Monoclonal antibodies to murine gamma-interferon which differentially modulate macrophage activation and antiviral activity

Four monoclonal IgG antibodies to purified, recombinant murine gamma-interferon (rIFN-gamma) have been produced by fusion of immune hamster splenocytes with HAT-sensitive murine myeloma cells. Specificity was confirmed either with an enzyme-linked immunosorbent assay (ELISA) that used immobilized rIFN-gamma or with a radioimmunoassay that employed soluble 125I-rIFN-gamma and heat-killed, fixed Staphylococcus aureus-bearing Protein A. Competition binding experiments suggested that the monoclonal antibodies (MoAb) displayed two distinct epitope specificities: one displayed by H1 and H2, and the other displayed by H21 and H22. By using murine-human recombinant IFN-gamma hybrid molecules, the H1/H2 epitope was shown to depend on the amino-terminus of IFN-gamma, whereas the H21/H22 epitope was formed by the carboxy-terminal amino acid sequence. The MoAb also reacted with natural IFN-gamma. When bound to a surface, all four MoAb, but not normal hamster IgG, removed 100% of the antiviral and MAF activities present in supernatants of cultures of the murine 24/G1 T cell hybridoma. In free solution, all four antibodies inhibited IFN-gamma dependent antiviral activity, but with different efficiencies. Soluble H21/H22 also blocked all of the 24/G1-derived activity that induces nonspecific tumoricidal activity in macrophages (MAF) while H1/H2 enhanced MAF activity. The differential inhibitory or enhancing activities of H21 or H1 reflected their ability to inhibit or enhance binding of 125I-rIFN-gamma to macrophages, respectively. Soluble H21/H22 and solid-phase H1/H2 inhibited 100% of the MAF, microbicidal, and Ia-inducing activities from lymphokine preparations produced by mitogen stimulation of normal murine splenic cells. These results help to establish definitive structure-function relationships for the IFN-gamma molecule, and indicate that IFN-gamma is the primary lymphokine responsible for inducing nonspecific tumoricidal activity and Ia antigen expression, and for enhancing microbicidal activity in macrophages.     

Anti-interferon-gamma antibodies in sera from HIV infected patients

High serum levels of antibodies to interferon-gamma (IFN-gamma) have been found in patients infected with human immunodeficiency virus (HIV). A radioimmunoassay (RIA) with a recombinant IFN-gamma protein or an affinity purified IFN-gamma preparation as antigens, was developed to detect the specific anti-IFN-gamma antibodies. Reactivity of sera to IFN-gamma was confirmed by Western blot analysis. These antibodies, however, do not seem to recognize the active site of the molecule, since they do not neutralize the antiviral IFN-gamma activity in a biological assay. These results enforce the hypothesis of the role of autoimmunization during the course of the disease.     

Protective efficacy of nonpathogenic nef-deleted SHIV vaccination combined with recombinant IFN-gamma administration against a pathogenic SHIV challenge in rhesus monkeys

We previously reported that a nef-deleted SHIV (SHIV-NI) is nonpathogenic and gave macaques protection from challenge infection with pathogenic SHIV-C2/1. To investigate whether IFN-gamma augments the immune response induced by this vaccination, we examined the antiviral and adjuvant effect of recombinant human IFN-gamma (rIFN-gamma) in vaccinated and unvaccinated monkeys. Nine monkeys were vaccinated with nef-deleted nonpathogenic SHIV-NI. Four of them were administered with rIFN-gamma and the other five monkeys were administered with placebo. After the challenge with pathogenic SHIV-C2/1, CD4(+) T-cell counts were maintained similarly in monkeys of both groups, while those of the unvaccinated monkeys decreased dramatically at 2 weeks after challenge. However, the peaks of plasma viral load were reduced to 100-fold in SHIV-NI vaccinated monkeys combined with rIFN-gamma compared with those in SHIV-NI vaccinated monkeys without rIFN-gamma. The peaks of plasma viral load were inversely correlated with the number of SIV Gag-specific IFN-gamma-producing cells. In SHIV-NI-vaccinated monkeys with rIFN-gamma, the number of SIV Gag-specific IFN-gamma-producing cells of PBMCs increased 2-fold compared with those in SHIV-NI-vaccinated monkeys without rIFN-gamma, and the NK activity and MIP-1alpha production of PBMCs were also enhanced. Thus, vaccination of SHIV-NI in combination with rIFN-gamma was more effective in modulating the antiviral immune system into a Th1 type response than SHIV-NI vaccination alone. These results suggest that IFN-gamma augmented the anti-viral effect by enhancing innate immunity and shifting the immune response to Th1.     

