Enzyme-linked immunosorbent assay for C1q in human serum by use of monoclonal antibodies

A sandwich ELISA system has been developed for the detection of C1q in human serum. It is specific, uses monoclonal antibodies, is sensitive into the nanogram range and is rapidly performed. Therefore, it may be a helpful tool for clinical routine diagnosis, e.g., detecting abnormal C1q levels in patients with rheumatic disorders. Various combinations of poly- and monoclonal antibodies were tested in a sandwich assay. One of these combinations, in particular, resulted in a highly reproducible standard curve: C1q bound to solid-phase polyclonal anti-C1q was detected by the monoclonal antibody 242 G3. In this assay, the C1q concentration in sera of normal individuals was found to be 160 micrograms/ml (mean value of 70 normal human sera). This ELISA detected nanogram levels of C1q and gave results comparable to those obtained by haemolytic C1q titration. One nanogram of C1q corresponded to ca. 2.6 X 10(10) effective C1q molecules. With this technique, selective C1q deficient sera as well as sera from patients with rheumatoid diseases were analysed.     

Detection of both hepatitis B e antigen and antibody in a single assay using monoclonal reagents

Monoclonal antibodies raised against HBeAg were used to develop a HBeAg and anti-HBe detection assay. Monoclones containing anti-HBe were used both for the coating of the solid phase and for the fluid phase label in a sandwich type assay. The percentage binding of 125I-labelled anti-HBe to serum HBeAg was much greater than that seen in a similar assay using only polyclonal reagents. Therefore it was possible to add a small quantity of HBeAg for neutralising any anti-HBe present in a test serum without affecting HBeAg detection. This small amount of serum HBeAg was incorporated into each test sample thus allowing the determination of the e status of a patient using only one aliquot of test serum. This single test assay could be performed either as a radioimmunoassay or as an ELISA. The sensitivity of these assays was found to be greater than the conventional polyclonal assay particularly with regard to sera containing anti-HBe.     

Enzyme-linked immunosorbent assay (ELISA) for detecting antisperm antibodies in mice with testicular autoimmunity

We developed an ELISA for measuring antisperm antibodies in the mouse by using serum samples obtained from mice immunized with murine testicular antigens in complete Freund's adjuvant (CFA) as well as from mice rendered vasectomized. Sperm antigens used were syngeneic epididymal spermatozoa and two types of soluble, murine testicular antigens prepared in our laboratory. This study deals with a) the sequential changes of antisperm antibody levels following immunization; b) determination of immunoglobulin classes of these antibodies; c) a correlation between the absorbance values and the endpoint titers of antisperm antibodies; and d) comparison of endpoint titers of antisperm antibodies detected by ELISA with those by immunoperoxidase staining method in immune and nonimmune sera. It is suggested that serum dilution as high as 1/800 or more is required for detecting antibody titers of immune sera, because nonimmune mouse sera reveal a definite, although low, level of absorbance value at a serum dilution of 1/400 or less.     

Characterization of monoclonal antibodies to Marburg virus nucleoprotein (NP) that can be used for NP-capture enzyme-linked immunosorbent assay

After the first documented outbreak of Marburg hemorrhagic fever identified in Europe in 1967, several sporadic cases and an outbreak of Marburg hemorrhagic fever have been reported in Africa. In order to establish a diagnostic system for Marburg hemorrhagic fever by the detection of Marburg virus nucleoprotein, monoclonal antibodies to the recombinant nucleoprotein were produced. Two clones of monoclonal antibodies, MAb2A7 and MAb2H6, were efficacious in the antigen-capture enzyme-linked immunosorbent assay (ELISA). At least 40 ng/ml of the recombinant nucleoprotein of Marburg virus was detected by the antigen-capture ELISA format. The epitope of the monoclonal antibody (MAb2A7) was located in the carboxy-terminus of nucleoprotein from amino acid position 634 to 647, while that of the MAb2H6 was located on the extreme region of the carboxy-terminus of the Marburg virus nucleoprotein (amino acid position 643-695). These monoclonal antibodies strongly interacted with the conformational epitopes on the carboxy-terminus of the nucleoprotein. Furthermore, these two monoclonal antibodies were reacted with the authentic Marburg virus antigens by indirect immunofluorescence assay. These data suggest that the Marburg virus nucleoprotein-capture ELISA system using the monoclonal antibodies is a promising technique for rapid diagnosis of Marburg hemorrhagic fever.     

