Natural antibodies in sera of patients with systemic lupus erythematosus

Sera obtained from fifty-five patients with active systemic lupus erythematosus (SLE) and from four patients with mixed connective tissue disease (MCTD) previously shown by immunofluorescence and by double immunodiffusion to possess antinuclear antibodies, were tested for the presence of natural antibodies of IgG, IgA, and IgM isotypes. Antibody activity to actin, myosin, DNA, TNP, albumin, and tubulin was examined, using an enzyme-linked immunosorbent assay (ELISA). It was found that, in comparison with the antibody titers in normal sera, most of the SLE and MCTD sera possessed statistically greater amounts of IgG, IgA, and IgM antibodies directed against all the antigens tested. Furthermore, the IgG, IgA, and IgM antibody activity to DNA and TNP, compared to that found with all the other antigens, was significantly higher. Antibodies reacting with a saline extract of calf thymus (ECT) were studied by ELISA and by immunodiffusion. No correlation was observed between the natural antibody titers and the serum antibody levels to ECT detected either by ELISA or by immunodiffusion.     

Relationship between Deoxyribonucleoprotein and Deoxyribonucleic Acid Antibodies in Systemic Lupus Erythematosus

A soluble preparation of nucleoprotein (sNP), a complex of native deoxyribonucleic acid (DNA) and histones, was isolated from calf thymus nuclei and labeled with [(125)I]iodide. Isotope-labeled antigen ([(125)I]sNP) was used in a primary binding radioimmunoassay method to detect antibodies to both sNP and native DNA. Sera with antibody to native DNA reacted with the DNA moiety of sNP and bound [(125)I] sNP, but this binding was completely inhibited by addition of unlabeled native DNA. Antibody to sNP which reacted with DNA-histone complex was not inhibited in the radioimmunoassay by addition of unlabeled DNA. Thus, antibodies to sNP and native DNA could be detected and differentiated by use of a single isotopically labeled antigen. In systemic lupus erythematosus (SLE), sera with binding to [(125)I]sNP was present in 21/36 (58%) patients. The majority (18/21) had antibodies to sNP and native DNA present simultaneously, one had antibody only to sNP and two had antibody only to DNA. In contrast, patients with other connective tissue diseases rarely showed binding to [(125)I]sNP. Serial studies on SLE patients showed that high serum binding to [(125)I]sNP paralleled renal disease activity as reflected by the degree of proteinuria. A fall in binding was observed with subsidence of renal disease and reappearance of increased binding coincided with exacerbation. In these patients, antibodies to sNP and DNA appeared or disappeared pari passu suggesting that in addition to the previously demonstrated role of antibody to native DNA, antibody to sNP might also be implicated in the pathogenesis of immunologically-mediated tissue lesions such as SLE nephritis.     

An ELISA procedure to detect anti-double-stranded and anti-single-stranded DNA binding antibodies in connective tissue disorders

A simple and sensitive enzyme-linked immunosorbent assay (ELISA) is described for the measurement of antibodies to native DNA (double-stranded) and to heat-denatured DNA (single-stranded) in sera of patients with connective tissue disorders. DNA bound to polyvinylchloride plates is incubated with serum samples, and antibodies to both single- and double-stranded DNA are detected by means of goat antihuman IgG alkaline phosphatase conjugate. The binding in individual sera is expressed as binding units per milliliter and refers to the absorbance in relation to the absorbance value obtained with a set of standards. The various parameters of the ELISA assay are described, and the results are compared with results of the Crithidia and Farr DNA antibody assays. Two case reports are discussed.     

Induction of cross-reactive anti-dsDNA antibodies in preautoimmune NZB/NZW mice by immunization with bacterial DNA.

To investigate the role of antigen drive in anti-double-stranded (ds) DNA production, the antibody response induced in lupus-prone NZB/NZW mice by E. coli (EC) dsDNA was evaluated. Preautoimmune NZB/NZW female mice were immunized with complexes of EC dsDNA with methylated bovine serum albumin (mBSA) in complete Freund's adjuvant; control mice received either mBSA complexes with calf thymus (CT) dsDNA or mBSA alone in adjuvant. IgG antibody responses were assessed by ELISA. Similar to normal mice, immunized NZB/NZW mice produced significant levels of anti-dsDNA when measured with EC dsDNA as antigen. Whereas normal mice produce antibodies which are specific for the immunizing bacterial DNA, NZB/NZW mice produced antibodies that bound crossreactively to CT dsDNA by ELISA. Furthermore, the induced antibodies resembled lupus anti-DNA in their fine specificity for polynucleotide antigens and reactivity with Crithidia luciliae DNA. Despite their response to EC dsDNA, NZB/NZW mice immunized with CT dsDNA failed to generate significant anti-dsDNA responses. These results provide further evidence for the enhanced immunogenicity of bacterial DNA and suggest that immune cell abnormalities in NZB/NZW mice promote the generation of crossreactive autoantibody responses when confronted with a foreign DNA.     

