抗人白细胞单克隆抗体的特异性及其特性鉴定

目的制备抗人白细胞单克隆抗体（ｍＡｂ），并对其识别抗原及组织的特异性进行鉴定。方法采用分离的人全白细胞悬液免疫Ｂａｌｂ／ｃ小鼠，取脾细胞与Ｓｐ２／０细胞常规融合，用间接ＥＬＩＳＡ筛选ｍＡｂ，流式细胞仪及免疫组化染色ＳＰ法鉴定其组织特异性。　结果成功地制备１株抗人白细胞　ｍＡｂ，命名为ＳＺ－１０５。ＥＬＩＳＡ法测定其腹水效价为１×１０－５。琼脂糖双扩散鉴定为ＩｇＧ１类。流式细胞仪间接免疫荧光技术及免疫组化ＳＰ法测定表明，ｍＡｂ　ＳＺ－１０５可选择性地与外周血液的粒细胞、单核细胞和淋巴细胞呈强阳性反应，而与红细胞及血小板不出现反应。该ｍＡｂ还可与正常人骨髓的白细胞前体起反应。另外，在肝、肺、脾、胸腺及淋巴结正常组织中的巨噬细胞表面，也有ＳＺ－１０５抗原表达。ＳＺ－１０５抗原经亲和层析纯化，再经ＳＤＳ－ＰＡＧＥ　和Ｗｅｓｔｅｒｎ　ｂｌｏｔ证实，ｍＡｂ　ＳＺ－１０５识别的抗原是相对分子质量（Ｍｒ）为７５　０００左右的单链蛋白多肽。结论　获得１株具有高度免疫学活性和组织特异性的抗人白细胞ｍＡｂ　ＳＺ－１０５，其对研究白细胞的分化、免疫功能，以及隐匿性急、慢性炎症疾病的放免显像诊断都具有一定的临床意义。     

应用合成肽研究柯萨奇B病毒衣壳蛋白VP1共的抗原表位

对柯萨奇Ｂ４病毒（ＣＶＢ４）衣壳蛋白ＶＰ１保守区的亲水性和二级结构进行分析和预测，选择可能代表优势抗原表位的肽段进行合成（ＶＰ１－１肽，ＲＩＹＦ ＫＰＫＨＶＫ ＡＹＶ），并截取了该肽段的前１２个残基（ＶＰ１－２肽）用同样方法进行合成。用ＶＰ１－１肽免疫家兔制备出高效阶抗体，应用酶联免疫吸附法（ＥＬＩＳＡ）检测，这些抗体与ＣＶＢ１－６病毒均有结合反应，ＶＰ１－１肽与型特异性ＣＶＢ１－６抗体均有良好的     

抗bFGF单克隆抗体的鉴定及其意义

用重组牛ｂＦＧＦ免疫Ｂａｌｂ／ｃ小鼠，通过细胞融合，以反向间接血凝和间接ＥＬＩＳＡ筛选，以及有限稀释法克隆化，建立了９株稳定分泌抗ｂＦＧＦ单克隆抗体（ｍＡｂ）杂交瘤细胞。对其中３株用Ｗｅｓｔｅｒｎｂｌｏｔ和生物活性抗体中和实验进行了鉴定。结果显示，ｍＡｂ可结合重组牛或人ｂＦＧＦ，并能抑制ｂＦＧＦ刺激Ｂａｌｂ／ｃ３Ｔ３细胞的生长；腹水的ＥＬＩＳＡ滴度为１∶３０００；用其中２株ｍＡｂ建立的双抗体夹心ＥＬＩＳＡ法灵敏度可达５０ｐｇ／孔。本文还讨论了抗ｂＦＧＦｍＡｂ的应用价值。     

降钙素基因相关肽单克隆抗体的研制及初步鉴定

以人工合成的降钙素基因相关肽（ＣＧＲＰ）－牛血清白蛋白（ＢＳＡ）偶联物（ＣＧＲＰ－ＢＳＡ）为免疫原，用微量脾内免疫法免疫ＢＡＬＢ／ｃ小鼠。取其免疫脾细胞与小鼠Ｓｐ２／０骨髓瘤细胞融合，经间接ＥＬＩＳＡ法筛选、３次克隆化后，获得３株能稳定分泌抗ＣＧＲＰ单克隆抗体的杂交瘤细胞３Ｂ４、７Ｅ１１和８Ｆ２。用琼脂双扩散法鉴定，３Ｂ４为ＩｇＧｌ亚类，７Ｅ１１和８Ｆ２均为ＩｇＭ类。杂交瘤细胞的染色体条数在９２￣     