超抗原葡萄球菌肠毒素与抗人大肠癌单链抗体ND-1 scFv融合基因的构建、表达及活性分析

目的:构建并表达超抗原葡萄(SEA)和鼠抗人大肠癌单链抗体ND-1scFv的融合蛋白,以提高SEA的靶向杀伤作用。方法:构建超抗原SEA和鼠抗人大肠癌单链抗ND-1scFv的融合基因ND-1 scFv/SEA的表达载体,转化到大肠杆菌E.coli M15中进行诱导表达。Ni-NTA亲和层析对表达产物进行分离、纯化。间接免疫荧光法检测融合蛋白的靶向结合活性,MTT法检测靶向杀伤效率。结果:成功构建了融合基因ND-1scFv/SEA,实现功能性表达,纯化的ND-1scFv/SEA融合蛋白与表达有ND-1相应抗原的大肠癌细胞CCL-187有高度亲和活性,通过激活外周血单核细胞,可特异性杀伤靶细胞,在4μg/mL浓度下对CCL-187的杀伤率达到91%,明显优于SEA的杀伤活性。结论:融合蛋白ND-1 scFv/SEA对大肠癌细胞CCL-187具有靶向结合和杀伤活性,为SEA用于靶向性的大肠癌治疗奠定了基础。     

Competitive direct ELISA based on a monoclonal antibody for detection of Ochratoxin A in dried fig samples

A monoclonal antibody (MAb) generated against Ochratoxin A (OTA) has been used in a competitive direct enzyme linked immunosorbent assay (cdELISA) for the detection of OTA in dried figs obtained from the Spanish retail market. Fifty per cent inhibition of the maximum binding was obtained with an OTA concentration of 2 ng/mL, and the detection limit for OTA in solution was 0.18 ng/mL, corresponding to 3.15 ng OTA per gram of sample. OTA was detected in 19 (54.3%) out of 35 samples of dried figs analysed, with concentrations that ranged from 3.15 to 277 ng/g. Five samples contained OTA concentrations above the tolerable level set by EC regulations for dried vine fruits (10 ng/g). The MAb-based cdELISA assay developed in this work could be effectively applied for OTA screening in dried figs.     

重组多肽酶联免疫吸附法检测抗LKM-1抗体的初步应用

目的：制备人自身抗原细胞色素P4502D6（CYP2D6）257-351位氨基酸片段融合蛋白作为自身抗原,探讨ELISA检测抗LKM-1抗体的敏感性和特异性。方法：以肝脏的cDNA混合文库为模板作PCR,将PCR产物与真核表达载体pEGH共同转化酿酒酵母Y258,碱裂解法进行质粒制备,PCR扩增鉴定。表达载体构建成功后,在半乳糖的诱导下表达产生重组融合蛋白,经GST亲和层析法进行纯化后,免疫印迹法鉴定抗原性,ELISA检测抗LKM-1抗体阳性血清及部分其他结缔组织病患者血清中的抗LKM-1抗体。结果：重组融合蛋白在宿主菌中获得表达,免疫印迹法鉴定表明其能与标准抗LKM-1抗体阳性血清反应,而与正常血清、其他抗血清无反应。在26份抗LKM-1抗体阳性血清中,6份抗HCV抗体阳性血清用重组多肽ELISA检测有5份呈阳性,其余20份血清用重组多肽ELISA检测均呈阳性；20份其他结缔组织病患者血清用重组多肽ELISA检测均为阴性。结论：重组的257-351位氨基酸片段是CYP2D6抗原的主要抗原表位区域,以重组多肽为基质ELISA检测抗LKM-1抗体的敏感性较高,为进一步研究抗体水平与临床病情变化的相关性奠定了基础。     

A monoclonal antibody, NCRC-11, raised to human breast carcinoma. 1. Production and immunohistological characterization

The production of a mouse monoclonal IgM antibody, NCRC-11, raised against human breast carcinoma is described. It has been characterized immunohistologically. The antigen recognised has a wide but highly specific distribution in normal tissues, being virtually confined to the surface of certain epithelial cell types. It is found in some forms of epithelial metaplasia and most epithelial malignancies, particularly adenocarcinomas. The heterogeneity of staining in mammary carcinomas is outlined and is of particular interest. The immunohistological staining distribution of NCRC-11 is similar to other antibodies, including anti-epithelial membrane antigen, which were raised against human milk fat globule membrane. A competition experiment with some of these antibodies, using a flow cytofluorimeter, showed competition with one antibody, LICR LON/M8.     

