Cell surface detection of HLA-E gene products with a specific monoclonal antibody.

An HLA-E-specific monoclonal antibody (mAb) was obtained by immunization of human beta2-microglobulin transgenic mice (M-TGM) with spleen cells from double transgenic mice expressing HLA-E molecules (EM-TGM). This mAb, designated V16, specifically recognizes in flow cytometry analysis the HLA-E expressing mouse cells, whereas it does not bind to mouse cells expressing various HLA class I molecules (HLA-A2, -A3, -A11, -A26, A29, -B7, -B27, -Cw3, -Cw7, and HLA-G). V16 mAb binds efficiently to human EBV-infected B lymphocytes, PHA blasts and PBL, thus establishing the surface expression of HLA-E in vivo on these cells.     

Immunoradiometric studies with a monoclonal antibody against an estrogen-receptor-related protein in human breast cancer

The concentration of P29, a phosphoprotein related to estradiol receptor (RE), was measured by an immunoradiometric assay in human breast cancer cytosol and the results compared with the levels of RE and progesterone receptors (RP), as measured by the classical ligand-binding assay. Good linear correlation was found between P29 and RE, but not RP. Tumors which were positive for both RE and RP had a higher P29 mean value than RE-negative, RP-negative tumors. Menopausal status of the patients influenced the result, since premenopausal women had lower mean concentrations or were P29-positive less frequently than postmenopausal women. RE-negative, RP-positive tumors tended to have positive P29 levels. The correlation between RE and P29 corroborates results obtained with other techniques and supports a role for P29 in predicting response to hormonal treatment.     

8-1A, a human monoclonal antibody that reacts with intact human chorionic gonadotropin

The incidence of choriocarcinoma has decreased over time and therapeutic results have improved about 90% complete remission in patients without extensive metastasis. However, some choriocarcinomas metastasize to other organs and show resistance to chemotherapy, having a poor prognosis despite multidisciplinary treatment. Better methods of early diagnosis for recurrence or micrometastasis, and treatment against cases with intractable gestational trophoblastic neoplasia (GTN) are needed to improve the prognosis. Human chorionic gonadotropin (hCG) is a glycoprotein hormone composed of two dissimilar subunits and a tumor marker to make a diagnosis and monitor therapeutic effect in GTN. Even when hCG levels in the serum become too low to measure with the hCG beta-CTP system which is the most sensitive assay, there are estimated to be approximately 10,000 trophoblastic cells in the body. Residual trophoblast cells may cause symptoms such as bleeding or undergo malignant transformation to choriocarcinoma. Since most monoclonal antibodies developed so far are murine, administration creates human anti-mouse antibodies, resulting in clinical failure. More recent mouse/human chimeric antibodies or humanized antibodies still possess substantial immunogenicity that makes repeated administration difficult. In the present study, KM mice that can produce completely human monoclonal antibodies were used to prepare hCG-specific human monoclonal antibody. This yielded 8-1A, a human monoclonal antibody capable of reacting with intact hCG. In the future, new diagnostic techniques and treatments for chorionic diseases may be developed using this kind of human monoclonal antibody.     

Sex selection with monoclonal H-Y antibody

H-Y antigen defined by antibody from male-sensitized female mice has been reported in male embryos of mouse, rat, cattle, goat, pig, and sheep. We now describe the use of monoclonal H-Y antibodies in identification of male and female during embryo transfer in cattle. Monoclonal H-Y antibodies were applied with fluorescein-isothiocyanate-conjugated goat antimouse immunoglobulin (Ig), and the embryos were scored for fluorescence under ultraviolet light. In alternative trials with 149 embryos, the method could be applied with 73% to 82% efficiency. Pregnancy rates for treated embryos were at least as high as those expected for untreated embryos. The calves that developed from the treated embryos are normal and growing.     

