Properties of mouse homocytotropic and heterocytotropic antibodies.

Mouse antisera were analyzed for the presence of homocytotropic and heterocytotropic antibodies. Two distinct populations of antibodies were detected by the homologous passive cutaneous anaphylaxis (PCA) reaction. The first was active 2 hr after injection, heat-stable, and partially reactive with antisera to mouse IgG1. The second was active 48 hr after injection, was heat-labile, and probably belonged to the IgE class of mouse immunoglobulins. In addition, it was demonstrated that there were also two antibodies active in the heterologous PCA reaction in rats, a heat-stable antibody and a heat=labile antibody. Contrary to results obtained with homocytotropic antibodies, none of the heterocytotropic antibodies detected reacted with antisera to mouse IgG1 or IgG2. These studies suggest that in addition to IgE there may exist another heterocytotropic antibody in mouse antisera and that caution should be employed when using the 2-hr PCA reaction in the rat as a sole criterion for detection of mouse IgE.     

Separation of mouse homocytotropic antibodies by biological screening

An attempt was made to separate mouse γ₁ antibody from mouse reaginic antibody by injecting mouse antiserum containing both antibodies into normal mice and then at 6 and 24 hours bleeding the animals and testing their sera for passive cutaneous anaphylaxis (PCA) activity. This was termed biological screening. The 2-hour homologous PCA activity was used as a measurement of mouse γ₁ and the rat PCA activity of the mouse antisera was used as a measurement of mouse reaginic antibody. These experiments showed that in vivo screening of mouse antisera containing both mouse and rat PCA activity results in removal of the rat PCA activity of these sera whereas the mouse PCA activity remains practically unchanged. It is concluded that a separation of the mouse serum PCA activity due to γ₁ antibody from that due to mouse reaginic antibody can be achieved by biological screening. Screening experiments using very early mouse antisera collected 8 days after a single antigenic stimulation resulted in the simultaneous disappearance of both homologous and heterologous PCA activity. Heating of these very early antisera resulted in complete inactivation of heterologous PCA activity and almost complete inactivation of homologous PCA activity. Absorption of these same antisera with rabbit anti-mouse γ₁ caused no change in homologous or heterologous PCA activity. It is suggested that the PCA activity of the very early antisera is due almost entirely to mouse reaginic antibody.     

Properties of rabbit antiragweed and antihorseradish peroxidase homocytotropic antibodies

Homocytotropic antibodies (HCA) were produced by the rabbit after primary injection of the dialysis residue of an aqueous extract of dwarf ragweed pollen (DRg) or a purified dwarf ragweed pollen extract (Pool C) only when the antigen was adsorbed on Al(OH)₃. The properties of rabbit ragweed-specific HCA were qualitatively similar to ragweed skin-sensitizing antibodies found in sera of ragweed-allergic human beings, namely: homocytotropic activity (72 hour homologous PC A reaction), heat lability at 56 °C., and inactivation by reduction with 2-mercaptoethanol followed by alkylation with iodoacetamide. The molecular weight of rabbit HCA was ~250,000 (Sephadex G-200), somewhat higher than human γE skin-sensitizing antibodies. Most rabbits immunized with ragweed antigen-Al(OH)₃ systems gave positive conjunctival and intradermal reactions. Intravenous challenge (8 mg. per kilogram) of these animals produced anaphylaxis. HCA were also elicited in response to the immunologic marker, horseradish peroxidase (HRP), when admixed with Al(OH)₃. The properties of HRP-specific HCA (molecular weight ~250,000) were similar to rabbit ragweed-specific HCA. The results suggest that the rabbit can be used as a model to study the biosynthesis of antibodies that have some physicochemical and biologic characteristics in common with human antibodies involved in immediate hypersensitivity.     

