Characterization and antigenic specificity of chlorpromazine-induced antinuclear antibodies

Prolonged treatment with chlorpromazine is often associated with the development of antinuclear antibodies, an immunoglobulin M lupus anticoagulant, and polyclonal serum IgM elevation, but not with clinical features of systemic lupus erythematosus (SLE). Sera from 62 long-term psychiatric patients given treatment daily with 100 mg or more of chlorpromazine for at least 1 year were screened for antinuclear antibodies by indirect immunoperoxidase assay using HEp-2 cells. In 26 samples, antinuclear antibody titers greater than or equal to 1:40 with a homogeneous pattern were seen when anti-human IgM was used as the second antibody, three sera samples reacted with IgG, and four samples reacted with both IgG and IgM antisera. The antinuclear antibody antigenic reactivity was investigated by using histone and nonhistone nuclear antigens by enzyme-linked immunosorbent assay and passive hemagglutination techniques. Forty serum samples reacted with histone. Twenty-five samples reacted with deoxyribonucleoprotein (DNP), 28 with single-stranded DNA, and two with double-stranded DNA. No reaction was obtained with the extractable nuclear antigens RNP or Sm. These results indicate that chlorpromazine-induced antinuclear antibodies, like the antinuclear antibodies induced by hydralazine and procainamide, react mainly with histone nuclear antigens. Unlike the hydralazine and procainamide response, in which both IgG and IgM antibodies are demonstrated, the chlorpromazine-induced autoantibodies are predominantly of the IgM class.     

ANTI-DNA ANTIBODIES IN HYPERIMMUNIZED RABBITS

Complement-fixing anti-DNA antibodies were detected in a minority of sera of rabbits hyperimmunized with killed Gram-negative bacteria. The C'-fixing property of DNA was lost after DNase treatment. Preferential reactivity with denatured DNA was observed. The antisera reacted with DNA preparations derived from rabbit bone marrow and thymus, calf thymus, pneumococci, salmon sperm, and Escherichia coli. E. coli DNA was less effective than preparations of mammalian and salmon sperm DNA in fixation of C'. Inhibition of DNA C' fixation by nucleotides and nucleosides was observed. The bulk of anti-DNA activity was associated with the low molecular weight antibody fraction.     

CARDIAC AUTOANTIBODIES : I. IMMUNODIFFUSION ANALYSIS OF MULTIPLE RESPONSES EVOKED HOMOLOGOUSLY AND HETEROLOGOUSLY

1. A high proportion of rabbits immunized with pooled rabbit heart homogenates in complete Freund's adjuvant responded with the production of multiple precipitating antibodies to soluble rabbit heart antigens. The most potent antisera revealed at least five distinct antigens. 2. Rabbit anti-rabbit heart antibodies reacted with similar antigens in other mammalian hearts, (human, rat, guinea pig, and bovine) with apparent "reactions of identity" by immunodiffusion. 3. Rabbits immunized with human, rat, or guinea pig heart homogenates also responded with multiple precipitating antibodies directed against rabbit cardiac antigens, although somewhat less intensively than animals immunized homologously. The specificities of these antibodies appeared to be the same as those evoked homologously. 4. The autoantibody nature of the homologously and heterologously induced responses was unequivocally demonstrated in several instances by reactions between the sera from immunized rabbits and their own hearts. 5. Many of the autoantibodies appeared to be directed against antigens restricted to the heart, judging by comparative immunodiffusion tests with other rabbit tissue extracts. This was convincingly confirmed by multiple absorption of potent antisera with several rabbit tissues. The cardiac-restricted antigens were also present in heart extracts of other mammalian species. In those instances where some of the cardiac-evoked autoantibodies reacted with other rabbit tissues, the tissue cross-reactions were quite variable. 6. Rabbits immunized with guinea pig heart homogenate suffered a high early mortality of undetermined cause, compared to animals immunized with rabbit, human, or rat hearts. 7. A small proportion of the anti-heart sera revealed immunodiffusion reactions with Group A streptococcal products, derived from organisms grown in antigen-free media. In these few instances, the reactions appeared unrelated to cardiac autoantibody responses.     

