Production and preliminary characterization of monoclonal antibodies to human liver-specific lipoprotein (LSP)

ABSTRACT— Monoclonal antibodies (mc/anti-LSP) have been prepared by polyethylene glycol fusion of P3/NSI/Ag4–1 myeloma cells with spleen cells from Balb/c mice hyperimmunized with human liver-specific membrane lipoprotein (LSP). Ten hybridomas, cloned by limiting dilution, produced mc/anti-LSP reacting (by ELISA) with human LSP but not with normal human plasma proteins nor with a variety of other proteins likely to co-purify with LSP. Three of these (A15/7, A9/63 and B20), producing high-titre IgG1 mc/anti-LSP, were biosynthetically radiolabelled and used as index antibodies. By competitive inhibition of binding of the index antibodies to LSP in an immunoradiometric assay, the ten hybridoma products were classified into four distinct groups according to their specificities for different epitopes in LSP. None of the index antibodies reacted, on ELISA, with glutaraldehyde-fixed PLC/PRF/5, Chang, Daudi or HSB-2 cell lines nor with human peripheral blood leucocytes. However, A15/27 (but not A9/63 or B20) reacted with saponin-permeabilized PLC/PRF/5 and Chang cells and also with rabbit LSP. The results emphasize the polyantigenic nature of LSP and indicate that at least one of the mc/anti-LSP (A15/27) recognises a species crossreactive antigen that is present in liver-derived cell lines.     

Anti—LSP—Antikörper bei Patienten mit verschiedenen Formen von Hepatitis—Nachweis mittels SPA—ELISA

Zusammenfassung Das Lipoprotein der Leberzellmembran, das LSP, wurde durch Chromographie nach der Methode, die von McFarlane beschrieben wurde, aus normalem Lebergewebe isoliert. Zur Charakterisierung des isolierten LSP-Pr?parates wurde Polyakrylamid-Gel-Elektrophorese durchgeführt. Enzym-gekoppeltes Staphylokokken-Protein A Immunosorbens Assay (SPA-ELISA) mit dem gereinigten LSP-Pr?parat als Antigen wurde zur Bestimmung von Anti-LSP-Antik?rper in Seren von Patienten verwendet. Insgesamt wurden 277 Serumproben untersucht. Die Resultate der Untersuchungen in der Hepatitis-Gruppe zeigten, da\ die Nachweisrate von Anti-LSP bei Patienten mit schwerer Hepatitis am h?chsten (8/9) und bei Patienten mit CAH die n?chsth?chste (75%) war. Die Nachweisrate von Anti-LSP in Seren von Patienten mit AH, CPH und von HBsAg-Tr?gern betrug 47,6%, 38,4% bzw. 30,4%. In den Seren von 46 Normalpersonen zeigte sich aber nur ein Fall Anti-LSP-positiv. In der Gruppe mit anderen Erkrankungen war Anti-LSP positiv in 8 von 13 F?llen mlt Leberzirrhose, 5 von 7 F?llen mit SLE und 2 von 7 F?llen mit chronischer Nephritis. Die Resultate der Untersuchungen zeigten, da\ SPA-ELISA eine empfindliche Methode ist, die in der Klinik leicht durchgeführt werden kann. Es bestand eine Korrelation zwischen den Nachweisraten bzw. den Titern von Anti-LSP und Lebersch?digung bei Hepatitis. Die Bestimmung von Anti-LSP dürfte deshalb eine brauchbare Methode zur Beurteilung der Lebersch?digung bei Patienten mit Hepatitis sein. Die immunopathologische Bedeutung von LSP und Anti-LSP-Antik?rper bei der chronischen Hepatitis wird diskutiert.

     

肝特异性膜脂蛋白抗原性多肽成份及其意义的研究

本文采用免疫转印技术分析了肝病患者血清与肝特异性膜脂蛋白(LSP)抗原性多肽成份的反应。结果表明,人LSP中的225kd、210kd、185kd、180kd,165kd和155kd左右的多肽,可能是肝病患者对LSP自身免疫反应所针对的主要抗原成份。并对这些多肽与兔LSP和人肾脂蛋白之间的关系、不同类型肝病患者血清对LSP多肽成份识别的差异等进行了观察和讨论。     

Methods for reducing non-specific antibody binding in enzyme-linked immunosorbent assays

Enzyme-linked immunosorbent assays were used to study (i) binding of rabbit antibodies (raised against litter mate liver plasma membrane fraction) to the immunizing membrane fraction, and (ii) binding of human antibodies to liver membrane fractions and to liver-specific lipoprotein (a liver membrane-derived antigen complex). When assays were conducted using the non-ionic detergent Tween 20 as blocking agent, high non-specific binding was encountered. With the low titre rabbit antisera high binding of non-immune test antibody and of second antibody (anti-rabbit IgG) to the immunogen, and also directly to the solid phase, was found. This was abolished by replacement of Tween 20 in the antibody diluent buffers by a non-reactive protein, casein proving to be a more effective blocking agent than either bovine serum albumin or gelatin. With human sera, high binding of human IgG to the solid phase was noted. This too was blocked by casein, but only when the anti-microbial agent Thimerosal was included in the casein buffer, and when Tween 20 in the wash buffer was replaced by casein-Thimerosal so that the solid phase was exposed to casein before incubation with the test serum. The casein buffers described may prove of general value in solid-phase assays where high non-specific binding is encountered.     

