COMPARISON OF IMMUNOFLUORESCENCE AND IMMUNOPEROXIDASE FOR DEMONSTRATION OF ANTINUCLEAR ANTIBODIES ON HEp-2 SUBSTRATE

A comparison of antinuclear antibody (ANA) detection on HEp-2cell substrate, using immunofluores-cence (IF) and immunoperoxidase(IP) techniques, was performed on 183 sera from 178 individuals,with and without connective tissue diseases. All sera were numbercoded and scored blindly by two independent observers. ANA wasdetected in 61% of sera by IF and 62% of sera by IP. Positiveand negative results corresponded between the two techniquesin 97%of sera. Patterns and titres corresponded in 85% of positivesera. The two independent observers scored ANA more consistentlywith IP than with IF. Both methods were technically simple toperform and produced consistent results with control sera. Thesedata show that IP provides results equivalent to the traditionalIF technique for demonstrating ANA on HEp-2 substrate. Fewerdiscordant results between two independent observers using IPsuggests that this technique has technical advantages for interpretationof ANA on HEp-2 substrate. KEY WORDS: Antinuclear antibodies, Immunofluorescence, Immunoperoxidase, Systemic lupus erythematosus, Rheumatoid arthritis, Scleroderma, HEp-2 cell line.     

快速检测抗日本血吸虫抗体的金标免疫渗滤法的建立及应用

以血吸虫虫卵浸出液为抗原，金标记兔抗人ＩｇＧ为显示剂，建立了检测抗血吸虫抗体的金标免疫渗滤法（ＤＩＧＦＡ）。以该法检测血吸虫病患者血清８７份，阳性检出率为９８．９％；非流行区血清１００份，阴性符合率为１００％；１５例肺吸虫病患者血清，１例出现阳性反应，交叉反应率为６．７％；１０例华支睾吸虫病患者和２５例乙型肝炎患者，均无交叉反应。本法与ＥＬＩＳＡ比较，符合率达９６．７％。揭示ＤＩＧＦＡ的敏感性和特异性与ＥＬＩＳＡ相近，但方法简便、快速、试剂稳定，且无需特殊设备，适合于基层单位和现场应用     

促甲状腺激素受体抗体放射受体分析法的建立及其临床应用

用0.5％ Triton X-100一次增溶猪甲状腺细胞膜,用固相Iodogen法标记bTSH再经受体亲合纯化获得了高比生物活性的可溶性TSH受体和~(125)I-bTSH,建立了TRAb放射受体分析法。制备的TSH受体和~(125)I-bTSH的结合可被非标记bTSH和Graves病患者血清中的TRAb呈剂量依赖性抑制。TRAb平均抑制率分别为15.4％和90.6％时,批间变异系数相应为12.6％和3.1％。试验和临床应用结果表明,本方法灵敏度高、特异性强、重复性好,且操作简便、快速,适于临床常规应用。     

抗dsDNA抗体两种检测方法的比较

比较抗双链DNA(dsDNA)抗体的两种检测方法。用胶体金快速斑点渗滤技术(DIGFA)及马疫锥虫间接荧光抗体染色法检测27名正常儿童和46例系统性红斑狼疮(SLE)病人和非狼疮病人血清抗dsDNA抗体,27名正常儿童和17例非狼疮病人血清抗dsDNA抗体经两种方法检测均为阴性;29例SLE患者用两种方法检测出抗dsDNA抗体的阳性符合率为94.44％(17/18)。说明两种方法具有一致的特异性和敏感性,DTGFA更简捷、快速、方便。     

A STUDY OF ANTIPOLYNUCLEOTIDE ANTIBODIES, ANTI-KLEBSIELLA (K30) ANTIBODIES AND ANTI-DNA ANTIBODY IDIOTYPES IN ANKYLOSING SPONDYLITIS

Recent studies have indicated that both ankylosing spondylitisand the anti-DNA antibodies found in systemic lupus erythematosusmay be related to Klebsiella surface antigens. In order to explorethese possible relationships further, the sera of 24 patientswith ankylosing spondylitis (AS), and 20 controls, have beenexamined for binding to a wide range of antipolynucleotide antibodies,antibodies binding to the Klebsiella pneumoniae polysaccharideK30 and two DNA antibody idiotypes designated 16/6 and 134.We report that although 21% of the AS patients had IgG ssDNAantibodies it is evident that the aetiopathogenesis of thisdisease is not through the mechanism of autoantibodies or thecommon DNA antibody idiotypes tested. KEY WORDS: Ankylosing spondylitis, DNA antibody idiotype, Klebsiella antibody     

