Detection of Antibody against Extractable Nuclear Antigen by an Enzyme-Linked Immuno-Sorbent Assay

Anti-ENA antibody determination by ELISA technique may offer a valuable diagnostic help in the discrimination of patients with mixed connective tissue disease (MCTD) from those with other chronic inflammatory connective tissue diseases. Determination of this antibody was performed in a prospective designed investigation among 101 blood donors, 154 patients with various non-rheumatic internal medical diseases, and 229 patients with chronic inflammatory connective tissue diseases, including five patients with MCTD. A positive titre of anti-ENA antibody was found in approximately 10% of blood donors and patients with various internal medical disorders. A highly positive anti-ribonucleoprotein (RNP) titre was found in the patients with MCTD, but was also observed in patients with other chronic inflammatory connective tissue diseases, giving a predictive value of 56% for MCTD. We conclude that highly positive anti-RNP antibody values do not automatically indicate the diagnosis MCTD. Other diagnostic possibilities should still be considered.     

抗人肿瘤细胞核单克隆抗体的制备及鉴定

根据Epstein等报告,肿瘤坏死部分的不溶性抗原可以作为放射免疫检测与治疗的靶部位。作者制备了抗人肿瘤细胞核单克隆抗体(McAbs),并对其性质进行了鉴定。放射性同位素标记的抗核McAds的初步生物分布试验表明,抗核McAds确可到达肿瘤坏死部位。     

Prevalence of systemic autoimmune rheumatic diseases and clinical significance of ANA profile: data from a tertiary hospital in Shanghai,China

It is necessary and useful to explore prevalence of various systemic autoimmune rheumatic diseases (SARDs) in patients with suspicion of having SARDs and to characterize antinuclear antibodies (ANA) profile for identifying different populations (SARDs and non‐SARDs). A total of 5024 consecutive patients with available medical records were investigated, whose sera had been tested for ANA profile, including ANA, anti‐dsDNA and anti‐extractable nuclear antigen (ENA) antibodies, between 31 January 2012 and 26 March 2014. Only 594 (11.8%) patients were diagnosed with SARDs of those suspected with SARDs. The prevalence of systemic lupus erythematosus (SLE) was highest (3.2%), followed by rheumatoid arthritis (RA) (2.5%), primary Sjögren's syndrome (pSS) (1.7%), ankylosing spondylitis (AS) (1.5%), etc. Of females, SLE also showed the highest prevalence (6%), while of males, AS showed the highest prevalence (1.9%). The prevalence of most SARDs was closely associated with age, except mixed connective tissue disease (MCTD), and the variation characteristics among different age groups were different among various SARDs. The prevalence of ANA was significantly increased in most SARD patients [especially in SLE, systemic sclerosis (SSc) and MCTD]. For anti‐ENA antibodies, in contrast to some autoantibodies associated with multiple SARDs (e.g. anti‐SSA, SSB, nRNP), others were relatively specific for certain diseases, such as anti‐dsDNA, Sm, histone, nucleosome and Rib‐P for SLE, anti‐SCL‐70 for SSc and anti‐Jo‐1 for polymyositis/dermatomyositis (PM/DM). Of note, ANA profile appeared to be of little significance for AS, ANCA‐associated vasculitis (AAV), polymyalgia rheumatic (PMR), adult‐onset Still's disease (ASD) and Behcet's disease (BD). The younger were more likely to have the presence of anti‐dsDNA, Sm, histone or Rib‐P for SLE, and anti‐SSA for RA or MCTD. No significant differences for frequencies of ANA and anti‐ENA autoantibodies were found between sexes in most SARDs, with the exception of RA and AS. The present study suggests that, of patients with SARDs‐like clinical manifestations, the proportion of those with true SARDS is small, for most of whom tests for autoantibodies are necessary and useful to help make a prompt and precise diagnosis.     

Significance of extractable nuclear antigens in childhood autoimmune liver disease.

