Quantification of circulating immune complexes by chicken anti-C4 micro ELISA

A quantitative assay for C4-containing immune complexes (IC) by a solid phase anti-C4 micro ELISA is described. It is based upon the use of an affinity purified chicken anti-human C4 antibody to capture the immune complex, and protein A-alkaline phosphatase for detection. The chicken antibody was chosen as capture antibody because it does not react with rheumatoid factor, does not activate the human complement system and is not detected by anti-mammalian IgG antibodies or protein A. Increased levels of C4 containing circulating immune complexes (CIC) were detected in sera from patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and lung cancer, when compared with normal sera. Normal levels of C4 containing immune complexes were found in sera from patients with Bell's palsy.     

Modified anti-C3 immune complex assay which avoids interference by anti-F(ab')2 antibodies

The present authors and Olds et al. reported that the anti-F(ab')2 antibodies (Abs) in serum interfere with the solid phase (SP) anti-C3 immune complex assay. The anti-F(ab')2 Abs in human sera bind solid phase F(ab')2 anti-C3 of rabbit or goat, and were measured erroneously as C3 bearing circulating immune complexes (CIC). Gel filtration analysis of SP anti-C3 assay revealed that C3 bearing CIC is detected only in heavy fractions and 7S CIC-like activity is not CIC but anti-F(ab')2 activity. As the molecular weight of such CIC is heavy enough to be precipitated by 5% polyethylene glycol (PEG) and IgG anti-F(ab')2 Abs and free C3 are not included in 5% PEG precipitates, 5% PEG precipitates of the test sera were used for SP anti-C3 (Modified SP anti-C3). CIC measured by modified SP anti-C3 were positive in 14/16 at active stage of SLE and positive only in 2/16 at inactive stage. CIC by this test were also correlated well to serum complement activity, and were thought to be clinically reliable and useful.     

两种血清循环免疫复合物测定方法的比较

目的　对测定血清循环免疫复合物 ( circulating immune complexes,CIC)的两种方法 ( PEG沉淀法及 CIC-C1 q ELISA法 )进行评价 ,进一步探讨 CIC测定的临床意义。方法　采用 PEG沉淀法和 CIC-C1 q ELISA法检测肾脏损害、系统性红斑狼疮 ( SLE)、类风湿性关节炎 ( RA)等 86例患者的血清 CIC。结果　 86例患者中 PEG沉淀法检测阳性 1 7例 ( 1 9.77% ) ,CIC-C1 q ELISA法检测阳性 3 8例 ( 4 4.1 9% ) ,差异有显著性 ( P<0 .0 5 )。CIC-C1 q ELISA法检测 SLE、RA和肾脏损害患者的 CIC阳性率分别为 1 1 /1 5、5 /8和 2 0 /61 ( 3 2 .79% )。结论　与 PEG沉淀法相比 ,CIC-C1 q ELISA法较敏感 ,适宜于临床推广应用。CIC的阳性与 SLE、RA和肾脏损害有关。     

Binding of complement component C5 to model immune complexes and the use of anti-C5 antibodies for determination of C5-containing circulating immune complexes

When normal human or mouse serum is added to micro ELISA plates coated with monomeric or aggregated IgG, complement component C5 binds to IgG. C5 binding was demonstrated with a specific chicken anti-C5 antibody. Hydrazine treatment of the serum or addition of EDTA to the serum abolished the binding of C5. C5-deficient mouse serum was negative for C5 binding, whereas the same serum supplemented with human C5 restored the binding of C5. Chicken anti-C5-coated plates were used for determination of C5-containing circulating immune complexes (CIC). Increased concentrations of CIC were found in sera from patients with rheumatoid arthritis and Bell's palsy.     

类风湿关节炎和系统性红斑狼疮患者血清基质金属蛋白酶2、9检测的意义

目的探讨基质金属蛋白酶（MMPs）在类风湿关节炎（RA）和系统性红斑狼疮（SLE）中的重要作用。方法采用酶联免疫吸附试验和酶谱分析了48例RA和27例SLE患者及186例健康对照者血清中MMP-2和MMP-9的浓度和活性。结果酶谱分析结果显示RA和SLE患者血清MMP-2和MMP-9的活性显著高于对照组（P〈0.01）。ELISA检测结果也表明RA、SLE组的MMP-2和MMP-9浓度明显高于对照组（P〈0.05）。结论MMP-2和MMP-9在RA和SLE患者血清中的水平及活性明显升高,表明MMPs在这些自身免疫疾病起一定的作用,可作为临床辅助诊断指标。     

