宫颈癌抗独特型单克隆抗体的制备和鉴定

目的：制备宫颈癌抗独特型单克隆抗体并鉴定其抗原模拟特性。 方法和结果： 以识别小鼠和人宫颈癌相关抗原分子共同表位的单克隆抗体AU14-1（Ab1）为免疫原在含免疫反应剂的无血清培养液中致敏小鼠脾细胞，并将其与SP2/0融合，经筛选和克隆化，建成一株能分泌抗独特型单克隆抗体（Ab2）的杂交瘤细胞系。Ab2的抗原模拟特性经ELISA、结合和竞争抑制试验以及免疫组化染色，表明为Ab2β，具有宫颈癌细胞膜表面抗原的“内影像”。 结论： 获得一株具有宫颈癌抗原内影像的抗独特型单克隆抗体。     

99m Tc 标记单克隆抗体Au14-1对荷宫颈癌小鼠的放射免疫显像研究

目的和方法：应用99mTc直接法标记抗小鼠宫颈癌（U14）单克隆抗体Au14-1,对荷瘤Km小鼠进行放射性分布和放射免疫显像的实验观察,探讨用99mTc－An14-1作宫颈癌导向诊断和导向治疗的可能性。结果：（1）99mTc－Au14-1注射后12h肿瘤灶已有放射性聚集,24h肿瘤组织的％ID／g值达8．79,呈明显特异性浓聚;（2）除肾脏外,各脏器的T／NT值在2．02~6．71之间,SPECT图像12h已见肿瘤显影,24h肿瘤影像更为清晰。结论：99TcTc－An14-1的体内分布与显像结果相一致,表明An14-1在体内具有良好的宫颈癌导向定位作用。     

New monoclonal antibody specific for Candida albicans germ tube

Hydrophobic components of the germ tube of the dimorphic pathogenic fungus Candida albicans were used as immunogens to prepare monoclonal antibodies (MAbs). Among the resulting MAbs, one (MAb 16B1-F10) was shown by indirect immunofluorescence to be specific to the surface of the mycelium phase of the C. albicans and C. stellatoidea species. No labeling of any other genera and Candida species tested was observed, including C. dubliniensis, a newly described species which has many phenotypic similarities to C. albicans. This phase-specific epitope resides on a protein moiety. The molecular mass of the antigen released by Zymolyase digestion was determined by gel filtration and ranges from 25 to 166 kDa. The antigen was also shown to be highly hydrophobic. This anti-C. albicans cell wall surface-specific MAb may be a good candidate for use in tests for the rapid differentiation of the two closely related species C. albicans and C. dubliniensis.     

宫颈癌单克隆抗体AU14-1介导效应细胞对靶细胞的细胞毒

目的：研究单克隆抗体AU14-1介导的细胞毒作用对小鼠宫颈癌（U14）细胞的杀伤效果。 方法： 以小鼠淋巴因子激活的杀伤细胞（LAK）、脾淋巴细胞及巨噬细胞为效应细胞，应用MTT比色法测试在单克隆抗体AU14-1介导下对小鼠宫颈癌（U14）细胞的体外细胞毒作用。 结果： 3种效应细胞对于经单克隆抗体AU14-1处理的肿瘤细胞的杀伤率显著高于未经AU14-1处理的肿瘤细胞（P＜0.01）；以LAK的ADCC作用最强，巨噬细胞次之，脾淋巴细胞最弱（P＜0.01）。 结论： 单克隆抗体AU14-1能通过抗体依赖细胞介导细胞毒（ADCC）发挥淋巴样细胞、巨噬细胞及LAK细胞对靶肿瘤细胞的杀伤作用；提示特异性宫颈癌单克隆抗体AU14-1介导的ADCC在治疗宫颈癌中可能是一种有潜在价值的方法。     

Mucin 16蛋白的表达纯化及单克隆抗体的制备与鉴定

目的:在原核生物中表达带有His标签的mucin 16N端重组蛋白(简称为His-mucin 16N),制备抗mucin 16的单克隆抗体(mAb)。方法:将mucin 16基因片段插入原核表达载体pET-32,在大肠杆菌中表达重组蛋白,用亲和纯化方法纯化后免疫BALB/c小鼠,并进行细胞融合。筛选可稳定分泌抗mucin 16抗体的阳性单克隆杂交瘤细胞株,用Westernblot、ELISA、免疫荧光和免疫组化等方法分析和鉴定抗mucin16的mAb。结果:表达并纯化了His-mucin 16N蛋白;筛选出几株可稳定分泌特异性抗人mucin 16 mAb的细胞株;挑选出效价高、特异性好的1株进行纯化。获得的抗mucin 16 mAb,可用于Western blot、ELISA、免疫组化、免疫荧光等检测,并鉴定该抗体亚型为IgG1。通过上述免疫学实验,分析了在不同肿瘤细胞中mucin 16的表达情况。结论:在原核生物中成功表达和纯化带His标签的mucin 16N重组蛋白,制备出具有高特异性的抗mucin16的mAb。     