Immunohistochemical detection of progesterone receptor in formalin-fixed and paraffin-embedded breast cancer tissue using a monoclonal antibody

Summary The potential for immunohistochemical detection of progesterone receptors (PRs) in routinely formalin-fixed and paraffin-embedded cancer tissues by use of the monoclonal antibody Mi 60-10 (mPR1, Dianova GmbH, Hamburg) was evaluated. The PR content of breast cancer tissue was investigated in 170 cases. A positive reaction to Mi 60-10 was found exclusively in the nuclei of benign or malignant epithelial cells. The distribution of PRs was heterogeneous. Immunohistochemical reaction was scored by multiplying the percentage of positive tumour cells by their prevalent degree of staining (Immunoreactive Score or IRS). The IRS values of formalin-fixed tissues (n=170) were compared with those in snap frozen tissues (n=82), with the PR content assayed by a DCC (dextran-coated charcoal) method (n=170), with histopathological grading according to Bloom and Richardson and with the menopausal status of the patient. There was an acceptable ranked correlation (r=0.74) between IRS in formalin-fixed and paraffin-embedded parts and snap frozen parts of the same carcinoma. A good correlation (r=0.72) was also found, when the semiquantitative results of immunohistochemical PR detection in formalin-fixed and paraffin-embedded tissues were compared to PR concentrations measured by a DCC method in tumor cytosols. There was an 80% concordance between the two methods for qualitative discrimination of PR-negative and PR-positive carcinomas. IRS correlated significantly with the degree of histological differentiation of the tumors (P<0.001) but not with the menopausal status of the women (P>0.05). Storage of paraffin-embedded tissues did not impair PR detection, for up to at least 5 years. Fixation of tissues in formalin only decreased the immunohistochemical detection rate if fixative acted for more than 24 h. Dedicated to the memory of the late Dr. H. Würz     

Preoperative diagnosis of ovarian carcinoma with a novel monoclonal antibody

OBJECTIVE: This study was undertaken to determine whether preoperative radioimmunoscintigraphy of complex ovarian masses with technetium Tc 99m MAb-170 (Tru-Scint AD; Biomira Inc, Edmonton, Alberta, Canada), a murine whole immunoglobulin G monoclonal antibody that has been found to have panadenocarcinoma affinity, would predict surgical findings. STUDY DESIGN: The age range of studied patients was 42 to 83 years (mean, 60.3 years). Planar computed tomographic imaging and single-photon emission computed tomographic imaging were performed at 15 minutes, 6 to 8 hours, and 18 to 24 hours after injection of 1000 MBq technetium Tc 99m MAb-170. Laparotomy was performed within 10 days. RESULTS: Eighteen patients had borderline or invasive ovarian cancers verified by histologic examination. All primary malignancies or deposits (including intrahepatic deposits) yielded positive results on radioimmunoscintigraphic imaging. Radioimmunoscintigraphy was able to identify serosal deposits not seen on computed tomographic or ultrasonographic scans. False-positive localization of the antibody was noted in 6 of the 9 patients with benign pathologic processes. CONCLUSION: It is possible to detect with technetium Tc 99m MAb-170 all patients who have cancer (including sites not seen on computed tomographic or ultrasonographic scan); however, the low specificity (33%) means that patients still require surgical verification of disease.     

Therapeutic effect of a radiolabeled monoclonal antibody on human ovarian cancer xenograft in nude mice

The therapeutic value of 131I-OC125, a radiolabeled monoclonal antibody directed against a human ovarian tumor associated antigen CA125, was examined in an ascites forming intraperitoneal human ovarian carcinoma nude mouse model. Nude mice were injected intraperitoneally with NIH:OVCAR3 cells. Twenty-one days after tumor transplantation, groups of animals were injected intraperitoneally as follows: Group 1 with 200 microCi of 131I-OC125 (n = 20), Group 2 with 200 microCi of 131I (n = 17), Group 3 with 200 microCi of 131I-IgG (n = 21), Group 4 with 60 micrograms of OC125 (n = 18), and Group 5 was left untreated (n = 21). Survival of the tumor-bearing animals was used as the endpoint of the experiment. Mean survivals were found to be 52 +/- 18 days for the 131I-OC125 group, 53 +/- 16 days for the 131I-IgG group, 49 +/- 13 days for the 131I group, 47 +/- 24 days for the OC125 group, and 47 +/- 15 days for the untreated control. These results would indicate no therapeutic advantage of 131I-OC125 over controls in this animal model. However, other approaches using single as well as multiple radiolabeled monoclonal antibodies need to be tested in this model in order to definitely establish the efficacy of this treatment modality.     