The relative virulence of different isolates of Mycobacterium avium has been linked to their capacity to trigger the secretion of TNF from the macrophages they infect. Smooth opaque (SmOp) variants of Myco. avium have been shown to trigger higher expression of TNF-alpha by macrophages in vitro than the smooth transparent (SmTr) variants. To analyse the role of TNF in resistance to infection by Myco. avium, we studied the infection by two different morphotypes of strain 2.151 of Myco. avium both in vitro and in vivo in the presence or absence of neutralizing antibodies to TNF. No effects were found in vitro regarding the growth of either isolate of Myco. avium. In vivo, only the virulent SmTr morphotype showed enhanced growth in the presence of the neutralizing antibodies. This enhancement occurred relatively late when priming for TNF secretion in vivo was evident. Among four isolates of Myco. avium, three virulent ones induced a marked priming for TNF release and one avirulent strain did not. Mycobacterium tuberculosis H37Ra, which is very active in inducing TNF release due to its lipoarabinomannan moiety, was used to compare with the previous results. The growth of H37Ra in macrophages was increased in vitro by the neutralization of TNF and neutralization of either TNF and/or interferon-gamma (IFN-gamma) enhanced the in vivo proliferation of this microbe in the spleen and liver of infected animals, whereas only the combination of both anti-TNF and anti-IFN-gamma enhanced bacterial proliferation in the lung. We conclude that resistance to the avirulent strains of Myco. avium did not involve TNF, but rather antimicrobial mechanisms expressed constitutively in the mononuclear phagocytes. In contrast, TNF plays an important role in the control of Myco. tuberculosis H37Ra infection.     

抗前列腺特异性膜抗原膜外区多肽单克隆抗体的研制及初步应用

目的建立前列腺特异性膜抗原（PSMA）膜外区多肽杂交瘤细胞株，并对其分泌的PSMA单克隆抗体进行初步鉴定，为PSMA的功能研究和人源化抗体的制备奠定基础，以求进一步用于前列腺癌的诊断和治疗。方法使用人工合成多肽免疫BABL／c小鼠，采用PEG融合技术建立杂交瘤细胞株，制备单克隆抗体。通过免疫荧光法、酶联免疫吸附法及斑点金标法确定单克隆抗体的交叉反应性、亲和力及免疫球蛋白的类型和亚类。结果获得两株可稳定分泌PSMA单克隆抗体的杂交瘤细胞，4F4为IsG1类，1F1为IgC3类。两株单抗均能识别LNCap细胞表达的PSMA蛋白，与不表达PSMA的PC-3、SP2／0等细胞无交叉反应。杂交瘤细胞株1F1培养上清效价为1：40。腹水效价为1：6400；而杂交瘤细胞株4F4培养上清效价为1：80。腹水效价为1：8000。结论成功地制备出两株抗PSMA单克隆抗体，均具有良好的特异性和亲和力，为进一步建立免疫分析方法。进行PSMA相关研究奠定了基础。     

抗独特型单抗NP30抗山羊血吸虫病的免疫研究

观察日本血吸虫单克隆抗独特型抗体NP30主动免疫对山羊血吸虫病的抗病免疫作用。山羊随机分为 2组。实验组 12只 ,NP30免疫剂量为 10 0 0 μg/只 /次 ,后腿肌肉注射 ,连续免疫 3次 ,最后一次免疫后 8周进行尾蚴攻击。对照组 10只 ,肌肉注射SP2 / 0腹水。 2组山羊于尾蚴攻击感染后第 12周处死。应用NYD 10 0 0型图像分析系统测量肝脏虫卵肉芽肿大小和纤维化程度。实验结果表明 ,实验组山羊肝脏虫卵肉芽肿的面积、体积减小 ,数量明显减少 ,肝纤维化程度减轻 ,山羊体重增加。本文提示NP30主动免疫对山羊血吸虫病可产生较好的抗病免疫作用 ,NP30可作为血吸虫病疫苗候选分子。     