兔抗人ClqIgG（Fab＇）_2的制备及鉴定

本文利用硫酸铵盐析及ＤＥＡＥ纤维素层析提取兔抗人ＣｌｑＩｇＧ．经胃蛋白酶消化及ＳＰＡ－Ｓｅｐｌｉａｒｏｓｅ４Ｂ层析，制备了兔抗人ＣｌｑＩｇＧ（Ｆａｂ＇）＿２．ＳＤＳ－ＰＡＧＥ分析分子量为９３ＫＤ；经Ｃｌｑ－ＥＬＩＳＡ滴定其效价为１０￣（－４）．与人ＩｇＧ无交叉反应；当该制剂预先经人Ｃｌｑ处理后，阻断其与Ｃｌｑ特异结合的能力，在合适的条件下可抑制Ｃｌｑ与Ｒａｊｉ细胞上ＣｌｑＲ的结合。表明用该法制备的兔抗人ＣｌｑＩｇＧ（Ｆａｂ＇）＿２具有较好的纯度及特异性，可用于ＣｌｑＲ功能的研究。     

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。     

肿瘤碱性蛋白单克隆抗体免疫学特性的研究及其应用

应用淋巴细胞杂交瘤技术，建立了４株抗人血清肿瘤碱性蛋白（Ｔｕｍｏｕｒｂａｓｉｃｐｒｏ－ｔｅｉｎ，ＴＢＰ）杂交瘤细胞株（１Ｃ３、４Ｆ４、５Ｆ４、３Ｇ９），并以制备的单克隆抗体（ＭｃＡｂ）对其抗原决定簇及免疫学特性进行了分析。Ｉｇ亚类测定：均为ＩｇＧ２ａ，腹水效价为１×１０－６～１×１０－８。特异性测定：ＴＢＰＭｃＡｂ与ＩｇＧ、ＩｇＡ、ＩｇＭ和Ａｌｂ无交叉反应。单抗相加试验证实：５Ｆ４、４Ｆ４和１Ｃ３为识别ＴＢＰ上同一抗原决定簇，３Ｇ９则为识别ＴＢＰ上另一抗原决定簇。分别利用单株和混合株ＭｃＡｂ标酶建立了可应用于人血清ＴＢＰ含量测定的ＥＬＩＳＡ双抗体夹心法，并用于人血清ＴＢＰ含量测定。     

抗金黄色葡萄球菌C_1型肠毒素McAb的制备及初步应用

金葡菌Ｃ１型肠毒素免疫ＢＡＬＢ／ｃ小鼠，取其脾细胞与Ｓｐ２／０小鼠骨髓瘤细胞融合选出了能分泌高滴度ＭｃＡｂ的１８个克隆株，对其中１０株进行了鉴定，６株属ＩｇＧ１，４株属ＩｇＧ３。用双抗体夹心法和特异性中和抑制试验比较了ＭｃＡｂ和ＰｃＡｂ的敏感性和特异性，并用制备的ＭｃＡｂ诊断试剂对ＳＥＣＩ污染食品进行了检测应用，可特异检出ｌｕｇＳＥＣ１／０．５ｇ食物／ｍｌ。     

血管内皮细胞生长因子受体KKR胞外配基结合区单？…

目的 研制能封闭血管内皮生长因子（ＶＥＧＦ）受体ＫＤＲ的单克隆抗体（ＭｃＡｂ）?探讨其抑制ＶＥＧＦ诱导的体内外活性。方法 以原核表达的ＫＤＲⅠ￣Ⅳ区融合蛋白免疫ＢＡＬＢ／ｃ小鼠，用杂交瘤技术制备抗ＫＤＲⅠ￣Ⅳ的ＭｃＡｂ，并用免疫双扩、ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ免疫印迹鉴定其亚类和抗原结合特异性，用ＶＥＧＦ刺激内皮细胞增殖及鸡胚血管形成实验检测该抗 的中和活性。结果 通过筛选和鉴定获得了２株ＫＤＲⅠ     