用PCR技术直接检测致病真菌的实验研究

目的　建立和评价从血标本中直接PCR检测病原真菌的方法。方法　用红、白细胞裂解液 ,基因释放剂等直接处理血标本 ,提取微量的靶DNA ,然后用真菌通用引物及种特异性引物进行PCR扩增。结果　在几种重要致病真菌制备的人血标本中检测出靶DNA ,其敏感性达 10个孢子 /ml以下 ,且从标本处理到报告结果仅需 6h。结论　用PCR方法可简便快速特异敏感地从血标本中直接检测病原真菌 ,提示可为临床快速诊断深部真菌病打下基础。     

快速免疫色谱测试法与免疫荧光法检测登革病毒IgG抗体的比较

目的：应用登革热快速免疫色谱测试法（Dengue Fever Rapid Immunochromatographic Test,DRIT)与间接免疫荧光法(Indirect Immunofluorescent Assay,IIF）检测登革病毒IgG抗体进行比较。方法：DRIT按说明操作,在Control线区出现紫红色线的前提下在IgG线区出现紫红色线表明IgG抗体阳性;IIF抗原片系应用本室分离的登革II型病毒感染C6／36细胞制备,操作按常规进行。结果：检查临床疑似登革热病人血清56份,IIF检测阳性30份,阳性率53.6％。DRIT检查阳性26份,阳性率46.4％,两法符合率91.07％,X^2=1.50,P＞0.05。检测无症状人血清68份,IIF检查阳性37份,阳性率为54.4％。DRIT检查阳性23份,阳性率为33.8％,两法符合率73.53％,X^2=9.39,P＜0.01。结论：检测病人血清两法无显著性差别,具有操作简单,时间较短,结果易观察等优点。IIF成本较低,检测病人或无症状人群IgG抗体均较敏感,仍不失为理想的常规方法。     

PCR 与 DIFA 检测沙眼衣原体的比较研究

用所建立的沙眼衣原体质粒引物聚合酶链反应技术（ＰＣＲ）检测１１６例泌尿生殖道沙眼衣原体，并与直接免疫荧光法（ＤＩＦＡ）进行比较。结果ＰＣＲ阳性３８例，ＤＩＦＡ阳性的３２例中有３１例ＰＣＲ阳性。ＰＣＲ敏感性为９６．９％，特异性为９１．７％。ＰＣＲ阳性者于治疗结束１～２周后复诊２１例，其中４例ＰＣＲ仍然阳性。结果显示，ＰＣＲ可快速、敏感、特异地检测泌尿生殖道沙眼衣原体ＤＮＡ，然而对治疗后短期内复诊患者，似乎不宜仅以ＰＣＲ阳性结果作为重复治疗的依据。     

人生长激素LAB—ELISA测定方法

本文报道了用LAB-ELISA技术测定尿中人生长激素的方法。本方法对人催乳素、人促甲状腺素、人促卵泡成熟激素和人促黄体生成激素均无明显交叉反应。精密度测试批内CV 4.72～12.00％,批间CV 4.89～11.17％,偏倚为0.20～12.00％,回收率达100±1.4％。本文对LAB-ELISA技术所涉及的因素作了较系统的研究,并提出了优化条件。通过LAB-ELISA技术测定正常和GHD儿童的尿标本,表明本测定技术具有灵敏度高、特异性强和稳定性好等优点。     

脑囊虫病患者脑脊液常规和生化检查与循环抗原检测的比较

本文对408例脑囊虫病患者脑脊液循环抗原检测与脑脊液常规检查和生化分析进行了比较.研究发现循环抗原强阳性组和弱阳性组脑脊液中出现嗜酸粒细胞者所占的比例高于阴性组(分别是50.43%、41.26%和17.65%).274例进行脑脊液生化分析,氯化物基本正常.循环抗原强阳性组葡萄糖含量降低的发生率(14.71%)高于阴性组(3.85%).脑脊液蛋白增高者所占的比例明显高于弱阳性组,弱阳性组又明显高于阴性组(分别为89.70%、71.43%和50.00%).23例原发性癫痫患者脑脊液中CAg均阴性,脑脊液常规和生化分析结果正常.     

