Immunoblotting identifies and antigen recognized by anti gp63 in the immune complexes of Indian kala-azar patient sera

In SDS-PAGE the immune complexes (IC) of kala-azar patient sera showed intense bands at 55 kDa and 20 kDa corresponding to heavy and light chains of immunoglobulins. In immunoblot experiment, kala-azar and normal IC after treatment with patient sera showed multiple bands of which the band at 55 kDa was most prominent in kala-azar IC. It is known that in kala-azar sera antihuman IgG is present, so the heavy band at 55 kDa region may be due to higher amount of IgG and/or other antigen(s) present at that region. Immunoblot experiments of kala-azar IC with anti gp63 also developed a major band at 55 kDa. It suggests that the antigen (55 kDa) and gp63 have common antigenic epitope (s). Normal IC did not react with anti gp63 indicating absence of this antigen in normal IC. Antigenic similarity between the IC antigen (55 kDa) and gp63 indicated that the former antigen may have been processed from gp63. In summary, identification of a parasite antigen (55 kDa) in IC of kala-azar patients sera may be useful in developing a serodiagnostic assay for visceral leishmaniasis. (Mol Cell Biochem130: 11–17, 1994)Abbreviations IC Immune Complexes - PEG Polyethylene Glycol (Mol wt 8000) - PBS Phosphate Buffer Saline - VL Visceral Leishmaniasis - AVL American Visceral Leishmaniasis - IgG Immunoglobulin G - TBS Tris Buffer Saline - SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis - gp63 A leishmanial surface glycoprotein of molecular mass 63,000 - TEMED N,N,N,N-Tetramethylethylenediamine     

Immune complex antigens as a tool in serodiagnosis of kala-azar

The 63 kDa surface antigen of Leishmania promastigotes is one of the most important virulent factors in establishing the host parasite relationship. This glycoprotein is revealed by surface iodination study as well as by metabolic labeling and immunoblot methods. In search of this specific antigen for serodiagnosis, immune complexes (ICs) were isolated from kala-azar patient sera and analysed by SDS-PAGE and Western immunoblotting. The immunoblot of kala-azar IC with patient sera, antipromastigote sera and anti gp63 sera detected the major antigen of 55 kDa. This recognition suggests that 55 kDa antigen and gp63 have common antigenic epitope(s). Normal IC did not react with anti gp63 sera indicating absence of this antigen in normal IC. To confirm the parasitic origin of the 55 kDa antigen of kala-azar IC, in vitro IC was formed with parasite antigen and acid dissociated kala-azar IC antibody. This indicated the antigenic similarity of the 55 kDa antigen and gp63 antigen of the parasite. This also suggested that the former antigen may have been processed from gp63. In summary, identification of parasite antigen (55 kDa) in IC of kala-azar patients' sera may be useful in developing a serodiagnostic assay of visceral leishmaniasis. Several other antigens are visualized in kala-azar IC when developed with patient sera. But specificity and efficacy of these antigens have not yet been evaluated in serodiagnosis of the disease.     

Variation of antigenicity and serological reaction to Pneumocystis carinii in Korea.

The present study observed the variation of antigenicity of Pneumocystis carinii and serum IgG antibody reaction to the antigens from different localities in Korea. Antigens of rat P. carinii and sera of inhabitants were collected at Chunchon. Chungju, Kwangju, and Seoul during 1995-1996. Enzyme-linked Immunosorbent Assay and immunoblot were used for immune reaction. Absorbance of 1,294 human sera ranged between 0.01 and 0.93. Sera from Chunchon showed higher absorbances than those from other areas. Immunoblotting revealed IgG antibody reactions to 116, 100, and 45-55 kDa antigenic bands of rat P. carinii, but the frequencies of positive reaction to individual bands were variable by localities. Total 62.6% of the sera showed the reaction to 116 kDa band while 37.7% reacted to 100 kDa band and 32.0% to 45-55 kDa bands. For the reaction to 116 kDa, the reaction rate was 60.0% to 82.6% by localities. It is found that the reaction rates of the human sera to rat P. carinii antigen are variable according to the localities. Also, the high molecular antigen of 116 kDa of rat P. carinii is the most frequent antigenic band reacting to human sera.     

