Analysis of Mouse Sperm Isoantigens Using Specific Monoclonal Antibodies

ABSTRACT: Female mice were isoimmunized with homologous spermatozoa of the same strain. Hybrid cells that secrete monoclonal antibodies to mouse sperm isoantigens were generated by modified hybridoma techniques using a semi-solid Iscove's modified Dulbecco's medium containing methylcellulose for the initial cloning. Out of more than 1,000 colonies that were initially recovered for subculture, 246 were shown to produce antibodies reacting with various cytological regions of mouse spermatozoa, when methanol-fixed sperm were employed in an indirect immunofluorescent assay. More than 75% of the generated monoclonal isoantibodies were shown to bind the acrosomal regions of mouse spermatozoa. Some were found to cross-react with spermatozoa from other mammalian species including those of human, rabbit, rat, and guinea pig. However, none were shown to cross-react with mouse lymphocytes. Two-thirds of the generated monoclonal antibodies can also bind live mouse spermatozoa. By an immunohistochemical technique using testicular sections, some of these monoclonal antibodies were shown to react with specific antigens expressed during different stages of spermatogenesis. It is concluded that these mouse sperm isoantigens are sperm-specific and appear uniquely during spermatogenesis. Monoclonal isoantibodies produced in the present study may have potential applications regarding the investigations of sperm iso- or autoimmunity, spermatogenesis, and fertility control.     

Immunohistochemical localization of the MHS-5 antigen in principal cells of human seminal vesicle epithelium

The location of the antigen recognized by monoclonal antibody MHS-5 in the human reproductive tract was examined by means of enzyme-linked immunosorbant assay (ELISA) and indirect immunohistochemistry employing the strepavidin-biotin-complex method. Homogenates of male reproductive tract tissues and other human organs assayed by ELISA demonstrated immunoreactivity of the MHS-5 monoclonal antibody specifically with human seminal vesicle extracts. Varying ratios of seminal protein and monoclonal antibody ascites were tested to determine the amount of antigen necessary to completely absorb the antibody in the ELISA assay. This ratio was subsequently used to obtain the absorbed negative control for histochemical localization studies. By light microscope examination of seminal vesicle tissue in paraffin section, the MHS-5 antigen was localized in principal cells of the seminal vesicle epithelium. Epididymal sperm, obtained from patients at orchiectomy and vasovasostomy were found to lack the MHS-5 antigen. Following incubation with seminal protein or fluid obtained from the lumen of the human seminal vesicle, epididymal sperm reacted with the MHS-5 antibody on ELISA. These findings indicate that the MHS-5 antigen, a novel protein previously shown to be a unique marker for human semen, is a secretory product of the human seminal vesicle epithelium and may be reconstituted on the surface of epididymal spermatozoa.     

抗人类免疫缺陷病毒gp41和丙型肝为病毒NS3杂交？…

目的 为检测血清中人类免疫缺陷病毒和丙型肝炎病毒抗原提供血清学指标。方法 用纯化的重组ＨＩＶ和ＨＣＶ抗原蛋白作为联合免疫原,免疫ＢＡＬＢ／ｃ小鼠,取脾细胞与Ｓ２／０小鼠骨髓瘤细胞融合,并采用挑单个细胞的方法进行一次克隆。结果 分别获得４株和６株能稳定分泌高效价抗ＨＩＶ和ＨＣＶ抗的蛋白单克隆抗体杂交瘤细胞株。     

抗人类免疫缺陷病毒gp41和丙型肝炎病毒NS3杂交瘤细胞株的建立

目的为检测血清中人类免疫缺陷病毒（ＨＩＶ）和丙型肝炎病毒（ＨＣＶ）抗原提供血清学指标。方法用纯化的重组ＨＩＶ（ｇｐ４１）和ＨＣＶ（ＮＳ３）抗原蛋白作为联合免疫原，免疫ＢＡＬＢ／ｃ小鼠，取脾细胞与Ｓｐ２／０小鼠骨髓瘤细胞融合，并采用挑单个细胞的方法进行一次克隆。结果分别获得４株和６株能稳定分泌高效价抗ＨＩＶ（ｇｐ４１）和ＨＣＶ（ＮＳ３）抗原蛋白单克隆抗体杂交瘤细胞株。初步建立了双抗体夹心法检测ＨＩＶ（ｇｐ４１）和ＨＣＶ（ＮＳ３）抗原的酶联免疫吸附试验。结论本方法是建立单抗杂交瘤细胞株的快速方法，所建杂交瘤细胞株特异性强，效价高，分泌抗体性能稳定，有推广价值。     