DETECTION OF ANTI-Ro(SSA) ANTIBODIES IN AUTOIMMUNE DISEASES: COMPARISON OF FIVE METHODS

Counterimmunoelectrophoresis (CIE), RNA precipitation, ELISAand immunoblotting against cytoplasmic HeLa cell extract (IB-HeLa)and erythrocyte extract (IB-RBC) were applied to detect anti-Ro(SSA)antibodies in 93 sera selected from patients with various autoimmunediseases [47 were anti-Ro(SSA) positive by CIE]. The RNA precipitationassay, which demonstrated the highest sensitivity was selectedas the reference method. CIE was found to be reliable with aspecificity of 100% and a sensitivity of 89%. ELISA showed acomparable specificity (95%) but somewhat lower sensitivity(72%). Antibodies to 52 or 60 kDa Ro(SSA) proteins by IB-HeLademonstrated a high specificity (95 and 97% respectively) buta low overall sensitivity (36 and 17% respectively). Anti-Ro(SSA)antibodies to 52, 54 and 60 kDa erythrocyte proteins by IB-RBC,had a variable overall specificity (95, 97 and 57%) and sensitivity(51, 13 and 34%). The anti-52 kDa antibodies detected by IB-HeLacorrelated to those found by IB-RBC (P<0.001) and occurredpredominantly in primary Sjögren's syndrome (P<0.001,sensitivity: 71 and 77%) as well as in sera with anti-Ro(SSA)and anti-La(SSB) antibodies (P<0.001). These findings confirm that RNA precipitation assay has thehighest sensitivity and specificity for anti-Ro(SSA) antibodydetection. However, until a more sensitive ELISA is available,CIE because of its reliability appears to be the method of choice.Finally IB-RBC was found to be more sensitive than IB-HeLa forthe detection of anti-Ro52 kDa antibodies. KEY WORDS: Autoantibodies, Counterimmunoelectrophoresis, RNA precipitation, Enzyme-immunoassay, Immuno-blotting     

CLINICAL EVALUATION OF A TWO-SITE IMMUNORADIOMETRIC ASSAY FOR ADRENOCORTICOTROPIC IN UNEXTRACTED HUMAN PLASMA USING MONOCLONAL ANTIBODIES

We have developed a sensitive two-site immunoradiometric assay (IRMA) for intact ACTH and its precursors, pro-opiomelanocortin and 22 kDa peptide in unextracted human plasma. The assay uses two monoclonal antibodies. Antibody 1A12, specific for ACTH 10–18, is radiolabelled and antibody 2A3 specific for the C-terminal region (ACTH 24–39), is coupled to Sephacryl S300 for the solid-phase. Samples are incubated for 18 h with labelled antibody followed by 2 h with solid-phase antibody. Separation employs the sucrose layering technique. Using human pituitary ACTH 1–39 (code 74/555) in diluent containing 10% horse serum to standardize the assay, the sensitivity (upper 99% confidence limit of zero standard) is 3.5 ± 0.8 ng/l ( n = 7). The mean coefficient of variation is 5.9% within-assay and 6.7% between-assay and is less than 10% between 22 and >5000 ng/l. Mean recovery of ACTH 1–39 added to dexamethasone-suppressed human plasma is 109% and endogenous ACTH behaves indistinguishably from standard ACTH on dilution. In normal subjects, mean plasma ACTH levels are 30 ng/l at 0730 h, and 15 ng/1 at 1630 h at rest. ACTH concentrations are between 60 and 330 ng/l, 8–10.5 h after metyrapone (2 g orally at 2300 h), between 140 and 320 ng/1, 30–60 min after insulin-induced hypoglycaemia, and < 4 ng/1, 8 h after dexamethasone (1.5 mg orally at 2300 h). In a range of pathological conditions ACTH concentrations accurately reflect the disorders of the pituitary-adrenal axis. Endogenous ACTH immunoactivity is stable in vitro at 22°C for at least 1 h in whole blood and at least 4 h in plasma. It is concluded that this two-site IRMA for ACTH in unextracted plasma offers a reliable assay for clinical purposes.     

抗－HEV免疫吸印法的建立及应用

应用ＨＥＶ基因结构区的重组融合蛋白，建立了免疫吸印法（ＷｅｓｔｅｒｎＢｌｏｔ．ＷＢ）检测抗－ＨＥＶＩｇＧ，并与现行的酶联免疫试验（ＥＩＡ）进行了比较。结果表明，ＷＢ检出戊型肝炎暴发点急性肝炎病人血清抗－ＨＥＶＩｇＧ的阳性率（６８／７３，９３．２％）高于现行的ＥＩＡ（５１／７３，７００％），对实验感染猕猴抗－ＨＥＶＩｇＧ检出率前者也高于后者（分别为７／７和５／７），且检出抗－ＨＥＶＩｇＧ时间较长，滴度也较高。对多种对照血清检测结果表明，除５０％（３／５８）的丙型肝炎血清呈阳性外，甲型、乙型、ＥＢＶ、ＣＷＶ肝炎及健康人血清均为阴性，说明该法具有良好的灵敏度和特异性，可望用于流行病学调查和基础研究，也可用于疑是戊型肝炎病例的确诊     