Usefulness of IgG4 subclass antibodies for diagnosis of human clonorchiasis

The present study analyzed serum IgG subclass antibody reaction to major antigenic bands of Clonorchis sinensis to investigate improvement of its serodiagnosis. Of the four subclass antibodies, IgG1 and IgG2 antibodies were produced but not specific, IgG3 antibody was least produced, and IgG4 antibody was prominent and specific. The serum IgG antibody reaction to any of 43-50, 34-37, 26-28, and 8 kDa bands was found in 65.5% of 168 egg positive cases while IgG4 antibody reaction was found in 22.0% of them. The positive rates of IgG and IgG4 antibodies were directly correlated with the intensity of infection. All of the sera from heavily infected cases over EPG 5,000 showed positive reaction for specific IgG and IgG4 antibodies. The specific serum IgG4 antibody disappeared within 6 months after treatment. The bands of 35 kDa and 67 kDa cross-reacted with IgG antibodies but not with IgG4 antibodies in sera of other trematode infections. The present findings suggest that serum IgG4 antibody reaction to 8 kDa band is specific but not sensitive. Any method to increase its sensitivity is required for improved serodiagnosis.     

Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not initiated by brain mononuclear cells. Purkinje cell death was not simply due to intraneuronal antibody accumulation.     

Immunoelectrotransfer blot assay in acute and chronic human trichinellosis

An immunoelectrotransfer blot assay (IETB) using excretory secretory products of muscle larvae of Trichinella spiralis (ML-ESP) and the avidin biotin system was developed in order to characterize reactivity against ML-ESP in sera from patients with acute and chronic trichinellosis. A complete pattern of up to 13 bands was developed by sera from individuals with trichinellosis where doublets, triplets, or single bands were shown to have molecular weights of roughly 66, 55, 45, 36, 29, 24, and 14 kDa. The bands at approximately 55, 36, 29, and 14 kDa proved specific for T. spiralis. The band at approximately 55 kDa was present in all trichinellosis sera, whereas the approximately 14-kDa band was present in only a small percentage of sera. The development of approximately 36- and 29-kDa bands suggests a modulation of the reactivity against ML-ESP over time. IETB proved more sensitive for the population of chronic trichinellosis under study than a conventional diagnostic enzyme-linked immunosorbent assay, allowing negative or borderline serum samples to be determined. Thus, this technique, when applied for human trichinellosis surveillance, should provide a useful tool in endemic areas.     

18 kDa microtubule-associated protein: identification as a new light chain (LC-3) of microtubule-associated protein 1 (MAP-1)

SDS gel electrophoresis of microtubule proteins obtained from bovine brain by polymerization cycles revealed a new protein of 18 kDa. This protein was copolymerized with tubulin and its stoichiometry to tubulin remained constant for at least 5 cycles of assembly. Moreover, this protein remained bound to microtubules stabilized with 10 μM taxol and pelleted through a 4 M glycerol cushion. The same 18 kDa protein was found in a purified preparation of the high molecular mass microtubule-associated protein 1 (MAP-1). The 18 kDa protein copurified with the MAP-1 heavy chains during column chromatography on phosphocellulose, DEAE-cellulose, hydroxyapatite and Bio-Gel A-15m. Incubation of the MAP-1 preparation with a mouse monoclonal antibody to the light chain 1 (LC-1) of MAP-1 and with a second precipitating antibody (a rabbit antibody to mouse IgG) immunoprecipitated from the solution all the known components of MAP-1 (heavy chains, LC-1, LC-2), as well as the 18 kDa protein. Immunoblotting showed, however, that this antibody does not interact directly with the 18 kDa protein. These results indicate that the 18 kDa protein forms a complex with all other components of MAP-1. This polypeptide, therefore, is a new light chain (LC-3) of M AP-1.     