抗hER合成肽单克隆抗体的制备与鉴定

用合成的人雌激素受体（ｈＥＲ）抗原决定簇多肽（氨基酸序列从１５１～１６５）与ＫＬＨ的偶联物为抗原，免疫ＢＡＬＢ／Ｃ小鼠，经鼠－鼠杂交，ＥＬＩＳＡ和免疫细胞化学法筛选，有限稀释克隆化，得到一株分泌抗ｈＥＲ合成肽的杂交瘤细胞株（２Ｂ１２）。用ＥＬＩＳＡ法测定。该纯化抗体１０ｎｇ／ｍｌ仍与抗原反应，其免疫球蛋白为ＩｇＧ２ａ．ｋ。与ＭＣＦ－７乳腺癌细胞溶解蛋白的Ｗｅｓｔｅｒｎｂｌｏｔ分析，在分子量６７ｋＤ     

抗人重组SCF单克隆抗体杂交瘤细胞系的建立及初步鉴定

本研究利用PEX31B作为载体,在大肠杆菌表达出重组人SCF融合蛋白。用该蛋白作为抗原,免疫BALB/c小鼠。通过鼠-鼠杂交瘤技术,成功地获得4株分泌抗人重组SCF单克隆抗体(下称单抗)的杂交瘤细胞系。经检测,它们所分泌的抗体亚类均为IgG1,免疫印迹试验和抗体特异性鉴定证实,4株单抗均能特异性地识别可溶性SCF。抗SCF单抗杂交瘤细胞系的建立,为深入研究SCF的生物学特性、功能以及SCF产品的纯化,提供了有力的工具。     

Analysis of the Neutralizing Antibody Response to the Measles Virus Using Synthetic Peptides of the Haemagglutinin Protein

Infection or immunization with measles virus induces a protective immune reaction including neutralizing antibodies against the haemagglutinin and fusion protein. The reactivity of the polyclonal IgG response of sera obtained from late convalescent donors was studied, using overlapping 15mer peptides covering the complete sequence of the measles virus haemagglutinin. Most sera reacted with a similar set of peptides generating a characteristic binding pattern. The reactive peptides correspond to a region mediating cell hemolysis (aa310–325), to regions which serve as targets to neutralizing antibodies and to a putative transmembrane region (aa35–58). The latter region contains also a human T-cell epitope providing evidence of a non-random association of T- and B-cell epitopes. We also immunized different strains of mice and rabbits with measles virus. In contrast to the human sera, animal sera with strong neutralizing activities did not react with any of the H-protein peptides. The mostly weak reactivities with the linear sequences contrast with the strong neutralizing activities of the human or animal antibodies, suggesting that these primarily recognize the fusion protein or conformational epitopes of the haemagglutinin protein.     

Monoclonal antibodies reacting with the interleukin 1 receptor define a multi-molecular complex

By sequential immunization with a variety of different interleukin (IL) 1 receptor (IL 1R)-bearing cells and by generating a large number of clones from a single fusion experiment, we have managed to produce two monoclonal antibodies which react with the lymphocyte IL 1R. The antibodies also react with the mouse fibroblast line, 3T3, but we have not yet defined their reactivity with cells carrying low-affinity receptors. Immunoprecipitation allows the IL 1R to be isolated from EL4 6.1 cell membrane preparations and shows that the IL 1R exists as a complex. The antibodies clearly interfere with IL 1 responses both in vitro and in vivo. These are the first anti-mouse IL 1R antibodies to be described which clearly and profoundly affect the immune response.     

Definition of linear antigenic regions of the HPV16 L1 capsid protein using synthetic virion-like particles.

Mice of three haplotypes (H-2d, H-2b, and H-2d/b) were immunized with synthetic HPV16 virus-like particles (VLPs), produced using a vaccinia virus doubly recombinant for the L1 and L2 proteins of HPV16. The resultant anti-VLP antisera recognized HPV16 capsids by ELISA assay and baculovirus recombinant HPV16 L1 and L2 protein on immunoblot. Overlapping peptides corresponding to the HPV16 L1 amino acid sequence were used to define the immunoreactive regions of the L1 protein. The majority of the L1 peptides were reactive with IgG from the mice immunized with the synthetic HPV16 capsids. A computer algorithm predicted seven B epitopes in HPV16 L1, five of which lay within peptides strongly reactive with the murine antisera. The murine anti-VLP antisera failed to react with the two peptides recognized by anti-HPV16L1 monoclonal antibodies raised by others against recombinant L1 fusion protein. We conclude that the immunoreactive epitopes of HPV16 defined using virus-like particles differ significantly from those defined using recombinant HPV16 L1 fusion proteins, which implies that such fusion proteins may not be the antigens to look for HPV16L1 specific immune responses in HPV-infected patients.     