A live cell enzyme-linked immunosorbent assay for detecting human hepatoma membrane antigens

Live cell enzyme-linked immunosorbent (ELISA) and fixed cell indirect immunofluorescence (IF) assays were compared to screen mouse hybridomas producing immunoreactive monoclonal antibodies against cell membrane antigens expressed on Ha22T, a human hepatoma cell line. While performing live cell ELISA, two parameters were tested to improve the viability of the target cells. The first parameter was the inclusion of growth medium in the assay buffers, and the second was performing the assay incubations at 37 degrees C in an incubator containing 5% CO2 in the air. Fixed cell IF detected and classified 46% of the hybridomas secreting monoclonal antibodies reactive with membrane, cytoplasm, cytoskeleton, and nuclear antigens of Ha22T cells. Fixed cell IF was able to reveal mixtures of two or more hybridomas growing in the same well secreting antibodies to different cell organelles. The live cell ELISA, on the other hand, identified 12 additional membrane reactive monoclonal antibodies from the hybridoma supernatants that were not reactive by IF. These results disclose that cell fixation procedures used for IF either completely or partially inactivated some of the cell membrane antigens. We, therefore, propose the use of a combination of immunoassays to select the maximum number of hybridomas secreting useful monoclonal antibodies from somatic cell fusions.     

基于基因免疫制备抗人绒毛膜促性腺激素β亚基单克隆抗体及其特性研究

用真核表达人绒毛膜促性腺激素β亚基((human chorionic gonadotrophinβ,hCGβ)的重组质TR421-hCGβ免疫BALB/c小鼠,采用B淋巴细胞杂交瘤技术进行细胞融合,ELISA法筛选阳性细胞株,经过多次的克隆化培养,最终获得10株持续、稳定分泌抗hCGβ单克隆抗体的杂交瘤细胞株。随机抽取6H1杂交瘤细胞进行抗体的制备、纯化及免疫学特性的分析。间接ELISA法证明6H1单抗属于IgG2a亚类。Western blot证明6H1单克隆抗体可以特异性结合hCGβ,间接免疫荧光和流式细胞仪等结果表明,6H1单克隆抗体能够不同程度的结合不同来源的肿瘤细胞膜上表达的hCGβ分子,为进一步研究其生物学功能奠定了物质基础。     

Monoclonal Antibodies to Porcine Zona Pellucida That Block the Initial Stages of Fertilization

ABSTRACT: Zona pellucida (ZP) is thought to be of utmost biological importance in the early stages of fertilization and implantation. Current hybridoma technology was used to produce monoclonal antibodies (MAbs) against specific antigens to porcine ZP. Two monoclonal antibodies (4F2 and 2D9) were raised that reacted against ZP antigens shared by human and porcine ZP. These antibodies were shown to block fertilization of human oocytes in in vitro fertilization (IVF). It is likely that MAb 4F2 recognized a protein epitope localized on the outer surface of ZP. These antibodies may be quite useful immunologic probes for studying the precise mechanisms of the early events of fertilization in mammals.     

Monoclonal antibodies used to isolate IgM from chicken bile and avian sera and to detect specific IgM in chicken sera

Four hybridoma cell lines (designated M1, M10, M11, M31) were established which secrete antibody specific for chicken IgM. The specificity for the IgM heavy chain was shown by using ELISAs to screen for antibodies to IgM, IgA and IgG. Radioimmunoprecipitation tests confirmed that the four monoclonal antibodies reacted with IgM and also showed that they combined with Protein A. An immunoadsorbent was made using one (M1) of the monoclonal antibodies. IgM was purified in a single step by affinity chromatography from chicken bile and chicken, turkey and duck serum. This is the first unequivocal demonstration of chicken IgM in bile. The Ml monoclonal antibody was used in an ELISA to detect the specific chicken IgM response to the inoculation of bovine serum albumin. This anti-IgM reagent may also be used to detect the IgM response to other antigens.     

