用斑点酶联免疫吸附试验筛选抗恶性疟原虫单克隆抗体及检测恶性疟抗原

以往筛选抗恶性疟原虫(Pf)单克隆抗体(McAb)常用间接免疫荧光(IFA)或ELISA,以培养的原虫为检测抗原。所得McAb检测培养的Pf抗原效果较好,但检测病人血中Pf抗原时阳性率较低。抗原分析显示这些McAbs多是针对滋养体、裂殖体或裂殖子抗原。为了得到能用于诊断的抗环状体期McAb,我们直接以病人血中Pf为检测抗原,用斑点酶联免疫吸附试验(DotELISA)进行筛选,结果如下。     

单克隆抗体双夹心纸模金银染色法检测疟疾循环抗原的初步研究

本文首次报道用McAb双夹心纸模金银染色法检测疟疾病人血清中的疟原虫循环抗原获得成功。将McAb吸附于硝酸纤维膜上,与待检血清反应后加入胶体金标记的第二McAb,再置银显影液中显影,阳性反应呈黑色斑点。用本法检测间日疟疾和恶性疟疾病人血清各15份,阳性率分别为86.6％和     

抗恶性疟原虫单克隆抗体的分析

在两次细胞融合中,经2—4次克隆化后已建成28株抗恶性疟原虫(FCC—1/HN)的杂交瘤。其中针对裂殖子和分裂体抗原的McAb有9株,仅对裂殖体特异的有4株,对裂殖体和滋养体期原虫产生荧光反应的有15株。 应用间接荧光抗体技术,检查这28株McAb对恶性疟原虫(安徽株),间日疟原虫、食蟹猴疟原虫,诺氏疟原虫、伯氏疟原虫、约氏疟原虫和鸡疟原虫的交叉反应。结果,仅有93D4和94B5属株特异性McAb,其余各株均对恶性疟原虫(安徽株)有明显荧光反应。有5     

用生物素-抗生物素酶联免疫吸附试验作单纯疱疹病毒分型并与限制性核酸内切酶分析和应用单克隆抗体免疫荧光法的比较

应用捕捉法生物素-抗生物素酶联免疫收附试验(Capture B/SA ELISA)测定单纯疱疾病毒(HSV)的型别是在用单克隆抗体(McAb)分型的基础上发展起来的。兔抗HSVIgG用作捕捉抗体,生物素连接的特异性鼠抗HSV-1单克隆抗体或兔抗HSV-1、HSV-2型特异性多克隆抗体作为检测抗体。被捕捉的抗原用抗生物素连接的碱性磷酸酶以ELISA法检测,该抗生物素可与检测抗体中生物素分子反应。将捕捉法B/SA ELISA分型的效率     

抗杜氏利什曼原虫犬分离株单克隆抗体的研制及用于病犬血清循环抗原的检测

本文报道用杜氏利什曼原虫大分离株前鞭毛体膜抗原免疫BALB/c小鼠的脾细胞与Sp2/0瘤细胞融合,得到多株分泌抗体较高且稳定的细胞株。进一步筛选出McAb C11-G10-A4可用于检测病犬血清循环抗原。用McAb-AST检测采自甘肃省黑热病流行区的30份犬血清,阳性率30％。与骨髓涂片查病原体的总符合率为86.7％,阳性符合率达100％。69份非流行区对照大血清检测结果,仅1份出现阳性反应,可见本试验的敏感性较高,特异性较强(98.55％)。     

肝癌相关抗原HAg18的研究及其应用

用抗人肝癌单克隆抗体HAb18亲和层析纯化相应抗原,经SDS-PAGE及免疫印迹试验证实其分子量为61kD。抗原活性条带经高碘酸钠氧化及氯仿-甲醇-冰乙酸处理抗原无丢失,但经胰蛋白酶酶解后失活。PAS及苏丹Ⅲ染色均为阴性。用HAb18ELISA双抗体夹心法可检测肝癌病人血清中抗原水平,阳性率为65.1％(250/384)。HAb18配伍另一肝癌单克隆抗体,制成ELA快速诊断盒,肝癌检出率提高至70.6％(271/384)。免疫酶斑点测定HAg18与AFP、铁蛋白、C-EA无交叉。故认为HAb18是一新的肝癌蛋白性抗原。     