Characterization of monoclonal antibodies to human interferon-gamma and their application to enzyme-linked immunosorbent assay (ELISA)

The authors obtained two mouse monoclonal antibodies, G-208 and G-166, to recombinant human interferon-gamma (rH-IFN-gamma). Immunologically, they were classified as IgG1-K subclass. G-208 neutralized the antiviral activity of natural and recombinant human IFN-gamma, but did not bind to heat-denatured rH-IFN-gamma. G-166 was able to bind to rH-IFN-gamma as well as to heat-denatured rH-IFN-gamma, but it did not bind to natural human IFN-gamma (nH-IFN-gamma). A sandwich enzyme immunoassay specific to H-IFN-gamma molecule was developed using polyclonal rabbit anti-nH-IFN-gamma antibody and G-208. This assay monitors only biologically active H-IFN-gamma molecule. Thus, this method may be used for the direct determination of H-IFN-gamma instead of determination of antiviral activity of H-IFN-gamma.     

Identification of the secreted macromolecular immunogens of Staphylococcus aureus by analysis of serum

The ability of sera to recognise secreted macromolecules of Staphylococcus aureus was examined by ELISA and Western immunoblotting. Individual secreted proteins were also studied using both human sera and sera from rabbits immunised with secreted macromolecules. Patients sera showed a wide range of IgG antibody titres to secreted macromolecules and whole bacteria. Controls showed a significantly lower IgG response. Western immunoblotting revealed that a significant number of secreted proteins were recognised by circulating IgG antibodies. Surprisingly, both the sera from controls and from patients recognised similar macromolecules including a number of potential virulence factors. The major difference was in the IgG binding to a 16-kDa component, which was recognised by the majority of the sera from infected individuals, but only by a small number of sera from healthy controls. The higher incidence of antibodies recognising the 16 kDa component may be related to our earlier finding that the major bone resorbing component of S. aureus is a heterodimeric protein containing a 16-kDa subunit, the activity of which could be blocked by sera.     

Monoclonal antibody-mediated inhibition of interferon-gamma-induced macrophage antiviral resistance and surface antigen expression

A monoclonal antibody (MAb) with specificity for murine interferon-gamma (IFN-gamma) was used as a probe for studying the effect of recombinant IFN-gamma (rIFN-gamma) on antiviral activity, Fc receptor expression, and Ia antigen induction in macrophages. Cultures of C3H/HeJ peritoneal exudate macrophages were used to allow direct comparison of all three functions in the same target cell system. Our data provide two major findings: the efficacy of the MAb is very different depending on whether murine fibroblasts or macrophages are used as the target cell in the antiviral assay, i.e., greater than 20 to 100 times more MAb was required to block antiviral activity in macrophage cultures; and 10 to 50 times more MAb was required to inhibit Fc receptor vs Ia antigen expression in response to rIFN-gamma. These latter findings confirm and extend previous observations, which indicate that the induction pathways of two important differentiation markers by IFN-gamma may be dissociable.     

Nude mice injected with DNA released by antigen stimulated human tlymphocytes produce specific antibodies expressing human characteristics

Nude mice were injected with DNA purified from the nucleoprotein complex released by T lymphocytes previously exposed in vitro to inactivated herpes or poliovirus. After five days the serum of these mice was tested for its virus neutralizing activity. Results show that injected nude mice synthesize antiherpetic or antipolio antibodies depending on the antigen used to sensitize the T lymphocytes in vitro. These antibodies were not found in the serum of uninjected control mice or mice injected with inactivated herpes or polio viruses. Mice injected with DNA release by human T cells produced antibodies carrying human allotypes since they could be neutralized by anti-allotype sera. Moreover their antiviral activity was inhibited by anti-human IgM or IgG. However, the mice which were injected with DNA released by antigen stimulated murine T lymphocytes produced antiviral antibodies which were not neutralized by anti-human allotype sera.     

Effects of human anti-IL-1 alpha autoantibodies on receptor binding and biological activities of IL-1.

