Comparison of epitope specificity of anti-heat shock protein 60/65 IgG type antibodies in the sera of healthy subjects,patients with coronary heart disease and inflammatory bowel disease

Previously, we reported on the presence of antibodies to linear epitopes of human and mycobacterial 60 kD heat shock proteins (HSP) in the sera of healthy blood donors. Since many recent findings indicate that the levels of these antibodies may be altered in coronary heart disease (CHD) and also inflammatory bowel diseases (IBD), it seemed worthwhile to compare the epitope specificity of the anti-HSP60 and anti-HSP65 antibodies in the sera of patients with these diseases to those in healthy subjects. The multipin enzyme-linked immunosorbent assay method was applied with a large overlapping set of synthetic 10-mer peptides covering selected regions of human HSP60 and Mycobacterium bovis HSP65. Sera of 12 healthy persons (HP), 14 CHD, and 14 IBD patients with the same concentration of total anti-HSP60 and HSP65 IgG antibodies were tested. We have identified CHD-specific epitopes in the equatorial domain of the HSP60 protein but in neither region of the HSP65 molecule, indicating that the formation of anti-HSP60 antibodies is not or only partially due to the cross-reaction between human HSP60 and bacterial HSP65. IBD-specific epitopes were found in many regions of the HSP60 and in even more regions of the HSP65 molecule including an IBD-specific T cell epitope in region X as well. These findings indicate that the epitope specificity of the anti-human and anti-mycobacterial HSP60 antibodies associated with various diseases is different.     

Increased concentrations of antibody against heat shock protein in patients with myeloperoxidase anti-neutrophil cytoplasmic autoantibody positive microscopic polyangiitis

To determine serum antibody against human and bacterial heat shock protein (HSP) 60/70 in myeloperoxidase (MPO)-specific anti-neutrophil cytoplasmic autoantibody (ANCA) positive microscopic polyangiitis (MPA), 58 patients with MPO-ANCA positive MPA, 48 with RA (rheumatoid arthritis) and 40 with SLE (systemic lupus erythematosus) were studied. Serum antibodies against HSP (human HSP 70, human HSP 60, Mycobacterium HSP 70, and Escherichia coli HSP 60) were measured by sandwich ELISA. The frequency of anti-human HSP 60/70 antibody positive patients was significantly greater in MPO-ANCA positive MPA than SLE and healthy controls. Anti-human HSP 60/70 antibody titers in patients with MPO-ANCA positive MPA were significantly higher than those of healthy controls; anti-bacterial HSP 60/70 antibody titers were also higher. There was a significant correlation between titers of anti-human HSP 70 antibody and anti-Mycobacterium HSP 70 antibody. A correlation was also found between titers of anti-human HSP 70 antibody and anti-human HSP 60 antibody. Anti-human and bacterial HSP 60/70 antibody titers changed in parallel with disease activity in patients with antibody positive MPA. The anti-HSP antibody titer was also increased in patients with RA and SLE. These results suggest that an immunological background via anti-HSP 60/70 antibodies might be associated with pathogenesis in MPO-ANCA positive MPA.     

Anti-heat shock protein 70 autoantibody epitope changes and BD091 promotes atherosclerosis in rats

It has been previously reported that the plasma levels of autoantibodies against heat shock protein 70 (HSP70) are elevated in atherosclerosis. The aim of the present study was to elucidate whether anti-HSP70 antibodies are involved in the pathogenesis of atherosclerosis. To determine this, we chose rats as an atherosclerosis model. Titers of plasma anti-HSP70 autoantibody were determined by ELISA. After the intravenous administration of antibody into the tail, the damaged areas of aorta were stained with Evans Blue, atheromatous plaque were stained by Oil Red O, and then they were measured and quantified with AxioVision computer software. The number of macrophages ( $ {{\hbox{M}}_\Phi } $ ), smooth muscle cells (SMCs), and T cells were determined by immunocytochemistry. The level of anti-HSP70 IgG1 antibody was apparently increased in the AS group at the tenth week, and one hybridoma of HSP70 antibody (BD091, IgG1, recognizing C-terminal) had the same binding epitope as plasma anti-HSP70 autoantibodies. After intravenous administration, the lesion area of aorta with BD091 was significantly larger than those of IgG^mouse and SPA-810. Moreover, injection of BD091 resulted in significant endothelium damage, followed by a greater accumulation of $ {{\hbox{M}}_\Phi } $ , T cells, and SMCs in lesions than in the control. In conclusion, BD091 reaction with HSP70 expressed on arterial endothelial cells inducing endothelium damage triggers the inflammatory response in the vessel wall that accelerates atherosclerosis in rats. BD091 shares the same binding epitope with HSP70 autoantibodies. These data indicated that a specific epitope of anti-HSP70 autoantibody participated in the pathogenesis of atherosclerosis.     

