抗人红细胞NADH—细胞色素b5还原酶单克隆抗体的建立

以重组人红细胞ＮＡＤＨ－细胞色素ｂ５还原酶为抗原免疫ＢＡＬＢ／ｃ小鼠；通过常规方法将小鼠脾细胞与Ｓｐ２／０骨髓瘤细胞融合，经ＥＬＩＳＡ筛选及有限稀释法克隆化，选出６株阳性克隆，其中两株分泌ＩｇＧ，其余均为分泌ＩｇＧ。免疫印迹分析表明，两株ＩｇＧ单克隆抗体对人红细胞ＮＡＤＨ－细胞色素ｂ５还原酶有较高的特异性和亲和力。这一研究为开展对人红细胞ＮＡＤＨ－细胞色素ｂ５还原酶的研究和遗传性高铁血经蛋白血症的     

抗人Ig轻链系列单抗的研制：Ⅲ抗人Ig游离λ型轻链共同抗原决定…

用多例提纯的ＢＪ－λ混合免疫ＢＡＬＢ／ｃ小鼠的脾细胞与骨髓瘤细胞Ｓｐ２／０融合，获得３株稳定分泌抗人Ｉｇ游离λ链ＭｃＡｂ的杂交瘤细胞株（ＡＨλＤ８、ＡＨλ１Ｃ１和ＡＨλ２Ｃｌ）。它们分泌的ＭｃＡｂ均能与我室所有１７例ＢＪ－λ起强反应，但不与结合的λ型轻链及Ｋ型轻链反应，表明这３株ＭｃＡｂ可能是抗人Ｉｇ游离λ链共同抗原决定簇的。３株ＭｃＡｂ均属小鼠ＩｇＧ１亚类。添加ＥＬＩＳＡ表明它们识别相同的表位。     

人心肌肌钙蛋白T单克隆抗体的研制及鉴定

以人心肌肌钙蛋白Ｔ（ｃＴｎＴ）为抗原，采用脾内免疫法，免疫ＢＡＬＢ／ｃ小鼠，取其脾淋巴细胞与小鼠Ｓｐ２／０细胞融合，经间接ＥＬＩＳＡ法筛选，三次克隆化后获得５株能稳定分泌抗ｃＴｎＴ单克隆抗体（ＭｃＡｂ）的杂交瘤细胞Ｇ３、Ｇ８、Ｇ１０、Ａ５和Ａ７。免疫球蛋白亚类鉴定其中１株为ＩｇＧ２ａ，４株为ＩｇＭ。染色体数目９２～１１０条。将Ｇ３、Ｇ８、Ｇ１０、Ａ５的单克隆抗体腹水做１：１００稀释与ＬＤＨ、ＣＫ、ＣＫＭＢ和ＧＯＴ等心肌酶均无交叉反应。５株ＭｃＡｂ的腹水效价为３．２×１０－６～１．６×１０－７。ＭｃＡｂ相加试验表明，Ａ５和Ｇ３可识别不同的抗原表位。     

抗淋球菌PIA单克隆抗体的制备和应用分析

用淋球菌全菌体免疫ＢＡＬＢ／ｃ小鼠，取脾细胞与Ｓｐ２／０骨髓瘤细胞融合，以纯化ＰＩＡ做ＥＬＩＳＡ间接法筛选，获得５株稳定分泌抗ＰＩＡ的ＭｃＡｂ的杂交瘤细胞株。５株ＭｃＡｂ中３株为ＩｇＭ类（２Ｈ１１，４Ｈ８，４Ｅ１０），另２株分别为ＩｇＧ１（１Ｃ２）和ＩｇＧ２ｂ（５Ａ５）亚类。２Ｈ１１和１Ｃ２为高亲和力抗体，１Ｃ２与５Ａ５识别的抗原表位相同。Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ试验表明，５株ＭｃＡｂ均能从复杂的淋球菌菌体崩解物中特异地识别分子量为３５ＫＤａ的ＰＩＡ抗原，与淋球菌ＰＩＢ无交叉反应。     

抗人IgG-抗HRP双特异性单克隆抗体细胞株的建立与鉴定

目的 制备人ＩｇＧ检测用双特异性单克隆抗体诊断试制。方法 用人ＩｇＧ免疫ＢＡＬＢ／ｃ小鼠脾细胞与抗ＨＲＰＭｃＡｂ杂交瘤细胞ＨＡＴ敏感株进行第２次融合。结果 获得５株能稳定分泌抗人Ｉｇ－抗ＨＲＰ的双特异性单克隆抗体的杂交－杂交瘤细胞,分泌的抗体亚类其中１株为ＩｇＧ２ａ／ＩｇＧ１,余为ＩｇＧ１／ＩｇＧ１。培养上清与腹水效价分别为２＾７、１０＾０４以上,经ＨＲＰ亲和层析有２个蛋白吸ＢｓＭｃＡｂ存在于第２     