IgG antibody responses in early experimental sparganosis and IgG subclass responses in human sparganosis

Antigenic components in the crude extracts of Spirometra mansoni plerocercoid were analyzed in early experimental infections and in IgG subclass observed in clinical sparganosis. By IgG immunoblot, sera obtained serially from experimental mice, fed 5 spargana each, were reacted with the crude extracts. Protein bands at 36-26 kDa and 103 kDa showed positive reactions since two weeks after infection. In a differential immunoblot, in which a monospecific antibody against sparganum chymase at 36 kDa was pre-treated, the reactions at 36-26 kDa disappeared, indicating that the sparganum chymase and its degradation products invoked IgG antibody reactions. When 69 patients sera of human sparganosis were examined for their IgG subclass responses, IgG4 levels showed the highest reaction which was followed by IgG1. The IgG4 antibody also reacted mainly with 36-31 kDa protease. These results indicate that 36 kDa chymase of S. mansoni plerocercoid is the main antigenic component inducing IgG antibody response in early stage of experimental sparganosis and for specific IgG subclass reactions in human sparganosis.     

IgG anti-IgE autoantibodies in visceral leishmaniasis

Procedures for IgG depletion in visceral leishmaniasis (VL) and schistosomiasis sera using Sepharose-protein G beads also deplete IgE. In this study, the presence of IgG anti-IgE autoantibodies in sera from patients with VL (n = 10), and hepatic-intestinal schistosomiasis (n = 10) and from healthy individuals (n = 10) was investigated. A sandwich ELISA using goat IgG anti-human IgE to capture serum IgE and goat anti-human IgG peroxidase conjugate to demonstrate the binding of IgG to the IgE captured was performed. VL sera had higher titers (p < 0.05) of IgG anti-IgE autoantibodies (OD = 2.01 +/- 0.43) than sera from healthy individuals (OD = 1.35 +/- 0.16) or persons infected with Schistosoma mansoni (OD = 1.34 +/- 0.18). The immunoblotting carried out with eluates from Sepharose-protein G beads used to deplete IgG from these sera and goat anti-human IgE peroxidase conjugate, showed a similar pattern of bands, predominating the 75 kDa epsilon-heavy chain and also polypeptides resulting from physiological enzymatic digestion of IgE. A frequent additional band immediately above 75 kDa was observed only in VL sera.     

Defining potential targets for immunotherapy in Burkholderia cepacia infection

Abstract Burkholderia cepacia has become a serious source of infection in patients with cystic fibrosis. Antibiotic therapy is difficult as the bacteria are intrinsically resistant to most antibiotics. The present study compared the antibody response by immunoblot of 50 negative control sera, 22 patients with cystic fibrosis and no evidence of B. cepacia , 9 clinically well patients with cystic fibrosis colonised by B. cepacia and 5 patients with cystic fibrosis and deteriorating or fatal B. cepacia infection. Nineteen antigenic bands varying in apparent molecular weights from 19 to 170 kDa were identified. Two bands at 19 and 21 kDa were only present when the organism was grown in an iron-deficient medium. The band at 30 kDa was identified as a porin and the possession of IgG antibody carried a statistically significant ( P = 0.00003) better prognosis. This antigen was thus a potential target for immunotherapy.     

Generation of low m.w., C3-bearing immunoglobulin in human serum

The generation of low m.w. C3-bearing immunoglobulin (lg) in normal human serum by an immune complex (IC) model was investigated in vitro by using discontinuous sucrose density gradient centrifugation (DGC) and an assay that measures C3-bearing Ig. In this method developed to measure circulating IC, all C3 and C3-bearing material is precipitated from serum by using anti-C3 sera in C3d antibody excess, and immune precipitated, C3-bearing Ig is quantitated by the uptake of 125I-5S-anti-IgG. When plasma from patients with clinically active systemic lupus erythematosus was assayed after DGC, most of the reactive material was low m.w. (7S), rather than greater than or equal to 19S as expected for IC, in agreement with a previous report. Low m.w., C3-bearing Ig was found in normal EDTA plasma after extended storage at -29 degrees C but not after storage at -70 degrees C. Such material was also generated in normal human serum during incubation at 37 degrees C and its generation was stimulated by the addition of an IC model, high m.w., heat-aggregated IgG (HMW-HAIgG). In experiments in which the participation of serum IgG was monitored by the addition of 125I-7S-IgG and 131I-HMW-HAIgG was used as an IC model, low m.w., C3-bearing Ig was generated exclusively from serum IgG and the amount generated was proportional to the concentration of 131I-HMW-HAIgG. No significant decrease in sedimentation of 131I-HMW-HAIgG was observed, but the ability of anti-C3 sera to precipitate 131I-HMW-HAIgG decreased 66% 4 hr after initial C activation. These results indicate that generation of nascent C3b in serum results in its interaction with monomeric serum IgG, producing low m.w., C3-bearing IgG. In addition, the data indicate that circulating IC that activate C have a brief time span during which they can be detected by methods that depend upon the binding of C3.     