Leukocyte-specific protein 1 (LSP1)

LSP1 is an F-actin bundling cytoskeletal protein expressed in hematopoietic lineage and endothelial cells. We investigated the function of this protein by generating and analyzing an LSP1-deficient mouse strain and in this review we describe our findings together with those of other investigators. The results show a complex function of LSP1 in regulating leukocyte recruitment to inflamed sites. Based on current evidence, we propose that the levels of LSP1 on the cytoskeleton and the type of integrin involved are some of the critical elements which affect LSP1 function in modulating the threshold for transmigration.     

肺结核合并肺部感染患者病原菌分布及内毒素、钙结合蛋白检测的临床意义

目的 分析肺结核合并肺部感染患者病原菌分布及内毒素(LSP)、钙结合蛋白(S100A9)检测的临床意义.方法 选取2017年1月至2020年6月我院收治肺结核患者216例,根据有无合并肺部感染分为感染组与未感染组.分析肺结核患者肺部感染及病原菌分布情况,对比感染组与未感染组LSP、S100A9蛋白、降钙素原、C反应蛋白...     

miR-196a2成熟区单核苷酸多态性对靶基因LSP1表达的影响

目的:研究miR-196a2成熟区单核苷酸多态性rs11614913(T/C)对预测靶基因LSP1表达的影响.方法:构建含有不同基因型miR-196a2的真核表达载体pCDNA3.1-miR196a2-C和pCDNA3.1-miR196a2-T;同时利用化学合成含有不同基因型的成熟miR-196a2探针miR-196a2-C和miR-196a2-U.将靶基因LSP1的3'UTR序列克隆至pMIR-REPORTTM Luciferase载体中而获得LSP1报告基因载体pMIR-LSP1.采用所构建的miR-196a2真核表达载体以及化学合成的成熟miR-196a2探针分别与靶基因LSP1报告基因载体组建两套转染体系.分别共转染于HEK293、A549和CHO 三种细胞后进行荧光素酶活性分析.结果:miR-196a2真核表达载体与靶基因LSP1报告基因表达载体共转染于HEK293、A549和CHO三种细胞后进行荧光素酶活性分析.pCDNA3.1-miR196a2-C等位基因荧光素酶相对活性与pCDNA3.1-miR196a2-T等位基因相比均有显著降低(P<0.05);含不同基因型的化学合成的成熟miR-196a2探针与靶基因LSP1报告质粒共转染HEK293、A549和CHO三种细胞,miR-196a2-C等佗基因荧光素酶活性与miR196a2-T等位基因相比也有显著降低(P<0.05).结论:荧光索酶活性强弱间接反映了miR-196a2与靶基因LSP1结合能力的大小.当miR-196a2成熟区rs11614913位点为C时,miR-196a2可能更为有效地与预测靶基因LSP1结合,从而在转录后水平调控靶基因表达.     

MAP3K1及LSP1基因单核苷酸多态与中国北方汉族绝经前妇女乳腺癌风险相关性

目的:探讨MAP3K1及LSP1基因单核苷酸多态与中国北方汉族绝经前妇女乳腺癌风险的关系。方法:采用多重单碱基延伸单核苷酸多态性分型技术(Snapshot)分析方法,检测280例绝经前乳腺癌患者和287例绝经前正常对照者MAP3K1基因rs889312和LSP1基因rs3817198多态性位点基因型,并比较不同基因型与乳腺癌风险的关系。结果:MAP3K1基因rs889312和LSP1基因rs3817198多态性位点基因型频率在乳腺癌和对照样本之间未存在显著差异(P=0.937、P=0.323)。Logistic回归分析结果显示,对于MAP3K1的rs889312位点,与AA携带者相比,AC携带者、CC携带者和AC+CC基因型携带者与乳腺癌的患病危险无关(OR=0.814,95%CI=0.537-1.236,P=0.335;OR=0.999,95%CI=0.627-1.594,P=0.998;OR=0.876,95%CI=0.591-1.298,P=0.509);对于LSP1的rs3817198位点,与TT携带者相比,CT携带者、CC携带者和CT+CC基因型携带者与乳腺癌的患病危险无关(OR=0.832,95%CI=0.565-1.223,P=0.349;OR=0.651,95%CI=0.108-3.936,P=0.640;OR=0.839,95%CI=0.573-1.229,P=0.369)。结论:上述两个基因MAP3K1和LSP1位点多态性与中国北方汉族绝经前妇女乳腺癌易感性之间无明显相关性。     

放射免疫沉淀法与酶联免疫吸附试验检测抗-LSP的双盲对照研究

研究表明,抗肝细胞膜特异性脂蛋白抗体(抗-LSP)在病毒性肝炎,特别是慢性活动性肝炎的发病机理中可能起较重要的作用。关于抗-LSP的检测,国内外文献中多