Antireceptor antibodies as probes of insulinlike growth factor receptor structure.

Insulin receptors and Type I insulinlike growth factor (IGF) receptors have a similar structure with a major binding subunit of Mr approximately 130,000 linked by disulfide bonds to other membrane proteins to form a Mr greater than 300,000 complex. Both insulin and Type I IGF receptors also interact with both insulin and IGF, although with different binding affinities. We used a panel of human and rabbit sera containing antibodies to insulin receptors to determine whether these sera also interact with Type I IGF receptors. Immunoglobulins from five of five human sera inhibited binding of 125I-insulin and 125I-IGF-I to insulin receptors and Type I IGF receptors in human placenta and human lymphocytes. The rank order of reactivity with both receptors was the same; two sera, however, appeared to be selectively less reactive with the Type I IGF receptor, especially in placenta. Sera from five of seven patients and from a rabbit immunized with purified insulin receptor effectively immunoprecipitated both placental insulin receptors and Type I IGF receptors. Of the remaining sera, one had only a low titer against the insulin receptor and did not immunoprecipitate the IGF receptor, whereas the second serum effectively immunoprecipitated cross-linked and surface-iodinated insulin receptors, but had negligible reactivity against the Type I IGF receptor. These results suggest that most antisera to the insulin receptor also contain antibodies to Type I IGF receptors. Whether both specificities are inherent in the same or different antibody molecules remains to be determined. These data support the hypothesis that the insulin and IGF-I receptors are separate but related molecules, although there remains a small possibility that both receptors are domains on the same protein.     

OCCURRENCE OF 19S AND 7S ANTI-IGGS DURING HYPERIMMUNIZATION OF RABBITS WITH STREPTOCOCCI

All 110 rabbits immunized with Group A, A-variant, and C streptococcal vaccines produced 19S anti-IgG in addition to antibodies to the streptococcal carbohydrates. 19S anti-IgG was detected by hemagglutination of rabbit red blood cells coated with rabbit anti-blood group F antibody. Antisera of 88 of these animals were also tested for 7S anti-IgG with a coprecipitation assay. This assay is based on the coprecipitation of 7S anti-IgG with complexes of streptococcal carbohydrate and anti-carbohydrate antibody. 50 of the 88 anti-Group C streptococcal antisera contained 7S anti-IgGs. In eight antisera the concentration was greater than 5 mg/ml. The data suggest a genetic influence on the occurrence of 7S anti-IgG. The eight rabbits which produced more than 5 mg/ml of 7S anti-IgG belonged to three related families. Moreover, there were families in which almost every member produced 7S anti-IgG and other families in which only 30% of the members manufactured 7S anti-IgG. The streptococcal vaccine was an especially efficient stimulus for the production of 19S anti-IgG, whereas the pneumococcal vaccine was much less effective in this respect. Furthermore, 7S anti-IgGs were not detected in antipneumococcal antisera, although the concentration of anti-capsular antibodies was similar to that of anti-carbohydrate antibodies in antistreptococcal antisera.     

THE INDIVIDUAL ANTIGENIC SPECIFICITY OF ANTIBODIES TO STREPTOCOCCAL CARBOHYDRATES

Although a single electrophoretically uniform antibody component with specificity for the group carbohydrate may comprise the bulk of the γ-globulin in rabbits immunized with streptococcal vaccines, this is not always the case. Not infrequently, electrophoresis may reveal multiple antibody components. Nevertheless, it has been feasible by various preparative procedures to isolate from a single antiserum at least two antibody components with similar reactivity for the carbohydrate both of which are electrophoretically monodisperse. Light chains from such antibodies reveal a restricted pattern when examined by disc electrophoresis. Antibodies to streptococcal carbohydrates have been examined for their individual antigenic specificity. Goats were immunized with isolated Group C and Group A-variant antibodies raised in rabbits. Individual antigenic specificity of these antibodies was brought out by absorption of the goat anti-antiserum with Fr II of pooled normal rabbit sera. Additional absorption of the goat anti-antisera with Fr II diminished but did not eliminate the reactivity for the homologous antibody. Immunoelectrophoretic studies with papain fragments of purified streptococcal antibodies localized the specificity to the Fab fragment. Specificity was not confined to the isolated light chains of the antibody.     