Rapid laboratory diagnosis of human cytomegalovirus infection using monoclonal antibody

The monoclonal antibody E. 13, recognized an early nonstructure antigen (68 KD) of human cytomegalovirus (HCMV), was used for early detection of HCMV in cell cultures. The virus antigen could be detected by the indirect immunofluorescence staining (IF) technique 6 hours after inoculation of HCMV AD169 and NTUH871233 strains into human embryonic lung fibroblast (HELF) cells growing in shell vials prepared by low speed centrifugation (700xg) at 25 degrees C for 1 hour. The intensity of fluorescence reached a peak 14 hours after virus inoculation and remained at the same level for 72 hours. The feasibility of application of this technique for rapid detection of HCMV in the clinical virology laboratory was tried. A total of 201 urine specimens requesting HCMV isolation were inoculated into HELF cells using the shell vial culture method. The virus growth in the shell vial cultures was detected by the indirect IF technique with the monoclonal antibody E.13 at 16 to 18 hours postinoculation. The results obtained were compared with those of the conventional tube culture method. The sensitivity and specificity of the test vs the conventional tube culture method for detection of HCMV in clinical specimens were 93.06% and 99.22%, respectively. The average time of virus isolation by the conventional culture method was 10.9 days, whereas final results with the shell vial culture method were obtained in only one day. The above results indicate that detection of HCMV early antigen in infected cells with monoclonal antibody E.13 is a sensitive and reliable method for rapid diagnosis of HCMV infections.     

Production of monoclonal antibody against a newly established human endometrial cancer cell line SNG-II

A cell line designated SNG-II was established from the operation specimen of human endometrial adenocarcinoma, and by means of an immunization procedure using intact SNG-II cells, a monoclonal antibody (Mab) named MSN-1 which reacts immunohistochemically with endometrial cancers was obtained. The cell line grew well without interruption for over 5 years, and, SNG-II cells produced tumors of cell differentiated adenocarcinoma in nude mice. The modal chromosomal number was diploid without a marker chromosome. The production of human chorionic gonadotropin and its beta-subunit, CA-125, tissue polypeptide antigen, and placental proteins such as PP6 and PP7 in SNG-II was confirmed. MSN-1 was of IgM subclass. As the antigenic reactivity was unchanged by trypsin treatment, but lost by periodic treatment, it was suggested that the antigen corresponding to MSN-1 was a carbohydrate sequence. Immunohistochemically MSN-1 reacted with about 70% cases of endometrial adenocarcinoma, but seldom with normal endometrium. Furthermore, the staining pattern of MSN-1 was different in benign cells from that in malignant cells: only the luminal surface of the normal endometrium was positive, whereas the cytoplasma was also stained in many of adenocarcinoma cells.     