Correlation of treponemicidal activity in normal human serum with the presence of IgG antibody directed against polypeptides of Treponema phagedenis biotype Reiter and Treponema pallidum, Nichols strain

Normal human serum (NHS) was shown to have complement-dependent treponemicidal activity against both Treponema pallidum and Treponema phagedenis biotype Reiter (TPR) by employing in vitro-in vivo neutralization and TPR plaque assays, respectively. The molecular basis of NHS treponemicidal activity was studied by immunoblot analysis in conjunction with treponemicidal assays. Five major T. pallidum polypeptide bands (47kDa, 35kDa, 33kDa doublet, and 30 kDa) and three major TPR polypeptide bands (47kDa and 33kDa doublet) bound IgG present in NHS. Absorption of NHS with TPR completely removed both TPR and T. pallidum treponemicidal activity; corresponding immunoblots demonstrated a significant removal of IgG antibody against all three TPR polypeptide bands as well as four T. pallidum polypeptide bands (30kDa, 33kDa doublet, and 35kDa). In contrast, T. pallidum absorption of NHS was found to remove treponemicidal activity against T. pallidum but not TPR; corresponding Western blots showed the complete removal of IgG antibody against all but one T. pallidum polypeptide band (47kDa) but no detectable loss in IgG antibody against the TPR polypeptides. These results suggest that antibody in NHS generated against nonpathogenic, indigenous treponemes is responsible for the T. pallidum treponemicidal activity. Furthermore, the treponemicidal activity against T. pallidum correlated with the presence of IgG antibody against T. pallidum polypeptides of 30kDa, 35kDa, and a 33kDa doublet.     

内蒙地区发热患者血清中冠状病毒抗体的检测

目的分析内蒙地区发热患者中冠状病毒的感染情况。方法以SARS冠状病毒感染Vero细胞涂片为冠状病毒抗原片,用间接免疫荧光法分别检测55例发热患者和68例正常人血清中冠状病毒的IgG、IgM抗体。结果发热患者血清中冠状病毒IgG抗体和IgM抗体阳性率分别为29.1%(16/55)和10.9%(6/55),而正常人血清中只检测到2.9%(2/68)的IgG抗体,且未检测到IgM抗体,2组患者的IgG和IgM抗体阳性率比较差异均有显著性;随机选取7例患者的IgG阳性血清进行SRAS冠状病毒的特异性抗体封闭实验,结果有6例血清仍为阳性,有1例血清转为阴性,说明冠状病毒IgG抗体阳性血清中85.7%为普通冠状病毒特异性,14.3%为SARS冠状病毒特异性。结论普通冠状病毒是内蒙地区发热患者的主要病原体之一,部分患者还存在SARS冠状病毒的既往感染。     

Antiglobulin factor in sera from patients with liver disease.

(1) An antiglobulin factor, non-neutralizable by human gamma-globulin, was demonstrated in sera of two patients with liver disease. (2) By absorption and elution techniques, two fractions were differentiated in a serum: one is reactive to rabbit antibody and the other seems cross-reactive to rabbit and human antibodies. (3) In double immunodiffusion test, the antiglobulin factor formed a precipitation band with heat-aggregated human IgG as did rheumatoid arthritis serum. (4) While the antiglobulin activity to rabbit antibody was demonstrated in both the IgM and IgG fractions, the reactivity to human antibody was localized in the IgM fraction. (5) From its selective reactivity to individual anti-D sera (sensitizers), at least a part of the specificity of the antiglobulin factor must be related to anti-Gm (1), and consequently can be regarded as auto-reactive.     