Use of the direct linear plot to estimate, in an immunoglobulin solution, the proportion which is specific monoclonal antibody against an unknown cell surface antigen

A Cornish-Bowden direct linear plot was used to assess the level of specific monoclonal antibody in a protein A preparation from mouse ascitic fluid. With this method, experimental observations are plotted directly as lines in parameter space and estimates of kinetic parameters are read directly from the plot without need for further calculation. This method is particularly well suited for the analysis of systems, such as the one outlined here, where conventional kinetic analysis is not possible because the preparation contains both specific and non-specific antibody. Results obtained with the direct linear plot showed that estimates of the level of specific immunoglobulin present in a given protein A preparation are consistent, not only within individual experiments, but also throughout a series of experiments using more than one labelled antibody preparation.     

Suppression of antibody responses to ricin A chain (RTA) by monoclonal anti-RTA antibodies

Balb/c mice treated with an immunotoxin constructed by conjugation of murine monoclonal antibody 791T/36 via a disulfide linker to ricin A chain generate a pronounced antibody response to peptide epitopes on ricin A chain. Monoclonal anti-RTA antibodies which recognize peptide epitopes have been developed and these have been used to down-regulate anti-RTA antibody responses in 791T/36-RTA immunotoxin-treated Balb/c mice. Of the five MAB tests, two (608/7 and 596/134) proved most effective, inhibiting anti-RTA antibody formation by up to 73%. MAB treatment was effective when initiated up to 3 days after immunotoxin treatment. Pharmacokinetic studies with 791T/36-RTA have shown that the immunotoxin is rapidly eliminated from the circulation, with no more than 4% remaining in blood after 24 hr. It is proposed that the down-regulation of anti-RTA antibodies is effected by MAB interfering with antigen processing.     

鼠抗人4-1BB单克隆抗体的研制及其生物学特性的鉴定

目的:鼠抗人4-1 BB分子功能性单克隆抗体(mAb)的研制及其生物学特性的鉴定.方法:以高表达人4-1 BB分子的转基因细胞293T/4-1 BB为免疫原,常规免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术进行细胞融合,并以293T/4-1BB为阳性抗体筛选细胞,293T/mock为阴性抗体筛选细胞,经免疫荧光标记和流式细胞术(FCM)分析、反复筛选和多次克隆化培养,筛选出特异分泌鼠抗人4-1BB分子mAb的杂交瘤细胞株;采用Western blot、间接免疫荧光法、竞争结合抑制试验、3H掺入和细胞凋亡分析等方法对mAb进行生物学特性的鉴定.结果:成功获得3株持续、稳定分泌鼠抗人4-1BB mAb的杂交瘤细胞株,命名为1G5、4B11和9F11.对mAb的生物学功能的研究结果表明,3株mAb均能识别活化T细胞和单核细胞来源的树突状细胞(Mo-DC)表达的4-1BB分子.mAb 4B11能有效地激发T细胞体外增殖、抑制其凋亡,并能促进Mo-DC体外扩增,上调CD80、CD83、CD86和CD1α的表达.结论:获得的3株分泌鼠抗人4-1BB mAb的杂交瘤,所分泌的抗体具有特异性地识别人4-1BB分子;其中4B1 1 mAb具有激发T细胞增殖及促进Mo-DC体外扩增与成熟的作用,为激发型4-1BB mAb.     

An anti-human μ chain monoclonal antibody: Use for detection of IgM antibodies to toxoplasma gondii by reverse immunosorbent assay