Immunoglobulin G (IgG) antibodies to interleukin 1 alpha (IL-1 alpha) are frequently found in the sera of healthy human individuals. The effects of these autoantibodies on receptor binding and biological activities of human IL-1 were tested. Using the murine T-lymphocyte line NOB-1, human thyrocytes and human foreskin fibroblasts, the antibodies competitively inhibited the biological activity of human recombinant IL-1 alpha (rIL-1 alpha). The degree of inhibition correlated with 125I-rIL-1 alpha binding to IgG in different immunoglobulin preparations and in individual sera. These antibodies also neutralized the IL-1 activity of isolated membrane fragments and lysates of human blood monocytes activated by lipopolysaccharide. In contrast, the supernatant IL-1 activity was not affected. Stronger inhibition of biological activity and cell binding of 125I-rIL-1 alpha was obtained with NOB-1 cells than with human thyrocytes. The antibodies failed to interfere with the biological activity of rIL-1 beta. It is concluded that IgG autoantibodies of IL-1 alpha in the sera of healthy humans selectively inhibit the biological activity of the soluble and membrane-associated forms of IL-1 alpha in vitro, and that the degree of biological inhibition afforded by these antibodies depends upon the target cell.     

Control of allergic reactivity in human filariasis. Predominant localization of blocking antibody to the IgG4 subclass.

Patients with chronic helminth infections, despite having abundant basophils and mast cells specifically sensitized with antiparasite IgE and often exposed repeatedly to parasite Ag, rarely manifest allergic symptoms. This control of clinical allergic reactivity likely results from Ag-specific IgG "blocking antibodies" shown previously to be abundant in the sera of such patients. In the present study we used two approaches to determine in which of the four IgG subclasses this blocking activity was localized. First, specific antifilarial antibodies of each of the four IgG subclasses were quantified in the sera of 28 patients with Bancroftian filariasis and correlated with the levels of blocking activity in these sera (determined by histamine release assays). A significant correlation with blocking activity was seen only for antibodies of the IgG4 subclass, and, indeed, the correlation was especially strong in the group of totally asymptomatic patients (but with microfilariae circulating in the blood) in whom blocking antibody levels were highest. Interestingly, however, if the analysis excluded these asymptomatic microfilaremic patients and focused instead on those with lymphatic inflammatory pathology (who had relatively low levels of both serum blocking activity and specific IgG4 antibodies), then the small amount of blocking activity found in these sera correlated only with the levels of IgG1 subclass antibodies. The second approach utilized selective depletion of IgG4 (by anti-IgG4 affinity columns) from the sera of three microfilaremic patients with high levels of blocking activity and demonstrated clearly that removal of IgG4 abolished the majority of the blocking activity in these sera (53, 78, and 81%). These two sets of findings demonstrate a predominant role for specific IgG4 antibodies in blocking IgE-mediated allergic responses to the parasite Ag in vitro, but they also indicate that in some situations IgG1 antibodies can block such reactions. Furthermore, the correlation demonstrated between patients' clinical presentations and the levels of both their specific IgG4 antibodies and serum blocking activity suggests that these antibodies play a similar role in vivo as well.     

Autoantibodies against human tropomyosin isoform 5 in ulcerative colitis destroys colonic epithelial cells through antibody and complement-mediated lysis

INTRODUCTION: Patients with ulcerative colitis (UC) have IgG1 antibodies in serum and colon against human tropomyosin isoform 5 (hTM5), a cytoskeletal microfilament protein found intracellularly and on the surface of colonic epithelial cells (EC). These antibodies may be pathogenic in UC. METHODS: Sera from patients with UC (n=110) or Crohn's disease (CD) (n=50) and from healthy individuals (Hl) (n=30) were preincubated with recombinant hTM5 or bovine serum albumin (BSA), then cultured for 4h with (51)Cr-labelled colonic adenocarcinoma cells (LS180). Cytotoxicity was determined by (51)Cr release assay. RESULTS: All serum samples lysed up to 36% of LS180 cells regardless of the source of the serum. However, adding hTM5 to UC, but not to CD or HI, sera reduced cytotoxicity by up to 75%. This hTM5-induced inhibition of cytotoxicity was found especially with sera from UC patients with active disease, and was found even after total colectomy. The hTM5-induced inhibition was mediated by purified IgG from UC sera. Complement was involved since hTM5-induced inhibition of cytotoxicity declined with either heat inactivation of the sera or premixing sera with Fc fragments. CONCLUSIONS: This study shows that hTM5-specific IgG autoantibodies present in UC sera destroy LS180 cells by antibody and complement-mediated lysis. Such a phenomenon was not seen in CD or HI. This suggests an autoantigenic role of hTM5 and anti-hTM5 antibodies in the pathogenesis of UC. This observation may lead to novel diagnostic and therapeutic possibilities.     