Detection of Neisseria gonorrhoeae by dot-enzyme immunoassay using monoclonal antibodies

A highly sensitive and specific dot-enzyme immunoassay for the detection of Neisseria gonorrhoeae was developed using a pool of monoclonal antibodies (MAbs). The MAbs were obtained following immunization of mice with lithium acetate extracted outer membrane (OM) preparations. Western immunoblotting experiments demonstrated that MAbs NG26 and NG38, both IgG2a, reacted with lipopolysaccharides (LPS) and with the major OM protein, P1, respectively, MAb NG28, an IgG3, did not react in Western immunoblotting, MAbs NG28 and NG38 failed to react with OM treated with proteolytic enzymes or with semi-purified preparation of LPS. MAb NG26 reacted with the same LPS preparation. Binding radioimmunoassay with live bacteria showed that all the MAbs adsorbed to cell surface-exposed antigenic determinants. The limit of detection of the dot-enzyme immunoassay was between 1 and 4 x 10(4) cfu per dot. Using a panel of 177 strains of N. gonorrhoeae, MAbs NG28 and NG38 recognized only P1A and P1B strains respectively. MAb NG26 reacted with P1A, P1B and non-typable strains. These MAbs did not react with other Neisseria species or other bacterial species. Using this pool, the dot-enzyme immunoassay had a sensitivity of 93.2% and a specificity of 100%.     

Identification of a sequential B-cell epitope on major allergen (Cry j 1) of Japanese cedar (Cryptomeria japonica) pollen in mice

BACKGROUND: Japanese cedar (Cryptomeria japonica; CJ) pollinosis is one of the most common allergic diseases in Japan. B cell epitopes on Cry j 1, a major allergen of CJ pollen, have been analyzed by the specific monoclonal antibodies to Cry j 1, and most of these epitopes may be conformational, but no previous report has addressed the analysis of sequential epitope mapping with synthetic peptides. The main purpose of the present study is to identify IgE and IgG B cell epitopes on Cry j 1 by using a synthetic peptide approach in mice. METHODS: We synthesized 35 overlapping peptides that cover the entire length of Cry j 1 and examined whether mouse IgE and IgG antibodies produced by immunization with Cry j 1 reacted to the Cry j 1 peptides. RESULTS AND CONCLUSION: We found that mouse IgE and IgG antibodies reacted strongly to Cry j 1 peptide No. 15 ((141)GVEPVHPQDGDALTLRTATN(160)), though those antibodies did not react with other peptides. IgE and IgG antibody binding to peptide No. 15 was completely inhibited by Cry j 1 and the peptide. To determine the minimum epitope in peptide No. 15, we conducted an ELISA inhibition test. IgE and IgG antibody binding to peptide No. 15 was inhibited by smaller peptides of this peptide. We found the core of the epitope to be (145)VHPQDGDA(152).     

Immunoglobulin Fc binding activity is associated with the mouse hepatitis virus E2 peplomer protein

Antigenic variation among murine coronaviruses is associated primarily with the surface peplomer protein E2 (180,000 Da). E2 is responsible for attachment of the virus to the host cell, MHV-induced cell fusion, and eliciting neutralizing antibody. We report here the molecular mimicry between E2 and Fc gamma receptor (Fc gamma R). Molecular mimicry between E2 and Fc gamma R may allow the escape of virus-infected cells from destruction by immunological mechanisms. Rabbit IgG, monoclonal rat IgG1 and IgG2b, monoclonal mouse IgG2a and IgG2b, and the rat anti-mouse Fc gamma R monoclonal antibody 2.4G2 immunoprecipitated from MHV-JHM-infected cells a polypeptide with a molecular mass identical to that immunoprecipitated by anti-E2 antibodies. F(ab')2 fragments of rabbit IgG did not immunoprecipitate any proteins from MHV-infected cells. All of these antibodies did not immunoprecipitate any proteins from uninfected cells. The anti-mouse Fc gamma R monoclonal antibody 2.4G2 immunoprecipitated from MHV-JHM-, MHV-3-, or MHV-A59-infected L-2 cells and 17CL-1 cells, or MHV-JHM-infected cultures of neonatal BALB/c brain cells, a protein with a molecular weight identical to that of MHV-JHM E2. The anti-Fc gamma R monoclonal antibody did not immunoprecipitate any proteins from uninfected cells. Furthermore, the 2.4G2 monoclonal antibody (mab), unrelated rat and mouse monoclonal antibodies, and a goat antiserum against E2, but not normal goat serum, immunoprecipitated a 75,000- to 77,000-Da molecule from uninfected WEHI-3 cells, a Fc gamma R bearing cell line. Several lines of evidence demonstrated that the protein immunoprecipitated by the anti-Fc gamma R mab from MHV-JHM-infected cells is the E2 glycoprotein: (1) Partial proteolytic maps obtained by Staphylococcus aureus V-8 protease treatment of the 180,000-Da proteins immunoprecipitated by the anti Fc gamma R mab and the anti-E2 mab were identical. (2) Sequential immunoprecipitation experiments from MHV-JHM-infected cells revealed that the same polypeptide chain was recognized by the anti-E2 mab and by the anti-Fc gamma R mab 2.4G2, (3) Actinomycin D did not influence the induction and expression of the 180,000-Da polypeptide chain that was immunoprecipitated by the anti-Fc gamma R mab, demonstrating that this protein is of viral origin.     