双单链抗体夹心法诊断汉坦病毒感染的初步研究

目的制备检测汉坦病毒感染的双单链抗体夹心ELISA诊断试剂,并予以评价。方法以抗汉坦病毒NP抗原单链抗体包被反应板,以辣根过氧化物酶(HRP)标记大肠埃希菌表达的抗NP抗原单链抗体,并制成双单链抗体夹心诊断试剂,检测56份早期肾综合征出血热(HFRS)患者血清及66份非HFRS对照血清。结果56份HFRS患者血清中,检测出29例阳性,阳性率为51.8%,对照组66例,检测结果均为阴性。结论研制的试剂特异性好,价格低廉。     

Reaction of systemic lupus erythematosus antinative DNA antibodies with native DNA fragments from 20 to 1,200 base pairs.

Double-stranded DNA fragments of varying sizes were isolated and tested for binding to systemic lupus erythematosus (SLE) antinative DNA antibodies. Fragments of 20-25, 40-50, 90-110, and 160-180 base pairs (bp), along with intermediate-size pieces were isolated by preparative gel electrophoresis of a limited micrococcal nuclease digest of calf thymus DNA. Larger helical polynucleotides of 160-200, 380, 600-1,000, and 1,200 bp were isolated by preparative gel electrophoresis of DNA from chicken erythrocyte nucleosomes and oligonucleosomes. The fragments behaved as base-paired structures as tested by thermal denaturation, resistance to S1 nuclease, and serological assays with antibodies to native or denatured DNA. At a concentration of 0.27 muM, fragments of 20-25 bp were able to react with two SLE sera in competition with native DNA. With these and two other sera, DNA of 40-50 bp was a much more effective competitor. One serum required DNA greater than 180 bp for competition in the concentration range tested. Denatured fragments were much less effective than native fragments. The results emphasize the heterogeneity of SLE antinative DNA antibodies, confirm that secondary structure of the antigen is important for specific binding to these antibodies, and support the suggestion that bivalent binding to one molecule may be important for high functional affinity.     

与α-烯醇化酶相关的抗内皮细胞抗体ELISA的建立及临床初步应用

目的建立与α-烯醇化酶（ENOI）相关的抗内皮细胞抗体（AECA）的酶联免疫吸附试验（ELISA）,并探讨ENOI-AECA与自身免疫性疾病的关系。方法人工合成获得人ENOI基因编码序列并克隆该片段构建pUC57-ENOI重组质粒,再将重组质粒亚克隆入原核表达载体pET28（a）,回收酶切产物并经T4DNA连接酶连接,获得的重组表达质粒pET28（a）-ENOI,在大肠埃希菌BL21（DE3）中诱导表达蛋白,Ni NTA亲和层析纯化重组ENOI蛋白。以纯化的重组蛋白为包被抗原,并对ELISA反应条件进行优化,初步建立检测ENOI-AECAELISA并作分析性能评价。用建立的方法分别检测健康人及系统性红斑狼疮（SLE）、类风湿性关节炎（RA）患者血清中的ENOI-AECA。结果获得了相对分子质量为47 000的高纯度人ENOI重组蛋白。ELISA抗原最佳包被浓度为5μg/mL,血清最佳稀释倍数为1∶50。以免疫印迹法为参照,所建方法的敏感性和特异性分别为76.0%和90.2%。SLE和RA患者血清中ENOI-AECA的阳性率分别为23.1%和57.1%;RA组ENOI-AECA阳性率与健康对照组比较,差异有统计学意义（P=0.000）。结论以人ENOI重组蛋白为抗原建立的检测ENOI-AECA的ELISA有较高的敏感性和特异性,可用于ENOI-AECA的检测。ENOI-AECA阳性提示患者可能患有自身免疫性血管炎。     

An enzyme-linked immunosorbent assay for the detection of antitetanus toxoid antibody using aluminum-absorbed coating antigen