B cell repertoire analysis identifies new antigenic domains on glycoprotein B of human cytomegalovirus which are target of neutralizing antibodies

Human cytomegalovirus (HCMV), a herpesvirus, is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and infected newborns. Efforts are underway to prepare effective subunit vaccines and therapies including antiviral antibodies. However, current vaccine efforts are hampered by the lack of information on protective immune responses against HCMV. Characterizing the B-cell response in healthy infected individuals could aid in the design of optimal vaccines and therapeutic antibodies. To address this problem, we determined, for the first time, the B-cell repertoire against glycoprotein B (gB) of HCMV in different healthy HCMV seropositive individuals in an unbiased fashion. HCMV gB represents a dominant viral antigenic determinant for induction of neutralizing antibodies during infection and is also a component in several experimental HCMV vaccines currently being tested in humans. Our findings have revealed that the vast majority (>90%) of gB-specific antibodies secreted from B-cell clones do not have virus neutralizing activity. Most neutralizing antibodies were found to bind to epitopes not located within the previously characterized antigenic domains (AD) of gB. To map the target structures of these neutralizing antibodies, we generated a 3D model of HCMV gB and used it to identify surface exposed protein domains. Two protein domains were found to be targeted by the majority of neutralizing antibodies. Domain I, located between amino acids (aa) 133-343 of gB and domain II, a discontinuous domain, built from residues 121-132 and 344-438. Analysis of a larger panel of human sera from HCMV seropositive individuals revealed positivity rates of >50% against domain I and >90% against domain II, respectively. In accordance with previous nomenclature the domains were designated AD-4 (Dom II) and AD-5 (Dom I), respectively. Collectively, these data will contribute to optimal vaccine design and development of antibodies effective in passive immunization.     

Performance of a Western immunoblot assay to detect specific anti-Toxoplasma gondii IgG antibodies in human saliva

Toxoplasma gondii represents the most prominent infectious parasitic organism found in humans. While normally asymptomatic in healthy individuals, toxoplasmosis can cause abortion in patients during pregnancy, or can be fatal in immunosupressed individuals such as persons suffering from acquired immunodeficiency syndrom (AIDS). Toxoplasma gondii infection in humans is routinely assesssed by serological means. Here, we show that detection of anti-T. gondii IgG is also possible using a non-invasive methodology employing saliva. Sera and saliva of 201 healthy volunteers were investigated for the presence of anti-T. gondii-IgG antibodies by immunoblotting. The sera of 59 (29.4%) individuals showed IgG antibodies against T. gondii by ELISA, Vidas, and immunoblotting; 58 (98.3%) of these were also positive for anti-T. gondii IgG in the saliva immunoblot, with diagnostic relevant bands of Mr of 32-35 kDa and 40-45 kDa. The saliva immunoblot test exhibits a specificity of 100% and a sensitivity of 98.5%. Thus, saliva could be used as an alternative, non-invasive means for the detection of specific anti-T. gondii IgG in humans.     

Serum enhancement of human immunodeficiency virus (HIV) infection correlates with disease in HIV-infected individuals.

The sera from 16 individuals infected with the human immunodeficiency virus (HIV) at different clinical stages were evaluated for antibody-dependent neutralization and/or enhancement of infectivity by HIV. The HIV isolate from each individual (homotypic) and established laboratory strains showing broad cellular host range and cytopathicity were used. All sera could neutralize one of the laboratory-passaged isolates, whereas only two could neutralize the corresponding homotypic strain. Seven homotypic isolates were enhanced by serum from the respective individual. This activity was primarily observed in patients with acquired immune deficiency syndrome. Moreover, the tropism for macrophages of four of these seven viral isolates was found to be enhanced by the homotypic sera. Finally, sequential pairs of HIV and sera obtained from five HIV-infected individuals with different clinical progression were studied over time. The enhancing activity of three of the five sera appeared to increase over time, indicating changes in both the host virus population and the type of antibodies produced. These results suggest that enhancing antibodies contribute to the spread and pathogenesis of HIV in vivo. They emphasize the necessity of studying further the association of enhancing antibodies and disease progression in infected individuals.     