PROGRESS STUDY: Progression of chronic kidney disease in children and heat shock proteins

Various molecular and cellular processes are involved in renal fibrosis, such as oxidative stress, inflammation, endothelial cell injury, and apoptosis. Heat shock proteins (HSPs) are implicated in the progression of chronic kidney disease (CKD). Our aim was to evaluate changes in urine and serum HSP levels over time and their relationships with the clinical parameters of CKD in children. In total, 117 children with CKD and 56 healthy children were examined. The CKD group was followed up prospectively for 24 months. Serum and urine HSP27, HSP40, HSP47, HSP60, HSP70, HSP72, and HSP90 levels and serum anti-HSP60 and anti-HSP70 levels were measured by ELISA at baseline, 12 months, and 24 months. The urine levels of all HSPs and the serum levels of HSP40, HSP47, HSP60, HSP70, anti-HSP60, and anti-HSP70 were higher at baseline in the CKD group than in the control group. Over the months, serum HSP47 and HSP60 levels steadily decreased, whereas HSP90 and anti-HSP60 levels steadily increased. Urine HSP levels were elevated in children with CKD; however, with the exception of HSP90, they decreased over time. In conclusion, our study demonstrates that CKD progression is a complicated process that involves HSPs, but they do not predict CKD progression. The protective role of HSPs against CKD may weaken over time, and HSP90 may have a detrimental effect on the disease course.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-021-01239-9.     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. I. Occurrence of antibodies to enterobacterial common antigen (ECA)

The antibodies against the Enterobacterial Common Antigen (ECA) were detected using the ELISA in 293 serum samples collected from 185 persons suspected for yersiniosis, as well as 115 serum samples from healthy individuals (blood donors). The presence of IgA antibody in diagnostically significant titres for ECA were detected by ELISA in 3.5%, IgG in 13.0%, and IgM in 5.2% of blood donors. Statistical analysis showed that the frequency of detecting antibodies for ECA among the patients with yersiniosis was significantly higher (p < 0.05) in relation to the blood donors. Most frequently the elevated antibody levels were detected among patients with reactive arthritis (IgA 29.2%, IgG 35.4%, IgM 16.7%) while the most infrequent among patients with abdominal pain in acute phase of yersiniosis (IgA 14.9%, IgG 25.3%, IgM 19.5%). The level of antibodies for ECA, together with age increased reaching its peak, on the average, among individuals aged 41 - 60 years. In majority of the individuals studied antibodies of the IgG class reached the level much higher in relation to those of the IgA and IgM classes. The obtained results showed that the detection of antibodies to ECA may be useful in serodiagnosis of Yersinia infections.     