Characterization and large production of human monoclonal antibodies against the HIV-1 envelope.

Peripheral blood lymphocytes from a volunteer immunized with a recombinant vaccinia virus VSC-25 expressing the gp160 env protein of HTLV-IIIB strain and from an asymptomatic HIV-infected individual were immortalized by Epstein-Barr (EBV). Clones which secrete human monoclonal antibodies from the two individuals (DZ, IgG1, lambda and C31, IgG1, kappa) were obtained and were stable for more than 2 years. The two monoclonals were directed against the gp160 env protein of HIV, DZ directed against the gp41 and C31 directed against the gp120. C31 was group-specific, whereas DZ was directed against the HTLV-IIIB and HTLV-RF strains. The epitope recognized by DZ was mapped to the carboxy terminus of the gp41, by expression of HIV DNA fragments in a yeast system and peptide analysis. The C31 epitope was not expressed by the yeast library and not present among the peptides which were tested. Monoclonal antibodies had no inhibitory effect in an HIV-induced cell fusion assay, but DZ showed a weak neutralizing activity against the HTLV-IIIB strain. Cloned EBV-transformed cell lines were fused to a murine myeloma, which allowed the heteromyeloma to be cultivated in serum-free medium. The monoclonal antibodies were produced in large quantity in a hollow-fibre reactor at defined culture conditions and purification procedures.     

Production and characterization of two new mouse monoclonal antibodies reactive with denatured mouse class II beta chains.

We report on the production and characterization of two murine peptide specific anti-major histocompatibility complex class II chain specific monoclonal antibodies. Two new mouse monoclonal antibodies reactive with two synthetic peptides corresponding to Abb amino acids (146-157) and Abs amino acids (146-157) were produced. KL295 is the mouse anti-Abb monoclonal antibody, which reacts with denatured beta chains of H-2b, H-2d, H-2p, and H-2q, but fail to react with spleen cell lysates from H-2f, H-2j, H-2k and H-2s mice. The mouse anti Abs monoclonal antibody, KL304 in contrast reacts with the denatured Class II beta chains of H-2f, H-2j, H-2k and H-2s mice, but fails to bind H-2b, H-2d, H-2p or H-2q beta chain, suggesting residues 153-155 of this molecule to be critical for the epitope. Both KL295 and KL304 bind B10.WB (H-2j) which suggests a unique epitope in this strain of mice. Neither KL295 or KL304 react with native mouse class II cell surface molecules.     

Partial purification of lymphoblasts after in vitro immunization increases the yield in Ig-producing hybridomas

Using in vitro immunization with a human plasma protein (apolipoprotein-A1) as antigen, we have shown that it is possible to prepare more monoclonal antibodies using a ten-fold lower concentration of antigen compared to in vivo immunization procedures (Weech et al., 1985). In addition, we can increase the number of Ig-producing hybridomas after in vitro immunization by a simple one-step separation of the lymphoblasts on a Percoll gradient before the fusion procedure. In order to apply this procedure to in vivo immunization techniques, it is necessary to expand the B-blast/plasma cell population by culturing the spleen cells for 4-6 days before fusion. Only antibodies of the IgM class were produced with the in vitro technique. However, by combining in vivo priming with in vitro immunization, it is possible to produce specific antibodies to both IgG and IgM classes.