Shinzo Isojima

Further studies on sperm-immobilizing antibody found in sera of unexplained cases of sterility in women.

Abstract The sperm-immobilization test and Franklin-Dukes and Kibricks sperm-agglutination tests were compared as methods for detecting antisperm antibody in sera of sterile, pregnant, and unmarried women. Positive reactions in the sperm-immobilization test were given by sera of 17.2 per cent of the patients with sterility of unexplained cause and not by those of normal pregnant and unmarried women. Franklin-Dukes and Kibricks tests gave rather higher perceniages of positive sperm-agglutination reactions in normal pregnani women than in cases of sterility of unexplained cause but a very low percentage of positive reactions in unmarried women. The sperm-immobilizing factor found in sera of cases of sterility of unexplained cause were analyzed immunologically. This factor appeared to be an antibody. It was present in the γG and γM fractions, and in most cases it seemed to be an antibody against seminal plasma-specific antigens.

Establishment and characterization of a human hybridoma secreting monoclonal antibody with high titers of sperm immobilizing and agglutinating activities against human seminal plasma.

Peripheral blood lymphocytes isolated from an infertile woman possessing strong sperm immobilizing and agglutinating antibodies were stimulated by culturing with poke-weed mitogen (PWM) and spermatozoa from a healthy donor for 5 days. The stimulated lymphocytes were fused with mouse myeloma NS-1 by PEG-1000. Fused growing hybrid cells were observed in 58 of 96 wells, and 22 of these showed the production of human immunoglobulin. Among the 22, one hybridoma clone (H6-3C4) was found to produce human IgM (lambda) with strong sperm immobilizing and agglutinating activities. The supernatant from the culture medium contained approximately 1.5 microgram IgM/ml and the antibody titers were 5000 SI50 units on sperm immobilization and 1:1600 dilutions on sperm agglutination. The hybridoma H6-3C4 has continuously produced high titers of antibody exhibiting sperm immobilizing and agglutinating activities over 8 months and contains chromosomes of acrocentric type from mouse and metacentric type from human. The monoclonal antibody (Mab) H6-3C4 reacted specifically to human seminal plasma, ejaculated spermatozoa and male accessory gland but not to testis, any other somatic tissues, or secreted fluids tested. Immunofluorescence staining indicated that the antigen corresponding to Mab H6-3C4 was present over the surface of ejaculated spermatozoa. The binding of Mab H6-3C4 to human spermatozoa was blocked by the serum of the patient from whom the lymphocytes were obtained for cell fusion.

Monoclonal antibodies to porcine zona pellucida antigens and their inhibitory effects on fertilization

Five hybridomas which produce monoclonal antibodies (Mabs) to porcine zona pellucida (ZP) were established. The immunoglobulin classes were IgG2a from hybridoma B11C8 and IgM from the other four. Using immunofluorescent staining, the Mab from B11C8 stained only porcine oocytes, the Mab from G10G5 stained oocytes of pigs and hamsters but Mabs from C6H1, D3H4, G10F9 stained oocytes of pigs, humans, hamsters, rats and mice. Mabs from B11C8 and G10F9 strongly blocked boar sperm binding to porcine ZP but other Mabs produced only slight blocking. However, no Mabs could block sperm penetration during in vitro fertilization (IVF) of hamster oocytes. When a goat antibody (IgG) to mouse serum gamma-globulin was applied after each Mab as a second antibody, only Mab from G10G5 impaired IVF of hamster oocytes.

Production and characterization of monoclonal antibodies to cross-reactive antigens of human and porcine zonae pellucidae.

Three monoclonal antibodies (Mabs), 3A4-2G1, 1D5-2B7 and 1F2-1B8 were produced against heat-solubilized porcine zona pellucida (ZP). Each Mab stained intact ZP but no other pig tissues using immunofluorescence staining. All three Mabs stained selectively zonae pellucidae (ZPe) from pigs and humans but not from hamsters, rats or mice, and showed no inhibitory effect on sperm binding to human oocytes. When goat antiserum to mouse gamma-globulin was added to human oocytes pre-treated with 3A4-2G1 or 1D5-2B7, sperm binding to oocytes was completely blocked with formation of immune precipitates around them. SDS-PAGE analysis of the immune precipitates of 125I-labeled porcine zona proteins and Mab showed that the antigen binding 3A4-2G1 was mainly composed of components with approximate molecular weights of 92,000, 65,000 and 23,000 and the antigen binding 1D5-2B7 contained two components with approximate molecular weights of 57,000 and 49,000, respectively. The epitope of ZP antigen, corresponding to 3A4-2G1, was found to be present in the molecule of 92,000 daltons as demonstrated by enzyme immunostaining of the proteins after blotting to nitrocellulose membrane from SDS-PAGE gels.

Localization of human seminal plasma No. 7 antigen (Ferrisplan) in accessory glands of male genital tract and spermatozoa.

The establishment of a hybridoma (1C4) producing sperm immobilizing monoclonal antibody to human seminal plasma No. 7 antigen (HSP No. 7 Ag.) and the isolation of the pure antigen by immunoaffinity chromatography bound monoclonal antibodies have been reported previously. In the present investigation, HSP No. 7 Ag. has been termed Ferrisplan and its distribution in male genital organs, spermatozoa and body fluids has been studied. The amount of Ferrisplan in the body fluids was determined by radioimmunoassay. Large amounts were detected in seminal plasma and milk, trace amounts in saliva, and none in the serum and urine. The concentration of Ferrisplan was highest in the seminal plasma of azoospermic patients and gradually decreased from oligospermia to normospermia. Using an immunofluorescent method with anti-Ferrisplan monoclonal antibody, strong staining was observed on the epithelial layers of human seminal vesicles, no staining on testes and bright staining on the post-nuclear cap and mid-piece segment of spermatozoa. These results indicate that Ferrisplan is excreted mainly from the seminal vesicle and adheres to the post-nuclear cap and mid-piece of the spermatozoa as a sperm-coating antigen.

