Rabia Hussain

The quantitation of parasite-specific human IgG and IgE in sera: evaluation of solid-phase RIA and ELISA methodology.

We have developed a non-competitive solid-phase radioimmunoassay (SPRIA) to quantitate both human IgE and IgG antibodies against soluble adult antigens of Brugia malayi (B.m.), a filarial parasite causing extensive infection throughout the tropics. Previously enzyme-linked immunosorbent assays (ELISA) had been used to detect microgram/ml levels of IgG anti-B.m., but IgE antibodies were difficult to detect in this system. Since the SPRIA successfully quantitates both IgG and IgE anti-B.m., we sought to examine the reasons for the SPRIAs apparent superiority in detecting IgE anti-B.m. by extracting specific IgG from sera with high levels of IgE and IgG anti-B.m. antibodies. IgE anti-B.m. was then quantitated in these sera using both the SPRIA and ELISA methods. Results indicate that IgG anti-B.m. does not interfere with detection of specific IgE antibody in the SPRIA but does interfere in the ELISA. While ELISA permits detection of IgE anti-B.m. in the absence of competing IgG anti-B.m., as levels of specific IgG increase, the IgE is no longer detectable. These differences between SPRIA and ELISA can be explained by the SPRIAs antigen excess conditions which assure that there are sufficient antigens both to detect all anti-B.m. antibodies present in the serum and to adequately represent all antigen specificities in the crude B.m. extract. Our findings commend the use of SPRIA methods over ELISA in assessment of B.m.-specific IgE antibody in filariasis and indicate a potential role for SPRIA methods in absolute quantitation of specific serum antibodies.

Use of monoclonal antibodies to quantify subclasses of human IgG: I. Development of two-site immunoenzymometric assays for total IgG subclass determinations

Dissection of the IgG antibody response into its subclass components has been difficult largely because of the lack of adequate supplies of specific reagents. The development of monoclonal antibodies (Mcab) promises to overcome this problem, but the use of such antibodies has certain inherent problems. It has been shown recently that Mcabs which were avid, potent and specific for well defined epitopes may partially or completely lose their activity depending on the assay system in which they were used. In order to identify Mcabs that would be specific and useful as capture antibodies in a simple two-site enzymometric assay, a panel of 18 Mcabs was screened and one Mcab to each of the four IgG subclasses was identified for quantitation of subclass levels in human serum.

Brugia malayi: Stage-specific expression of carbohydrates containing N-acetyl-d-glucosamine on the sheathed surfaces of microfilariae

Microfilariae, infective larvae, and adult worms of Brugia malayi were incubated with a panel of seven lectins in order to study the expression of surface carbohydrates. Infective larvae and adult worms did not bind any of the lectins utilized. Microfilariae, on the other hand, bound wheat germ agglutinin. The binding of this lectin was saturable and specific, and attributed to the presence of N-acetyl-D-glucosamine. In addition, microfilariae derived in vitro bound concanavalin A, indicating the presence of glucose and/or mannose on this stage of the parasite. The fact that similar concanavalin A binding was not seen on microfilariae recovered directly from the infected host implies that there is masking or loss of parasite surface antigens as microfilariae mature in vivo.

Comparison of immunoblot and immunoprecipitation methods for analyzing cross-reactive antibodies to filarial antigens.

Qualitative analysis of antibody responses in helminth infections is essential not only for developing better immunodiagnostic antigens but also for understanding immune recognition and its relevance to immunopathogenesis and protective immunity. In this study 2 qualitative analytic methods (immunoprecipitation and immunoblotting) were compared for the ability to define the extent of cross-reactivity in the serum antibodies from patients with various forms of filariasis (caused by Brugia malayi, Wuchereria bancrofti, Loa loa and Tetrapetalonema perstans) or other non filarial helminth infections (ascariasis, strongyloidiasis, trichinosis, echinococcosis and schistosomiasis). Our results demonstrated that the spectrum of cross-reactive antibodies identified by immunoprecipitation was limited because of the selective radiolabeling of particular filarial antigens, while immunoblotting was able to detect a much wider range of cross-reactive antibodies in both filarial and non-filarial serum pools. In addition, this latter procedure was easily adapted for simultaneous analysis of different antibody isotopes (e.g., IgE and IgG) to the same antigens in individual sera. Immunoblotting thus provides an excellent tool for studying the spectrum of antibodies of different isotypes evoked during helminth infections and for discriminating between those responses that are species-specific and those that are cross-reactive.

Parallel recognition of filarial antigens by IgE and IgG4 antibodies.

IgE responses are highly modulated in human filariasis and allergic symptoms usually associated with IgE antibodies are seldom observed in individuals with Elephantiasis and Microfilaremia. Only a minor group with tropical pulmonary eosinophilia (TPE) suffer from nocturnal asthma. One possible mechanism may be the presence of IgG blocking antibodies which can block IgE mediated histamine release in vitro. Using immunoblot analysis we have further shown that remarkably similar pattern of antigen recognition were obtained with IgE and IgG antibodies which would be a major prerequisite for the blocking activity. In the present study, we have tried to determine if a particular IgG subclass is responsible for the parallelism seen with IgE antibodies. Immunoblotting with autoradiographic analysis was used to study IgE and IgG subclasses using highly characterized radiolabeled antibodies in 24 sera representing all three groups mentioned above. The most striking observation was that in 23/24 sera, the only antibody that showed consistent and parallel binding was IgG4. No consistent pattern of response was seen with any of the other three subclasses. IgG1 and 2 when present were usually directed predominantly to the high molecular weight antigens and were detected with greater frequency in Elephantiasis group. These results suggest that IgG4 may be an ideal candidate for the blocking activity seen in these sera. Such modulation of IgE responses may have important survival value for the host in filarial infection which tend to evoke rigorous IgE responses.