妊娠期高血压患者血清热休克蛋白70水平与心功能及免疫球蛋白的关系研究

目的:探究妊娠期高血压患者血清热休克蛋白70(HSP70)水平与心功能及免疫球蛋白的关系。方法:选择2017年4月至2018年4月在陕西省人民医院诊治的200例妊娠期高血压患者作为妊娠期高血压组,同时选择同期在我院进行孕检的200名健康孕妇作为对照组。采用酶联免疫吸附法检测血清HSP70水平,采用全自动生化分析仪检测免疫球蛋白G(Ig G)、免疫球蛋白M(Ig M)、免疫球蛋白A(Ig A)、免疫球蛋白D(Ig D)和免疫球蛋白E(Ig E)水平,采用多普勒超声心动图监测两组的左心室后壁厚度(LVPWT)、左心室舒张末期内径(LVEDD)和室间隔厚度(IVST)、左心室收缩末期内径(LVESD)、左心室射血分数(LVEF)和每搏指数(SVI),计算左心室质量(LVM)、二尖瓣舒张早期血流速度峰值与二尖瓣环侧壁舒张早期运动峰速度的比值(E/Em)、舒张早期与舒张晚期血流速度峰值的比值(E/A)。采用spearman相关性分析血清HSP70水平与血清免疫球蛋白水平及心功能指标的相关性。结果:与对照组相比,妊娠期高血压组的血清HSP70水平明显升高,而Ig G和Ig M水平明显下降,并且LVESD、LVEF、E/A也明显下降(P0.05)。血清HSP70水平与Ig G、Ig M、LVESD、LVEF、E/A均呈负相关性(P0.05)。结论:妊娠期高血压患者的血清HSP70水平明显升高,并且血清HSP70水平与妊娠期高血压患者免疫功能和心功能下降存在相关性,在妊娠期高血压患者的诊断和治疗中具有一定临床价值。     

Low prevalence of antibodies against heat shock protein 10 of Chlamydophila pneumoniae in patients with coronary heart disease

In this study the prevalence of antibodies against the heat shock protein 10 (HSP10) of Chamydophila pneumoniae (CP) (as assessed by ELISA) in patients with coronary heart disease (CHD) and seropositive or seronegative to CP, as assessed by microimmunofluorescence (MIF), was investigated. The controls were age- and sex-matched healthy subjects. The HSP10 preparation used throughout this study was a 6-his-tagged recombinant protein preliminarily shown to be immunogenic in mice. Low level IgG reactivity against CP-HSP10 was detected in 19 out of 200 and 5 out of 100 CHD patients and controls, respectively. No IgM or IgA isotypes were found. Furthermore, there was no difference in the frequency or level of anti-HSP10 IgG between CP-positive and CP-negative sera either in patients (11/140=7.9% vs. 8/60=13%) or in healthy subjects (3/40=7.5% vs. 2/60=3.3%). Overall, our data indicate that CP-HSP10, at variance with CP-HSP60, to which it is genetically and physiologically linked, should not be regarded as a major expressed immunogen or a marker of infection by CP in CHD patients.     

HSP70iQ435A to subdue autoimmunity and support anti-tumor responses

Developing immunosuppressive therapies for autoimmune diseases comes with a caveat that immunosuppression may promote the risk of developing other conditions or diseases. We have previously shown that biolistic delivery of an expression construct encoding inducible HSP70 (HSP70i) with one amino acid modification in the dendritic cell (DC) activating moiety 435–445 (HSP70i_Q435A) to mouse skin resulted in significant immunosuppressive activity of autoimmune vitiligo, associated with fewer tissue infiltrating T cells. To prepare HSP70i_Q435A as a potential therapeutic for autoimmune vitiligo, in this study we evaluated whether and how biolistic delivery of HSP70i_Q435A in mice affects anti-tumor responses. We found that HSP70i_Q435A in fact supports anti-tumor responses in melanoma-challenged C57BL/6 mice. Biolistic delivery of the HSP70i_Q435A-encoding construct to mice elicited significant anti-HSP70 titers, and anti-HSP70 IgG and IgM antibodies recognize surface-expressed and cytoplasmic HSP70i in human and mouse melanoma cells. A peptide scan revealed that the anti-HSP70 antibodies recognize a specific C-terminal motif within the HSP70i protein. The antibodies elicited surface CD107A expression among mouse NK cells, representative of antibody-mediated cellular cytotoxicity (ADCC), supporting the concept, that HSP70i_Q435A-encoding DNA elicits a humoral response to the stress protein expressed selectively on the surface of melanoma cells. Thus, besides limiting autoimmunity and inflammation, HSP70i_Q435A elicits humoral responses that limit tumor growth and may be used in conjunction with immune checkpoint inhibitors to not only control tumor but to also limit adverse events following tumor immunotherapy.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-021-01229-x.     