肾综合征出血热(HFRS)病毒McAb在临床早期诊断中应用的研究

本研究用间接荧光抗体试验检测77例临床诊断为HFRS病人外周血白细胞中病毒抗原。阳性率为50.7％,在早期病例中(第2—5病日)阳性率为64.3％(27/42),并发现病日越早、病情越重,阳性率越高。在间接免疫荧光中应用了抗HFRS病毒MCAb 4B_9、4E_7、3H_4株。抗原阳性者与3种McAb表现为5种反应类型,不同McAb对抗原的检出率也不相同。将McAb组合可提高阳性检出率。在抗原阳性早期患者中,急性期血清特异性IgM抗体阳性率为96.3％。将HFRS抗原检测和急性期IgM测定联合应用,可提高HFRS早期诊断的阳性率和准确性。     

免疫酶斑点法检测汉坦病毒抗原的研究

目的 建立一种新的诊断肾综合征出血热(HFRS)的实验手段。方法 采用R22株、陈株和湖北株3种汉坦病毒接种家兔，制备兔抗汉坦病毒多克隆抗体(抗HTV-IgG)。然后采用混合的3种抗体进行免疫酶斑点法实验(IEDA)，检测患者血清和尿液中的汉坦病毒抗原。同时采用间接免疫荧光法(IFA)检测患者血清中抗汉坦病毒-IgM作对照。结果 经用相关分析，IEDA与IFA检测的结果高度相关。血中汉坦病毒抗原检出率为73．68％，尿中为65．00％。发病5d内，血中检出率为94．34％，尿中检出率为83．33％。5病日内的早期诊断率前者明显高于后者。结论 IEDA与IFA相比，阳性率比后者有较大提高，特别是5病日内的早期阳性率的提高明显，值得用于临床HFRS的早期诊断。     

用生物素—抗生物素酶联免疫吸附试验作单纯疱疹病毒分型并与限制性核酸内切酶分析和应用单克隆抗体免疫荧光法的比较

应用捕捉法生物素-抗生物素酶联免疫吸附试验(Capture B/SA ELISA)测定单纯疱疹病毒(HSV)的型别是在用单克隆抗体(McAb)分型的基础上发展起来的。兔抗HSV IgG用作捕捉抗体,生物素连接的特异性鼠抗HSV-1单克隆抗体或兔抗HSV-1、HSV-2型特异性多克隆抗体作为     

恶性疟原虫保护性抗原复合基因-痘苗病毒重组活疫苗株在换人血猕猴模的抗攻击试验

目的 探讨恶性疟原虫保护性抗原复合基因－痘苗病毒重组活疫苗修选株在实验动物的抗疟原虫攻击能力,为下一步进入夜猴及人体试验奠定基础。方法 利用多次部分换人血猕猴模经静脉输血法进行了抗疟原虫红内期虫体的攻击试验。结果 在免疫后１个月攻虫,重组痘苗病毒免疫猴从第３天一直到第１２ｄ的采血检查中均未发现疟原虫;非重组痘苗病毒（对照病毒）免疫猴等３天后原虫感染率上升,第６天原虫感染率最高达到６．０％,而后原虫     

Cytochemical profile of megakaryoblastic leukaemia: a study with cytochemical methods, monoclonal antibodies, and ultrastructural cytochemistry.

A cytochemical study using: Sudan black B; alpha-naphthyl acetate (ANAE) staining; estimation of alpha-naphthyl butyrate (ANBE) esterase activity; acid phosphatase activity; and 5' nucleotidase activity was carried out in 15 cases of megakaryoblastic leukaemia. These included cases of M7 acute myeloid leukaemia and blast crises of chronic granulocytic leukaemia. The megakaryoblastic nature of the blasts was first established using two monoclonal antibodies against platelet glycoproteins, and by estimating the platelet/peroxidase reaction at ultrastructural level. Our findings suggest that megakaryoblasts have a typical cytochemical profile comprising positive ANAE staining and acid phosphatase activity with a predominant localisation in the Golgi zone and negative or weak ANBE activity. A similar positive cytochemical pattern was also found in five cases of erythroleukaemia (M6). The specificity of the 5'nucleotidase activity for megakaryoblasts was not confirmed. In most cases of megakaryoblastic leukaemia there was no 5'nucleotidase activity only two cases showed positive reactions--reactions were positive in several cases of myeloblastic and lymphoblastic leukaemia. We suggest that cytochemical methods may be useful in diagnosing M6 and M7 acute leukaemia because less than 40% of leukaemic cells react with specific monoclonal antibodies.     

