Zoltan Ovary

PCA reactions with mouse antibodies in mice and rats.

Mouse IgE was titrated in rats. The sensitization period was 2 h. The results were consistent and corresponded to the titers obtained in young adults SJL mice using a sensitization period of 48 h. With longer sensitization periods in rats, higher antibody titers were obtained. The optimum sensitization period in rats was found to be 48 h. Old CFW mice are inadequate for mouse IgE titration. IgG1 will not give a PCA reaction in rats. IgG1 titers are higher in SJL or A/J mice than in young CFW mice, and markedly higher than in old CFW mice.

Immunological Specificity of the Secondary Response with Dinitrophenylated Proteins.

Summary The data presented clearly indicate that anamnestic response in both systems studied is related to the immunological backbone of the carrier protein as well as the hapten, and that the carrier protein alone injected secondarily does not cause production of antibodies directed against the hapten used for primary immunization.

High Molecular Weight Antibodies

Despite the fact that the existence of high molecular weight antibodies has been known for a long time, only recently have they received attention commensurate with their significance. Several explanations may be cited for this fact. First and perhaps foremost are the fallacious concepts that these antibodies occur in only a few species (particularly the horse) and that the rabbit and the human produce only low molecular weight antibodies in response to antigenic stimulus. This was based on early work with antipneumococcal antibodies, in which few animals were examined. It now appears that not only do many horses make low molecular weight antibodies but that rabbits can make the high molecular weight type, even to pneumococci.’ Of essential importance are the stage of immunization and variations among individual animals. Another factor hindering their recognition was the belief that high molecular weight antibodies were artifacts produced by the aggregation of y globulin under chemical treatment. The failure of Cann2 to find this species in human y globulin isolated by electrophoresis convection contributed significantly to this concept. I t is now clear that y globulin isolated by other methods of electrophoresis does contain a high molecular weight component, representing as much as 10 per cent of the total y globulin! The high molecular weight material is a clearly definable entity in human and rabbit serum, with physical and chemical properties totally different from ordinary7 globulin. Antibodies belonging to this class also show these very different properties. It is no longer possible to say that a given material is or is not an antibody on the basis of its properties; the class of antibody, whether the ordinary 7s antibody or high molecular weight type, must be considered. In addition to these, a third group of “immunoglobulins,” recently defined by Heremans, contains antibodies of still different properties, the &A proteins! There undoubtedly are further subgroups. Before discussing the various special characteristics of the high molecular weight immunoglobulins, it might be well to consider the various antibodies and other materials from human serum that have been classified in this fraction. A summary of the types of antibodies and antibodylike substances of human serum that have been found to belong, at least in part, to the high molecular weight class is given in TABLE 1. Of course, the prototype of these proteins is the Waldenstrom macroglobulins. Some of these appear to have specific antibodylike activity. Cold agglutinin activity6 and complement fixation activity with human cellular antigensa have been described. In addition, we have recently encountered two cases of typical Waldenstrom macroglobulinemia in which the pathological proteins had rheumatoid factor activity. One of these7 precipitates at very high dilutions with aggregated y globulin. It also gives some of the other rheumatoid factor reactions, such as those of the =-sensitized human cell and the Fr. I1 chromium-treated red cell. I t does

Studies on Murine IgE with Monoclonal Antibodies

Rat monoclonal antibodies were constructed by fusion of immunized rat spleen cells with a nonsecreting mouse myeloma cell. Two monoclonal antibodies (6HD5 and HMK-12) were selected for further study. Both reacted with various IgE molecules of different specificities and different allotypes, but did not react with immunoglobulins of other isotypes and with light chains. These antibodies were therefore anti-isotypic (IgE) and not anti-allotypic or anti-idiotypic. It was shown by competition studies that these antibodies recognize different epitopes on the FcR epsilon fragment. A sensitive ELISA for the quantitation of murine IgE was developed with these monoclonal antibodies; the sensitivity was between 2 and 250 ng/ml for detection of serum IgE levels. Good correlation was obtained with protein amounts as determined by enzyme-linked immunosorbent assay (ELISA) and passive cutaneous anaphylaxis (PCA) activities. Both monoclonal antibodies were used to study anaphylactic reactions elicited by IgE antibodies. Both could inhibit PCA reactions and both could elicit reverse PCA reactions.

Characteristics of Guinea Pig IgE

Anti-dinitrophenyl (DNP) IgE antibody was produced in guinea pigs (GP) by immunization and repeated boosters with 1 mug DNP-Ascaris extract in A1 (OH)3. The isoelectric point was determined to be 6.48 and the molecular weight to be 185,000 daltons. In absorbtion chromatography, rabbit anti-GPIgG1 could not absorb the anaphylactic activity of the IgE. The activity of IgE was destroyed by heating at 56 degrees C for 1 h. IgE persisted in the skin for at least 3 weeks.