Heterogeneity of allergens from, and homocytotropic antibody to, a gastro-intestinal nematode of rabbits

More than one homocytotropic (reagin-like) antibody response in rabbits was demonstrated to a natural parasite, Trichostrongylus retortaeformis, a nematode which occurs only in the gastro-intestinal tract. The responses were detected by the homologous passive cutaneous anaphylaxis (PCA) reaction, three days after skin sensitization. Isoelectro-focusing of aqueous worm larval extract provided fractions in which more than one allergen was recognized. The separate allergen-homocytotropic antibody reactions were found with whole sera from different animals which had been infected by larvae of different ages, but it was not shown in this work whether this difference caused the separate antibody responses.     

Heterogeneity of guinea-pig lymphokines revealed by parallel bioassay

This paper describes the application of parallel bioassay to the measurement of lymphocyte mitogenic, inflammatory and macrophage migration inhibitory activities present in guinea-pig lymphokine preparations. Seven lymphokine preparations were made under similar conditions by antigen activation of sensitized guinea-pig peritoneal exudate cells; and one of these was prepared in sufficient quantity as a `working standard' for the repeated assay of the three lymphokine activities in the other six `test' preparations. Mean potency ratios of the separate lymphokine activities were calculated for the test preparations by reference to those of the standard preparation (designated as unity). Although the seven preparations were made under operationally similar circumstances, χ² analysis of the assay results revealed that the three separate lymphokine activities occurred in different absolute amounts and relative proportions in the different preparations. This demonstration by parallel bioassay of heterogeneity and dissociation of lymphokine activities implies that these biological activities cannot be ascribed to a single substance.     

Role of mouse IgG and IgE homocytotropic antibodies in passive cutaneous anaphylaxis.

The role of the mouse homocytotropic antibodies in passive cutaneous anaphylaxis reaction was investigated. One class of antibody was heat stable, detected at 2 h but not at 48 h after passive transfer, and belonged to a sublcass of mouse IgG. The other was heat labile, detected at 2 h and 48 h after passive transfer, and belonged to the IgE class of mouse immunoglobulins. In the presence of IgG, IgE homocytotropic antibody was not detected early after passive transfer. This was thought to be due to a masking of IgE by IgG antibodies rather than a competition for mast cell surface receptors, since inhibition studies with rat IgE myeloma protein suggested that mouse IgE and IgG1 may have different receptor sites on mast cell surfaces.     

Heterogeneity of anti-idiotypic antibodies to anti-DNA antibodies in humans.

Serum antibodies in some patients with systemic lupus erythematosus (SLE) were found to have specificity to idiotypes (Id) of 0-81 (human monoclonal anti-single-stranded DNA (ssDNA) antibody) but not to Id of NE-1 (human monoclonal anti-double-stranded DNA (dsDNA) antibody) or pooled human IgM. The interaction of the antibodies and 0-81 was blocked by the co-existence of free ssDNA. Some of SLE sera also showed preferential binding to Id determinants of NE-1, which included the antigen-binding sites of the dsDNA antibody. Some other SLE sera reacted with both Id of 0-81 and NE-13. Thus, there was heterogeneous population among human anti-Id autoantibodies to anti-DNA antibodies. The anti-Id activity was commonly detected in inactive SLE sera, and less frequently in normal controls, suggesting some regulatory role for anti-Id antibodies in the production of autoantibodies.     

Half-lives of two types of rat homocytotropic antibodies in circulation and in the skin.

The half-lives of two classes of rat immunoglobulins with homocytotropic properties, i.e., IgE and IgG2a, in the circulating blood and in the skin were studied. The catabolism of both normal (reaginic) and pathological (myeloma) IgE proteins in circulation was found to be extremely rapid with a half-life of 12 h. In contrast, the half-life of IgE antibody in the homologous skin was calculated to be 7.4 days. On the other hand, the half-life of IgG2a in circulation was about 5 days regardless of normal or pathological origin. IgG2a protein was, however, rapidly cleared from the injected skin site with a half life of 2.4 days, a value not longer than that obtained with nonskin-sensitizing goat IgG. The results indicate that rat IgE has an extremely short half-life in circulation despite of its sensitization period in tissues, and that the affinity for the target cells, as well as the mode of sensitization, of the two classes of homocytotropic antibodies, IgE and IgG2a, is different.     