Comparison of monoclonal and polyclonal antibodies directed against immunoglobulin light and heavy chains in non-Hodgkin's lymphoma

A study comparing the usage of monoclonal and polyclonal antibodies specific for immunoglobulin light and heavy chains was performed on frozen-tissue sections of 30 B-cell non-Hodgkin's lymphomas. In 16 cases, monotypic staining for an immunoglobulin light chain was demonstrated with monoclonal antibodies using a three-step avidin-biotin peroxidase complex (ABC) method; 13 cases were positive for kappa. In 14 cases, no immunoglobulin light-chain production was demonstrated. Repeat staining of 11 of these 14 cases with polyclonal anti-sera by a direct immunoperoxidase method demonstrated monotypic staining for light chain in 10 cases, 9 of which were positive for lambda. In 22 of 30 non-Hodgkin's lymphomas, an immunoglobulin heavy chain was identified using monoclonal anti-sera. In eight cases, however, no heavy chain was found. Repeated staining with polyclonal sera of additional sections in three of eight cases demonstrated heavy-chain production in each case. Decreased sensitivity, especially for the detection of the lambda light chain, rendered this particular lot of monoclonal antibodies unsuitable for immunophenotyping non-Hodgkin's lymphomas. Variability of antigenic sites on the immunoglobulin molecule seems a likely explanation for these observations.     

Comparison of monoclonal time-resolved fluoroimmunoassay with monoclonal capture-biotinylated detector enzyme immunoassay for adenovirus antigen detection.

A sensitive time-resolved fluoroimmunoassay (TR-FIA), adapted from TR-FIA procedures already described, was developed with monoclonal antibodies and compared with several enzyme immunoassays (EIAs) for detecting adenovirus antigens in clinical specimens. The most sensitive EIA was an all-monoclonal assay with biotin-labeled detector antibody and streptavidin-peroxidase conjugate. All tests were evaluated with nasopharyngeal aspirate specimens from respiratory illness, with tissue homogenates from patients with systemic infection, and with stool specimens from gastrointestinal illnesses. For respiratory and tissue specimens, the TR-FIA detected adenovirus in 85% of the specimens positive by culture, which was a sensitivity similar to those of the all-monoclonal biotin-avidin EIA (79%) and the polyclonal-capture biotin-avidin EIA (88%). For stool specimens, the TR-FIA detected adenovirus in 100% of the specimens positive by culture, which was a decidedly higher sensitivity than either EIA format (78 and 75%, respectively). The TR-FIA was shown to be an efficient, flexible, and specific test for large numbers of clinical specimens.     

Immunohistochemical determination of HER-2/neu expression in invasive breast carcinoma

Numerous methods exist for HER-2/neu assessment; however, technical and interpretive standardization is virtually absent. We evaluated 2 commercially available antibodies on routinely fixed paraffin-embedded tissue sections to establish our own guidelines. Thirty-three cases of infiltrating breast carcinoma were evaluated simultaneously with monoclonal and polyclonal antibodies. Only membranous staining, no matter how focal, was considered positive. An additional 32 tumors were studied subsequently using only the polyclonal antibody. Of all carcinomas, 13.0% showed immunohistochemical evidence of HER-2/neu overexpression. High-grade tumors were more often positive. There was no HER-2/neu gene expression in the benign epithelium that generally was present in the tissue section or in any of the well-differentiated tumors tested. The polyclonal antibody proved more sensitive than the monoclonal antibody. While true cytoplasmic staining was present occasionally, it did not create substantial difficulty in interpretation. The polyclonal antibody cost substantially less than the monoclonal antibody. Fluorescence in situ hybridization assay for HER-2/neu gene amplification performed on 32 of 65 cases showed concordant results in 31 cases. The immunohistochemical assay for HER-2/neu gene overexpression, using our methods, is accurate, economic, and easily integrated into the laboratory.     