A precipitating anti-human μ chain monoclonal antibody (designated Tibi 82 McAb) was produced by the cell fusion technique. This McAb (isotype: IgG1κ) reacted by radioimmunoassay with all 10 human IgM proteins tested. In contrast, no reactivity was observed with IgG, IgA, IgE, λ and κ chains. 19 S IgM proteins were precipitated by Tibi 82 McAb using the Ouchterlony method under standard conditions. Hence specificity of this McAb for the Cμ2 domain was characterized by inhibition of precipitin reactions using human IgM fragments. Despite its narrow specificity for the Cμ2 domain, such a McAb coulb be used for IgM capture in the detection of specific IgM to Toxoplasma gandii employing the IgM immunosorbent agglutination assay (IgM-ISAGA). Tibi 82 McAb was compared with 3 anti-human IgM polyclonal reagents in the routine analysis of 117 sera. With 2 of them, a correlation coefficient of 0.976 was obtained and Tibi 82 McAb was more sensitive than the third polyclonal reagent tested. The IgM-ISAGA technique was shown to be reproducible using Tibi 82 McAb and similar anti-human μ chain McAbs could permit the wider development of reverse immunosorbent methods for the detection of specific IgM in various infectious diseases.     

Increased sensitivity in detecting tumor-associated antigens in sera of patients with colorectal carcinoma

A monoclonal antibody defining the Lewis blood group determinant was used to immobilize antigen in sera of patients with adenocarcinoma of the gastrointestinal tract, and a second radiolabeled antibody, which defines a gastrointestinal cancer-associated antigen (GICA), was used to detect the immobilized antigen. With this approach, elevated antigen levels were found in 34 of 49 (69%) of sera from patients with advanced colorectal carcinoma as compared with 9 of 292 (3%) of sera from patients with non-malignant gastrointestinal diseases and of healthy donors. For early primary colorectal carcinoma, the combination of anti-Lewis and anti-GICA monoclonal antibodies was more sensitive in detecting GICA than using anti-GICA antibody alone. Double determinant radioimmunoassay revealed the glycolipid determinant lacto-N-fucopentaose (LNF) III circulating in colorectal carcinoma patients' sera. 53% of patients older than 65 years had elevated levels of the LNF III determinant compared to none of age-matched, apparently healthy donors or patients with benign gastrointestinal tract lesions, and 18% of patients with inflammatory gastrointestinal tract diseases. In younger patients, the differences were less marked. Our results suggest the potential usefulness of Lewis and LNF III determinants as markers for the detection of gastrointestinal tract malignancies.     

A novel rabbit monoclonal antibody arginase-1 is highly specific and highly sensitive in hepatocellular carcinoma

Identification of a sensitive diagnostic marker to differentiate primary hepatocellular carcinoma (HCC) from metastatic cancers to the liver is of immense clinical significance in the management of HCC. Diagnostic markers such as alpha-fetoprotein, HepPar1, and glypican-3 are available, but can suffer from poor specificity or lack of sensitivity. A rabbit polyclonal arginase-1 (ARG-1) has been identified as a sensitive and specific marker for HCC. Recently, a new rabbit monoclonal ARG-1 has also been developed; however it has not been tested directly against the referenced rabbit polyclonal ARG-2. Hepatocellular carcinoma and various normal and neoplastic tissues were evaluated by immunohistochemistry for specificity and sensitivity using rabbit monoclonal ARG-1. Additionally, a comparison study with the rabbit polyclonal and rabbit monoclonal ARG-1 was evaluated in normal tissues and in HCC. The result showed both ARG-1 antibodies stained normal kidney, liver, and pancreas. All other normal tissues were negative. In HCC, the rabbit monoclonal ARG-1 stained 82% (160/195) and rabbit polyclonal ARG-1 stained 81% (157/195). The rabbit monoclonal ARG-1 was negative in all other cancers (n?=?704), except in 3/64 prostate adenocarcinoma and in 1/12 cholangiocarcinomas. In conclusion, the rabbit monoclonal ARG-1 was a highly sensitive and specific marker for HCC. Rabbit monoclonal ARG-1 represents a new potential marker for the differential diagnosis of primary HCC versus tumors of unknown origin; and, its monoclonal properties offer reproducibility and eliminate batch to batch variation found in polyclonal antibodies.     

A Novel Anti-Human Syndecan-1（CD138） Monoclonal Antibody 4B3： Characterization and Application

Syndecan-1 （CD138）, a member of integral membrane heparin sulfate proteoglycans, is an essential matrix receptor for maintaining the normal morphological phenotypes. In this study, we generated a specific mouse anti-human syndecan-1 monoclonal antibody （mAb） 4B3 and identified it by competition assay with the available syndecan-1 mAb （BB4）. Stained by 4B3, the expression of syndecan-1 was detected on tumor cell lines, such as 8226, U266, XG-1, XG-2, Daudi and Jurkat. The expression was also found on neuron stem cells. It was established that 4B3 mAb could inhibit XG-1 and XG-2 proliferation. The data not only determined that 4B3 mAb was a functional anti-human syndecan-1 mAb, but also indicated that syndecan-1 might be a valuable surface antigen and play an important role in regulation of tumor pathology and differentiation of neural stem cells. This novel antibody 4B3 may be value of study of tumor proliferation/survival mechanism and contributes to diagnosis and treatment of diverse diseases. Cellular ＆ Molecular Immunology.     

