自身免疫病患者抗精浆免疫抑制物抗体的测定

用ＥＬＩＳＡ法测定血清中抗精浆免疫抑制物抗体（ＳＰＩＭ－Ａｂ），发现系统性红斑狼疮、类风湿性关节炎、混合性结缔组织病、重症肌无力和甲状腺疾病患者血清ＳＰＩＭ－Ａｂ水平与检出率均显著高于对照组（Ｐ＜０．０１）；抗核抗体、抗ｄｓ－ＤＮＡ抗体、抗Ｓｍ、抗ＲＮＰ抗体和类风湿因子与ＳＰＩＭ－Ａｂ无相关性，而抗甲状腺球蛋白抗体（Ａ－ＴＧ）和抗甲状腺微粒体抗体（Ａ－ＴＭ）阳性患者ＳＰＩＭ－Ａｂ水平均显著高于Ａ－ＴＧ、Ａ－ＴＭ阴性者（Ｐ＜０．０１）。     

系统性红斑狼疮病人外周血单个核细胞NF-κB活性的研究

目的;研究体外培养的系统性红斑狼疮（ＳＬＥ）病人外周血单个核细胞（ＰＢＭＣ）ＮＦ－κＢ的活化表达情况及其与ＰＢＭＣ分泌免疫球蛋白,自身抗体的关系。方法：ＮＦ－κＢ活性检测采用电泳迁移率改变法（ＥＭＳＡ）,细胞培养上清免疫球蛋白和抗ｄＳＤＮＡ抗体采用微量ＥＬＩＳＡ测定。结果;发现ＳＬＥ病人外周血ＰＢＭＣ ＮＦ－κＢ活性显著高于正常对照组,与其ＰＢＭＣ培养上清及血清ＩｇＧ和抗ｄｓＤＮＡ抗体含量成正相关     

用噬菌体表面表达技术筛选及表达抗重组人促红细胞 …

目的 获得抗重组人红细胞生成素（ｒｈＥＰＯ）的单链抗体（ＳｃＦｖ），为得到纯度高、活力强的重组人红细胞生成素（ｒｈＥＰＯ）产品和制备红细胞生成素（ＥＰＯ）检测试剂盒奠定基础。方法 采用噬菌体表达表达和重组抗体技术，从已建立的鼠抗ｒｈＥＰＯ杂交瘤细胞株Ｄ３中克隆出抗ｒｈＥＰＯ单链抗体ＳｃＦｖ基因片段，经噬菌体表面呈现，与固相抗原ｒｈＥＰＯ结合，“吸附－洗脱－富集”，酶联免疫吸附法（ＥＬＩＳＡ）检测，     

分泌性中耳炎中耳积液和血清中sIL－2R水平的初步研究

应用酶联免疫吸附法（ＥＬＩＳＡ）对３０例正常人血清（对照组）和６０例分泌性中耳炎患者（ＳＯＭ组）中耳积液和血清中可溶性白细胞介素２受体（ｓＩＬ－２Ｒ）进行了检测。结果示ＳＯＭ组血清ｓＩＬ－２Ｒ水平明显高于对照组，ＭＥＦ中其含量明显高于血清；鼻咽癌组血清及ＭＥＦ中ｓＩＬ－２Ｒ水平明显高于─般ＳＯＭ组；粘液组高浆液组；慢性组高于急性组（均Ｐ＜０．０１）。提示：血清及ＭＥＦ中ｓＩＬ－２Ｒ水平的测定有助于对ＳＯＭ患者免疫状态的评估；ＭＥＦ中ｓＩＬ－２Ｒ可能主要由局部中耳粘膜产生；ＭＥＰ中高浓度的ｓＩＬ－２Ｒ存在可能是ＳＯＭ迁延不愈的─个原因。     

用抗体捕捉ELISA法检测肾综合征出血热患者唾液中特异性IgM抗体

用抗体捕捉ＥＬＩＳＡ法检测肾综合征出血热患者唾液中特异性ＩｇＭ抗体孙晓芬，崔桂兰（成都军区后勤部军事医学研究所，成都６１００６１）诊断肾综合征出血热病毒（ＨＦＲＳＶ）感染通常依靠测定血清中特异性ＩｇＧ、ＩｇＭ抗体。本实验采用ＩｇＭ抗体捕捉ＥＬＩＳＡ法...     