The specificity of skin-sensitizing antibodies in the guinea-pig

In this paper the specificity of the skin-reactive guinea-pig immunoglobulin in the homologous PCA test has been reconsidered. Extensive purification of this immunoglobulin by chromatographic methods results in the isolation of a small fraction of medium speed immunoglobulins that contains all skin reactivity. No correlation could be found with the 7S immunoglobulins IgGγ1 and Igγ1A, suggesting a separate class of biologically active immunoglobulins. The PCA active antibody belongs to either an IgG subclass or is a heat stable homocytotropic antibody.Rabbit and chicken antisera against isolated fractions of guinea-pig immunoglobulins show the existence of 7S subclasses. Some suggestions for a revised nomenclature are given.     

The immunological response of mice infected with Trichinella spiralis: Biological and physico-chemical distinction of two homocytotropic antibodies

(1) Infection of mice with Trichinella spiralis induced the appearance in serum of two homocytotropic antibodies that could be distinguished by their biological and chromatographic behaviour.

(2) Biologically, the antibodies could be distinguished by their ability to persist in homologous skin after passive transfer. One antibody was able to induce PCA only when a short latent period was used, whereas the other was able to induce PCA even after 72 hours.

(3) They could also be separated when antiserum was passed through a DEAE-cellulose column. Antibody present in the first eluates was able to induce PCA only if a short latent period was used whereas antibody present in the subsequent eluates was able to induce PCA 72 hours after sensitization.

(4) Both antibodies appeared in the circulation 5 weeks after infection and reached their highest levels around the 9th week. Later, the 72-hour PCA antibody disappeared from the serum in some animals, whereas the 4-hour PCA antibody remained.

(5) Re-infection resulted in an increase in the levels of both antibodies.

(6) In animals subjected to repeated reinfections the reagin-like antibody either decreased or disappeared from the serum. On the other hand, the 4-hour PCA antibody increased.

(7) Immunization with `dead' T. spiralis antigen led to the appearance of both antibodies in the serum. A second dose of antigen resulted in increases in the levels of both antibodies, but further injections resulted in a high level of 4-hour PCA antibody and in the disappearance of the reagin-like antibody.

     

Further studies on the biological properties of guinea-pig IgG1 antibodies: anti-lymphocyte antibodies

Anti-rat lymphocyte serum was raised in guinea-pigs in order to study the ability of two 7S immunoglobulin classes to prolong the survival of rat skin homografts. Both classes, complement-fixing IgG2 and anaphylactic IgG1, are active, although the threshold for IgG2 activity is higher. This threshold can be lowered when a mixture of both classes is used. Thus, in this system, complement activation is not a requisite, and different mechanisms must come into play according to the class of anti-lymphocyte antibodies used. Moreover, these results have bearings on the postulated regulatory function of IgG1—antigen complexes.     

Production of guinea pig IgG1 homocytotropic antibodies to hapten-conjugated homologous serum albumin with different adjuvant combinations.

The effects of various antigen-adjuvant combinations on the production of IgG1 antibody was investigated with p-aminobenzoate conjugated to guinea pig serum albumin. The results demonstrate the importance that initial exposure of antigen-adjuvant combination has on IgG1 production. Further, there is a synergistic effect when antigen adsorbed to alum and antigen with complete Freund's adjuvant were used in conjunction with one another. The result of this effect is discussed within the context of T and B cell activation by antigen-adjuvant combinations.     

Heterogeneity of the antigenic centres of albumin toward chicken antibodies

Pooled chicken antiserum to bovine serum albumin contained antibodies capable of binding eighteen sites on the albumin molecule. Quantitative study of the reactivity of albumin following gradual coupling to tyrosyl, histidyl and lysyl residues, indicated that these residues are in close relationship to the antigenic centres, and that the centres and their respective antibodies are heterogeneous.