大鼠动脉粥样硬化过程中抗HSP70抗体水平变化及其意义

目的：观察高脂诱导大鼠动脉粥样硬化形成过程中血浆HSP70抗体水平及其与动脉粥样硬化的关系。方法：将28只大鼠分为正常组和高脂组,动态测定了大鼠血清TC、TG、LDL,HE染色观察大鼠主动脉弓病理改变;通过ELISA方法检测大鼠血浆HSP70抗体水平及抗体亚型的变化。结果：2周时,高脂组大鼠血清TC、LDL显著升高,与对照组比较具有显著性差异（P〈0．01）;而TG水平显著降低（P〈0．01）。血浆HSP70抗体在第四周开始显著升高（与对照比较P〈0．01）,随着动脉粥样硬化的进程逐渐升高;HSP70抗体IgM亚型第四周达到峰值（与对照比较P〈0．01）;而IgG亚型第四周开始升高（与对照比较P〈0．01）,后逐渐升高。第12周时主动脉出现粥样斑块典型的动脉粥样硬化的病理改变．结论：高脂可以诱导大鼠动脉粥样硬化形成,高脂组大鼠血浆HSP70抗体IgG亚型水平随着疾病的进程逐渐升高,与动脉粥样硬化具有显著的相关性,表明血浆HSP70抗体与动脉粥样硬化发生发展具有密切关系。     

Humoral Response against Small Heat Shock Proteins in Parkinson’s Disease

Introduction

In the light of evidence for the increased heat shock proteins (HSP) expression in neurodegenerative disorders, the presence of the adaptive humoral response of the immune system can be expected. The aim of the study was to check whether Parkinson’s disease (PD) has the ability to elicit immune response against small heat shock proteins.

Methods

IgG and IgM autoantibodies against alpha B-crystallin were assessed in 26 PD patients 26 healthy subjects. For the assessment of anti-HSP IgG autoantibodies serum samples from 31 parkinsonian patients and 31 healthy control subjects were collected. Serum samples from PD patients and healthy control subjects were collected twice, at baseline and after mean of 13 months follow up.

Results

Both IgM and IgG autoantibodies against alpha ß-crystallin in PD patients were significantly higher compared to healthy controls (p<0.05). We also found statistically significant increase in antibodies titers against alpha ß-crystallin over the time of 13 months, both for IgG (p = 0.021) and for IgM (p<0.0001). Additionally, PD patients presented higher levels of anti-HSP IgG autoantibodies than healthy controls (p = 0.02).

Conclusions

Increase of IgG and IgM autoantibodies against alpha B-crystallin in PD patients over time may suggest their involvement in the disease pathogenesis and progression. Further studies are required to confirm the role of this antibody as a biomarker of the disease progression.     

Heat shock protein HSP90 and its association with the cytoskeleton: a morphological study.

To investigate the cellular localization of the 90-kilodalton heat shock protein (HSP90) and its interaction with the cytoskeleton, we performed single- and double-staining immunofluorescence microscopy of cytoskeletal proteins and HSP90 in the absence and presence of cytoskeletal inhibitors. As a model, we used a human endometrial adenocarcinoma cell line (Ishikawa cells), which expresses HSP90. We confirmed the recently reported colocalization of HSP90 with microtubules. However, Ishikawa cells treated with 10(-5) M of the antimicrotubule agents colchicine or triethyl lead showed residual filamentous structures stained with anti-HSP90 antibodies, while no microtubules were visualized with anti-tubulin antibodies. In the presence of 10(-5) M cytochalasin B, the microfilament staining of the cells disappeared, while residual filamentous structures were labeled with anti-HSP90 antibodies. Furthermore, Ishikawa cells treated with 10(-5) M triethyl lead and stained with anti-HSP90 antibodies demonstrated residual filamentous structures, clearly different from those of reorganized vimentin intermediate filaments. Conversely, similar reorganized morphology of filamentous structures stained with both anti-HSP90 and anti-cytokeratins antibodies were observed when Ishikawa cells were treated with 2 x 10(-5) M cytochalasin B and 2 x 10(-5) M colchicine. HSP90 was also present in Ishikawa cell preparations of the Triton X-100 insoluble cytoskeleton. In addition, Triton-insoluble cytoskeleton treated with 10(-5). M triethyl lead and double stained with anti-HSP90 and anti-vimentin antibodies demonstrated clearly different filamentous patterns, when exposed on the same photographic plaque.(ABSTRACT TRUNCATED AT 250 WORDS)     

The 90-kDa heat shock protein, HSP90, binds and protects casein kinase II from self-aggregation and enhances its kinase activity.