抗人Ig轻链系列单抗的研制Ⅲ抗人Ig游离λ型轻链共同抗原决定簇单克隆抗体的制备和初步鉴定

用多例提纯的ＢＪ－λ混合免疫ＢＡＬＢ／Ｃ小鼠的脾细胞与骨髓瘤细胞Ｓｐ２／０融合，获得３株稳定分泌抗人屹游离λ链ＭｃＡｂ的杂交瘤细胞株（ＡＨλＤ８、ＡＨλ１Ｃ１和ＡＨλ２Ｃ１）．它们分泌的ＭｃＡｂ均能与我室所有１７例ＢＪ－λ起强反应，但不与结合的λ型轻链及Ｋ型轻链反应，表明这３株ＭｃＡｂ可能是抗人Ｉｇ游离λ链共同抗原决定簇的．３株ＭｃＡｂ均属小鼠ＩｇＧ１亚类。添加ＥＬＩＳＡ表明它们识别相同的表位。用于检测正常人血清和尿中的游离λ链及多发性骨髓瘤患者的ＢＪ－λ均具有很好的特异性和敏感性．本文还讨论了有关的免疫问题．     

重组人红细胞生成素单克隆抗体的制备

本文应用常规淋巴细胞杂交瘤技术制备了４株能稳定分泌抗人重组红细胞生成素（ｒＨｕＥＰＯ）单克隆抗体（ＭｃＡｂ）的小鼠杂交瘤细胞系ＢⅡ１Ｂ５、ＤⅡ６Ｂ９、ＭⅡ１Ｈ４和ＧＩ３Ｅ７。用鼠单克隆抗体分型试剂盒鉴定，其分泌的ＭｃＡｂ的类分别是ＩｇＭ、ＩｇＭ、ＩｇＧ１和ＩｇＧ２ａ。间接ＥＬＩＳＡ法测定细胞上清的效价为１×１０－２～１．２５×１０－４，腹水效价为１×１０－２～１×１０－８。培养上清经ＥＬＩＳＡ鉴定，与ＩＬ－２、ＧＭ－ＣＳＦ、ＩＦＮ－α等细胞因子均无交叉反应，只与ｒＨｕＥＰＯ特异性结合。     

人醛缩酶A单克隆抗体的制备及应用

纯化并鉴定了人醛缩酶Ａ、Ｂ、Ｃ（ｈＡＬＤ－Ａ、Ｂ、Ｃ）。用ｈＡＬＤ－Ａ免疫Ｂａｌｂ／Ｃ小鼠，将免疫的脾细胞和Ｐ＿３－Ｘ＿（６３）－Ａｇ８．６５３骨髓瘤细胞用ＰＥＧ进行融合，用ＥＬＩＳＡ法筛选，ｈＡＬＤ－Ｂ、Ｃ作阴性对照，将阳性反应的杂交瘤细胞进行克隆，获得３株杂交瘤细胞株。其ＭｃＡｂ的亚类分别为ＩｇＧ２ｂ，ＩｇＧ１、ＩｇＭ，亲合常数分别为７．５×１０￣（１０）、３．５×１０￣９、２．３×１０￣９。３株细胞株分泌的单抗通过Ｉｍｍｕｎｏｂｌｏｔｔｉｎｇ得到证实。用亲合层析法从人肝癌细胞中提纯了ＡＬＤ－Ａ，ＳＤＳ－ＰＡＧＥ显示为单一区带，但其电泳迁移率较ｈＡＬＤ－Ａ滞后。     

抗肾综合征出血热病毒人单克隆抗体杂交瘤细胞株的建立及初步鉴定

以亲和层析技术纯化的肾综合征出血热病毒（ＨＦＲＳＶ）结构蛋白（５５０００，６７０００）为特异性抗原，体外免疫正常人外周血淋巴细胞（ＰＢＬ），然后将体外免疫的淋巴细胞与人－鼠种间杂交瘤细胞（Ｋ６Ｈ６／Ｂ５）融合，经ＥＬＩＳＡ间接法筛选出２株（２Ｄ５，１Ｂ７）分泌抗－ＨＦＲＳＶ人单克隆抗体（Ｈ－ＭｃＡｂ）杂交瘤细胞株，４次克隆化后，１００％阳性。对２Ｄ５株初步鉴定表明，其抗体类型为ＩｇＧ１；培养上清中抗体浓度为２０～３０μｇ／ｍｌ；特异性免疫荧光反应证明２Ｄ５株Ｈ－ＭｃＡｂ是ＨＦＲＳＶ特异性；中和效价为１∶１６０；体外连续传代６个月仍稳定分泌抗体。     