间接ELISA法的改进并用于肾综合征出血热病人血清的初步分型

采用超声、差速离心法制备肾综合征出血热病毒（ＨＦＲＳＶ）抗原。通过与免疫荧光法（ＩＦＡ）和捕获ＩｇＭＥＬＩＳＡ法（ＭａｃＥＬＩＳＡ）比较，分析了病人临床四个期血清抗体阳性率，对３组不同地区的正常人群ＨＦＲＳＶ抗体阳性血清作抑制试验，以及比较ＩＦＡ和ＥＬＩＳＡ血清抗体几何平均滴度证实本方法是特异和灵敏的。用这一方法对野鼠型和家鼠型ＨＦＲＳ病人血清，与两型病毒作交叉ＥＬＩＳＡ试验，结果呈单向反应；采用胞膜荧光反应为主的感染细胞制备抗原，可提高其分型效果。     

IL－2R水平在骨髓增生异常综合征中的意义

为了对ＭＤＳ的发生发展和免疫学异常进一步了解，我们用双抗体夹心ＥＬＩＳＡ法检测２０例ＭＤＳ患者血清中ｓＩＬ－２Ｒ水平；采用ＡＰＡＡＰ桥联酶免疫染色观察９例ＭＤＳ患者ＰＢＭＣ中ｍＩＬ－２Ｒ的表达，发现ＭＤＳ患者血清中ｓＩＬ－２Ｒ较正常人增高。在ＭＤＳ亚型中ＲＡＥＢ，ＲＡＥＢ－ｔ组较ＲＡ组增高显著，Ｐ＜０．０１；校再障组也增高。ｍＩＬ－２Ｒ阳性细胞百分率也较正常人高。血清中ｓＩＬ－２Ｒ释放水平与ＰＢＭＣ中ｍＩＬ－２Ｒ的表达比较，两者无明显相关。研究结果表明，ＭＤＳ除髓系细胞累及外，ＩＬ－２Ｒ水平增高可能是ＭＤＳ淋巴细胞异常，免疫系统功能紊乱的一种表现。     

重症肌无力患者血清非Ig成分对突触前,后膜受体及其相应抗体反应？…

利用盐析、免疫亲和吸附及ＰｒｏｔｅｉｎＧ ＦａｓｔＦｌｏｗ完全去除２０例抗乙酰胆碱受体抗体和抗突触前膜受体抗体阳性重症肌无力（ＭＧ）、２０例抗体阴性ＭＧ、１５例其它神经科疾病患者和１５例正常对照血清的Ｉｇ部分，并以ＡＢＣ－ＥＬＩＳＡ观察应用上述方法提取的血清非Ｉｇ成发对ＡｃｈＲ，ＰｓｍＲ抗原抗体反应的影响。结果显示除抗体阴性ＭＧ血清经非Ｉｇ成分作用后，ＡｃｈＲ、ＰｓｍＲ与其各自抗体反应反而增强外     

可溶性PTA1分子的发现及其检测方法的建立

目的 建立检测ｓＰＴＡ１的方法,探讨正常人和肿瘤患者血清中是否存在可溶性ＰＴＡ１（ｓＰＴＡ１）分子。方法 以亲和层析法从血小板纯化的天然ＰＴＡ１分子作为免疫原,制备抗ＰＴＡ１分子的多克隆抗体（ＰｃＡｂ）,并以此建立检测ｓＰＴＡ１的双抗体夹心ＥＬＩＳＡ法。结果 建立的双抗体夹心ＥＬＩＳＡ法敏感性达１００ｎｇ／Ｌ,应用此方法从经ＴＰＡ,ＰＨＡ及抗ＣＤ３ ｍＡｂ活化的人ＰＢＭＣ细胞培养上清中检测到ｓＰＴ     