Induction of antibodies to nuclear antigens in rabbits by immunization with hydralazine-human serum albumin conjugates.

The antihypertensive drug hydralazine can induce in man a syndrome similar to spontaneous systemic lupus erythematosus (SLE). The pathogenesis of this drug-induced syndrome is not understood. In this investigation, five groups of rabbits were studied: group I, 10 rabbits hyperimmunized with hydralazine conjugated to human serum albumin (HSA) in complete Freund's adjuvant (CFA); group II, four rabbits with HSA in CFA; group III, four rabbits with CFA alone; group IV, five rabbits with hydralazine conjugated to rabbit serum albumin (RSA); and group V, four rabbits with a major metabolite of hydralazine conjugated to HSA. The rabbits immunized with hydralazine-HSA developed rising titers of antibodies to hydralazine and progressively increasing amounts of antibodies to both single-stranded and native DNA. The antibodies to DNA were cross-reactive with hydralazine as determined by inhibition of DNA binding and DNA hemagglutination tests. Similar results were obtained in rabbits immunized with the metabolite-HSA compound except the major hapten antibody response was to the metabolite. The DNA antibodies in this group were also capable of being absorbed by metabolite-HSA as well as hydralazine-HSA, indicative of the cross-reactivity between hydralazine and its metabolite. Immunization with hydralazine-RSA caused rabbits to produce antibodies to hydralazine but not to DNA, indicating the requirement for an immune response to the carrier protein in order for antibodies reactive with DNA to be produced. Thus, hyperimmunization of rabbits with hydralazine-protein conjugates may provide a useful animal model of SLE. The data suggests that an immune response to hydralazine may be important in human hydralazine-induced SLE.     

Naturally induced auto-anit-idiotypic antibodies. Induction by identical idiotopes in some members of an outbred rabbit family

Naturally induced auto-anti-idiotypic (AAI) antibody responses specific for antimicrococcal antibody idiotypes were detected in 42% of the rabbits in a family immunized with Micrococcus lysodeikticus. The natural AAI response of each rabbit recognized only a portion (11-41%) of that individual's total antimicrococcal antibody population. Cross-reactions of idiotypes were observed within the group of rabbits exhibiting natural AAI responses. Examination of the basis for the cross-reactions showed that the natural AAI antisera recognized identical idiotopes on the antimicrococcal F(ab')2 fragments from each rabbit that made an AAI response. The cross-reactive idiotopes were shown to be of paternal origin and were found in the antimicrococcal antibodies of each offspring. The data strongly support the idiotypic network concept that naturally induced AAI responses may occur routinely in outbred normal individuals as a result of antigenic stimulation. Further, the data suggest that the induction of regulatory AAI antibody responses in outbred rabbits may depend on the expression of particular germ line idiotopes.     

IDIOTYPY OF RABBIT ANTIBODIES : I. COMPARISON OF IDIOTYPY OF ANTIBODIES AGAINST SALMONELLA TYPHI WITH THAT OF ANTIBODIES AGAINST OTHER BACTERIA IN THE SAME RABBITS, OR OF ANTIBODIES AGAINST SALMONELLA TYPHI IN VARIOUS RABBITS

