Anti-tumor x anti-CD3 heteroconjugates direct human peripheral blood lymphocytes to lyse colon tumor cells

T lymphocytes normally recognize antigens through the antigen receptor complex (TCR/CD3) but can be redirected to bind and lyse unrecognized tumor cells by anti-tumor X anti-CD3 heteroconjugates. Chemical coupling of an antibody directed against the T cell receptor complex and an antibody directed against tumor antigen produces a conjugated antibody that activates the T cell lytic mechanism and bridges the T cell and tumor cell. We tested the lytic activity of heteroconjugate-treated cultured human peripheral blood lymphocytes (PBLs) in 4-h radioactive chromium release assays with human colon tumor cell targets. PBLs were enriched for T cells by the depletion of Leu11a+ and Leu19+ cells, prior to culture in rIL-2 and anti-CD3. Cultured human PBLs depleted of Leu11a+ and Leu19+ cells produced low levels of tumor cell lysis in the absence of antibodies. Anti-tumor X anti-CD3 heteroconjugates significantly enhanced tumor cell lysis by cultured PBLs when tested against four different colon tumor cell lines (P less than 0.005), but, heteroconjugates in the absence of PBLs did not augment tumor cell lysis. Cultured PBLs treated with monoclonal anti-tumor antibody, with monoclonal anti-CD3 antibody, or with irrelevant heteroconjugate did not enhance tumor cell lysis. We conclude that heteroconjugate-directed lysis is mediated by PBLs and that both the anti-tumor antibody and the anti-CD3 antibody are essential for heteroconjugate function. In addition, heteroconjugate-enhanced tumor cell lysis is mediated through a mechanism other than antibody-dependent cellular cytotoxicity or nonspecific T cell receptor crosslinking.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibodies to human urothelial cell lines and hybrids: production and characterization

Eleven independent monoclonal antibodies, the LBS series, were isolated after immunization of mice with RT112 cells, a continuous cell line derived from a transitional cell carcinoma of the human bladder. These antibodies were tested by indirect immunofluorescence on a panel of 28 human cell lines, of which 17 were urothelial carcinoma-derived, 4 of non-urothelial carcinoma origin, 3 fibroblast cell lines, 4 lymphoblastoid lines and 7 murine cell lines. Also tested were 7 somatic cell hybrid clones derived by fusion of human RT112 cells with murine bladder carcinoma MB63T/H cells. None of the LBS antibodies reacted with mesenchyme-derived cells, although all reacted with RT112 cells. On the basis of reactivity with the cell line panel, the antibodies were divided into 3 groups. Group I (LBS-1 and 19) reacted with all human epithelium-derived cell lines. Group II (LBS-2, 8, 15 and 17) reacted only with human urothelium-derived cells, tending to recognise the least anaplastic types. Group III antibodies (LBS-10, 20A, 20B, 21 and 34) were urothelium-specific on the human continuous cell line panel, but additionally reacted with murine urothelial and epithelial cell lines. The 6 human-specific antibodies (Group I and II) were used for preliminary analysis of human gene expression in a series of 7 mouse X human urothelial somatic cell hybrids. Each hybrid reacted with at least 1 LBS antibody, although there were changes in gene expression with time in culture, indicating both loss and unmasking of human genes. These data suggest the LBS-series antibodies recognise different determinants associated with epithelial and urothelial cell differentiation, and thus may be valuable probes in the study of normal differentiation and malignant transformation in human urothelial cells.     

Metastatic potential of colon carcinoma. Expression of ABO/Lewis-related antigens

Four monoclonal antibodies specific to various Lewisx-related antigens were tested for binding to monolayers and detergent extracts of the human colon carcinoma cell line HT-29 P and its metastatic variant HT-29 LMM. Only monoclonal antibody FH6 (antisialyl-dimeric Lex) bound more to the metastatic variant compared with HT-29 P. Detergent extracts of human tissues were then assayed for their FH6-binding activity. Normal colonic mucosa displayed less FH6 binding than either Dukes stage B or D primary colon carcinomas. Liver metastases from colorectal carcinoma demonstrated greater binding than stage B and stage D primary tumors, and more than normal liver. Expression of sialyl-dimeric Lex increases as the metastatic potential of colorectal carcinomas increases, and is related to the progression of colorectal cancer.     