系统性红斑狼疮患者3种血清免疫因子水平检测的意义

目的 探讨系统性红斑狼疮(SLE)患者血清中白细胞介素-6(IL-6)、IL-10、肿瘤坏死因子-α(TNF-α)的水平变化和意义.方法 分别用放射免疫分析和酶联免疫吸附试验(ELISA)对26例SLE患者IL-6、IL-10、TNF-α进行检测,并与20例健康者作比较.结果 SLE患者IL-6、IL-10和TNF-α水平明显高于健康组,差异具有统计学意义(P<0.01).结论 检测SLE患者血清IL-6、IL-10、TNF-α水平的变化对SLE患者的临床诊断和临床观察有临床价值.     

Pro- and antioxidant blood system in patients with rheumatoid arthritis and systemic lupus erythematosus

AIM: To estimate the levels of prooxidants (malonic dialdehyde-MDA and nitric oxide-NO), Zn, activity of antioxidant enzymes (superoxide dismutase--SOD and glutathione peroxidase--GPO) in the blood of patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). MATERIAL AND METHODS: Pro- and antioxidant system was assessed in 45 RA patients and 32 SLE patients by content of MDA estimated by reaction with thiobarbituric acid, NO (Boehringer Manheim kits, Germany), Zn (Unicam SP 190/191, Great Britain), activity of SOD and GPO (kits Ransod, Ransel; Randox, Great Britain). RA activity was evaluated by DAS index, SLE--by SLEDAI. RESULTS: MDA and NO concentrations were found elevated while SOD and GPO activity low in RA and SLE. The level of Zn was subnormal in RA. The activity of RA and SLE did not influence the above indices significantly. CONCLUSION: RA and SLE patients have high levels of prooxidants (MDA and NO) whereas their antioxidant enzymes (SOD and GPO) activity was low. This may promote oxidant stress.     

类风湿关节炎患者血液中PADI4表达的研究

目的探索肽基精氨酸脱亚胺基酶4（PADI4）在早期类风湿关节炎（RA）患者血液中的表达。方法收集早期RA（发病六个月）和早期骨关节炎（发病六个月）（OA）患者的血清,EusA方法检测患者血清中的PADI4,anti—CCP和RF的水平,同时检测超过三年的RA患者血浆中这三种物质的水平。另外还采集了健康者以及系统性红斑狼疮（SLE）患者的血样进行分析。作为对照。结果68．7％的早期RA患者的PADI4水平明显高于早期OA患者。长时间患RA病人的PADI4表达量高于SLE病人,但与健康人水平没有多大的差剐。在RA患者血液中,PADI4的水平与RF和anti—CCP的水平密切相关。蛄论PADI4可能在RA发病起始过程中发挥作用,PADI4的表达水平可能与疾病活动争一些临床表现有关。     

抗Ⅱ型胶原短肽（260-272）抗体ELISA测定方法的建立与初步应用

目的了解类风湿关节炎（RA）,其他自身免疫病和健康对照组血清抗Ⅱ型胶原短肽,即CⅡ（260-272）抗体水平,探讨其在RA疾病诊断和病情监测中的价值。方法将合成的CⅡ（260-272）短肽与牛血清白蛋白（BSA）耦联得CⅡ（260-272）-BSA复合物,用其作为抗原包被建立抗CⅡ（260-272）抗体的ELISA测定方法,经方法学考核后,对临床病例标本检测结果进行评价。结果建立的抗CⅡ（260-272）抗体的ELISA法实验结果具有较好的稳定性和特异性,其批内和批间变异系数分别为8.4%和9.6%,特异阻断抑制率最高达76.3%。临床检测结果发现抗CⅡ（260-272）抗体在RA中的阳性率为75.0%（30/40）,显著高于其他自身免疫病和健康对照组（P〈0.01）。以测定吸光度（A）0.213为临界值时,抗CⅡ260-272抗体对RA诊断特异性91.0%,阳性预测值63.8%,阴性预测值94.5%,阳性似然比8.33,阴性似然比0.275,Youden指数0.66。ROC曲线分析结果显示该抗体对RA诊断准确度为中等偏上水平（AUCROC值=0.890）。未发现抗CⅡ（260-272）抗体与类风湿因子（RF）水平相关（在13例RF阴性RA患者中,该抗体阳性10例,阳性率76.9%）,但抗CⅡ（260-272）抗体与抗环瓜氨酸肽（CCP）抗体显著相关（P〈0.01）,且该抗体水平随患者临床症状的改善有逐渐降低趋势。结论成功建立抗CⅡ（260-272）抗体ELISA测定方法,用其检测抗CⅡ（260-272）抗体水平可作为RA疾病诊断和病情监测的一种新的参考指标。     