Immunohistochemical and immunoblot analyses of ras-p21 expression in lung carcinomas

Immunohistochemical and immunoblot analyses were performed on 49 cases of surgically resected primary lung carcinoma to examine the expression of ras oncogene product using monoclonal antibodies to ras-p21. Two different monoclonal antibodies, NCC-RAS-001 and RAP-5 were used for immunohistochemical study. Cancer cells of 16 cases (33%) and 15 cases (31%) were strongly positive for NCC-RAS-001 and RAP-5, respectively. The staining pattern of antibodies was heterogenous among cancer cells, even in the same case. Among various histologic type of lung cancers, squamous cell carcinomas and well-differentiated adenocarcinomas had a tendency to react more intensively than other histologic types of carcinoma. Immunoblot analysis using monoclonal antibody NCC-RAS-004 revealed the presence of ras-p21 not only in cancer tissues but also in non-cancerous tissues in all cases analysed. In 13 cases (27%), cancer tissue expressed more than twice as much ras-p21 as non-cancerous tissues. Our study showed that the over-expression of ras-p21 in lung carcinomas compared with non-cancerous tissues was a relatively common phenomenon, especially in squamous cell carcinomas and adenocarcinomas.     

Immunohistochemical and Immunoblot Analyses of ras-p21 Expression in Lung Carcinomas

Immunohistochemical and immunoblot analyses were performed on 49 cases of surgically resected primary lung carcinoma to examine the expression of ras oncogene product using monoclonal antibodies to ras -p21. Two different monoclonal antibodies, NCC RAS 001 and RAP 5 were used for immunohistochemical study. Cancer cells of 16 cases (33%) and 15 cases (31%) were strongly positive for NCC RAS 001 and RAP 5, respectively. The staining pattern of antibodies was heterogenous among cancer cells, even in the same case. Among various histologic type of lung cancers, squamous cell carcinomas and well-differentiated adenocarcinomas had a tendency to react more intensively than other histologic types of carcinoma. Immunoblot analysis using monoclonal antibody NCC RAS 004 revealed the presence of ras p21 not only in cancer tissues bus also in non-cancerous tissues in all cases analysed. In 13 cases (27%), cancer tissue expressed more than twise as much ras p21 as non cancerous tissues. Our study showed that the over expression of ras -p21 in lung carcinomas compared with non cancerous tissues was a relatively common phenomenon, especially in squamous cell carcinomas and adenocarcinomas. Acta Pathol Jpn 39: 643 647, 1989.     

Carcinoembryonic Antigen and Nonspecific Cross-reacting Antigen in Medullary Carcinoma of the Thyroid

Carcinoembryonic antigen (CEA) and nonspecific cross-reacting antigen (NCA) were studied immunohisto-chemically in formalin fixed, paraffin embedded tissues of 73 cases of medullary carcinoma of the thyroid (MTC) using 2 polyclonal antibodies (CEA antisera cross-reactive with or without NCA), 3 monoclonal antibodies recognizing epitopes only on CEA, and one monoclonal antibody against NCA. The staining patterns of the 5 antibodies against CEA in MTCs were not different, and they reacted with 86.3% of all cases. With regard to the effects of fixatives on the staining patterns, samples fixed with formalin or 4% paraformaldehyde demonstrated CEA immunoreactivity in both the cell membrane and cytoplasm. In Bouin fixed tissue, the immunoreactivity was predominant on the cell membrane, whereas cytoplasmic positivity predominated in alcohol fixed specimens. Thus the difference in fixatives used in previous studies does not appear to be a major reason for the difference in the reported incidence of CEA positive MTCs. It is concluded that CEA is still a useful tumor marker for MTC and that it is detectable only in thyroid tumors originating from C cells, as seen in our series. The epitope defined by monoclonal antibody F106 88, present only on NCA, was found in 42.5% of all cases (49.2% of CEA positive MTCs). The NCA immunoreactivity was located in the tumor cell cytoplasm as globular aggregates, which were also labeled for CEA.     