化学发光免疫测定抗dsDNA抗体的实验条件分析和初步应用

采用正交设计确定了化学发光免疫测定抗ｄｓＤＮＡ抗体的最适实验条件，并与免疫荧光法（ｌＦ）、酶联免疫吸附测定法（ＥＬＩＳＡ）和放射免疫测定法（Ｆａｒｒ）比较。结果表明，最适条件：ｄｓＤＮＡ包被浓度为１０μｇ／ｍｌ、ＡＢＥＩ－ＳａＨＩｇＧ结合物使用发光强度为１．０×１０￣４ｍｖ坎德拉，氯化高铁血红素（Ｈｅｍｉｎ）浓度为５μｍｏｌ／Ｌ，Ｈ＿２Ｏ＿２浓度为０．３％，５６份ＳＬＥ患者血清中，化学发光免疫测定法（ＣＬＩＡ）检测抗ｄｓＤＮＡ抗体阳性率为８３．９％，高于ＩＦ和ＥＬＩＳＡ法，接近Ｆａｒｒ法水平。阻断试验证明，本方法检测抗ｄｓＤＮＡ抗体具较高特异性。     

A latex slide agglutination test for rapid detection of antimyeloperoxidase antibody

AIM: To develop and test a new latex slide agglutination test (MPO-LSAT) to detect antimyeloperoxidase (anti-MPO) antibody in serum. METHODS: Latex bead coating was adjusted to give maximum sensitivity by attending to latex size, MPO to latex ratio for coupling, ratio of diluted serum to MPO-latex, reaction time and temperature for coupling, and reaction time for agglutination. Inhibition studies were performed using MPO, proteinase 3, bactericidal/permeability increasing protein, and lactoferrin. RESULTS: There was very good correlation between this test and the conventional anti-MPO enzyme linked immunosorbent assay (ELISA): 81% of sera positive in the ELISA were positive by MPO-LSAT. MPO-LSAT results correlated better with IgM anti-MPO than with IgG anti-MPO. CONCLUSIONS: MPO-LSAT is a simple diagnostic test that is potentially useful in the clinical laboratory as a rapid screening tool for vasculitic diseases.     

Difference in antigenic determinant profiles between human and rat myeloperoxidase

We tested whether rat and human MPO have similar antigenic determinants using 36 human MPO-ANCA positive sera, one mouse anti-rat MPO and four mouse anti-human MPO monoclonal reagents. Purified rat and human MPO were used in ELISA, with or without crossinhibition by preincubation with human MPO or irrelevant antigen in the liquid phase. Only one human MPO ANCA positive serum exhibited significant binding in rat MPO ELISA. This binding was poorly inhibited by preincubation with human MPO in the liquid phase, but was conserved after adsorption of non specific anti-rat activity in a chromatography column. Three mouse anti-human MPO IgG monoclonal antibodies did not recognize rat MPO. Only one mouse anti-human MPO IgA monoclonal antibody bound to rat MPO. This binding was poorly inhibited by preincubation with human MPO (35% at 2 micro g/ml). Conversely, the mouse anti-rat MPO monoclonal did not bind human MPO. We have concluded that: (1) Most human MPO-ANCA recognize antigenic determinants on human MPO which are absent on rat MPO. Therefore, human auto-antibodies bind to epitopes which recently appeared after species evolution; (2) Inversely, the mouse anti-rat MPO monoclonal do not bind human MPO. Therefore, rat MPO epitopes have been altered during species evolution; (3) Mice injected with human MPO preferentially develop antibodies against xeno-epitopes which are not present in rodents. Therefore, human MPO may not be the best antigen to raise ANCA in animal models and (4) A comparison of the amino acid sequences of rat and human MPO may help elucidate the major antigenic epitopes.     