Clinical significance of ELISA positive and immunofluorescence negative anti-dsDNA antibody

BACKGROUND: Positivity for anti-dsDNA antibody is a diagnostic criterion of systemic lupus erythematosus (SLE). In the present study, the significance of ELISA positive and Crithidia luciliae immunofluorescence test (CLIFT) negative anti-dsDNA sera was evaluated. METHODS: There were 371 consecutive serum samples submitted to for anti-dsDNA testing that were assayed using anti-dsDNA ELISA and CLIFT. Sera showing discrepant results were collected and then examined using 3 commercial anti-dsDNA and anti-ssDNA ELISA kits and by Farr assay. Medical records were reviewed for those patients who were ELISA positive and CLIFT negative for anti-dsDNA. RESULTS: Fifty-two patients of 100 anti-dsDNA ELISA positive patients were negative by CLIFT. For ELISA positive and CLIFT negative sera, Farr assays showed the highest positive rate (72.7%) for the 4 different anti-dsDNA assays (3 commercial kits and the Farr assay). Nearly 80% of 44 ELISA positive and CLIFT negative patients met >or=3 of the SLE classification criteria (excluding the anti-dsDNA criterion). CONCLUSION: Some anti-dsDNA ELISA kits have diagnostic efficiencies that are similar to that of the Farr assay. Moreover, the study identifies a group of patients that are ELISA positive but CLIFT negative for anti-dsDNA, and indicates that the majority of these patients have clinically relevant SLE.     

Elevated serum immune complex levels in Pogosta disease, an acute alphavirus infection with rash and arthritis

Circulating immune complexes (CIC) were studied in Pogosta disease, an acute alphavirus infection with fever, rash and arthritis. The disease is caused by a virus antigenically closely related to Sindbis virus. 75 serum specimens from 25 patients with serologically verified infection were obtained from 1-87 days after the onset. Six different CIC detection methods were used and CICs were observed in all patients at least with one test. Tests based on CIC binding onto human platelets followed the natural course of the disease and maximal values were observed between 10-15 days after onset. Slightly elevated levels were observed 2-3 months after onset. The mean conglutinin binding test values were slightly elevated during the whole follow-up period. The severity of arthritis did not directly correlate to CIC levels. C3c and C1q-binding test were positive only in a few cases. Latex and enzyme immunoassay tests for rheumatoid factors gave low positive values in some of the sera. Agarose gel electrophoresis of serum proteins revealed non-specific changes in alpha 1-alpha 2 interzone characteristic of an acute infectious disease. The presence of CIC in the sera of patients with Pogosta disease may indicate body's natural clearange mechanisms of viral antigens. CIC may have a pathogenic role in the prolonged arthritis, even though no direct correlation with CIC levels and severity of arthritis was observed.     

系统性红斑狼疮患者sCD40L水平变化及临床意义

目的对系统性红斑狼疮（SLE）患者血清中可溶性CD40配体（sCD40L）的含量变化及临床意义进行讨论。同时分析血小板减少组SLE患者与血小板正常组SLE患者血清中sCD40L的含量差别。初步探讨血清和血浆中sCD40L浓度有无差异。分析SLE患者血清中sCD40L的含量与系统性红斑狼疮疾病活动度评分（SLEDAI评分）有无相关性。方法用酶联免疫吸附法检测25例SLE患者和15例正常人血清中及18例正常人血浆中sCD40L的浓度。结果①血小板正常组SLE患者血清中的sCD40L浓度明显高于正常对照组和血小板减少组SLE患者。②SLE患者血清sCD40L浓度、血小板正常组SLE患者血清中sCD40L浓度、血小板减少组SLE患者血清中sCD40L浓度与SLEDAI评分均无相关性。③SLE患者血清中sCD40L浓度与血小板计数呈正相关。④发现正常人血清中sCD40L的浓度明显高于血浆中的sCD40L的浓度。结论 SLE患者血清中sCD40L浓度较正常人高可能参与SLE的发生发展过程。血小板计数对血清中sCD40L浓度有一定程度的影响,且SLE患者血清中sCD40L与SLEDAI评分无相关性,在临床上sCD40L不适合作为监测SLE疾病的活动度的指标。     