Sera of rabbits immunized against Salmonella typhi have been studied for the idiotypy of certain of their components, i.e., the property of these components to possess an antigenic specificity which is different in individual rabbits, and which varies with the antigens against which these rabbits have been immunized. The reagent used (precipitating anti-idiotypic sera) have been prepared by injecting rabbits with bacteria agglutinated by anti-S. typhi sera (immunizing sera) as was done in the first observations by the authors of the phenomenon in the rabbit. These first observations have been confirmed and extended. In contrast to allotypy, the anti-idiotypic sera precipitate the corresponding immunizing sera, but not the sera taken in the immunizing rabbits prior to their immunization against S. typhi, nor the immunizing sera absorbed with the somatic antigen of S. typhi, demonstrating that idiotypes are antibodies. The idiotypic specificities of the antibodies of one rabbit against S. typhi are not detected in the antibodies of the same rabbit against another noncross-reacting Salmonella (S. tranoroa) and vice versa; nor are they detected in the anti-pneumococcal antibodies of the same rabbit. Each anti-idiotypic serum fails to precipitate anti-S. typhi sera of rabbits other than the immunizing one except for certain extremely faint reactions, the significance of which has not been established. The idiotypic specificities of anti-S. typhi antibodies of three rabbits were not found in anti-S. typhi antibodies of their parents. This lack of a sign of hereditary transmission of idiotypic specificities contrasts with allotypy. The apparent role of random chance in the determinism of the idiotypic patterns or of the idiotypic determinants has been discussed. Unless it were admitted that antibodies with similar functions do not exist in different individuals, idiotypy apparently adds an order of magnitude to the antibody variability which had been previously envisaged. In one given individual, the heterogeneity of the idiotypic specificities seems to be less extended than that of the antibody functions. The possible relationships between these two levels of molecular variability and between the corresponding levels of cellular variability have been discussed.     

ELECTROPHORETIC STUDY OF ANTIVIRAL SERA

1. Sera of animals immunized against Japanese B encephalitis, Venezuelan equine encephalomyelitis, and Western equine encephalomyelitis viruses were fractionated by electrophoresis. 2. Electrophoretic patterns of rabbit sera before and after immunization against Japanese B virus showed no consistent change traceable to antibody formation. 3. To determine the antibody content, the electrophoretic fractions of the respective sera were mixed in varying dilutions with infected mouse brain suspensions, and the neutralizing titers of the fractions were compared. 4. In all instances serum fractions containing γ-globulin were protective, whereas in no case did serum albumin show any virus-neutralizing activity. The Japanese B encephalitis antibody appeared to be associated entirely with the γ-globulin. The Venezuelan and Western equine encephalomyelitis antibodies were associated with the β- and γ-globulins and probably possessed an average electrophoretic mobility between that of β- and γ-globulins. 5. Normal rabbit serum similarly separated electrophoretically showed no neutralizing properties. 6. Chickens, whose electrophoretic serum pattern is markedly different from that of rabbits, were also immunized against the Japanese B encephalitis virus. Their antisera were electrophoretically fractionated and similarly subjected to neutralization tests. The specific neutralizing capacity of chicken serum was considerably lower than that of rabbit serum and no neutralizing activity was found in the fractions containing the faster moving components. The antibody appeared to be associated with component 4 which had a mobility of approximately 2.3 x 10^–5 cm.²/volt/sec.     

EXPERIMENTAL ALLERGIC GLOMERULONEPHRITIS INDUCED IN THE RABBIT WITH HOMOLOGOUS RENAL ANTIGENS

Rabbits immunized to several homologous renal antigens developed a variety of autoantikidney antibodies. Some of these antibodies reacted with the host's glomeruli and appeared to cause glomerulonephritis. Passive transfer of sera from some of these nephritic rabbits into normal, unilaterally nephrectomized rabbits led to the induction of nephritis. The production of autoantibody to glomeruli was transitory in most instances in spite of continued immunization. In some rabbits immunized to whole kidney homogenate, extracts or sediment, antibodies were found fixed to renal tubular basement membranes where an ultrastructural lesion was demonstrated. Rabbits also produced antikidney antibody apparently to tubular cytoplasmic components which did not fix to kidney in vivo and were of no pathogenetic significance. Immunization with autologous renal basement membranes induced a small autoantibody response in half the rabbits. This response was not associated with detectable renal injury.     