Encephalitozoon cuniculi Isolated from the Urine of an AIDS Patient, which Differs from Canine and Murine Isolates

ABSTRACT. A species of Encephalitozoon has been isolated from the urine of a patient with the acquired immunodeficiency syndrome and maintained in vitro in Madin Darby Canine Kidney cells. When examined by random amplified polymoprhic DNA polymerase chain reaction the new isolate was found to differ from E. hellem and to have amplified products in common with murine and canine E. cuniculi . However, it more closely resembled the canine than the murine isolate. Sodium dodecylsulphate polyacrylamide gel electrophoresis differentiated between all three isolates of E. cuniculi , with a band at 42–45 kDa present in the murine isolate only, bands at 52 kDa present in the canine and human isolates but not the murine, and a single band at 60 kDa (murine) and 65 kDa (canine) replaced by two bands at 55 and 70 kDa in the human isolate. The 55 kDa and 70 kDa antigens were also revealed as characteristic bands of the human isolate by Western blotting. The study has thus revealed that the species Encephalitozoon cuniculi is not a homogeneous entity.     

IgM and IgG significant reactivity to Borrelia burgdorferi sensu stricto, Borrelia garinii and Borrelia afzelii among Italian patients affected by Lyme arthritis or neuroborreliosis

Abstract This survey evaluates the specificity of band patterns in immunoblot of sera taken from clinically defined cases of Lyme arthritis and neuroborreliosis, towards three locally isolated strains of Borrelia burgdorferi , belonging to the three species: Borrelia sensu stricto, Borrelia garinii and Borrelia afzelii . To assess specificity, patient sera were statistically ( χ ², P ≤ 0.05) compared with blood donors sera samples. Both IgG and IgM antibodies were considered. The overall reactivity of the three Borrelia strains in IgG immunoblots indicated that ten protein bands were significant, with a different prevalence of some of them in the two groups of patient sera: bands at 60-58, 30–33, 36–37 and 28-27 kDa were markers for neuroborreliosis sera; proteins at 100-83, 72-70 and 18-17 kDa behaved like markers for Lyme arthritis. The IgM Immunoblots revealed significant bands at 100-83, 72-70, 51, 24-21 and 18-17 kDa only with neuroborreliosis sera. Though there were variable band reactivities in each strain, a correlation emerged between the three genospecies and the clinical symptoms: in fact B. afzelii and B. garinii were prevalent in Lyme arthritis sera, (IgG Immunoblots); B. garinii was associated to neuroborreliosis (IgG and IgM Immunoblots); B. sensu stricto was strongly reactive with neuroborreliosis in IgM immunoblots. These data indicate that the three locally isolated strains of Borrelia representing the three genospecies should be used together in immunoblot to detect antibodies elicited in neuroborreliosis and Lyme arthritis.     

Nature of antigens and antibodies in immune complexes isolated by staphylococcal protein A from plasma of melanoma patients

Summary Immune complexes (IC) were isolated from plasma of melanoma patients by absorption to staphylococcal protein A and subsequent elution with MgCl₂. The isolated ICs were purified by precipitation with polyethylene glycol and sucrose density gradient ultracentrifugation after radioiodination with ¹²⁵I. The purified ICs were dissociated and radiolabeled antigen/antibody components were separated by ultracentrifugation at low pH (2.6). Under these conditions, about 72% radioactivity of the purified IC remained in the light-density region as a wide band. After neutralization, 26%–60% radioactivity in the region of 5S sedimentation bound to immobilized autologous immunoglobulins, as opposed to a maximum of 23% to immobilized immunoglobulins from human normal serum. Significant levels (73%–77%) of radioactivity in 7S region bound to rabbit anti-human IgG immunobeads. Immunoprecipitation of the antigen fraction by allogeneic anti-melanoma and rabbit anti-melanoma antibodies followed by SDS-polyacrylamide gel electrophoresis revealed the presence of a fetal antigen (FA) and a melanoma tumor-associated antigen (TAA). In addition, the presence of auto-antigen(s) was indicated by using autologous antibody in immunoprecipitation. Immunoglobulins (IgG) isolated from purified IC bound to cultured melanoma, sarcoma, and normal fibroblasts, although the binding to sarcoma and normal fibroblasts could be inhibited by preincubation of isolated IgG with soluble FA but not with soluble melanoma TAA. Thus, results of this investigation provide evidence that circulating IC in melanoma patients are composed of at least IgG and different antigens, and some of these antigens are produced by their tumor.     