An enzyme-linked immunosorbent assay (ELISA) for the detection of antitetanus antibodies was developed using aluminum-absorbed tetanus toxoid as the coating antigen. The assay was tested by measuring antitetanus antibody levels in serum obtained from subjects before and after immunization with the aluminum-absorbed tetanus suspension. The specificity of the antibodies for tetanus antigen was tested both before and after antitetanus activity was removed with tetanus antigen-coated beads. Also, the activities of the sera were compared with those of a commercially available test. Our results indicated that the aluminum-absorbed tetanus suspension can be coated onto standard polystyrene ELISA plates and used to measure antibody titers to tetanus toxoid.     

梅毒螺旋体嵌合抗原的克隆表达及其临床应用

目的 在大肠埃希菌中表达梅毒螺旋体Tp15—17嵌合抗原,用于临床检测梅毒感染。方法 利用PCR法从Tp全基因组中分别扩增目的基因Tp15和Tp17,用T4DNA连接酶连接后,克隆进表达载体pGEX-6P-1中,构建重组质粒pGEX-6P-1＋Tp15-17,在大肠埃希菌中表达重组Tp嵌合蛋白,重组蛋白经亲和层析柱纯化后包被微孔板,初步建立间接ELISA法检测血清中的抗Tp抗体。结果 重组嵌合抗原获得了高效表达,免疫印迹试验显示重组抗原具有很强的抗原性。用重组嵌合抗原建立间接ELISA法,对梅毒螺旋体抗体阳性参比血清及梅毒患者血清的检测符合率100％。结论 重组梅毒螺旋体嵌合抗原Tpl5—17能够作为梅毒螺旋体诊断性ELISA抗原。     

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.     

Antimyenteric neuronal antibodies in scleroderma.

The pathogenesis of gastrointestinal (GI) dysmotility in scleroderma is incompletely understood, although previous studies have proposed a neuropathic mechanism. We studied patients with scleroderma as compared with other connective tissue disease patients and normal controls for the presence of circulating antibodies to myenteric neurons. Serial dilutions of sera were overlaid on rat intestine, double-labeled with antineurofilament antibody as a myenteric plexus marker, and imaged using indirect immunofluorescence techniques. High titer sera (> or = 1:50) from 19 out of 41 scleroderma patients stained myenteric neurons, whereas none of 22 normals or 5 patients with idiopathic GI dysmotility were positive. Although 6 out of 20 SLE and 6 out of 10 mixed connective tissue disease patients' sera stained myenteric plexus neurons, when positive sera were absorbed with calf thymus extract to remove antinuclear antibody, 15 scleroderma sera, 0 SLE, and 2 mixed connective tissue disease patients retained positive staining of myenteric neurons. Western blotting using actin and neuronal intermediate filament preparations failed to show immunoreactivity with scleroderma sera containing antimyenteric neuronal antibodies. Paraneoplastic sera associated with GI dysmotility stained myenteric neurons in a different pattern than seen with scleroderma sera. A positive correlation between the presence of Raynaud's phenomenon and antimyenteric neuronal antibodies was observed in scleroderma patients. Our results indicate that IgG antibodies reacting with myenteric neurons are present in many patients with scleroderma. Although the neuronal antigen has not yet been identified, the presence of myenteric neuronal antibodies in patients with GI dysmotility and scleroderma suggests a neuropathic process.     

噬菌体模拟抗原替代亲环素A在测定自身抗体中的应用

目的 探讨噬菌体展示肽库来源的模拟抗原替代重组人亲环素A(rhCyPA)在测定自身免疫病人血清抗亲环素自身抗体中应用价值。方法 以抗人亲环素A单克隆抗体(McAb)D4和E6作捕获抗体对噬菌体展示肽库进行淘筛，通过序列测定推断出模拟抗原氨基酸序列。再以筛出的模拟抗原包被微孔板，与重组人亲环素A抗原进行对比，用ELJSA法检测系统性红斑狼疮患者血清抗亲环素自身抗体。结果 从12肽库淘筛出的10个噬菌体克隆中9个共有一致氨基酸序列HWEYTISPHPRL。56例SLE患者血清用12肽模拟抗原的ELISA测定，CyPA自身抗体阳性率为50％(28／56)，用rhCyPA作包被抗原测定阳性率为43％(214／56)；两法检测34例其他自身免疫病的阳性率均为11％，两法结果符合率为95．6％。通过试验比较，12肽模拟抗原用于检测CyPA自身抗体优于重组抗原和7肽模拟抗原。结论用抗CyPA单克隆抗体从噬菌体展示肽库淘筛出的特异12肽能模拟亲环素A抗原表位信息，可以代替亲环素A用来检测其自身抗体。     