Characterization of autoantibody activities in sera anti-DNA antibody and circulating immune complexes from 12 systemic lupus erythematosus patients

To examine autoantibodies present in patients with active systemic lupus erythematosus (SLE), sera, circulating immune complexes (CIC), and antibodies purified on DNA-immunoadsorbent were tested by enzyme immunoassay. A panel of self-antigens, including DNA, histones (HIS), glomerular basal membrane (GBM), thymus cell extract (TCE), actin (ACT), myosin (MS), and tubulin (TUB), was used to define their specificities. IgM antibodies against all antigens of the panel were detected in sera, CIC, and in antibodies eluted from the DNA-immunoadsorbent and demonstrated a large polyreactivity. IgG antibodies showed restricted activities against DNA, HIS, GBM, and TCE in sera and a large polyreactivity in CIC. Inhibition experiments were performed to assess their mono- or polyreactivities. Among the IgG autoantibody population recognizing DNA, two populations of IgG antibodies were detected in the sera and in the affinity purified anti-DNA: one recognizes DNA, HIS, and GBM, and the other binds to DNA and to cytoskeletal proteins. These autoantibody populations were found in CIC, which also often contained high amounts of IgG antibodies recognizing ACT and MS. A third population of IgG antibody that recognizes only TCE and could not be inhibited by DNA or other antigens was found in serum and CIC. Our data demonstrate the existence of several populations of autoantibody in serum and CIC of SLE patients: (1) IgM polyreactive autoantibodies, (2) IgG polyreactive autoantibodies recognizing DNA and cytoskeletal proteins, (3) IgG specific to DNA, which cross react with HIS and GBM, and (4) IgG specific to TCE antigens. © 1996 Wiley-Liss, Inc.     

活动性类风湿性关节炎患者血清葡萄糖-6-磷酸异构酶含量分析

目的研究活动性类风湿性关节炎(RA)患者血清中葡萄糖-6-磷酸异构酶(GPI)含量。方法应用ELISA方法检测82例活动性RA患者、20例系统性红斑狼疮患者、20例2型糖尿病患者和30例健康志愿者血清GPI含量,同时检测活动性RA患者血清抗CCP抗体、AKA、RF、CRP、C3和C4。结果 活动性RA患者组血清中GPI含量明显高于SLE患者组、2-DM患者组和健康对照组。82例活动性RA患者中有8例血清GPI含量大于20.0mg/L,8例患者均伴有关节肿胀疼痛的急性加重。在活动性RA患者中,GPI与RF、C3、C4以及病情活动指标之间存在相关性。在抗CCP抗体阴性的8例患者中有5例GPI阳性,在AKA阴性的19例病人中有15例GPI阳性。结论活动性RA患者血清GPI抗原含量明显增高,而且RA患者有高浓度的GPI存在时,常伴有严重的关节肿胀疼痛。     

Differences in specificities of anti-C3d sera raised to C3d antigens prepared in different ways

Anti-C3d sera were raised to different C3d antigens or to the same C3d antigen by different methods. Although identical by immunoprecipitation studies, the various anti-C3d sera showed differences in specificities against bound C3d antigen. Such differences were observed wih red blood cells coated with C3d in vivo and in vitro. Antisera made to the C3d− KAF antigen detected fewer molecules/cell on C3d−tryp cells than did antisera made to the C3d−tryp antigen. The converse was true for C3d− KAF cells. "Saturation" experiments indicated that different anti−C3d detected different "subpopulations" of bound C3d. C3d bound to red blood cells in vivo was, in at least one case, detectable by some anti- C3d sera but not by others. Such differences in anti-C3d specificity may be important in determining the optimal characteristics of anti-C3d antiglobulin serum for routine laboratory use.     

Circulating immune complexes in pulmonary tuberculosis

The presence of circulating Immune complexes (CIC) in sera from patients with pulmonary tuberculosis was demonstrated by 3 techniques (a) latex agglutination (b) 3.5% PEG precipitation and determination of optical density (O.D.) at 280 nm and (c) radioimmunoassay (RIA) of CIC using bovine spermatozoa. 40 normal control sera and 100 T.B. patients sera were included in the study. 12% cases were positive for CIC by all the 3 methods mentioned above, 13% were negative by all the 3 methods and the remaining patients were positive by one or more methods of detection. To correlate the levels of CIC as detected by different techniques with the activity of the disease, patients were broadly grouped as (a) sputum positive and (b) sputum negative. Higher levels of CIC were obtained in sputum positive cases than sputum negative by all the 3 methods studied. While IgG, IgA and IgM were elevated in the CIC of T.B. patients, and IgG and IgA were also present in controls, IgM immunoglobulins were detected only in patients and not in controls. The effect of antitubercular treatment on the levels of CIC was also evaluated and it was found that the levels of CIC remained unchanged even after prolonged chemotherapy.     