抗人肝再生增强因子单克隆抗体的研制

目的　利用纯化的重组人肝再生增强因子(hALR)制备抗hALR的单克隆抗体,建立hALR的检测方法。方法 采用杂交瘤技术制备单克隆抗体,经ELISA和免疫印迹证明其特异性。结果获得了4株分泌抗hALR特异性抗体的杂交瘤 细胞系;无血清培养液效价为1×10-2,腹水效价为1×10-5;亚类鉴定表明2株单克隆抗体为IgGl,另2株为IgG2b;免疫印迹 显示抗体结合抗原的分子量与hALR相符,为特异性抗hALR抗体。结论通过杂交瘤技术,获得了抗hALR的单克隆抗体 为以后的工作奠定了基础。     

抗HIV-1核心抗原p24单克隆抗体的制备及特性的初步鉴定

目的 :建立分泌抗HIV 1p2 4单克隆抗体 (mAb)的杂交瘤细胞株 ,并对其特性进行初步鉴定。方法 :以纯化的基因工程制备的p2 4抗原免疫BALB/c小鼠 ,取免疫小鼠的脾细胞与Sp2 /0骨髓瘤细胞融合 ,经HAT、HT选择培养及有限稀释法进行克隆化后 ,用间接ELISA法及Dotblot对其进行筛选和特性鉴定。结果 :筛选到 2株可分泌抗HIV 1p2 4mAb的杂交瘤细胞 ,其腹水效价为 1×10 -5,亲和力为 1.7× 10 4～ 1.8×10 4mol/L ,mAb的Ig亚类均为IgG1。两株mAb与HBcAg、HCVRNA阳性血清及HIVgp4 1等均无交叉反应 ,只与HIV 1p2 4抗原阳性血清产生特异反应。结论 :成功地建立了 2株可分泌抗HIV 1p2 4mAb的杂交瘤细胞 ,为进一步研制HIV 1p2 4抗原的ELISA检测试剂盒奠定了基础     

抗巨细胞病毒早晚期抗原单克隆抗体制备及在病毒培养物鉴定中的初步应用

目的 制备抗CMV早晚期抗原单克隆抗体并在病毒培养物鉴定中初步应用.方法 CMV感染MRC-5细胞24h、48h和72h后甲醛灭活的可溶性抗原免疫BALB/c小鼠,小鼠脾细胞与骨髓瘤细胞NS-1进行融合.酶免法筛查阳性杂交瘤并有限性稀释法进行克隆.免疫荧光及印迹对筛选后的克隆进行鉴定,最终获得的杂交瘤制备腹水并使用Protein G进行纯化.纯化后单抗应用于CAP室间质评标本以及临床标本CMV培养物的鉴定.结果 3只小鼠脾细胞与骨髓瘤细胞融合后初步筛查出约110株阳性克隆.剔除与其它抗原有交叉反应、抗体效价低、非全覆盖早晚期抗原的克隆最终获得抗CMV单抗杂交瘤1株,即23B5-1.免疫荧光显示23B5-1株单抗与CMV感染后3h-120h的MRC-5细胞均反应,即覆盖即刻、早期和晚期抗原.免疫印迹试验显示23B5-1株单抗与CMV抗原25KD-50KD之间的5个蛋白条带结合.23B5-1株单抗免疫球蛋白亚型为IgG1.Protein G纯化腹水后的单抗效价≥1∶12800.纯化单抗染色鉴定6份室间质评及10份临床标本CMV培养物全部符合.结论 初步应用显示制备的抗CMV早晚期抗原单克隆抗体性能良好.     