Detection of autoantibodies to ss-a/ro by indirect immunofluorescence using a transfected and overexpressed human 60 kd ro autoantigen in hep-2 cells

The objective of the study was to determine the sensitivity and specificity of an indirect immunofluorescence (IIF) assay using transfected HEp-2 cells to detect anti-SS-A/Ro autoantibodies in human sera. Seventy-three sera having SS-A/Ro autoantibodies as determined by double immunodiffusion (ID) and immunoblotting (IB) were tested by IIF on a HEp-2 cell substrate that had been transfected with a full-length cDNA encoding a human 60 kD SS-A/Ro autoantigen. Controls included 30 normal human sera and 50 sera with a variety of other antinuclear antibodies. Prototype human and rabbit sera directed against the 60 kD SS-A/Ro antigen produced intense speckled nuclear and nucleolar staining of transfected cells. Sixty-nine of 73 (95%) SS-A/Ro positive sera also produced this characteristic staining pattern. The endpoint autoantibody titers on transfected cells was fivefold greater than on untransfected cells. The 30 normal human sera and the 50 sera with other antinuclear antibodies did not produce this characteristic staining. Six of 32 (19%) unselected sera that were sent for autoantibody testing had reactivity with transfectants by IIF. Four of the six sera were confirmed to have anti-SS-A/Ro antibodies by ID and 5/6 by IB. By contrast, only three of these sera were scored as having a staining pattern compatible with SS-A/Ro antibodies by IIF on standard HEp-2 substrates. We conclude that SS-A/Ro autoantibodies can be detected by an IIF assay using a HEp-2 cell substrate transfected with a SS-A/Ro cDNA. This new substrate detects SS-A/Ro antibodies that were not identified on standard HEp-2 substrates and by other immunoassays.©1995 wiley-Liss, inc.     

Induction of antibodies to hyaluronic acid by immunization of rabbits with encapsulated streptococci

The immunogenicity of hyaluronic acid was investigated. Rabbits were immunized with encapsulated group A and C streptococci. Intact long-chain hyaluronate was conjugated to BSA for use as antigen in an ELISA. Antibodies to the hyaluronate-BSA conjugate were detected in peak immune sera. The specificity of the antibodies for both mammalian and streptococcal hyaluronate was shown by inhibition studies. To further confirm the presence of antihyaluronate antibodies, hyaluronidase-digested streptococcal hyaluronate was conjugated to biotin and used as an antigen in the ELISA. A clear immunization effect was shown for each rabbit by the study of preimmune and postimmunization bleedings. Titers for each rabbit increased by greater than 32 - 256 - fold. Inhibition studies using hyaluronidase-digested hyaluronate and periodate-treated hyaluronate showed that the immunodominant site of antibody reactivity was a terminal glucuronic acid residue. Further studies showed that the carboxyl group of the terminal glucuronide was the major immunoreactive site. Both mammalian and streptococcal hyaluronate inhibited the immune rabbit sera reaction to streptococcal hyaluronate, demonstrating crossreactivity of these molecules. Thus, hyaluronate was shown to be immunogenic in rabbits.     

ANTIBODY FORMATION AGAINST TREPONEMA PALLIDUM—AGGLUTINATION

It has been shown by our experiments that the serum of rabbits treated with emulsions of Treponema pallidum contains agglutinating substances. Normal rabbit serum also possesses agglutinating power for this organism, but, as in the case of normal bacterial agglutinins, to an extent very much inferior to that possessed by the sera of immunized animals. Normal human sera will agglutinate similar pallidum emulsions, as will the sera of certain syphilitic patients with positive Wassermann reactions. Whether or not there is a quantitative difference of diagnostic value between the sera of normal human beings and those of syphilitics remains to be seen. The sera of rabbits immunized with strain A agglutinate Noguchi''s strain 9 in dilutions as high as 1 to 500. We regard as the most important result of these experiments the demonstration of definite antibodies in the circulation of animals treated with dead emulsions of Treponema pallidum. Since it is our belief that the agglutinating effect is due to an antibody essentially the same as that which produces bactericidal, precipitating, and opsonic effects, i. e., that there is probably one type of antibody only, we believe that the demonstration of agglutinins establishes the fact that in syphilis as in bacterial diseases the host responds by the formation of antibodies or sensitizers specific for the treponema. Spirocheticidal experiments with these sera, both in vitro and in vivo, are in progress.     