口腔颌面部肿瘤单克隆抗体研究的初步报告

作者用人牙龈癌细胞系Ca9-22免疫的BALB/c小鼠脾细胞与小鼠骨髓瘤Sp2/0细胞融合,制备出2株单克隆抗体(McAbs)C-D11和C-E9。2株McAbs的类型均为IgG2b型。杂交瘤培养上清液和腹水的效价分别为1:128～256和1:10~4～10~6。C-D11与13种培养的瘤细胞中的7种反应;C-E9与其中的10种瘤细胞反应;但2株McAbs与13种正常细胞几乎不反应。C-D11和C-E9相应抗原能耐受蛋白水解酶和一些有机试剂的处理,但不耐受过碘酸的氧化作用。     

甲型和乙型流感单克隆抗体用于碱性磷酸酶桥联酶标快速诊断

作者将甲型和乙型流感单克隆抗体(McAb)用于桥联酶标技术快速诊断流感抗原125例,以IFA技术作对照,获得满意效果。其阳性符合率是87.5％;阴性符合率是96.1％。由于碱性磷酸酶主要存在于肠道,而呼吸道细胞几乎不含此酶,因此没有内源酶的干扰,结果在一般光学镜下格外清晰易辨。此技术诊断流感快速,2h内可得到准确结果。染色封片后半年内细胞着色不变。有利于实验室之间的质量控制和作回顾性研究。     

系统性红斑狼疮皮肤表皮抗核抗体的探讨

本文对１９１例ＳＬＥ患者的“正常”皮肤进行了免疫荧光的研究，结果发现６．８％（１３／１９１）的患者伴有“体内”ＡＮＡ或称ＥＮＳ，其中以Ｓ型ＩｇＧ在表皮细胞核内沉积最为多见。同时与其他检测结果做了比较，未发现ＥＮＳ的核型或荧光强度受血清ＡＮＡ，抗ｄｓ－ＤＮＡ抗体，ＢＭＺ荧光染色的影响，因此我们认为，ＥＮＳ的出现对ＳＬＥ具有协助诊断价值。     

Production and Characterization of Monoclonal Antibodies against the Extracellular Domain of CA 125

Carcinoma antigen 125 (CA 125) is overexpressed in ovarian cancer and antibodies against it are widely employed for diagnostic purposes. The rarity of CA 125 antigenic domains and its highly glycosylated structure, however, is a problem that may prevent immunized mice from developing a diversified population of anti-CA 125 antibodies. In this study a prime-boost strategy, which potentially could augment the humoral immune responses against rare and poorly immunogenic determinants, was used for immunization of mice and monoclonal antibodies (mAbs) were produced by hybridoma technology. Reactivity of mAbs was then assessed by ELISA, western blotting, immunoprecipitation, immunohistochemistry and immunofluorescence staining of OVCAR-3 cell line. Altogether, 10 clones were produced, 3 of which had IgG isotype and the rest were IgM. Two-third of clones recognized cognate antigen in fixed and living cells and had strong immunoreactivity in IHC staining. In Western blotting, our antibodies recognized CA 125 as high molecular weight antigen mostly migrated in the 3% stacking gel. Immunoprecipitation of OVCAR-3 cell lysate by mAbs resulted in a very similar migration pattern that reconfirmed their specificities. The mAbs produced in this study are invaluable tools in diagnosis and research fields for assessment of CA 125 expression in cancerous ovarian tissues.     

单克隆抗体用于APAAP桥联酶标技术快速检测副流感病毒3型抗原的研究

作者将副流感病毒3型(PIV-3)单克隆抗体(McAb)用于APAAP桥联酶标技术快速检测患儿PIV-3抗原100例,并与IFA技术及血凝抑制试验(HAI)作了比较,获得满意效果。其与IFA的阳性和阴性符合率均>96％。与HAI的阳性符合率为88％,阴性符合率为100％。由于呼吸道细胞内很少含有碱性磷酸酶,因此没有内源酶的干扰。另外,此技术不需荧光显微镜,一般光学镜下染色格外清晰,两小时可得到准确结果。     

IgE Anti-IgG Antibodies in Patients with Juvenile and Adult Rheumatoid Arthritis Including Felty's Syndrome