Immunogenicity of a 92kDa component of porcine zona pellucida isolated using a monoclonal antibody (3A4-2G1) exclusively cross-reactive with porcine and human zonae pellucidae

Three monoclonal antibodies (Mabs) to solubilized porcine zona pellucida (s-PZP) were produced. They reacted exclusively to porcine and human zonae pellucidae (ZPe) and two of them blocked sperm penetration of ZP in humans by collaborating with goat anti-mouse gamma-globulin serum as a second antibody. The antigen corresponding to one of these Mabs (Mab 3A4-2G1) was fractionated from s-PZP by immunoaffinity chromatography bound Mab 3A4-2G1. The antigen corresponding to Mab 3A4-2G1 was further fractionated by SDS-PAGE and the protein band corresponding to 92kDa was found to react to Mab 3A4-2G1, as demonstrated by Western blotting. The 92kDa fraction of s-PZP was isolated and injected into mice with complete and incomplete Freunds adjuvants to obtain an antiserum. It was shown that the antiserum to the 92kDa molecule reacted in a tissue-specific manner to human ZP and exhibited a strong inhibitory effect on sperm penetration of ZP of human oocytes.

Stable production of recombinant human sperm immobilizing antibody using cDNA expression vectors.

PROBLEM: Sperm immobilizing antibodies present in sterile women may be one of the principal causes of immunological infertility. We already established cell lines that secrete recombinant human IgG sperm immobilizing antibody using class‐switched (from IgM to IgG) genomic immunoglobulin genes. However, these transfectants produced a small quantity of antibody and required continuous use of a medium with selective reagents. We have now constructed cell lines that stably secrete the antibody in large quantities using immunoglobulin cDNAs and cDNA expression vectors.

Microimmunofluorescence Using Terasaki Plates and Direct Plate Freezing Method—Rapid and Reliable Screening System of Hybridomas

Microimmunofluorescence using Terasaki plates and a direct plate freezing method were combined for effective screening of hybridoma supernatants. The microplates, in which the fused cells (myeloma and spleen cells) were cultured and hybridoma colonies were growing, were frozen after harvest of supernatants and saved at −80 C for several weeks without affecting antibody production ability of hybridomas. Microimmunofluorescence was performed in Terasaki plates on which target cells were attached by poly‐l‐lysine and glutaraldehyde or by short time culture of the cells in Terasaki plates. The direct plate freezing method prevented initial hybridoma cells from changes or disappearance of antibody productions during screening of hybridoma supernatants; the microimmunofluorescence staining method permits fast and detailed estimation of specificity of antibodies of hybridomas by saving time and minimal consumption of supernatant for checking. The combination of these two methods is a powerful tool for obtaining desired monoclonal antibodies.

Establishment of a murine monoclonal antibody against human clear cell carcinoma and analysis of the corresponding antigen.

A murine monoclonal antibody (Mab) 4B6, immunoglobulin M (IgM), lambda chain, against human clear cell carcinoma of the ovary was established. Mab 4B6 reacted specifically to clear cell carcinomas, but failed to react to other types of ovarian carcinomas, such as mucinous, serous carcinoma, and also failed to react to human normal organ tissues. Mab 4B6 recognized antigenic determinants located on the membranes of carcinoma cells. Antigenic substances corresponding to Mab 4B6 were detected in sera of patients with ovarian clear cell carcinoma by using Sandwich radiometric assays performed with Mab 4B6-coated microplates and 125I-Mab 4B6. Gel filtration on Sephacryl S-300 revealed that the antigen corresponding to Mab 4B6 possessed a molecular weight of 50 to 60 kDa. Furthermore, after periodic acid and enzyme treatments, it was suggested that the antigen epitope corresponding to Mab 4B6 contains a carbohydrate moiety.

Urinary hCG Excretion Patterns of Chorionic Tumor Patients during and after Chemotherapies

In the treatment of chorionic tumor patients, the most difficult point for physicians and surgeons was to know when the chemotherapies should be finished. Many chorionic tumor patients in treatments were examined of daily urinary hCG radioimmunoassay using polymerized anti-hCG serum and the following patterns were discovered. i) Urinary hCG after delivery of hydatidiform mole usually dropped to LH level (150 IU/24 hrs) within 4 weeks, when patients received three successive intra uterine curettages with one week interval. ii) During or within 4 days after a course of chemotherapy, the temporary rises of urinary hCG were found in many cases (Cellular Effect). When the chemotherapies were discontinued eventhough the Cellular Effect was positive, the diseases were recurrent. iii) When daily assays of low titers of urinary hCG were carried out, the intermittent sharp rises of urinary hCG were found in some patients, though the hCG level were beeing kept in LH level. We call this phenomenon as Intermittent Peak and it was found that the recurrence did occur in patients with Intermittent Peak when the chemotherapies were discontinued. iv) In the course of hCG dropping by chemotherapies, the hCG titer showed large daily fluctuation. Thus, when the hCG titers dropped close to LH level, daily assay of low titer became necessary to prevent the errors. v) From the above findings, the remission of the disease must include, at least, the following conditions; a) the fall of urinary hCG to LH level, b) no cellular effect by chemotherapy, c) no hCG intermittent peak. Even the above three conditions could be seen in patients after treatments, further two courses of chemotherapies should be given. vi) Eventhough the urinary hCG did not drop to LH level over a long period of chemotherapies, therapy should be continued while the daily large fluctuation of hCG could be seen, because there were cases of hCG dropping to LH level in 10 months following repeated chemotherapies. (See pp. 1424,,,1435)