Study of penicillin antibodies by fluorescence polarization and immunodiffusion

1. Antibodies prepared in the rabbit to penicilloyl-rabbit serum albumin or to penicilloyl-bovine serum albumin were demonstrated by immunodiffusion and absorption methods to be apparently directed exclusively against the penicilloyl moiety. The antibodies could precipitate heavily substituted penicilloyl polylysine, as well as each other, with "reactions of identity". All could mutually deplete detectable antibodies by cross-absorption. 2. Small haptens containing both fluorescein and the penicilloyl group were synthesized. They were also capable of completely absorbing the antipenicilloyl antibodies from rabbit antiserum, as evidenced by immunodiffusion tests. The haptens were used in fluorescence polarization tests with gamma globulin from normal and immunized rabbits, and from normal and allergic humans. The rabbit antipenicilloyl gamma globulins in concentrations as low as 5 to 10 µg of protein/ml could significantly increase the polarization of the haptens. Normal gamma globulin had no effect at the highest concentrations tested, 1200 µg/ml. In all tests, rabbit and human antibody reacted similarly. 3. Fluorescence polarization titration curves for both human and rabbit antipenicilloyl gamma globulins were analyzed by computer and the antibody concentrations, avidities, and heterogeneity constants were determined. For the human antibody, the latter two values were 3.0 x 10⁷ M ^–1 and 0.78, while for the rabbit, they were 8.7 x 10⁶ M ^–1 and 0.71. The data were employed to estimate the limit of sensitivity of fluorescence polarization for detecting antipenicilloyl antibodies. Under the conditions employed, this value was roughly 0.4 µg antibody/ml. 4. When the whole rabbit sera were tested for penicilloyl antibodies by fluorescence polarization, both normal (preimmune) and immune sera revealed striking and equivalent increases in polarization with the penicilloyl haptens. This non-specific binding was shown to be due at least in part to serum albumin. Indications were obtained that it might be significantly reduced by increasing the pH or the salt concentration of the medium, or by addition of certain anions.     

STUDIES ON RABBIT LYMPHOCYTES IN VITRO : I. STIMULATION OF BLAST TRANSFORMATION WITH AN ANTIALLOTYPE SERUM

Rabbit lymphocytes may be stimulated in vitro with specific antiallotype sera to transform into "blast" cells and to synthesize DNA. This transformation only occurs when the donor cells are obtained from a rabbit having a given γ-globulin allotype (As4) and these cells are cultured in the presence of an antiserum prepared against the given allotype (As4). Heterologous (sheep, goat, and guinea pig) anti-rabbit γ-globulin sera also induce significant blast transformation and DNA synthesis in rabbit lymphocytes. Allotypic transformation and DNA synthesis are due to 7S antiallotype antibodies and do not require complement. The degree of transformation and rate of DNA synthesis is related to the concentration of antibody. Incubation of the appropriate cells with the antiallotype antibody for as short a time as 15 minutes results in a significant degree of "blast" transformation, indicating that the recognition of the antiallotype specificity in the cells and stimulation of the cellular changes leading to eventual transformation is rapid. The activity of the antiallotype sera as measured by transforming or haemagglutinating capacity, may be absorbed by lymphocytes of the appropriate allotype, but is not absorbed by lymphocytes from a donor rabbit not having the allotype to which the antiserum is directed. Transformation does not occur with mixtures of lymphocytes from different rabbits even if 1 donor is immunized against an allotype present in the other donor. Peripheral rabbit lymphocytes can also be induced to undergo "blast transformation" in vitro by phytohaemagglutinin and staphylococcal filtrate. The lack of demonstrable leucoagglutinins in staphylococcal filtrate and antiallotype serum indicates that agglutination is not a necessary prerequisite to the induction of blast transformation.     