Activation of tissue macrophages from AIDS patients: in vitro response of AIDS alveolar macrophages to lymphokines and interferon-gamma

To test the hypothesis that tissue macrophages from AIDS patients have no intrinsic defects in either antimicrobial activity or in the capacity to respond to T cell-derived activating stimuli, alveolar macrophages from 11 patients were treated with crude lymphokines produced by healthy donors. After 72 hr of pretreatment with 10% mitogen- or antigen-induced crude lymphokines (which contained 300 U/ml of interferon-gamma [IFN-gamma]), AIDS alveolar macrophages generated twofold to threefold more H2O2 and readily inhibited the replication of the intracellular pathogens Toxoplasma gondii and Chlamydia psittaci. These responses were indistinguishable from those displayed by activated alveolar cells from 12 non-AIDS patients and three healthy volunteers. As judged by the abrogating effects of a neutralizing anti-human IFN-gamma monoclonal antibody, lymphokine-induced alveolar macrophage activation appeared to be largely IFN-gamma-dependent; thus, macrophages were also stimulated with recombinant (r)IFN-gamma alone. Seventy-two hours of treatment with 300 U/ml of rIFN-gamma resulted in both enhanced oxidative and antimicrobial activity comparable to that achieved by crude lymphokines, and the responsiveness of AIDS alveolar macrophages to rIFN-gamma was identical to control cells. These in vitro results suggest that tissue mononuclear phagocytes from AIDS patients a) are free of apparent defects in intracellular antimicrobial activity, b) are fully responsive to activating T cell products, and c) support the use of IFN-gamma as a potential macrophage-activating immunotherapeutic agent in AIDS-related opportunistic infections.     

Enhancement of a secondary antibody response to vesicular stomatitis virus "G" protein by IFN-gamma treatment at primary immunization

IFN-gamma is a secreted polypeptide product of stimulated T lymphocytes with immunomodulatory properties as well as antiviral activity. We have investigated the effects of IFN-gamma treatment on a neutralizing antibody response to vesicular stomatitis virus (VSV) when administered in conjunction with immunization using purified envelope glycoprotein "G" of VSV. Administration of rIFN-gamma to mice or cattle at the time of primary immunization with VSV G glycoprotein enhanced the magnitude of a secondary virus-neutralizing antibody response after a booster administration of the same Ag without IFN-gamma treatment. Enhancement was statistically significant and occurred at relatively low doses of IFN-gamma in the absence of any additional adjuvants. Furthermore, cattle treated with IFN-gamma at the time of a single primary immunization were more resistant to VSV challenge than those immunized without IFN-gamma treatment. IFN-gamma treatment in conjunction with a single primary immunization may therefore provide a practical means of enhancing protection from a viral challenge without the use of inflammatory adjuvants or booster immunizations.     

Sera from patients with multiple sclerosis react with human cell T lymphotropic virus-I gag proteins but not env proteins--Western blotting analysis

To study the possible involvement of human T cell lymphotropic virus type I (HTLV-I)-related agent in Japanese multiple sclerosis (MS), we performed a Western blotting analysis, using purified viral antigens, on sera from 46 patients with MS, nine patients with other neurologic diseases, and 11 healthy controls. Of 46 MS patients, 11 (24%) had antibodies reactive with antigens corresponding to the group-specific antigen (gag) proteins (p15, p19, and p24), although the prevalence was lower than that reported in a recent study using an enzyme-linked immunosorbent assay (ELISA). Despite the lower frequency of immunoreactivity, Western blotting technique had merits of identification of multiple antigens and higher specificity for detection of antibodies than ELISA. Those sero-positive patients consisted of four cases with IgG antibodies reactive mainly to the gag p24 and/or p15, four with IgM antibodies mainly to the gag p24 and/or p19, and three with both IgG and IgM antibodies. These immunostaining patterns of MS sera were clearly distinguishable from those of adult T cell leukemia patients who had antibodies to the envelope (env) proteins and its precursors in addition to the gag proteins. The antibody in MS sera was generally of low titer and reactive at a high serum concentration (1/10 dilution). None of the sera from patients with other neurologic diseases and healthy controls had the viral antibodies. These findings indicate that at least one quarter of Japanese MS patients have antibody responses to a hitherto unidentified agent related to HTLV-I, which possibly plays a part, primarily or secondarily, in the pathogenesis of those patients.     