Multinational comparison of diagnostic clues for uterine cervical lesions among cytotechnologists in Asian countries

Vaccination has been underway in several countries for sexually inactive young girls or women against HPV 16 and 18 to prevent them from infection of these HPV types and concurrent cancer development. However, uterine cervical cancers may remain uncontrolled among some Asian countries, where other types of HPV infection are more frequent. A sensitive cancer screening system would remain important for detection of the earlier stage cervical cancers in Asian countries. In this study, 130 cytotechnologists (CTs) in Asian countries (Taiwan 80, Japan 18, Korea 15, Thailand 11, Singapore 3, Bhutan 2, and Mongolia 1) participated in the vote. Selected 10 cervical Pap smears that would be adequate to identify the diagnostic clues especially for atypical squamous cells (ASC) with two or three representative pictures for each case were displayed on the website. The percentages of consistent diagnosis voted by certified CTs with ≥5 years of experience were compared among 10 cervical cases or among Asian countries enrolled. As results, low consistency for ASC cases and high consistency for squamous intraepithelial lesion (SIL) were observed. Examining specimens for the diagnostic clues of ASC in TBS is crucial to maintain the high sensitivity and positive predictive value of SIL in Asian countries. Diagn. Cytopathol. 2010. © 2010 Wiley‐Liss, Inc.     

Generation and characterization of a murine monoclonal antibody to cervical glandular epithelium using mice rendered tolerant to cervical squamous epithelium

Murine monoclonal antibodies that distinguish glandular from squamous epithelia in human tissue were generated using a procedure that involved tolerization prior to immunization. Tolerization was achieved by injection of newborn (24 hrs old) Balb/c mice with extract of normal cervical tissue containing squamous epithelium (the tolerogen). Three weeks later, mice showing no evidence of antibodies to tolerogen in their sera were immunized with an extract of cervical tissue containing both glandular and squamous epithelia. Following immunization, the sera from mice subjected to this treatment showed strong reactivity with glandular cells but not with squamous cells in sections of frozen tissue examined by an indirect immunohistological method. Spleen cells from mice showing this pattern of serum reactivity were used as fusion partners with a mouse myeloma cell line in order to generate monoclonal antibodies. Following extensive screening, one monoclonal antibody (designated anti-GEA.49) was selected for further study on the basis of reactivity with high affinity to glandular epithelium and a complete absence of staining of squamous and connective-tissue cells. Detailed tests of specificity and patterns of reactivity indicate that the antigen detected by the antibody is expressed on the apical plasma membrane of glandular epithelia and is a glycoprotein with an apparent molecular weight of 49 kilodaltons. Both immunohistological and biochemical methods demonstrated the expression of the antigen on glandular epithelia but not on squamous epithelia from several sources, underlining the usefulness of tolerization/immunization approach for generating antibodies with particular specificity requirements.     

Detection of a mucin marker for the adenoma-carcinoma sequence inhuman colonic mucosa by monoclonal antibody AM-3.

The monoclonal antibody AM-3 was raised against mucins extracted from human colorectal carcinomas. It reacted strongly with sections of paraffin wax embedded colorectal carcinoma. In colonic adenoma tissue the percentage of cells expressing the epitope detected by AM-3 correlated with the degree of dysplasia. In contrast to immunohistochemical staining, which did not show the presence of the antigen in histologically normal mucosa, the more sensitive enzyme linked immunosorbent assay (ELISA) and immunoblot assays showed that it was weakly expressed in this tissue. AM-3 reacted with variable frequency with several normal and malignant human tissues, indicating that the detected epitope is not restricted to colonic tissue. In colonic carcinomas it is present on a sialomucin of apparent relative molecular mass of more than 440,000. These data suggest that the antigen detectable with AM-3 may be useful in the assessment of premalignant changes in colonic adenomas.     