抗人重组IL—1单克隆抗体的制备及初步鉴定

以重组人ＩＬ－１免疫ＢＡＬＢ／ｃ小鼠，取其脾细胞与小鼠骨髓瘤细胞６５３融合，得到一组分泌抗人重组ＩＬ－１单克隆抗体的杂交瘤细胞。对其中的３株 ＹＡ１３３、ＹＡ１４０和ＹＡ１６３进行了初步鉴定，抗体类别为ＩｇＧ，亚类均为ＩｇＧ１，免疫转印显示，３株抗体均能特异性地识别分子量为１７．５ｋＤ的ＩＬ－１蛋白条带，ＥＬＩＳＡ法证明与其它细胞因子如：ＩＬ－２、ＩＬ－６、ＴＮＤ－α，ＩＦＮ－γ和ＧＭ－ＣＳＦ均无     

抗人心肌肌凝蛋白轻链单克隆抗体的制备和鉴定

从人心脏中提取心肌肌凝蛋白轻链（ＣＭＬＣ１），免疫ＢＡＬＢ／ｃ小鼠，取其脾细胞与Ｓｐ２／０骨髓瘤细胞做常规融合，获得了２２株分泌抗ＣＭＬＣ１单克隆抗体的杂交瘤细胞株。经对其中某些株作ＩｇＧ分型、核型、腹水效价、表位和亲和常数测定，表明所获单克隆抗体的质量良好，为建立分析轻链的方法奠定了基础。     

Preparation of Hybridomas producing monoclonal antibodies against human interferon

To prepare hybridomas secreting monoclonal antibodies (MoAb) against human alpha-interferon (alpha-IFN), BALB/c mice were immunized with IFN produced in Namalwa cells. Native alpha-IFN, as well as partially purified or on cellulose adsorbed alpha-IFN preparations were used for immunization. Seven hybridomas continuously secreting IgG against human alpha-IFN were prepared by fusion of splenocytes from immunized donors with the mouse myeloma cells. MoAb reacted in ELISA as well as in neutralization test with human lymphoblastoid, leukocytic and recombinant alpha-IFN.     

Monoclonal antibodies against hepatitis B e antigen: production, characterization, and use for diagnosis

Five different hybridoma clones secreting anti-HBeAg antibody were constructed by fusing cells of mouse myeloma line SP2/0 with splenocytes from BALB/c mice immunized with recombinant HBeAg. The monoclonal antibodies obtained were characterized immunologically and one was used to develop ELISA for detection of HBeAg and anti-HBeAg antibody. These monoclonal assays enabled the detection of 3 U HBeAg/ml and 1 U anti-HBeAg/ml with reference to standards of the Paul Ehrlich Institute, Frankfurt, F.R.G. Both assays compared well with a commercially available kit (Abbott Laboratory) and were used for detection of HBeAg and anti-HBeAg antibody in clinical serum samples.     

Production and characterization of monoclonal antibodies specific to sweet clover necrotic mosaic virus

Monoclonal antibodies were produced in vitro by fusing mouse myeloma cells P3-X63-AgU1 with spleen cells derived from BALB/c mice immunized with purified sweet clover necrotic mosaic virus (SCNMV). Twenty-one out of 47 clones which secreted monoclonal antibodies of high titres against SCNMV were injected intraperitoneally into mice previously primed with Pristane. The ascites fluid harvested 10 to 14 days later showed a strong and specific anti-SCNMV activity in reverse passive haemagglutination inhibition, passive haemagglutination and indirect enzyme linked immunosorbent assay. The monoclonal antibodies of the 21 clones did not react with red clover necrotic mosaic virus (Swedish isolate). The class and subclass of immunoglobulins of the monoclonal antibodies secreted by established cultures were determined to be IgG2a for 15 clones and IgG3 for 6 clones.     

Mink-mouse hybridomas that secrete mink immunoglobulin G

Optimum conditions were established to obtain mink-mouse interspecific hybridomas secreting mink IgG in fusions of mouse myelomas with mink immune spleen cells. Minks were immunized with allogeneic IgG, and the spleen cells were fused with three mouse myeloma lines P3-X63-Ag8.653, NSO and Sp2/0-Ag14. Of these, P3-X63-Ag8.653 and NSO were found to be the best fusion partners giving the highest yield of hybrid clones and number of IgG secreting clones. Cloning of mink-mouse hybridomas was efficient when BALB/c nu/nu peritoneal and spleen cells were used as feeders. The ten clonal lines produced secreted intact mink IgG molecules as shown by SDS-PAGE and subsequent immunoblotting. The secretion level of IgG ranged from 5 to 200 ng/ml in the clonal lines.     