Human antibodies to phosphocholine. IgG anti-PC antibodies express restricted numbers of V and C regions

We examined the IgG subclass composition and isoelectric focusing (IEF) spectrotype pattern of naturally occurring human IgG antibodies that bind phosphocholine (PC) and found direct evidence for restricted expression of both V and C regions among these antibodies. In most individuals, the isotype of these IgG anti-PC antibodies was primarily IgG2. However, serum from some individuals contained significant amounts of IgG1 and IgG3 anti-PC antibodies. We also found that in individual sera, anti-PC antibodies are pauciclonal, as demonstrated by restricted spectrotypic patterns of the anti-PC antibodies. The IEF pattern of these antibodies were for the most part unique for each individual. In some sera, certain anti-PC antibodies with isoelectric points of basic pH bound PC conjugated to bovine serum albumin (PC-BSA) but did not bind pneumococcal C-carbohydrate bearing PC determinants. In two individuals, we found that the spectrotypes that bound only PC-BSA were of the IgG1 subclass. Taken together, these findings demonstrate that within individual sera, human antibodies to PC are quite restricted in both V and C region expression, and furthermore, these V and C regions of human Ig may not randomly associate.     

Activation of mouse peritoneal macrophages in vitro and in vivo by interferon-gamma

To determine the role of IFN-gamma in the activation of resident mouse peritoneal macrophages, crude macrophage-activating lymphokines were incubated with a monoclonal anti-murine IFN-gamma antibody. This treatment abolished the capacity of mitogen-induced lymphokines to enhance either H2O2 release or activity against the intracellular protozoa Toxoplasma gondii and Leishmania donovani. All macrophage-activating factor detected by these assays was also removed by passing the lymphokines over a Sepharose column to which the monoclonal anti-IFN-gamma antibody had been coupled. Therefore, pure murine rIFN-gamma was tested both in vitro and in vivo as a single activating agent. After 48 hr of pretreatment in vitro with 0.01 to 1 antiviral U/ml, macrophage H2O2-releasing capacity was enhanced an average of 6.4-fold; half-maximal stimulation was induced by 0.03 U/ml. Resident macrophages infected with T. gondii half-maximally inhibited parasite replication after 24 hr of preincubation with 0.14 U/ml of rIFN-gamma, and near complete inhibition was achieved by pretreatment with 100 U/ml. Half-maximal leishmanicidal activity was induced by 0.08 U/ml of rIFN-gamma, and 67 to 75% of intracellular L. donovani amastigotes were killed after macrophages were preincubated with 10 to 100 U/ml. Eighteen hours after parenteral injection of rIFN-gamma, peritoneal macrophages displayed a dose-dependent enhancement of H2O2-releasing capacity and antiprotozoal activity. Half-maximal enhancement required 85 to 250 U or rIFN-gamma given i.p. Peritoneal macrophages were also activated by rIFN-gamma injected i.v. and intramuscularly. These results suggest that, in the mouse model, IFN-gamma is likely to be a primary factor within mitogen-induced lymphokines responsible for activating macrophage oxidative metabolism and antiprotozoal activity, and indicate that rIFN-gamma is a potent activator of these effector functions both in vitro and in vivo. These findings provide a rationale for evaluating rIFN-gamma in the treatment of systemic intracellular infections, and indicate that murine models are appropriate for such studies.     

Composition of immunoglobulins in brucellosis patients using DEAE-cellulose column chromatography.

The composition of immunoglobulins in patients with brucellosis was studied. The method of ion-exchange chromatography on DEAE-cellulose columns was used to define more precisely the physico-chemical character of cysteine-resistant antibodies. The study of IgM, IgA and IgG fractions obtained from the patients sera showed the IgG fraction to possess the greatest serological activity in the agglutination reaction, in the passive haemagglutination reaction and in Coomb's test. Specific antibodies in the remaining 2 fractions (IgA and IgM) were found only in single patients in low titres, mainly in Coomb's test (incomplete antibodies). The study of IgM, IgA and IgG serum fractions before and after cysteine treatment revealed cysteine-resistant antibodies to be usually IgG globulins. The presence of specific IgG antibodies in the sera of patients was found to correlate with active clinical manifestations of brucellosis.