A method for detecting specific anti-C100 protein antibodies of IgM isotype in hepatitis C virus infection

Summary A method for the determination of specific IgM antibodies to C100 protein, a hepatitis C virus-associated antigen, was developed which employed fraction of serum proteins by gel chromatography and an enzyme-linked immunosorbent assay. Detection of IgM anti-C100 proved to be specific and reproducible in purified IgM fractions. Separation of IgM from IgG was necessary before IgM could be measured, because of the detrimental effect of the simultaneous presence of IgG antibodies with anti-C100 rectivity on IgM determination. IgM anti-C100 was not found in sera containing rheumatoid factors or IgM antibodies to other hepatotropic viruses. IgM anti-C100 was detected in 19 (44%) of 43 patients with hepatitis C virus-related chronic liver disease. When compared with the histological picture of liver disease, IgM anti-C100 was absent in patients with minimal changes and was most common (66.7%) in patients with progressive disease. It is suggested that IgM anti-C100 could reflect an active state of hepatitis C virus infection.     

56例女性系统性红斑狼疮患者血清肿瘤标志物检测分析

目的探讨女性系统性红斑狼疮（SLE）患者血清肿瘤标志物甲胎蛋白（AFP）、癌胚抗原（cEA）、肿瘤抗原CA125和CA19—9含量变化及临床意义。方法用微粒子酶免疫分析方法测定56例女性SLE患者血清中AFP、CEA、CA125和CA19—9水平,并与健康对照组进行比较。结果女性SLE患者血清AFP、CA125和CA19—9含量显著高于健康对照组,差异有统计学意义（P〈0．05）;CEA水平与健康对照组比较差异无统计学意义;SLE患者CA125阳性率显著升高。结论SLE患者某些血清肿瘤标志物含量较健康对照组增高,可能与器官受累有关。     

4种结缔组织病患者血管内皮细胞生长因子检测的分析

目的检测4种结缔组织病患者血清中血管内皮细胞生长因子(VEGF)水平,并探讨其意义。方法115例患者中,类风湿关节炎(RA)40例,系统性红斑狼疮(SLE)30例,多发性肌炎/皮肌炎(PM/DM)25例,系统性硬化症(SSc)20例,正常对照30例,采用酶联免疫吸附试验(ELISA)方法检测了血清中的VEGF(ng/L)的含量。结果血清VEGF水平(单位ng/L)分别为RA558.58±348.45,SLE244.63±87.44,PM/DM355.12±258.56,SSc362.72±177.52,类风湿关节炎、系统性红斑狼疮、多发性肌炎/皮肌炎、系统性硬化症患者血清VEGF水平均明显高于正常对照(P<0.01,P<0.05)。结论血清VEGF水平的测定对结缔组织病病情监测可能具有非常重要意义。     

Catalytic autoantibodies as a new molecular instrument in rheumatological practice

AIM: To compare clinicopathogenetic value of DNA-hydrolizing autoantibodies or DNA-abzymes in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). MATERIAL AND METHODS: We studied sera from 180 patients with SLE, 180 RA patients and 128 healthy donors matched by age and gender; assessed catalytic and cytotoxic activity of DNA-abzymes in patients with different variants of SLE and RA course. RESULTS: The highest catalytic and cytotoxic activities of DNA abzymes were observed in SLE patients. In SLE catalytic and cytotoxic activities of DNA abzymes ranged widely and their mean values depended on SLE activity in patients with systemic lesions. DNA-abzymes in RA patients showed lower catalytic and cytotoxic activities in relation to substrate DNA and target cells than in SLE. DNA-abzymes occurred most frequently in patients with high activity of RA, slow-progressive and lingering course of RA, especially in early development of visceral (extra-articular) pathology. Characteristic for DNA-abzymes in RA and SLE is the phenomenon of wide-range fluctuations due to factors determinating probability of induction of function of Ab-mediated catalysis and, therefore, incidence rates of DNA-abzymes, probably catalytic autoAb of the other specificity in a population of patients with systemic autoimmune diseases. CONCLUSION: The data indicate the validity of DNA abzymes use in clinical practice for realization of diagnostic and therapeutic programs in SLE and RA.