Immunoperoxidase studies by monoclonal antibody B72.3 applied to breast aspirates: diagnostic considerations

The purpose of this study was to determine the reactivity of monoclonal antibody (MAb) B72.3 when applied directly to aspiration biopsy cytology (ABC) of the breast in the following conditions: (1) infiltration lobular carcinoma; (2) fibrocystic disease; (3) fibroadenoma; and (4) apocrine cysts. Nine of ten aspirates from infiltrating lobular carcinoma were positive in these assays, while 21 of 22 benign cases reacted negatively. The single false-positive benign aspirate manifested a staining pattern characteristic of apocrine cells. This study demonstrates that the MAb B72.3 can be employed as a potentially valuable diagnostic adjunct. It can be used on stained aspirates to assist in the interpretation of ABC from breast lesions.     

A novel diagnostic monoclonal antibody specific for Helicobacter pylori CagA of East Asian type

Yasuda A, Uchida T, Nguyen LT, Kawazato H, Tanigawa M, Murakami K, Kishida T, Fujioka T, Moriyama M. A novel diagnostic monoclonal antibody specific for Helicobacter pylori CagA of East Asian type. APMIS 2009; 117: 893–9. Molecular biological and epidemiological studies have suggested that Helicobacter pylori producing East Asian CagA protein variant is more virulent than that producing Western CagA. In the present study, we developed and validated an enzyme‐linked immunosorbent assay (ELISA) using a monoclonal antibody specifically recognizing East Asian CagA‐positive H. pylori. A total of 32 H. pylori strains were tested and the data were subjected to receiver‐operator characteristic (ROC) curve analysis. The accuracy of the test, determined by calculating the area under the curve, was 0.96, which indicated a high level of accuracy. At the ROC optimized cutoff, the sensitivity and specificity of our ELISA method were 88.0% and 100%, respectively. The validated ELISA showed good performance in terms of sensitivity and specificity. These results suggest that this test is suitable for the diagnostic detection of East Asian CagA carrying strains. We also analyzed the localization of the CagA protein in H. pylori‐infected gastric mucosa with fluorescence immunohistochemistry, and found that CagA protein expression was up‐regulated by adhesion to epithelial cells.     

人粘蛋白糖链单抗的制备及在胃化生粘膜和胃肿瘤中的应用

制备抗胃肠道粘蛋白糖链单抗,并研究其抗原在小胃粘膜、化生粘膜及肿瘤中的表达,方法：将肠道粘膜冻干粉碎,ＣｓＣｌ平均密度离心法提取人胃肠粘蛋白,免疫ＢＡＬＢ／ｃ小鼠,取鼠脾细胞与ＳＰ２／Ｏ－Ａｇ１４鼠的骨髓瘤细胞融合成杂交瘤,克隆制备单克隆抗体,并应用ＬＳＡＢ法检测其抗原在胃粘膜,化生取粘膜和肿瘤中的表达。结果制备抗人胃粘蛋白单抗（ＨＧＭ７２,７５）和抗人肠粘蛋白单抗（ＨＣＭ１４,２１）均为抗粘蛋白     

Evaluation of a monoclonal antibody test to detect chlamydia in cervical and urethral specimens.

The MicroTrak Chlamydia trachomatis Direct Specimen Test (MT; Syva Co., Palo Alto, Calif.) was compared with cell culture in two patient populations. The sensitivity of the MT for a low-prevalence group was significantly lower (59.6%) than that for a high-prevalence group (84.4%). The results underscored the need to run the MT in parallel with culture initially if the prevalence of chlamydial infections is unknown and questioned the usefulness of the MT as a screening test for chlamydia in low-prevalence populations.     

A monoclonal antibody to cellular transglutaminase

A cellular enzyme-linked immunosorbent assay was developed for estimating cellular transglutaminase in situ using a monoclonal antibody produced to tissue transglutaminase. The minimum level of detection of TGase was 2-5 ng. The enzyme was present in greater amounts in WI-38 and IMR90 cells than in their simian virus-transformed counterparts. The levels of TGase in the virus-transformed cells increased significantly when the cells were grown in the presence of sodium butyrate to induce enzyme activity. Staining of confluent WI-38 cells by indirect immunofluorescence using the monoclonal antibody showed microscopic fibers suggesting that the enzyme may be associated with detergent-insoluble components.     

Pan-leukocyte monoclonal antibody L3B12. Characterization and application to research and diagnostic problems

In this report, the authors describe a murine anti-human monoclonal antibody, L3B12, which defines a pan-leukocyte cell surface antigen of approximately 180,000 m.w. Extensive screening against a variety of tissues indicates that L3B12 is sensitive and specific for leukocytes, related cells of bone marrow lineage, and their corresponding neoplasms. Unlike many lymphoid antigens that are not detectable following routine fixation and embedding, those recognized by L3B12 and related antibodies are variably preserved. L3B12 has proven useful in studying the antigen expression of normal leukocytic elements, lymphomas, and related disorders, and in enriching or depleting leukocytes from heterogeneous cell populations. From a diagnostic standpoint, L3B12 staining of tissue sections or cell suspensions is useful for distinguishing large cell lymphomas from undifferentiated carcinomas and in distinguishing lymphomas and leukemias from other small round cell tumors of childhood.     