Anti-IgG; antihodies (anti-IgG) of the IgE class were studied in sera from patients with juvenile rheumatoid arthritis (JRA). rheumatoid arthritis (RA) and patients with Felty's syndrome (FS) by use of an indirect immunofluorescence technique. Forty-two percent of 26 patients with JRA had IgE anti-IgG in serum all in low titers. Positive reactions prevailed in patients with multiple joint involvement. Sixty-three percent of 30 patients with RA and 80% of 20 patients with FS had IgE anti-IgG, the titers found in FS patients being significantly higher. In JRA and FS patients the IgE anti-IgG titers were correlated to the titers of anti-IgG of the IgG class, and for FS patients also with the IgM and IgA classes of anti-IgG. In six of 10 patients with RA the synovial fluid samples from both knees contained IgE anti-IgG. In four of these patients the titers of IgE anti-IgG were higher than in the corresponding serum sample, pointing to a local production. After G-200 Sephadex chromatography IgE anti-IgG were demonstrated in the void volume indicating the presence of these autoantibodies in immune complexes. IgE anti-IgG may be involved in the pathogenesis of JRA and RA by eliciting Type I and III reactions.     

Sperm,Nuclear, Phospholipid,and Red Blood Cell Antibodies and Isotype RF in Infertile Couples and Patients With Autoimmune Rheumatic Diseases

PROBLEM: To determine if measuring of nonorgan-specific autoantibodies is useful for better understanding and management of unexplained infertility. METHODS: Sera were obtained from 70 infertile couples, 57 rheumatic patients, and 76 fertile donors. Sperm antibodies (SA) were detected by the tests of Kibrick and Friberg, anti-histones, anti-cardiolipin antibodies, and RF isotypes by ELISA, antinuclear antibodies by indirect immunofluorescence, and anti-red blood cell antibodies by Capture-R. RESULTS: Multiple autoimmune reactivity (both partners positive and/or more than one type of autoantibody involved), higher than naturally occurring in fertile individuals, was found in 55% of the idiopathically infertile couples. IgA-RF was the dominant autoimmune marker. SA revealed similar rates in patients with rheumatic diseases and in infertiles with or without other autoantibodies. CONCLUSION: Although no single autoimmunity marker could predict occurrence of SA, the coincidence of enhanced polyclonal autoimmunity in both partners of infertile couples might potentiate their negative effect on reproduction.     

Anti-ENA Antibody in Serum Determined by ELISA-Technique

An enzyme linked immune sorbent assay, ELISA, for determination of serum antibodies against two different extractable nuclear antigens, the RNAse sensitive ribonucleoprotein (RNP), and the RNAse resistant Sm-antigen is described. The technical variables are systematically evaluated and a recommended procedure selected and described.     

Antibodies to hepatitis E virus among chinese patients with acute hepatitis in Taiwan

The prevalence of antibodies to hepatitis E virus (anti-HEV) was investigated in patients with acute hepatitis, and correlated with the clinical features. Sera from 110 patients with acute hepatitis and 60 healthy controls were tested for anti-HEV, antibody to hepatitis C virus (anti-HCV), and hepatitis B surface antigen (HBsAg). There were significant differences in the prevalence of anti-HEV, anti-HCV, and HBsAg between patients and controls (21.8% vs. 0%, 16.3% vs. 1.6% and 58.1% vs. 18.0%, respectively). Anti-HEV was detected in 6 (25.0%) of 24 patients with anti-HCV, 6 (9.3%) of 64 patients with HBsAg, and another 6 (22.2%) of 27 patients with acute hepatitis non-A, non-B, non-C. Anti-HEV was found in 15 men and three women, whose ages ranged from 34 to 75 (median, 57) years old. The median age of patients with anti-HEV was older than that in patients without this antibody (57 vs. 38 years; P = 0.001). The prevalence of anti-HEV in patients with anti-HCV alone (35.2%) was higher than that (11.1%) in patients with HBsAg alone (P = 0.03). Compared to patients without anti-HEV, HEV-infected patients had a higher frequency of travel to a foreign country (P = 0.0001), had a lower HBsAg rate (P = 0.019), and had higher serum alkaline phosphatase levels (P = 0.04) and gamma-glutamyl transpeptidase levels (P = 0.01). In conclusion, HEV infection occurs in 22.2% of patients with acute hepatitis non-A, non-B, non-C. HEV superinfection may occur in patients with chronic hepatitis B or C virus infection. © 1994 Wiley-Liss, Inc.     