We found that a preparation of the 90-kDa heat shock protein, HSP90, purified to apparent homogeneity, contains a serine/threonine kinase which phosphorylates HSP90. The protein kinase was identified as casein kinase II (CKII) according to its properties. The protein kinase was separable from HSP90 by adsorption to heparin-Sepharose or phosphocellulose. CKII was coimmunoprecipitated with HSP90 by anti-HSP90 antibodies from cell extracts. Sucrose density gradient centrifugation analysis revealed that an addition of anti-HSP90 antibodies to cell extracts induces a shift of the sedimentation peak of CKII toward the bottom of a centrifuge tube. These results suggest that CKII is associated with HSP90 in cell lysates at low salt conditions. Furthermore, the CKII.HSP90 complex was reconstituted from purified HSP90-free CKII and CKII-free HSP90. In a buffer at low ionic strength, CKII forms large aggregates, but HSP90 dissociates the aggregates. Finally, we found that HSP90 activates CKII; an addition of HSP90 to CKII dramatically increased phosphorylation of exogenous substrates as well as the CKII beta subunit. Taken altogether, these observations suggest that CKII is structurally and functionally active when it forms a complex with HSP90.     

A heat shock protein70 fusion protein with alpha1-antitrypsin in plasma of type 1 diabetic subjects

The recent observation that heat shock proteins (HSPs), mostly glucose regulated protein94 (Grp94) and HSP70, are present in plasma of Type 1 diabetic subjects as complexes with immunoglobulins, prompted us to investigate the nature and extent of this association, whether it represents HSP-induced activation of the immune system. Two complementary affinity chromatography procedures followed by immunoprecipitation and immunoblot analyses of HSP-enriched, plasma-purified peaks, revealed that HSPs were inextricably linked with IgG in SDS-resistant complexes from which proteins dissociate partially under reducing treatment. HSP70 was found also closely linked with alpha1-antitrypsin (alpha1AT) in a single protein having the mass of alpha1AT but elution characteristics different from those of normal alpha1AT. Immunoprecipitation with anti-HSP70 antibodies led to co-immunoprecipitation of the alpha1AT species linked to HSP70, thus confirming fusion of the proteins. The additional finding of circulating antibodies against the HSP70-alpha1AT protein supported its immunogenic properties with implications for diabetes and its complications.     

Serum immunoglobulins IgG, IgA and IgM in patients with oral lichen ruber

The aim of this study was to determine level of serum IgA, IgG and IgM in patients with OLR as indicators of humoral immunity which might reflect cell-mediated immunity. This study was conducted on 30 patients (age 60.17 +/- 11.75) with clinically and histopathologically confirmed diagnosis of OLR and 30 healthy controls (age 56.16 +/- 11.82) Determination of serum IgA, IgG and IgM was performed by use of standard laser nephelometry in both patients and controls. Statistical analysis was done using Mann-Whitney U test and the level of significance was determined as p values lower than 0.05. Serum IgA and IgM in patients with OLR were significantly increased in comparison to the control group, while serum IgG levels were higher in patients with OLR but they did not reach significance. We might conclude that elevated levels of serum IgA and IgM show that humoral immunity is implicated in the pathogenesis of OLR.     

Role of Salmonella and Yersinia in the pathogenesis of spondyloarthropathies and rheumatoid arthritis. II. Antibodies to Salmonella and Yersinia in sera and synovial fluids

IgA, IgG and IgM antibodies against Yersinia Yop proteins, Yersinia LPS and Salmonella LPS from different serogroups were determined by enzyme-linked immunosorbent assay (ELISA) in a 885 serum samples and 92 synovial fluids. The control group consisted of 200 healthy blood donors. Compared with control subjects, patients with arthritis showed significantly increased titres of antibodies against Yersinia Yop, Yersinia LPS and Salmonella LPS appropriately in 21.7%, 44.0% and 56.0% serum samples. The prevalence of positive antibody levels was highest in Yersinia serogroup O3 and Salmonella serogroup B and D antibodies. The IgA titres were found to be much higher in adults than in children and youngsters but IgM titres consequently decreased with age. Investigation of synovial fluids obtained from patients with arthritis showed that Yersinia and Salmonella antibodies in synovial fluid mirror those in serum by concentration, by specificity and by distribution in classes.     