FITC标记单克隆抗体检测icIL-1ra的方法研究

目的 建立用单克隆抗体技术检测细胞IL-1ra的方法。方法 制备异硫氰酸荧光素(FITC)标记的抗人IL-1ra单克隆抗体，使其进入U937细胞内与icIL-1ra结合，然后用流式细胞仪(FACS)检测荧光强度，并比较U937细胞在刺激前后荧光强度的变化情况是否与icIL-1ra表达的变化情况相一致。结果 在一定剂量范围内，经PMA分化和LPS刺激的U937细胞的icIL-1ra表达量增加，其检测荧光强度相应增加。结论 用单克隆抗体检测细胞内icIL-1ra是一种快速、准确的方法，本实验方法能够满足实际研究工作中对icIL-1ra定量检测的要求。     

A monoclonal antibody against hamster tracheal mucin, which recognizes N-acetyl-galactosamine containing carbohydrate chains as an epitope

Airway mucin that is present in airway secretion, plays an important role in host-defense by trapping airborne particles and removing them by mucociliary transport system. For the study of mucin, it is crucially important to have antibodies specific against mucin because other commonly used methods such as histologic stain for the detection of mucin usually suffer from varying levels of nonspecificity. In this study, we produced a monoclonal antibody (MAb) against hamster airway mucin, which is one of the most commonly used animal species for the study of mucin in vitro, and characterized its immunological properties along with the determination of the epitope it recognizes. The MAb, which was named MAb HTA, was IgM isotype and specific against mucin from both in vitro cell culture and in vivo airway secretion. In Western blot, MAb HTA specifically recognized high molecular weight airway mucin, which was also confirmed by the appearance of peak profile of immunological signal only on void volume fraction in Sepharose CL-4B gel filtration chromatography. It also immunoprecipitated high molecular weight hamster airway mucin with the aid of antimouse IgM agarose. In immunohistochemical stain of hamster trachea, it showed strong signal on airway epithelium and also on the mucin secreting goblet cell granules. The immunological signal was greatly increased by the treatment of endotoxin, which has been reported to cause airway secretory cell metaplasia. The MAb HTA recognized carbohydrate chains containing N-acetyl-galactosamine, one of the linking sugars of airway mucin, as an epitope. Treatment of mucin with N-acetyl-galactosaminidase caused great reduction of immunological signal. To the best of our knowledge, this is the first to report a MAb that recognizes N-acetylgalactosamine, a linking sugar of airway mucin. The specificity of MAb HTA against airway mucin and the clear demonstration of the epitope it recognizes should greatly aid the pharmacological and biochemical study of mucin in various physiological and pathological situations.     

Comparison of monoclonal antibody staining and culture in diagnosing cervical chlamydial infection.

We compared a fluorescein-conjugated monoclonal antibody (FA) direct specimen test (MicroTrak; Syva Co., Palo Alto, Calif.) with culture (TC) in McCoy cells (vials, with blind passage and iodine staining of inclusions) for diagnosis of Chlamydia trachomatis infection in the cervix. Duplicate specimens were collected from 1,230 women, but for 262 of these subjects, both results were unavailable (150 FA smears were inadequate, indicating a need for clinical training in specimen collection), leaving 968 comparisons. Prevalence of chlamydiae by culture was 13% (126/968). Compared with TC results, the sensitivity of FA was 70% (88/126) and the specificity was 94% (795/842). There was a 91% agreement (883/968). The predictive value of a positive FA test was 65% (88/135), and that of a negative FA was 95% (795/833). We reexamined 38 smears for which paired results were discrepant, and the reread would have changed the result in only 5 of these. TC is less than 100% sensitive and some FA-positive, TC-negative specimens represent positive specimens not detected by TC. Unfortunately, it is not possible to identify which results in this group are truly false-positive. Clearly, the FA procedure has a performance profile which would make it a useful tool in screening high-risk populations (particularly when TC is not available) but it is less suited to screening low-risk populations, for which false-positive results are more important. The greater utility of the FA procedure in a venereal disease clinic was confirmed by testing 172 evaluable specimen pairs, of which 34 (20%) were Chlamydia isolate positive. The FA sensitivity was 76% (26/34) and specificity was 96% (133/138), giving a predictive value of 84% (26/31) for a positive test.