抗HSPC238单克隆抗体的制备和初步鉴定

目的制备抗HSPC238的单克隆抗体，为HSPC238的功能研究奠定基础。方法以纯化的重组蛋白HSPC238为抗原，免疫BALB／c小鼠，运用杂交瘤技术制备HSPC238单克隆抗体，并用间接ELISA法和Western—blot法对单克隆抗体的特性进行鉴定。结果成功建立两株稳定分泌抗HSPC238的单克隆抗体杂交瘤细胞株，分别命名为E001和E002。ELISA检测抗HSPC238单克隆抗体的腹水效价为1：12800和1：25600。两株单克隆抗体的免疫球蛋白亚类均为IgG1。通过Western—blot实验证实，两株单克隆抗体均能特异性结合真核细胞内源性HSPC238蛋白。结论成功制备了两株效价高、特异性好的抗HSPC238单克隆抗体，制备的抗HSPC238单克隆抗体可用于HSPC238蛋白的鉴定，为HSPC238蛋白的生物学功能研究奠定了基础。     

Enhancement of limiting dilution in cloning mouse myeloma-spleen hybridomas by human low density lipoproteins

The limiting dilution technique is a critical step in the cloning of hybridomas for the preparation of monoclonal antibodies. We have found that culture medium supplemented with human plasma low density lipoproteins (LDL) markedly enhanced the yield of hybridoma clones derived from P3 X 63 Ag or FO mouse myeloma cell lines upon limiting dilution. Such enhancement was dependent on the concentration of LDL employed, being optimal at 1-10 micrograms/ml. At LDL concentrations greater than 20 micrograms/ml, the increase in yield of hybrid clones was not significant. The mechanism by which LDL enhances the yield of hybrid clones was partially elucidated by the demonstration that LDL could increase the DNA synthesis of hybridomas as assessed by [3H]thymidine incorporation. The data suggest that LDL play a role in the proliferation of hybridomas. It also indicates that LDL can be utilized for limiting dilution to increase the yield of desired clones. Since LDL is one of the most abundant lipoprotein fractions (approximately 500 micrograms/ml) in human plasma and the isolation procedure is simple, hybridoma culture medium supplemented with human LDL will prove to be a valuable reagent for investigators currently employing monoclonal antibody technology.     

HLA expression on human germinal cells

Ejaculated human seminal plasma cells obtained from 46 healthy men and 48 infertile patients were tested for expression of HLA class I and II antigens by complement-mediated cytotoxicity, cell-binding radioimmunoassay (CB-RIA), and an ELISA involving monoclonal antibodies (mAbs). In a group of healthy men tested for HLA expression on human spermatozoa, 4 of 46 were positive for class I and II HLA antigens. However, the results were negative in a second examination of the same donors, possibly on account of alterations with time in the expression of various subpopulations of cells which are antigen positive or antigen negative. In a group of infertile patients, we found positive expression of HLA class I and II antigens in 7 of 48 men. The samples contained immature and mature sperm but no contaminating leucocytes or epithelial cells.     

Monoclonal antibodies against the GP-2 subunit of laminin

Two stable rat X mouse hybridoma lines have been isolated. These hybridoma lines produce IgG antibodies directed against the polypeptide portion of the GP-2 subunit of laminin. Antibodies produced by the hybridomas have been shown to be IgG 2b (lambda) and IgG 2a (kappa), respectively. In competition ELISA assays the monoclonal antibodies exhibited different binding affinities for laminin. Furthermore, the two antibodies were partially additive in their reactivity to laminin. Preliminary results also indicate that the antibodies recognized different antigenic determinants in laminin as determined by their reactivity to basement membranes in human and mouse tissues. The monoclonal antibody designated LAM-I stained a broad spectrum of human and mouse tissues; the other monoclonal antibody LAM-II reacted with mouse, but not human tissues. The results indicate that these monoclonal antibodies could be utilized to explore the organization of laminin in basement membranes of different tissues and species.     