IgG1 and IgG2 antibody responses to Plasmodium falciparum exoantigens correlate inversely and positively, respectively, to the number of malaria attacks

Abstract In Manarintsoa, near Antananarivo, Madagascar, two groups of patients were defined in terms of malaria clinical immune status: Group MA⁺ consisted of 36 patients who suffered from between one to four malaria attacks (MA) during the 20-week study, and Group MA⁻ who comprised of 48 persons who did not have any malaria attacks during this time. In group MA⁺, IgM and IgG antibody levels to Plasmodium falciparum exoantigens (E-Ag) were inversely related to the number of malaria attacks. The level of IgM antibodies were significantly higher in group MA⁺. In contrast, IgG, IgG1, IgG2, IgG3 and IgG4 antibodies to E-Ag were significantly higher in group MA⁻. The level of IgG1 antibodies was inversely correlated, and IgG2 antibodies were positively correlated to the number of malaria attacks.     

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.     

Epitope mapping human heat shock protein 90 with sera from infected patients

Abstract Epitope mapping with sera from a range of infected patients showed that antibodies are commonly produced which cross-react with a number of epitopes on human heat shock protein 90 (HSP 90). Such autoreactive antibodies were particularly frequent in patients suffering from systemic candidiasis (9 patients), invasive aspergillosis (6 patients), ABPA (2 patients), a patient with aspergilloma and one with malaria. The patient with malaria recognised similar epitopes to those with invasive aspergillosis and candidiasis including the highly conserved epitope LKVIRKVIRK and an epitope NNLGTI which was otherwise only recognised by patients with candidiasis. Crossreacting antibodies to relatively few epitopes occurred in patients with Enterococcus faecalis and Corynebacterium jeikeium endocarditis. This was contrasted with the results from 6 patients with systemic lupus erythematosus (SLE) who were positive on immunoblot against fungal HSP 90. These did not react with the above epitopes but reacted with other areas within human HSP 90.     

Natural and immune human antibodies reactive with antigens of virulent Neisseria gonorrhoeae: immunoglobulins G, M, And A

Natural and immune human antibodies reactive with heat-labile and heat-stable antigens of virulent Neisseria gonorrhoeae were studied by use of an indirect fluorescent-antibody (IFA) procedure. The immunoglobulin class of the reactive antibodies was identified by using fluorescein-conjugated antisera specific for human IgG, IgA, or IgM in the IFA procedure. The effects of heat and mercaptoethanol on IFA reactivities were also studied. It appeared that antibodies of the IgG, IgM, and IgA classes present in the sera of both infected persons (immune antibodies) and normal persons with no history of gonococcal infection (natural antibodies) react with heat-stable somatic antigens. Immune IgG antibodies, however, were distinguishable from natural IgG antibodies by their ability to recognize heat-labile surface antigens. The distinction between natural and immune IgM antibodies was less obvious. IgM antibodies from both infected and normal persons appeared to react with heat-labile antigens. Some, but not all, infected persons had immune IgA antibodies to heat-labile as well as to heat-stable antigens. Treatment of sera with mercaptoethanol had no effect on IgG antibodies. The IFA activity of IgM antibodies was decreased, but not abolished. The effects of mercaptoethanol on IgA antibodies were variable. Some sera showed a decrease in IgA titer, and others showed an increase in IgA activity to certain antigens. Immune IgG antibodies were more resistant to heating than were natural IgG antibodies. Natural and immune IgM antibodies appeared equally sensitive to heating. IgA activity, on the other hand, was increased by heating sera at 60 C, but was decreased at higher temperatures. Thus, it appears that natural and immune human IgG antibodies to N. gonorrhoeae may be distinguished by their interactions with heat-labile antigens and by their resistance to heating.