Expression of Vimentin and Epithelial Membrane Antigen in Human Malignant Lymphomas

Immunoreactivity with monoclonal antibodies against the intermediate filament protein, vimentin, and epithelial membrane antigen (EMA) was examined in 330 cases of lymphoma (317 non-Hodgkin's and 13 Hodgkin's lymphomas), 12 reactive lymph nodes and mononuclear cells of the peripheral blood using either indirect immunoperox-idase staining or the avidin-biotin immunoperoxidase complex technique. The cell origin of each tumor was established using a panel of monoclonal antibodies against lymphocyte differentiation antigens. There were 41 T cell, 247 B cell and 29 undetermined lymphomas, and 13 cases of Hodgkin's disease in the series. Vimentin was expressed in 24 T-cell lymphomas (58.5%) and 60 B cell lymphomas (24.2%). This difference in frequency was statistically significant. Vimentin expression in follicular lymphomas was less frequent than in diffuse B-cell lymphomas. In diffuse lymphomas, small and medium cell types were more reactive with anti-vimentin than large cell types. Reed-Sternberg cells (R-S cells) in Hodgkin's disease were positive for vimentin in 11 cases (84.6%). The frequency of EMA reactivity in lymphomas was low, particularly in T cell lymphomas. No positive cases were found among follicular lymphomas. In diffuse non Hodgkin's lymphomas, EMA was expressed only in mixed and large cell types, but never in smaller ones. In conclusion, monoclonal antibodies against vimentin and EMA appear to be of limited usefulness for the diagnosis of non Hodgkin's lymphomas, but anti vimentin antibody may be used as an adjunct to the diagnosis of R-S cells in Hodgkin's disease.     

Expression of vimentin and epithelial membrane antigen in human malignant lymphomas

Immunoreactivity with monoclonal antibodies against the intermediate filament protein, vimentin, and epithelial membrane antigen (EMA) was examined in 330 cases of lymphoma (317 non-Hodgkin's and 13 Hodgkin's lymphomas), 12 reactive lymph nodes and mononuclear cells of the peripheral blood using either indirect immunoperoxidase staining or the avidin-biotin immunoperoxidase complex technique. The cell origin of each tumor was established using a panel of monoclonal antibodies against lymphocyte differentiation antigens. There were 41 T-cell, 247 B-cell and 29 undetermined lymphomas, and 13 cases of Hodgkin's disease in the series. Vimentin was expressed in 24 T-cell lymphomas (58.5%) and 60 B-cell lymphomas (24.2%). This difference in frequency was statistically significant. Vimentin expression in follicular lymphomas was less frequent than in diffuse B-cell lymphomas. In diffuse lymphomas, small and medium cell types were more reactive with anti-vimentin than large cell types. Reed-Sternberg cells (R-S cells) in Hodgkin's disease were positive for vimentin in 11 cases (84.6%). The frequency of EMA reactivity in lymphomas was low, particularly in T-cell lymphomas. No positive cases were found among follicular lymphomas. In diffuse non-Hodgkin's lymphomas, EMA was expressed only in mixed and large cell types, but never in smaller ones. In conclusion, monoclonal antibodies against vimentin and EMA appear to be of limited usefulness for the diagnosis of non-Hodgkin's lymphomas, but anti-vimentin antibody may be used as an adjunct to the diagnosis of R-S cells in Hodgkin's disease.     

Detection of Chlamydia trachomatis inclusions in Mccoy cell cultures with fluorescein-conjugated monoclonal antibodies.

We compared two methods for identification of Chlamydia trachomatis inclusions in McCoy cell monolayers: conventional iodine staining and immunofluorescence staining with monoclonal antibodies against the species-specific major outer membrane protein antigen of C. trachomatis. Among 878 urethral and cervical specimens tested in parallel, the immunofluorescence method detected eightfold more inclusions per monolayer, identified a higher proportion of positive specimens on first passage (98 versus 62% by iodine staining; P less than 0.01), and improved overall sensitivity (98% of total positive specimens detected versus 84% by iodine staining; P less than 0.01). Improved sensitivity was most evident in specimens with low numbers of inclusions. Compared with conventional iodine staining, immunofluorescence staining with monoclonal antibodies improves sensitivity and offers more rapid detection of chlamydial inclusions in cell culture.