Daunorubicin conjugated to a monoclonal anti-CA125 antibody selectively kills human ovarian cancer cells

The present study was designed to test the in vitro efficacy for human ovarian cancer cells of daunorubicin (DNR) conjugated to a monoclonal antibody (OC125). The OC125 antibody specifically binds to the antigenic protein CA125 from human ovarian carcinoma. New analogs of DNR containing various linker groups were conjugated to mouse monoclonal anti-CA125 antibody (DNR-OC125); nonspecific murine IgG1 (DNR-IgG1); or bovine serum albumin (DNR-BSA). The DNR-protein conjugates were all stable for several days in neutral solutions at room temperature. The DNR-OC125 conjugates selectively killed dividing cell populations but not nondividing cell populations of two human ovarian cancer cell lines (SK-OV-3 or OVCAR-3) that express the CA125 antigen. Equivalent concentrations of DNR-IgG1 or DNR-BSA conjugates were neither toxic to the dividing nor the nondividing populations of SK-OV-3 or OVCAR-3 cells. Only those DNR-protein conjugates linked to OC125 were cytotoxic to dividing cell populations of both cell lines. This indicates that cytotoxicity is dependent on OC125 antibody-CA125 antigen binding which concentrates DNR on the ovarian cancer cells. We advance the hypothesis that following antibody-antigen binding, DNR is released from the conjugate and it intercalates in DNA by a mechanism similar to that of the unmodified DNR. The new DNR-OC125 conjugate may be useful for delivering DNR to ovarian tumors that express the CA125 antigen because the drug-antibody conjugates (1) retain the cytotoxic characteristics of the unmodified drug: (2) specifically kill the human ovarian cancer cells that express the CA125 antigen; and (3) are completely stable for days in neutral solutions at room temperature.     

Alpha-adrenergic receptors in human myometrium during pregnancy

The distribution and mechanisms of alpha-adrenergic receptors have been studied in myometrial preparations from women delivered by cesarean section at term. With the use of the radioligand tritiated dihydroergocryptine, the number of alpha-adrenergic receptors was 210 fmol/mg of protein in the uterine fundus and 195 fmol/mg of protein in the lower uterine segment. Competition experiments showed that 60% of the alpha-adrenergic receptors had properties as alpha 1-adrenergic receptors and 40% as alpha 2-adrenergic receptors. In vitro tension studies verified the existence of physiologically active alpha 1- and alpha 2-adrenergic receptors in the myometrial preparations. alpha 2-Adrenergic receptor stimulation resulted in lowered levels of intracellular cyclic adenosine monophosphate. The intracellular cyclic adenosine monophosphate was further reduced by additional alpha 1-adrenergic receptor stimulation, probably secondary to an activation of calcium-calmodulin-dependent phosphodiesterase.     

Identification and characterization of human acrosomal antigen defined by a monoclonal antibody with blocking effect on in vitro fertilization.

A monoclonal anti-human sperm antibody (Mab 1A1) has been produced by fusion of myeloma cells with splenocytes from a BALB/c mouse immunized with in vitro capacitated human spermatozoa. Immunofluorescence studies with Mab 1A1 show that it recognizes an antigen(s) (Ag 1A1) which is located in the acrosome of human spermatozoa. As shown by Western blotting experiments, 1A1 antigen represents a family of proteins with Mr ranging from 20 kDa to 34 kDa. Immunofluorescence observations on epitope exposure and location suggest that during in vitro capacitation of human spermatozoa, Mab 1A1 epitope-bearing molecules are concentrated in regularly arranged granules in the acrosome. After long-term incubation the epitope is exposed on the apical acrosome surface exhibiting a spot-like arrangement. The 1A1 epitope is widely distributed among mammalian species: boar, ram, mouse and rat acrosome is intensively stained by Mab 1A1. The antibody inhibits in vitro fertilization mainly by blocking sperm attachment to and penetration through the zona pellucida when included in the medium for the in vitro fertilization of mouse, porcine and human oocytes.     

