 × 10 −9 M, at half-displacement) and high specificity for glyceryl phosphate inositol residues. Other phospholipids (PL) were not recognized by the anti-PtdIns Ab induced by malignant cell transformation.

Humoral immunity in myasthenia gravis: clinical correlations of anti-receptor antibody avidity and titer.

Antibody to human acetylcholine receptor (AChR Ab) in myasthenia gravis (MG) correlates with clinical (Osserman) classification. Patients in remission R) or with ocular only (I) symptoms differed significantly from those with generalized disease (IIA, IIB, III, IV) (p less than 0.01, p less than 0.05 respectively). Patients with mild generalized disease (IIA) differed significantly from those with acute severe (III) or chronic severe (IV) disease (p less than 0.01). However, within each clinical class titers ranged over two or three orders of magnitude. This variation in AChR Ab titer for patients with similar diseases severity was not explained by differences in immunoglobulin class. All patients produced IgG AChR Ab and occasional patients produced IgM or IgA at less than 10% of their IgG titer. No IgM to IgG switch was identified. In MG patients negative for AChR Ab by immunoprecipitation assay, blockade of toxin binding to extracted human AChR could still be identified indicating antibody specificity to the toxin binding site. The avidity of AChR Ab for receptor assayed in six myasthenic patients with differing severities of disease, varied widely with T1/2 (time to half-maximal binding) ranges from 25 to 81 minutes. However, differences in AChR Ab avidity did not explain differences in severity of disease in patients with similar titers. AChR Ab was fractionated in six patients into IgG kappa and IgG lambda; in four patients AChR Ab activity could be demonstrated in both fractions. Thus, differences among MG patients as a group are due to production of several AChR Ab idiotypes, with individual patients being oligoclonal or polyclonal as well. Differences in IgG subclass (complement fixation) and site of attachment of AChR Ab to receptor subunits may resolve these differences.