Human insulin autoantibody fine specificity and H and L chain use

Fine specificity and H and L chain isotypes of insulin autoantibodies in sera from 11 subjects were examined. None of these 11 subjects was treated with exogenous insulin. Two patterns of fine specificity were found. In one, the autoantibodies were specific for human insulin, with a requirement for threonine at B30. The conservative substitution in pork insulin (threonine to alanine) abrogated IgG binding by these sera. Insulin autoantibodies in other sera cross-reacted with beef, pork, and human insulin; not requiring threonine at B30. Reciprocal competitive inhibition experiments showed that epitopes recognized by the human specific insulin autoantibodies were exclusively on the B chain, whereas the cross-reactive sera contain autoantibodies that recognize both the B chain and combinatorial (A and B chain) epitopes. The fine specificity of cross-reactive insulin autoantibodies are thus similar to insulin antibodies from insulin-treated subjects. When IgG subclasses and L chains of insulin autoantibodies were examined, however, restricted C region usage was found. The hierarchy was IgG3 greater than G1 greater than G2 greater than G4; with one subclass dominant in each serum, although others were used. L chain use was similarly restricted. There was no correlation between isotype and fine specificity or between H and L chain type. It is concluded that heterogeneity of insulin autoantibodies is restricted. The response is probably more oligo- or pauciclonal than insulin antibody from insulin-treated subjects.     

Nature of antigens and antibodies in immune complexes isolated by staphylococcal protein A from plasma of melanoma patients

Immune complexes (IC) were isolated from plasma of melanoma patients by absorption to staphylococcal protein A and subsequent elution with MgCl2. The isolated ICs were purified by precipitation with polyethylene glycol and sucrose density gradient ultracentrifugation after radioiodination with 125I. The purified ICs were dissociated and radiolabeled antigen/antibody components were separated by ultracentrifugation at low pH (2.6). Under these conditions, about 72% radioactivity of the purified IC remained in the light-density region as a wide band. After neutralization, 26%-60% radioactivity in the region of 5S sedimentation bound to immobilized autologous immunoglobulins, as opposed to a maximum of 23% to immobilized immunoglobulins from human normal serum. Significant levels (73%-77%) of radioactivity in 7S region bound to rabbit anti-human IgG immunobeads. Immunoprecipitation of the antigen fraction by allogeneic anti-melanoma and rabbit anti-melanoma antibodies followed by SDS-polyacrylamide gel electrophoresis revealed the presence of a fetal antigen (FA) and a melanoma tumor-associated antigen (TAA). In addition, the presence of auto-antigen(s) was indicated by using autologous antibody in immunoprecipitation. Immunoglobulins (IgG) isolated from purified IC bound to cultured melanoma, sarcoma, and normal fibroblasts, although the binding to sarcoma and normal fibroblasts could be inhibited by preincubation of isolated IgG with soluble FA but not with soluble melanoma TAA. Thus, results of this investigation provide evidence that circulating IC in melanoma patients are composed of at least IgG and different antigens, and some of these antigens are produced by their tumor.     

Molecular studies of rheumatoid factor using pseudobioaffinity membrane chromatography

Using pseudobioaffinity ligand L-histidine immobilized to poly(ethylene vinyl alcohol) hollow fiber membrane is an interesting approach for the purification of total IgG and its subclasses from untreated serum in a single step. Gentle adsorption and elution conditions of this chromatography system allow efficient recovery of the protein in its native form. This approach was employed for the recovery and molecular study of rheumatoid factor (RF), an anti-IgG autoantibody (AAb) that form immune complexes with autologous IgG Abs in the sera. The purity of the recovered molecule was analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), revealed a 150-kDa IgG band and an additional approximately 300 kDa band which may be RF bound IgG complex. Since RF is an AAb, the purified protein was studied for its catalytic functions like peptide, DNA, and RNA hydrolyzing activities. The substrate Pro-Phe-Arg-4-methyl-coumaryl-7-amide (PFR-MCA) hydrolyzing activity by total IgG from different patient sera was found to be greater than healthy controls. In an effort to identify the subclass specificity for the proteolytic function, the pre-purified total IgG fractions from rheumatoid arthritis (RA) sera were subjected to rechromatography using a discriminating buffer. In this experiment, the activity was found in the non-retained fractions suggesting IgG2 specificity for the catalytic function. A comparative study between different catalytic functions was performed for IgG separated from individual patient.