鞘内注射强啡肽A(1-17)对不同功能运动神经元群树突的影响

目的:探讨强啡肽A对大鼠趾长伸肌(EDL,快肌)、比目鱼肌(SOL,慢肌)两种不同功能运动神经元(Mn)群树突的影响。方法:采用脊髓蛛网膜下隙给予强啡肽A,以霍乱毒素B亚单位结合辣根过氧化酶(CB-HRP)逆行标记EDL-Mn、SOL-Mn群,Mesulam-TMB法显示两运动神经元群被标记的树突。结果:与相应对照组比,强啡肽A致一过性后肢瘫大鼠用药后1h,位于腰4~5脊髓节段腹角的EDL-Mn、SOL-Mn群树突的分布范围减小,尤以EDL-Mn群为甚;EDL-Mn平均树突长度比对照缩短59.5%,而SOL-Mn平均树突长度比对照缩短35.6%。永久性后肢瘫大鼠用药后3h,EDL-Mn群仅见胞体和近端树突;与之相比,SOL-Mn群仍保留有较长的树突和较广的分布范围。结论:强啡肽A致一过性后肢瘫大鼠两群运动神经元树突明显受累,且以EDL-Mn群为甚,其差别可能与两群运动神经元所接受强啡肽A信息传入不同有关。     

Comparative evaluation of the prognostic value of MUC1, MUC2, sialyl-Lewis(a) and sialyl-Lewis(x) antigens in colorectal adenocarcinoma

AIMS: The significance of MUC1, MUC2 and sialylated Lewis blood group antigens as prognostic markers in colorectal adenocarcinoma was investigated in a large series of patients because previous investigations revealed inconsistent results due to unrelated tumour samples from different patient groups and methodological differences. METHODS AND RESULTS: Tissues from 243 patients with colorectal adenocarcinoma were stained immunohistochemically. MUC1 showed a strong immunoreactivity (in more than 35% of the tumour area) in 32.5%, MUC2 in 51.0%, sialyl-Lewis(x) in 67.9% and sialyl-Lewis(a) in 73.7% of the cases, respectively. MUC1 immunoreactivity displayed a significant correlation with tumour progression as reflected by advancing pTNM staging and poor differentiation. MUC2 expression was significantly stronger in mucinous adenocarcinomas. Sialyl-Lewis(x) immunostaining correlated with the extent of lymph node metastasis as well as low cytological differentiation. According to univariate and multivariate analysis (P < 0.0001) only MUC1 reactivity represented a marker of worse survival probability, opposed to the sialylated Lewis antigens that did not exert a predictive value. CONCLUSIONS: According to our data, MUC1 and sialyl-Lewis(x) immunoreactivity exhibit statistically significant correlations with established markers of tumour progression. However, only MUC1 presents as an independent prognostic factor of colorectal adenocarcinoma.     

大肠癌患者血清可溶性P-选择素和细胞间黏附分子-1的表达及意义

目的 探讨大肠癌患者手术前后血清可溶性P-选择素（sP-selectin）、细胞间黏附分子-1（sICAM-1）的表达及临床意义。方法 采用ELISA检测30例大肠癌患者术前、术后15日、术后30日及20例对照组血清sP—selectin、sICAM-1的表达水平。结果 ①大肠癌患者血清sP—selectin、sICAM-1的表达水平均显著高于对照组，P〈0.05；术后15日下降，仍高于对照组水平，P〈005；术后30日降至对照组水平，P〉0．05；②大肠癌患者血清sP-selectin、sICAM-1的表达水平与淋巴结转移和临床分期相关，P〈0．05，与年龄、性别、肿瘤大小厦组织学分级无明显相关。P〉O．05。结论 sPselectin、slCAM-1参与了大肠癌侵袭转移过程，是反映大肠癌的发生、发展及预后的一个重要指标。