Monoclonal antibodies against hepatitis B s antigen: production, characterization, and use for diagnosis

Three different hybridoma clones secreting anti-HBsAg antibody were constructed by fusing cells of mouse myeloma line Ag8-X63 with splenocytes from BALB/c mice immunized with recombinant HBsAg and natural HBV. The monoclonal antibodies obtained were characterized immunologically, and two were used to develop UMELISA for detection of HBsAg. This monoclonal assay enabled the detection of 0.1 UPE/mL with reference to the standard of the Paul Ehrlich Institute (Frankfurt, Germany). The assay compared well with a commercially available kit (UMELISA HBsAg) and was used for detection of HBsAg in blood donors.     

Monoclonal antibodies to the idiotypic determinants of a human IgG paraprotein: evidence for complexity of the anti-idiotype response.

BALB/c mice were immunized with a human gamma 2k paraprotein and hybridomas prepared by the Kohler & Milstein technique. Ten hybridoma clones producing high levels of antibody competed reacting specificity with the immunogen were isolated. Six of the monoclonal antibodies competed strongly with each other for sites on the antigen. In reverse passive haemagglutination tests immunoglobulins reacting with the antibodies were not detectable in normal or pathological sera. Polyclonal antisera to two of the monoclonal antibodies were prepared in rabbits; they were rendered specific by adsorption with Sepharose-normal mouse IgG and tested against the other monoclonal antibodies. A weak cross-reaction of one of the mouse antibodies with the immunogen was observed with one antiserum but none with the other. Furthermore, no repetition of the idiotypes of the two immunogens was observed in polyclonal responses of BALB/c mice immunized with the same paraprotein antigen. These results with monoclonal antibodies confirm those obtained with polyclonal antisera in showing that the antibody response of one species to the private idiotype determinants of an immunoglobulin of another species is highly heterogeneous.     

Monoclonal xenogeneic antibodies to murine cell surface antigens: identification of novel leukocyte differentiation antigens.

Hybrid myeloma cell lines secreting monoclonal antibodies to mouse cell surface antigens have been prepared. Spleen cells from a DA rat immunized with B10 mouse spleen cells that had been enriched for T cells were fused to cells from a nonsecreting mouse myeloma line (NSI). The presence in the culture supernatants of antibodies binding to mouse spleen cells was tested by a binding assay with 125I-labeled anti-rat IgG. From a large number of positive cultures, ten independent hybrid clones were purified, each secreting a different antibody. Each antigenic target was analyzed by (a) gel electrophoresis of immunoprecipitated 125 I-labeled cell surface molecules, (b) heat stability, (c) strain and species distribution and (d) cross-inhibition of binding of different monoclonal antibodies. It was concluded that the ten monoclonal antibodies regognized four types of antigen. One was the heterophile, heat-stable, Forssman antigen. The second (mol.wt. 210 000) appears to be a major 125I-labeled lymphoid cell surface protein. The third, a minor component of spleen cells, was precipitated as two polypeptides of mol.wt. 190 000 and 105 000. Five IgG-secreting clones identify the fourth antigen, a heat-stable, possibly glycolipid component expressed on mouse red blood cells and also on thymocytes. Cross-inhibition studies suggest that these last monoclonal antibodies bind to overlapping, but not identical, determinants. The class and chain composition of the monoclonal antibodies were studied by gel electrophoresis, isoelectric focusing and ability to lyse red blood cells and thymocytes.     

Recent insight into class-specific properties of murine immunoglobulins obtained with the help of monoclonal antibodies

BALB/c and C57BL/6 mice were immunized with monoclonal anti-DNP IgE, obtained by fusion of PX63AG8-6-5-3 cells with spleen cells from immunized C57BL/6 or BALB/c mice, respectively. With the antiallotype sera thus produced the allotype of IgE (i.e. 7) could be defined since BALB/c is of the Igh-a and C57BL/6 of the Igh-b allotype. The antiallotype 7b does not cross-react with other allotypes. Antiallotype 7a recognizes in addition to allotype 7a also allotypes 7j, 7d, and 7e as expected. Both the complement fixing and the sensitizing activities of murine anti-TNP and anti-DNP monoclonal antibodies were examined. The minimal amount/ml for complement fixation was 0.46 micrograms from one of the IgG1 antibodies, 0.46 from IgG2a, 12.5 ng from IgM, 0.7 micrograms from IgG3. Complement fixation by IgG1 was unexpected. The minimal amount/ml for mouse PCA was 0.5-0.7 micrograms from IgG1, 1.44-2.3 micrograms from IgG2a, 5.6 micrograms from purified IgG2b and about 15 ng from IgE. Sensitization by IgG2a and IgG2b were new findings. Minimal amount/ml for guinea pig PCA was 0.23 micrograms from IgG2a. Minimal amount/ml for rat PCA was 2 ng from IgE.