Multi-subtype gp160 DNA immunization induces broadly neutralizing anti-HIV antibodies

A highly desirable feature for an human immunodeficiency virus type 1 (HIV-1) vaccine is the ability to induce broadly reactive anti-envelope antibodies that can neutralize primary HIV-1 isolates. Two immunizations with an HIV-1 envelope-encoding plasmid together with recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) resulted in high antibody titers in mice. The antibody induction was further enhanced after immunization with genes encoding HIV-1 envelopes originating from subtypes A, B and C. The sera from these animals were able to neutralize A, B and C viral isolates, whereas the sera from animals immunized solely with subtype B DNA neutralized only subtype B virus. The combined DNA vaccine gave serum antibodies with broad recognition of HIV-1 envelope epitopes as determined by peptide mapping. Cell-mediated immunity was not compromised by the increased humoral immunity. This demonstrates the ability of multiple envelope genes to induce the desired antibody response against several subtypes. Moreover, it documents the ability of rGM-CSF to enhance the potency of such a vaccine when given simultaneously. The strategy may be useful for making an HIV vaccine more potent and broadly effective against strains of different clades.     

IMMUNOLOGICAL STUDIES CONCERNING THE NEPHRITIS OF SYSTEMIC LUPUS ERYTHEMATOSUS

Antibodies were eluted from the isolated glomeruli prepared from the kidneys of 10 patients with the nephritis of systemic lupus erythematosus. Antibodies reacting primarily with buffer extracts of nuclei were eluted by acid treatment, and antibodies reacting mainly with DNA and nucleoprotein were eluted with deoxyribonuclease. Quantitative immunochemical studies revealed a high concentration of antinuclear antibody per milligram of γ-globulin in glomerular eluates compared with that in the corresponding serums. The γ-globulin of two eluates was found to consist predominantly of antinucleoprotein antibody. The selective elution of antinuclear antibodies was also indicated by the absence of other serum antibodies in the eluates. DNA antigen was demonstrated in the glomeruli of two kidneys with nephritis by means of isolated anti-DNA antibody labeled with fluorescein. In one of these cases, anti-DNA antibodies were also found concentrated in the glomeruli and, in the second, circulating anti-DNA antibodies were demonstrated in the patient's serum. The immunochemical evidence for the high specific activity of antinuclear antibodies and the association of DNA antigen with DNA antibody in glomeruli add further support for the antigen-antibody complex hypothesis for renal injury in systemic lupus erythematosus.     

ANTIGENIC PROPERTIES OF THE TYPE-SPECIFIC SUBSTANCE DERIVED FROM GROUP A HEMOLYTIC STREPTOCOCCI

1. A substance extracted from group A hemolytic streptococcus is described, which induces active immunity in mice, and in rabbits gives rise to precipitins and to protective antibodies passively transferable to mice. 2. The active immunity in mice is principally type-specific, but some degree of non-type-specific immunity is also developed. The passively transferable protective antibodies are type-specific with only a slight suggestion of non-type specificity. In the precipitin test, the rabbit immune sera give both type-specific and non-type-specific reactions which have not been fully analyzed serologically. 3. Substances contained in the extract absorb the protective antibodies from the serum of rabbits immunized with whole hemolytic streptococci. 4. The most satisfactory method of extraction so far developed is fully described. Chemical tests on the material are consistent with the presence of protein and nucleic acid. 5. The type-specific M substance, prepared as previously described, was compared in some of its antigenic properties with the above mentioned substance. It was found capable of inducing active immunity in mice and of absorbing protective antibody from anti-bacterial immune serum in a manner qualitatively similar to that obtained with the preparations made by the newer methods.     

QUANTITATIVE EXPERIMENTS WITH ANTIBODIES TO SPECIFIC PRECIPITATES. II

1. Antisera have been produced in chickens with specific precipitates from Type II pneumococcus horse and rabbit antisera. 2. Specific precipitates from anti-Types I and II pneumococcus horse sera removed the same amount of antibody from the chicken anti-horse specific precipitate serum. Specific precipitates from horse antisera to diphtheria toxin and to crystalline egg albumin removed about one-half of the antibody. 3. Specific precipitates from anti-egg albumin, antipneumococcus C substance, and anti-Type II pneumococcus rabbit sera removed the same amount of antibody from the chicken anti-rabbit specific precipitate serum. 4. No antibody was removed from the chicken anti-horse specific precipitate serum by rabbit specific precipitates or from the chicken anti-rabbit specific precipitate serum by horse specific precipitates. 5. It is concluded that the antigenic specificities of antibodies from the horse and rabbit are not influenced by their particular antibody functions.