Study of Anti-Neutrophil Cytoplasmic Antibodies in Patients with Thromboangiitis Obliterans (Buerger’s Disease)

The presence of antineutrophil cytoplasmic antibodies (ANCA) and their specificity to myeloperoxidase, lactoferrin and elastase were studied in 27 patients with thromboangiitis obliterans (Buerger’s disease), including nine in mild stage of the disease (group I) and 18 in active or severe stage (group II). Serum samples were investigated for ANCA prevalence by indirect immunofluorescent technique and fixed neutrophil ELISA; the reactivity to myeloperoxidase, lactoferrin and elastase was measured by ELISA. ANCA were found in 55.5% (15/27) of the patients by indirect immunofluorescence and in 59.25% (16/27) by fixed neutrophil ELISA. A highly significant positive association was observed between the values obtained by the two methods. Only six of 15 ANCA-positive sera reacted with myeloperoxidase and four of 15 with lactoferrin. One ANCA-negative serum reacted with elastase. Antimyeloperoxidase, antilactoferrin and antielastase autoantibodies were found only in the severe group. There was statistically significant difference in the frequency of ANCA in sera of patients with thromboangiitis obliterans versus control sera. ANCA were predominantly found in group II (66.7%; 12/18) compared to group I (33.3%; 3/9) and the levels of antimyeloperoxidase, antilactoferrin and antielastase antibodies correlated with disease severity. Therefore, detection of ANCA can be a helpful serological test for the diagnosis of thromboangiitis obliterans.     

Native and recombinant proteins to analyze auto-antibodies to myeloperoxidase in pauci-immune crescentic glomerulonephritis

The prevalence of Anti-Neutrophil Cytoplasmic Antibodies (ANCA) directed against myeloperoxidase (MPO) in pauci-immune necrotizing crescentic glomerulonephritis (NCGN) is dependent on the assay(s) used. We investigated the frequency of MPO-ANCA as detected by different assays for MPO-ANCA in a large cohort of patients with biopsy-proven pauci-immune NCGN. Sera from 121 consecutive untreated patients presenting with pauci-immune NCGN were tested for ANCA directed to proteinase-3 (PR3) at diagnosis. PR3-ANCA negative sera were tested by direct ELISA using recombinant or native MPO and by capture ELISA using two different specific monoclonal antibodies directed to MPO and three different antigenic sources. Sera from 80 relevant disease controls were tested to explore the specificity of the different assays. Thirty-eight out of 121 patients (31%) with pauci-immune NCGN did not have PR3-ANCA. Sufficient amounts of serum from 30 of these 38 PR3-ANCA negative patients were available for further testing. Recombinant and native MPO were recognized by similar numbers of sera in a direct ELISA (recombinant MPO: 93%, native MPO: 93%) and a capture ELISA (recombinant MPO: 77-87%, native MPO: 93%). Sera of patients with PR3-ANCA positive pauci-immune NCGN and disease controls were less frequently positive for MPO-ANCA in a capture ELISA (recombinant MPO: 3-7%, native MPO: 6-7%) than in a direct ELISA (recombinant MPO: 25%, native MPO: 13%). Both direct and capture ELISA assays using either native or recombinant MPO are sensitive techniques to detect MPO-ANCA in patients with pauci-immune NCGN. A capture ELISA performs better than a direct ELISA because it combines a higher specificity with a comparable sensitivity. Recombinant MPO is a good alternative for native MPO when used as antigen in a capture ELISA, but not when used in a direct ELISA because of lower specificity in this latter assay.     