Adjuvant Effects of Amorphous Silica and of Aluminium Hydroxide on IgE and IgG1 Antibody Production in Different Inbred Mouse Strains

The adjuvant effects of an amorphous silica (Aerosil) and of A1(OH)3 on primary and secondary IgE and IgG1 immune responses to small doses of OA were studied comparatively in BALB/c, C57BL, DBA/1, AKR, DBA/2 and SJL inbred mouse strains. During the primary response, all mouse strains produced both IgG1 and IgE antibodies when antigen was given with A1(OH)3, whereas only DBA/1, BALB/c and DBA/2 mice produced IgG1 and/or IgE antibodies when Aerosil was used as the adjuvant. A booster effect, higher than that obtained by A1(OH)3, was induced by Aerosil in all mouse strains. The adjuvant effect of Aerosil was more selectively directed to the production of IgE antibody.

An improved method for the detection of IgE antibody of defined specificity by ELISA using rat monoclonal anti-IgE antibody

An improved method for the detection of IgE antibody of defined specificity by ELISA using rat monoclonal anti-IgE antibodies is described. The innovation consists of coating the plates first by a monoclonal rat anti-murine IgE antibody, adding the sera to this antibody-coated plates and then adding the biotin-conjugated antigen after the sera. The plates are then reacted with streptavidin-peroxidase and developed. This procedure eliminates possible competitions with other isotypes of the same specificity. The method is useful especially to quantitate IgE with defined specificity in the presence of high amounts of isotypes of the same specificity.

Effect of Nippostrongylus brasiliensis infection on anti-hapten IgE antibody response in the mouse. I. Induction of carrier specific helper cells.

Abstract Inoculation of infective larvae of Nippostrongylus brasiliensis into A/J, BALB/c, and SJL mice primed intraperitoneally (ip) 3 weeks before infection with 1 μg of dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) mixed with 1 mg Al(OH)3 induced a carrier effect on anti-DNP IgE and IgG1 antibody responses when the experimental mice were secondarily immunized with an ip injection of 1 μg of DNP-coupled N. brasiliensis extract (DNP-Nb) plus alum 2 weeks after infection. The magnitude of the hapten specific antibody response did not correlate rigidly with the number of larvae in the inoculum. Thus, a dose of 100 larvae was as effective in inducing the carrier effect as a dose of 800 larvae. Kinetic studies in A/J and BALB/c mice revealed that the anti-DNP IgE antibody response reached a maximum titer 7 days after the secondary immunization. These studies also showed that the enhanced IgE antibody response persisted for more than 40 days, while the response in all control groups terminated prior to that time. Using the adoptive transfer system, it was demonstrated that lymphoid cells obtained from the spleens or the mesenteric lymph nodes of infected mice cooperated with DNP-KLH primed cells to produce hapten specific IgE and IgG, antibodies when the challenge was made with DNP-Nb but not when it was made with 1 μg DNP-ovalbumin, clearly indicating carrier specificity. The helper activity of the cells obtained from infected mice was completely abolished or greatly reduced by the in vitro treatment with anti-θ serum and complement. The helper cells with maximum activity were present as early as 14 days after inoculation. The level of helper activity gradually decreased after 14 days. The results indicate that N. brasiliensis infection is effective in inducing carrier specific helper cells of thymic origin (T cells) in anti-DNP antibody responses. These results confirm those obtained by other investigators and add the new observation that N. brasiliensis infection elicits special helper T cells which induce an enhancement as well as a prolongation of anti-DNP IgE antibody response.

Production of Monoclonal Mouse IgE Antibodies with DNP Specificity by Hybrid Cell Lines

IgE anti-DNP antibody-secreting hybridomas were obtained by polyethylene glycol fusion of murine myeloma cells with spleen cells from mice, primed with DNP-KLH and challenged with DNP-Nippostrongylus brasiliensis extract. The in vivo and in vitro continuously growing hybridomas are producing high amounts of monoclonal IgE anti-DNP antibodies, which are heat-labile, hapten-specific and have rat mast cell sensitizing capacity.

Effect of Nippostrongylus brasiliensis infection on anti-hapten IgE antibody response in the mouse: II. Mechanism of potentiation of the IgE antibody response to a heterologous hapten-carrier conjugate

Nippostrongylus brasiliensis infection was capable of potentiating the primary and secondary mouse IgE antibody response to a hapten coupled to a nonhelminthic antigen (ovalbumin, Ov). When mice were infected 14 or 5 days before an ip injection of 1 μ g dinitrophenylated Ov (DNP-Ov) mixed with 1 mg Al(OH) 3 , the anti-DNP IgE antibody response was greatly enhanced. The infection did not enhance anti-DNP IgG 1 antibody response. Potentiation of the primary IgE antibody response coincided with the generation of Ov or N. brasiliensis antigen (Nb) specific lymphoid cells of thymic origin (T cells). Moreover, it was found in adoptive transfer experiments that the potentiated response against DNP determinants can be obtained by concomitant administration of Nb with DNP-Ov into recipients that have been given the combination of lymphoid cells from infected and DNP-primed donors. These results suggest that Nb specific T cells could provide a certain signal, probably a soluble factor, upon stimulation with the worm antigen.