合成肽与重组抗原检测丙型肝炎病毒抗体的对比研究

采用丙型肝炎病毒（ＨＣＶ）蛋白相对应区段合成肽和重组抗原各４条，分别以直接ＥＩＡ和重组免疫印迹试验（ＲＩＢＡ）检测抗－ＨＣＶ，并用逆转录－套式聚合酶链反应（ＲＴ－ｎｅｓｔｅｄ－ＰＣＲ）检测ＨＣＶＲＮＡ作对比。发现合成肽和重组抗原检测抗－ＨＣＶ的符合率以核心（Ｃ）区最高（９０．６５％～９４．７３％），非结构（ＮＳ）３区最低（３３．７８％）。单一合成肽抗－ＨＣＶ的检出率１２．５０％～８７．５０％，联合多区段合成肽可使检出率提高到９３．７５％～９６．８８％。结果提示合成肽可以构成重组抗原所含的抗原表位，但应用ＮＳ３区合成肽检测抗－ＨＣＶ可能受到限制。联合多区段合成肽检测对减少假阴性的发生有重要意义。     

THE ASSAY OF GRAVES''IMMUNOGLOBULINS: A COMPARISON OF DIFFERENT METHODS

A multiplicity of techniques has arisen for the measurement of the immunoglobulins specifically associated with Graves' hyperthyroidism, which has led to difficulty in assessing the clinical significance of these immunoglobulins. In this study of untreated toxic Graves' disease we have taken two assays (LATS protector and thyrotrophin binding inhibiting immunoglobulin, TBII) which depend upon the measurement of binding to thyroid cell membranes, and correlated them with a bioassay which measures as the index of activity the biologically relevant end point of T3 secretion from thyroid tissue. The frequency of positive responses obtained with each method was similar to that of previous studies; combining the results a positive response was found in 100%. No significant correlation was found between any of these three assays. T3 to assess which method relates most closely to circulating thyroid hormone levels in vivo. A highly significant correlation was obtained for TSAb measured by T3 secretion with both total serum T3 and total serum T4 levels. A positive correlation was also found for LATS protector, but not for TBII. We conclude that although these assays are specific for Graves' immunoglobulins, they are measuring closely related but not identical immunoglobulin activities.     

ARTHRITIS AND INTESTINAL DISEASES: A COMPARISON OF TWO FAMILY STUDIES

A comparison has been made between the results of two familystudies. Ninety-one patients with ulcerative colitis, 116 patientswith Crohn's disease and 449 of their relatives and spouseswere examined clinically and radiologically. A further 96 relativesand spouses were reviewed radiologically only. Intestinal synovitiswas found more frequently in patients with Crohn's disease thanin those with ulcerative colitis. This result was in accordwith published evidence and theoretical considerations. Sacro-iliitiswas found in similar proportions of probands, first-degree relatives,second-degree relatives, and spouses in each study. More ofthe subjects in the ulcerative colitis study were diagnosedas having ankylosing spondylitis, while a greater proportionof subjects with sacro-illtis in the Crohn's disease study werefemale. The possible interrelationship between these facts hasbeen discussed. ^*Formerly William Hewitt Research Fellow: Honorary Senior Registarin Rheumatology, Now Consultant Rheumatologist, South TeessideHospital Group Formerly Research Fellow Professor of Rheumatology     

DEVELOPMENT AND APPLICATION OF A MID-REGION SPECIFIC ASSAY FOR HUMAN PARATHYROID HORMONE

A new peptide spanning residues 28-54 of human parathyroid hormone (PTH) was synthesized and used to develop a homologous immunoradiometric assay specific for the mid-region of human PTH. The peptide was coupled to cellulose and used to absorb mid-region antibodies from a goat antiserum against intact human PTH. This assay has been applied to the measurement of circulating PTH in man: in normal subjects the concentration in serum ranged from undetectable (less than 40 pg/ml) to 70 pg/ml, the reference standard being the human PTH 28-54 peptide. In patients with primary hyperparathyroidism concentrations ranged from 120 to 1800 pg/ml. Hormone was not detected in patients with hypoparathyroidism. In normal subjects and in patients with primary hyperparathyroidism the mid-region PTH concentrations were similar to those obtained in an amino-terminal specific assay. By contrast, carboxy-terminal PTH concentrations were markedly higher being 10-fold greater in both groups studied. In patients with primary hyperparathyroidism undergoing parathyroidectomy and in chronic renal failure patients who were infused with calcium, mid-region and amino-terminal PTH disappeared much more rapidly than carboxy-terminal PTH. However, although mid-region PTH was initially cleared as quickly as amino-terminal PTH, it then reached a plateau and remained at a higher level. Thus the mid-region specific assay described here is proving to be of value in the study of the secretion and metabolism of PTH.