Increased production of antibodies to spermatozoa and seminal fluid in rabbits used as semen donors

Sera of 20 male rabbits that were used as frequent donors of semen and age-matched normal male and female rabbit controls were examined by an enzyme-linked immunosorbent assay (ELISA), with sperm and seminal fluid as antigens. Five of the 20 semen donors developed an especially high humoral response to seminal fluid and spermatozoa, similar to that observed in some female breeders. The antibodies increased gradually during the 9 months of semen collection. The specificity of the antibodies was demonstrated by total or partial absorption with seminal fluid or spermatozoa. The antibodies were virtually all IgM and were directed against the acrosomal cap region of the spermatozoa, but variable fluorescence was also observed in the postnuclear region and tail.     

Production and characterisation of monoclonal antibodies against native and disassembled human catalase.

Catalase isolated from human erythrocytes was used to immunise mice, in order to generate hybridomas producing specific monoclonal antibodies to the enzyme. Hybridomas secreting anti-(catalase) antibodies were identified by a modified enzyme-linked immunosorbent assay (ELISA) using either monomer/dimer catalase or native, tetrameric enzyme. Three stable hybridoma clones were selected and the characteristics of the antibodies produced were investigated by ELISA, immunofluorescence, immunoprecipitation and immunoblotting experiments. One monoclonal antibody (17E10) was shown to interact with both native tetramer catalase and--to a lesser extent--with monomer/dimer catalase. Two monoclonal antibodies (10B12H9, 13A10) were found to react only with completely denatured catalase or with monomer/dimer catalase but not with native catalase.     

Sperm-immobilizing monoclonal antibody to human seminal plasma antigens.

Rat spleen cells immunized to human azoospermic semen (a mixture of seminal plasma components) and mouse myeloma cells (P3/X63 Ag8U1; P3U1) (Marguilies et al., 1976) were successfully fused with polyethylene glycol (PEG 1500) and 19 of 89 fused cell cultures were found to produce sperm-immobilizing antibody. The cells that produced antibody indicating the highest sperm-immobilizing activity were distributed into wells for further recloning and 10 clones producing sperm-immobilizing antibody were established. The clone (1C4) producing the highest antibody titre was found to produce a large amount of IgG in culture supernatants and to contain a mixture of rat and mouse chromosomes. It was proved by immunodiffusion test that the monoclonal antibody was produced to the human seminal plasma antigen No. 7 which is common to human milk protein. Using this hybridoma which produced a large amount of monoclonal sperm-immobilizing antibody, a new method could be developed for purifying human seminal plasma antigen by immunoaffinity chromatography with bound antibody from the hybridoma.     

Monoclonal antibodies to rat renal antigens

We have developed hybridoma cell lines which secrete monoclonal antibodies to some rat renal antigens, namely the brush border of proximal tubular epithelium and the cytoplasm of tubular cells. The immunoglobulin class of the hybridoma was found to be IgG1. Specific antibody activity against either glomerular basement membrane (GBM) and tubular basement membrane (TBM) or Bowman's capsule and a part of TBM was observed, although these hybridoma cell lines have not yet been successfully established. In particular, the hybridoma secreting antibodies to TBM did not remain stable during antibody production, and was lost during the culture and cloning procedures. These monoclonal antibodies should be of value in research on the pathogenesis of human glomerulonephritis.     

Use of Microimmobilization and Microagglutination Assays for Attempted Detection of HLA Antigens and β2 Microglobulin on Human Sperm

Microtechniques for detecting sperm agglutinating and immobilizing antibodies are described. These assays are proved to be useful in the study of anti-sperm antibodies in the sera of vaseectomized men and the serum of a rhesus monkey immunized with human sperm. However, using various antisera against beta2 microglobulin and HLA region antigens, including Ia antigens, in these assays, very little, if any, activity was found against sperm. Absorption and inhibition tests also could not show a significant amount of these antigens on sperm. Although no HLA region antigens could be detected in the seminal plasma by inhibition tests, a large amount of beta2 microglobulin was found. It is suggested that some beta2 microglobulin could be adsorbed onto sperm, since mouse sperm was shown to pick up beta2 microglobulin after incubation with human seminal plasma. The maximum amounts of HLA region antigens and beta2 microglobulin in the seminal plasma are estimated.