Allergen-Specific IgE Antibodies against Antigenic Components in Cow Milk and Milk Substitutes

Crossed radioimmunoelectrophoresis (CRIE) was used to study the presence of serum IgE against antigenic components of cow milk in 21 selected milk-allergic patients. The amount of each IgE specificity was estimated by a scoring system. The milk-allergic children had mainly IgE against alpha-lactalbumin, beta-lactoglobulin, albumin and immunoglobulin, the four major proteins of bovine whey, as well as IgE against three casein components. A serum pool from 1000 normal adults had IgE against the same whey protein, but in smaller amounts, and no IgE against the casein components. Eight cow milk-based formulae, commonly used for infant feeding, and goat milk were studied by the same method. It was found that six of the milk substitutes did not differ significantly from cow milk in antibody binding, but the two hydrolysed casein products, Nutramigen and Pregestimil, consisted of such small molecules that the rabbit antisera could not precipitate the hydrolysed proteins in the gels on the CRIE plates. It was therefore not possible to study their IgE binding, if any, by this method.     

抗TNF和抗CALLA三体瘤的研制及其分泌抗体的初步纯化

抗CALLA一次杂交瘤79经8-杂氮鸟嘌呤诱导,成为HAT敏感型细胞系79A,79D;与TNF免疫的脾细胞融合,经筛选鉴定得到了几株分泌抗CALLA和抗TNF双特异性单克隆抗体的三体瘤。然后用羟基磷灰石柱对其中一株E6所分泌的抗体进行了初步纯化,与高压液相法相比,在普通常压色谱条件下取得了较为满意的效果。     

抗宋内氏志贺菌脂多糖单克隆抗体对小鼠免疫保护作用的初步研究

本文用宋内氏志贺菌加热致死后的菌体免疫ＢＡＬＢ／ｃ小鼠，以纯化脂多糖筛选，获得２株稳定分泌单克隆抗体的杂交瘤细胞株，并以小鼠腹腔感染作为实验动物模型，进行单克隆抗体被动保护实验。结果表明，在攻击前输入单克隆抗体，对同型菌感染有显著保护作用，保护率随抗体剂量增加而增高。提示识别脂多糖分子上某一抗原决定簇的抗体，能够对感染起到良好的保护作用。脂多糖是志贺菌毒力相关因子之一。     

Monoclonal antibodies against carcinoembryonic antigen (CEA) used in a solid-phase enzyme immunoassay. First clinical results

A solid-phase enzyme immunoassay using both mouse monoclonal and goat polyclonal antibodies against carcinoembryonic antigen (CEA) was developed. The assay detects 0.6 to 1.2 ng of CEA per ml of serum and has 3 incubation steps which can be performed in 1 day. Polystyrene balls coated with polyclonal goat anti-CEA antibodies are first incubated with heat-extracted serum samples. Bound CEA is then detected by addition of mouse monoclonal antibodies, followed by goat IgG anti-mouse IgG1 coupled to alkaline phosphatase. Results with this enzyme immunoassay using monoclonal antibodiies (M-EIA) have been compared with those obtained by the conventional inhibition radioimmunoassay (RIA) using goat antiserum. Three hundred and eighty serum samples from 167 patients with malignant or non-malignant diseases and from 134 normal individuals with or without heavy smoking habits were analyzed by the 2 assays. Excellent correlation between the results of the 2 assays was obtained, but the M-EIA, using monoclonal antibodies from a single hybridoma, did not discriminate better than the conventional RIA between CEA produced by different types of carcinoma and between CEA associated with malignant or non-malignant diseases. Follow-up studies of several patients by sequential CEA determinations with the 2 assays showed that the M-EIA was as accurate as the RIA for the detection of tumor recurrences.