An Immunologic Precipitin System between Soluble Nucleoprotein and Serum Antibody in Systemic Lupus Erythematosus

Double diffusion in agarose was employed for the characterization of a soluble nucleoprotein antigen that gave precipitin reactions with sera from patients with systemic lupus erythematosus. The soluble antigen that was isolated from calf thymus nuclei was demonstrated by enzyme degradation and ultracentrifugation studies, and by immunologic analysis, to be a complex of deoxyribonucleic acid and a moiety susceptible to proteolytic digestion. Antibody to soluble nucleoprotein did not react with the DNA portion of the complex released after proteolytic digestion or with purified calf thymus DNA. Immunologic reaction of identity was obtained between the soluble nucleoprotein antigen and synthesized DNA-histone complex, suggesting that the protein moiety of soluble nucleoprotein might in part be composed of histone. Antibody to soluble nucleoprotein was present in 51% of systemic lupus erythematosus sera examined and was more common than antibody to deoxyribonucleic acid.     

利用重组抗原检测抗SSA＼Ro—52kD自身抗体及其临床意义

目的 重组表达ＳＳＡ／Ｒｏ－５２ｋＤ融合蛋白,并用于检测自身抗体。方法 应用ＤＮＡ重组技术构建ＳＳＡ／Ｒｏ－５２ｋＤ工程菌,并表达ＳＳＡ／Ｒｏ－５２ｋＤ融合蛋白,经谷胱基肽巯基转移酶（ＧＳＴ）柱层析纯化后,作为抗原,分别用免疫印迹法（ＩＢＴ）和酶联免疫吸附试验（ＥＬＩＳＡ）检测２０份ＳＳ患者血清、６０份ＳＬＥ患者血清和３０份正常人血清。结果 重组抗原检测抗 ＳＳＡ／Ｒｏ－５２ｋＤ自身抗体敏感性较高     

Characterization of a distinct nuclear acidic protein antigen (MA) and clinical findings in systemic lupus erythematosus patients with MA antibodies.

Circulating antibodies against certain nuclear acidic protein antigens have been shown to have diagnostic and prognostic importance in connective tissue disease. We describe a new precipitin system found in the sera of patients with systemic lupus erythematosus. The antigen, called MA, was prepared from calf thymus nuclei, and was shown to be distinct from other nuclear acidic protein antigens by physicochemical and immunologic techniques. MA antibodies were detected in the serum of 12 of 66 lupus patients and in none of 554 sera from normal controls or patients with other rheumatic diseases. Lupus patients having MA antibodies had more severe disease than did lupus patients with Sm or native DNA antibodies, manifested by recalcitrant skin rashes and a significantly greater incidence of hypocomplementemia, serious renal disease, hypertension, hepatosplenomegaly, lymphadenopathy, and neurological disease (P values range from 0.025 to 0.005). The presence of circulating MA antigen was demonstrated in three lupus patients immediately before a flare of nephritis. These data suggest that MA is a nuclear acidic protein antigen that may identify a subset of lupus patients with very severe disease. The presence of the antigen in the circulation before clinical flares suggests a possible biologic role for the MA system in an immune complex nephritis.     

重组抗原检测抗干燥综合征A 抗原的自身抗体

目的 建立便捷的检测抗干燥综合征（ＳＳ）Ａ抗原（分子量为６００００的多肽成分）的自身抗体（抗ＳＳＡ）的方法,以利用疾病的早期诊断和病程监控。方法 构建基因工程菌,表达ＳＳＡ－６００００融合蛋白。经ＧＳＴ柱层析法纯化后,分别经免疫印迹（ＩＢＴ）法和酶联免疫吸附法（ＥＬＩＳＡ）检测２０份ＳＳ患者血清、６０份系统性红斑狼疮（ＳＬＥ）患者血清和３０份正常人血清。结果 利用重组抗原检测抗ＳＳＡ自身抗体敏感性     

Decreased 19S antibody response to bacterial antigens in systemic lupus erythematosus