HMOCC-1, a human monoclonal antibody that inhibits adhesion of ovarian cancer cells to human mesothelial cells

OBJECTIVES: Ovarian carcinoma is one of the most common gynecologic cancers and shows the worst prognosis since current therapies are not sufficiently effective at achieving and maintaining remission. To develop new treatment, a monoclonal antibody recognizing human ovarian cancer cells was raised in KM mice. METHODS: A human monoclonal antibody targeting RMG-I (an ovarian carcinoma cell line) was established with hybridomas of myeloma cells and spleen cells from KM mice. The immunohistochemical reactivity of various types of ovarian carcinoma and other tumors was investigated. RMG-I cells were treated with N-glycosidase F, NaOH, H(2)SO(4), and Gal NAC-alpha-benzyl to investigate the target antigens by Western blotting. The effect of HMOCC-1 on adhesion of RMG-I cells to cultured human mesothelial cells was also investigated. RESULTS: The new human monoclonal antibody, HMOCC-1, was an immunoglobulin M that recognized ovarian epithelial carcinoma. Immunohistochemical staining revealed HMOCC-1 positivity in 83.2% of ovarian carcinomas. The antigen recognized by HMOCC-1 was apparently a glycoprotein since Western blotting yielded a broad band (34.8-49.1 kDa). HMOCC-1 inhibited the attachment of RMG-I cells to monolayers of cultured peritoneal mesothelial cells in a concentration-dependent manner. CONCLUSIONS: This new human monoclonal antibody reacted with most ovarian cancers tested. The antigen recognized by HMOCC-1 is a glycoprotein located on the cell membrane. Inhibition of the attachment of RMG-1 cells to mesothelial cells by HMOCC-1 suggests a potential role for this antibody in the treatment of ovarian cancer.     

A monoclonal antibody cross-reactive with porcine and human zona pellucida recognized the protein's steric structure

The epitopes recognized by two monoclonal antibodies (Nos. 1 and 2) against solubilized porcine zona pellucida (Zp) were chemically characterized. No. 1 reacts with porcine but not human Zp, whereas No. 2 not only reacts with both but also inhibits the binding of sperms to oocytes in the human system. By Western blotting, both No. 1 and No. 2 were found to react with a glycoprotein in the 55 kDa family of porcine Zp. Treatment of this protein with trypsin, sodium periodate, glycosidases and organic solvents, followed by measurement of its reactivity against No. 1 and No. 2 by competitive inhibition in an enzyme-linked immunosorbent assay (ELISA), found that the epitopes recognized by the two antibodies belonged to the peptide but not carbohydrate portion of the protein. The two epitopes were different in that the one recognized by No. 1 was resistant to protein denaturing conditions, whereas the other recognized by No. 2 was destroyed by such conditions. These results suggested that No. 1 recognized a porcine Zp-specific primary structure, while No. 2 recognized a steric structure of the protein which is similar in both human and porcine Zps.     

Influence of human antimurine antibody on CA 125 levels in patients with ovarian cancer undergoing radioimmunotherapy or immunoscintigraphy with murine monoclonal antibody OC 125

Human antimurine antibody responses interfere with CA 125 antigen determinations by crosslinking the murine antiovarian carcinoma monoclonal antibody OC 125 with the second murine radiolabeled antibody used in the CA 125 radioimmunoassay. Serial CA 125 levels in 22 patients with epithelial ovarian carcinoma undergoing either radioimmunotherapy or radioimmunoscintigraphy with iodine 131-labeled F(ab')2 fragments of OC 125 were followed up for up to 96 weeks after infusion. Fourteen radioimmunoscintigraphy patients received 131I-labeled monoclonal antibody by the intraperitoneal (n = 5) or intravenous (n = 9) route: 10 of 14 had sera drawn at appropriate time points for human antimurine antibody detection; 8 of 10 had 1.3- to 363-fold increases in CA 125; 4 of 8 had detectable human antimurine antibody (18.5 to 22 and 575 to 36 micrograms/ml). Eight radioimmunotherapy patients received 131I-labeled monoclonal antibody by the intraperitoneal route: 8 of 8 displayed an apparent 4.8- to 3725-fold increase in CA 125 levels within 7 to 42 days after monoclonal antibody infusion; 6 of 8 had detectable human antimurine antibody (13 to 4 and 319 to 31 micrograms/ml). Adsorption of immunoglobulin G resulted in a 21% to 98% reduction in CA 125 antigen levels in 4 of 4 patients tested. In patients with demonstrable human antimurine antibody, CA 125 antigen levels obtained by the clinical CA 125 radioimmunoassay are spuriously elevated.     