Ultrastructural myeloperoxidase activity and expression of myeloid-associated antigens in acute lymphoblastic leukemia

Ultrastructural myeloperoxidase (MPO) activity and myeloid-associated antigen (MyAg) expression were investigated in 12 adult patients with acute lymphoblastic leukemia (ALL). Ultrastructural MPO was detected by 3 different methods. Immunophenotyping was performed by flow cytometry, using a series of monoclonal antibodies. Ultrastructural MPO-positive blast cells were detected in 6 patients (50%). In 5 of these 6 patients, the methods detecting both MPO and platelet peroxidase (PPO) activities found MPO-positive blast cells more frequently than those detecting MPO activity alone. In 2 patients (17%), at least one kind of MyAg was positive. Ultrastructural MPO activity was detected more frequently than MyAg expression in ALL patients. This method of detecting PPO and MPO is recommended for detection of ultrastructural MPO-positive ALL.     

Oral fluid antibody detection in the diagnosis of Helicobacter pylori infection.

The aim of this study was to evaluate an enzyme-linked immunosorbent assay (ELISA) for the detection of anti-Helicobacter pylori specific IgG antibodies in specimens of oral fluid. Antral biopsy specimens, serum and oral fluid samples were collected from 81 patients attending for upper gastrointestinal endoscopy. The presence or absence of current H. pylori infection was determined by culture, histology and urease detection. Anti-H. Pylori specific IgG was detected in serum by an established in-house ELISA and in oral fluid by an ELISA developed for this study. In all, 34 (42%) of 81 patients were positive for H. pylori by one or more of the 'gold standard' tests (culture, histology and urease detection). The oral fluid ELISA had a sensitivity of 94% and specificity of 85% with regard to current H. pylori infection. The serum ELISA had a sensitivity and specificity of 91%. There was an overall agreement of 88% between serum and oral fluid antibody detection. The detection of anti-H. pylori specific IgG in oral fluid by ELISA is comparable in sensitivity and specificity with serum-based methods. Oral fluid-based ELISA could provide a reliable, non-invasive method for the diagnosis of H. pylori infection, and may be of particular benefit for population surveys.     

Rapid diagnosis of echovirus type 33 meningitis by specific IgM detection using an enzyme linked immunosorbent assay (ELISA)

During an outbreak of meningitis in France (in the Lyon area), from June to October 1982, serum and stool samples were collected from 227 patients. An enzyme-linked immunosorbent assay (ELISA) for titrating IgG and IgM antibodies anti-echovirus type 33 was developed and compared with the virus isolation technique, and with the titration of neutralizing antibodies. In 39 patients excreting echovirus 33 in faeces, the ELISA test allowed a positive serodiagnosis in 85% of the cases by detection of specific IgM (64% of the cases) and by seroconversion (21%). Compared with the neutralization (Nt) test, ELISA was found to be more sensitive. The antibody titres in ELISA were over 50 times higher and detected earlier than the neutralizing antibodies. This early immune response allowed a rapid diagnosis by specific IgM detection in the acute sera collected within 8 days after the appearance of the clinical symptoms in more than 50% of the 97 patients examined, whereas the Nt test allowed a positive serodiagnosis in only 32% of the patients. The use of a caesium chloride purified antigen insured the specificity of the reactions.     

Detection of mitochondrial antibodies directed against the primary biliary cirrhosis (M2) antigen by an enzyme-linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of 1 subtype of mitochondrial antibodies (AMA) specific for chronic cholestatic inflammatory liver diseases. AMA were detected by ELISA in 16 of 16 patients with primary biliary cirrhosis (PBC) and in 2 of 31 patients with chronic active hepatitis. These 18 positive sera were positive for AMA by indirect immunofluorescence (IF) and by radioimmunoassay (RIA). No AMA were detected by ELISA in 2 patients with the pseudolupus erythematosus syndrome, who were positive for AMA by IF, 2 patients with secondary syphilis, positive for cardiolipin antibodies, 1 patient with systemic lupus erythematosus, positive for AMA by IF, 58 further patients with various hepatic and non-hepatic diseases and 10 healthy blood donors. The titers obtained by ELISA, ranging from 1:20 to 1:62,500, correlated well with those obtained by IF and RIA. The ELISA detected an AMA directed against one determinant of a mitochondrial antigen bearing the characteristics of the so-called PBC antigen (M2 antigen). The ELISA described is a sensitive and specific test for the detection of AMA directed against the PBC (M2) antigen and may be used not only as a standard method assaying this clinically important subtype of AMA but also as a tool for further purification and characterization of the PBC (M2) antigen.     