The antibody response to immunization with Brucella and the levels of natural antibody to Escherichia coli and Shigella were compared in patients with systemic lupus erythematosus and control groups. After Brucella immunization, SLE patients showed a significantly lower antibody response in whole serum and in the macroglobulin antibody fraction separated by sucrose density gradient centrifugation.Sucrose gradient fractionation of natural antibodies to E. coli and a polyvalent Shigella antigen showed a significant decrease in macroglobulin antibody against four of the five E. coli antigens tested and the Shigella polyvalent antigen in SLE patients when compared with a group of normal individuals and a matched control group with pulmonary tuberculosis. Whole serum natural antibody titers against 5 of 13 Shigella antigens were significantly lower in the SLE patients when compared with the normal group, and against 7 of 13 when compared with the matched tuberculosis controls. Whole serum titers against 8 of 13 E. coli antigens were significantly lower in the SLE patients when compared with normal subjects.The observed decreased antibody response to bacterial antigens in SLE patients, occurring mainly in the macroglobulin fraction, is discussed in relation to the increased incidence of infection commonly observed in these patients.     

Association of autoantibodies to different nuclear antigens with clinical patterns of rheumatic disease and responsiveness to therapy

Using a hemagglutination test which can detect antibodies to (a) native and denatured deoxyribonucleic acid (DNA) and (b) an extractable nuclear antigen (ENA), a comparative study of patterns of autoantibody formation has been done in systemic lupus erythematosus (SLE) and related rheumatic diseases. Antibody to native DNA was present in the serum in 96% of patients with active SLE and disappeared during remissions. Antibody to ENA was found in 86% of those patients with SLE nephritis who responded to treatment but in only 8% of those who did not. The highest titers of antibody to ENA were found in patients having a mixed connective tissue disease syndrome with features of SLE, scleroderma, and myositis. The latter syndrome was notable for the absence of renal disease and for a striking responsiveness to corticosteroid therapy. Hemagglutination testing of 277 sera from normal persons and patients with a wide variety of acute diseases other than SLE revealed the presence of antibody to native DNA in only 1.4% and antibody to ENA in only 0.4%.These results yield significant correlations among the pattern of autoimmune reactivity, the clinical form of the rheumatic disease, and responsiveness to treatment. They implicate the qualitative nature of the patient's immune response as a conditioning factor in the type of disease. Together with other correlations they may allow classification of rheumatic diseases into more biologically meaningful groups and lead to more selective methods of therapy.     

Development of an indirect ELISA with artificially synthesized N protein of PPR virus

The full-length gene encoding the nucleocapsid (N) protein of the virus (PPRV) responsible for an outbreak of peste des petits ruminants in Tibet in 2007 was synthesized in two stages using overlapping PCR without the need for viral genomic cDNA as template. The full-length N gene was successfully expressed in Escherichia coli, and the purified gene product bound to monoclonal antibody raised against PPRV N protein. Furthermore, it was able to replace recombinant B-N antigen as the coating antigen in a commercial ELISA kit prepared with another PPRV strain. Recombinant protein was employed as the coating antigen to develop an indirect ELISA for PPRV antibody detection in the sera of infected small ruminants. Antibody detection was optimal at a 1:200 serum dilution and an antigen concentration of 3.2 μg/ml, and the positive threshold (cutoff) value of the assay was 2.18. Analysis of 697 serum samples revealed the sensitivity and specificity of the indirect ELISA to be 96.7 and 96.1%, respectively, compared with a commercially available ELISA test.     

Antinuclear antibody with distinct specificity for polymyositis.

In the course of studying antinuclear antibodies in the rheumatic diseases, a new precipitin reaction (provisionally referred to as PM-1) was observed between calf thymus nuclear extract and polymyositis sera. Objectives of this study were to further define the immunologic nature of this reaction and to determine its specificity for polymyositis. Immunodiffusion studies using calf thymus nuclear extract revealed the PM-1 precipitin line in 17 of 28 patients with polymyositis. This reaction was not produced by sera of 460 patients with other diseases. Enzyme and heat treatments of the nuclear extract showed that PM-1 was distinct from native DNA, ribonucleoprotein, and Sm antigens. Fractionation of PM-1-positive serum by 30% ammonium sulphate and Sephadex G-200 chromatography revealed that the factor producing the PM-1 precipitin reaction was in a serum fraction which showed only IgG by immunoelectrphoresis against anti-whole human serum. Because of the apparent strong specificity, the PM-1 system may represent a marker antibody for polymyositis.