Reverse passive hemagglutination assay for human chorionic gonadotropin in urine using monoclonal antibody

Aiming to find a urinary hCG immuno-assay which is specific, sensitive and easy to perform, a reverse passive hemagglutination reaction was studied by using sheep red blood cells (SRBC) coupled with monoclonal antibodies (Mab) to hCG. Three Mabs (5D4, 6E4, 2F8) with different specialty were used for the study. Mab 5D4 reacted to hCG, hCG-beta, and LH but not to hCG-alpha. Mab 6E4 reacted to hCG, hCG-alpha and LH, but not to hCG-beta. Mab 2F8 reacted to hCG but not to hCG-alpha, hCG-beta, or LH. All three Mabs were IgG1. SRBC were treated with glutaraldehyde and then with tannic acid. These treated SRBC were coupled with IgG(2mg/ml) of each anti hCG-Mab. For assays, 30 microliters of 1:1 mixtures of two different Mab-coupled SRBC and 30 microliters of standard hCG or urine samples were mixed in wells of microtiter plates and reacted for 60 min at room temperature. Among three different combinations, the couple 5D4-SRBC and 2F8-SRBC were most sensitive and specific for hCG assays and the minimum amount of hCG and LH detected in this combination assays was 12.5 mIU/ml and 800 mIU/ml, respectively. Some clinical data obtained by applying this assay were presented.     

GDA-J/F7 monoclonal antibody: a new marker for sperm cell precursors in human semen

Germ cells isolated from semen of oligospermic human donors were found to react with GDA-J/F7 monoclonal antibody (MoAb). Their reactions with this antibody were demonstrated by using fluorescein activated cell sorter (FACS) analysis and indirect immunofluorescence (IIF) test. In the IIF test, the MoAb recognised an antigen on the surface of the sperm cell precursors (SpP) as well as on mature spermatozoa. The specificity of the antibody reaction with the SpP was further confirmed by immunoelectron microscopy. The MoAb did not react with peripheral blood lymphocytes or polymorphonuclear cells but did show cross-reactivity with monocytes. This antibody therefore provides the first marker for the SpP and could be used as a probe for their distinction from leucocytes. This could have clinical application in seminal analysis.     

Use of indium-111-labeled OC-125 monoclonal antibody in the detection of ovarian cancer

This is a preliminary study to evaluate the utility of using the monoclonal antibody (CO-125) labeled with indium-111 to image recurrences of ovarian cancer. This technique has been investigated in 23 patients with ovarian cancer and the results have been compared with blood OC-125 levels, CT scans, and findings at second-look surgery. Following infusion of 1 mg of F(ab')2 fragments (1-2 mCi 111In), quantitative SPECT and planar imaging was obtained daily for 72 hr along with analysis of serum. The nuclear medicine scans of the tumor site recurrences were technically excellent. When compared to second-look laparotomy, there were 2 true negatives, 2 false positives, 14 true positives, and 2 false negatives by nuclear imaging. CT scans correlated less well with surgery, but serum OC-125 levels correlate more closely with nuclear scans and second-look surgery. Those with multiple small metastatic implants showed a pattern of diffuse uptake which increased with time, whereas those with nodal or larger recurrences showed a more focal uptake. The combination of favorable biodistribution and positive images, especially in patients with normal antigen levels and negative CT scans, suggests a role for OC-125 monoclonal antibody imaging in their clinical management. However, further investigation is needed to determine whether nuclear scans can replace second-look surgery. If it can show that enough 111In-labeled antibody accumulates in the tumor site to justify radioimmunotherapy, then 90Y (a pure beta emitter) could be exchanged for 111In. This is potentially a method of radioimmunotherapy for recurrent ovarian carcinoma.