Cockroach allergen detection and cockroach allergens of allergic Thai patients

Hybridomas secreting monoclonal antibodies (MAb) specific to American cockroach (Periplaneta americana) were produced through a fusion of immune splenocytes of a BALB/c mouse immunized with crude cockroach (CR) extract and mouse myeloma cells. Two hybridomas namely 38G6 and 3C2 were established. These specific hybridomas secreted IgG1 monoclonal immunoglobulins with antigenic specificities to CR protein components of over 207 to 72 kDa and 45 to 40 kDa, respectively. The monoclonal antibodies were applied to select their specific epitopes out of the crude CR extract using affinity chromatography. A Prausnitz-Kustner test revealed that these epitopes were allergens which caused wheals and flares of the skin of a guinea-pig previously sensitized with a pool of serum samples from CR allergic patients. The monoclonal antibodies were also used in a capture ELISA to detect specific IgE in serum samples of allergic Thai patients. It was found that 72% and 76% of the patients had IgE antibodies to the epitopes of MAb 38G6 and MAb 3C2, respectively, indicating that the two epitopes are major CR allergens among the CR allergic Thai patients. An antibody-sandwich ELISA was developed for quantitative detection of CR allergens using the two monoclonal antibodies as a capture reagent and rabbit polyclonal antibodies to crude CR extract as a detection reagent. The assay could detect allergenic epitopes contained in as little as 122 pg of crude cockroach extract, and has high potential for direct measurement of the marker allergens in extracts of environmental samples.     

Immunoglobulin Isotypes of Anti-Myeloperoxidase and Anti-Lactoferrin Antibodies in Patients with Collagen Diseases

To investigate the prevalence and clinical relevance of immunoglobulin (Ig) isotypes of antimyeloperoxidase (MPO) and antilactoferrin (LF) antibodies in collagen diseases, enzyme-linked immunosorbent assay was employed to detect the Ig isotypes of both antibodies. The purified proteins of MPO and LF were used as two major representative antigens for anti-neutrophil cytoplasmic antibodies (ANCA) with a perinuclear staining pattern by an indirect immunofluorescent technique. We examined 131 serum samples from 79 patients with rheumatoid arthritis (RA), 32 with systemic lupus erythematosus (SLE), 14 with progressive systemic sclerosis (PSS), 6 with polymyositis/dermatomyositis (PM/DM), and 5 with idiopathic crescentic glomerulonephritis who served as positive controls and 36 healthy subjects who served as controls. A limited number of patients with RA (4–10%), SLE (6–9%), and PSS (7–14%) but not PM/DM showed positive IgG or IgA anti-MPO antibody (MPO-ANCA) but not IgM MPO-ANCA. However, 10–20% of RA, 40–60% of SLE, 20–36% of PSS but none of the PM/DM patients showed positive IgG, IgA, or IgM anti-LF antibody (LF-ANCA). MPO- and LF-ANCA positivity in RA patients was correlated with markers of disease activity such as the erythrocyte sedimentation rate, C-reactive protein, and serum Ig levels. IgG LF-ANCA but not MPO-ANCA positivity in SLE patients also was correlated with the disease activity index but not with clinical features. Neither MPO- nor LF-ANCA positivity in PSS patients was correlated with any clinical features. Overall, both MPO- and LF-ANCA were found mainly in RA, SLE, and PSS patients but not in PM/DM patients. The Ig isotypes of MPO- and LF-ANCA frequently belonged to both IgG and IgA and rarely to the IgM class. Both MPO- and LF-ANCA positivity reflected disease activity in RA and SLE rather than specific organ involvement.