Human monoclonal antibody constructed by lymphocytes from pancreatic cancer patients

The lymphocytes from pancreatic cancer patients were fused with human lymphoblastoid cell line H0323 and three hundred and twenty eight human hybridomas were constructed. Among them, human monoclonal antibody OC2D (IgM) reacted with two of seven pancreatic cancer cell lines and two of four colon cancer cell lines and one hepatocellular cancer cell line, but not with four normal cell lines. OC2D reacted rather specifically with malignant tissues such as pancreatic cancer, colon cancer and gastric cancer by immunoperoxidase staining. Antigen recognized by OC2D appeared to be non-secreting and was mainly in cell surface. Various chemical studies showed that its epitope was a carbohydrate structure which did not contain sialic acid. These results suggested that human monoclonal antibody OC2D was useful for an imaging and targeting therapy.     

A new series of monoclonal antibodies against pancreatic cancer cells

A new series of monoclonal antibodies (Span 1-7) was produced by immunizing mice with SW 1990 human pancreatic cancer cells. Span 1-4 antibodies (Ab) reacted with 4-5 of 8 pancreatic cancer cell lines tested and with 5-6 of 9 colon cancer cell lines and some lung cancer cell lines. Span 1-4 antigens (Ag) were detected not only on cell surface but also in cultured spent medium of SW 1990 cells by ELISA. They were also found in the fractions of a cesium chloride gradient of SW 1990 xenograft homogenates which have the highest molecular weight, density and carbohydrate content. Their immunoreactivity is dependent upon sialic acid because prior digestion with neuraminidase abolished their immunoreactivity. Span 5,6,7 Ab reacted with only 3 of 8 pancreatic cancer cell lines tested and did not reacted with any other cell lines such as colon cancer, lung cancer and melanoma. The epitopes which were recognized by Span 5,6,7 Ab did not contain sialic acid. These results suggest that Span 1-4 Ab has potential application in the detection of gastrointestinal cancers and that Span 5,7 may be useful to detect the origin which is unknown by using immunohistochemistry method for metastatic lesions.     

Generation of human monoclonal antibodies against colon cancer

Lymphocytes from regional lymph nodes of patients with colon cancer were fused with a human lymphoblastoid cell line with or without in vitro immunization. The efficacy of these two protocols for the generation of human monoclonal antibodies against colon cancer was investigated. The hyperplastic lymph nodes adjacent to the tumor were the best source of B lymphocytes. Fusion frequency and the number of tumor-reactive clones were markedly increased when the in vitro immunization protocol was applied prior to fusion. As a stimulant in in vitro immunization, the supernatant of pokeweed mitogen-stimulated T lymphocytes was superior to the supernatant of mixed lymphocytes culture. Carcinoembryonic antigen at 20 micrograms/L seemed to be the optimal dose for in vitro immunization. The reactivities of human monoclonal antibodies thus generated were measured by enzyme-linked immunosorbent assay and confirmed by immunoperoxidase staining. Combining in vitro immunization with lymphocytes of cancer patients may lead to the successful production of clinically useful human monoclonal antibodies.     

Development and characterization of a monoclonal antibody to human embryonal carcinoma

A monoclonal anti-testicular carcinoma antibody was obtained via the somatic cell fusion technique by immunization of BALB/c mice with freshly prepared single cell suspension from a patient with testicular embryonal carcinoma with choriocarcinoma components. The hybridoma supernates were screened against the testicular carcinoma cells used in the immunization as well as normal mononuclear white blood cells isolated from the same patient. An antibody (5F9) was selected which bound to fresh tumor cells from two patients with embryonal testicular carcinoma and failed to bind to fresh tumor cells from 24 patients (2 seminoma, 2 melanoma, 3 neck, 2 esophageal, 1 ovarian, 3 colon, 1 prostate, 2 breast, 1 liposarcoma, 3 endometrial, 1 kidney, 1 adrenal, 1 larynx and 1 bladder tumors) or cell suspensions prepared from normal liver, lung, spleen, ovary, testes, kidney, red blood cells or white blood cells. The antibody was tested for its binding to several well established cancer cell lines, and was found to bind to the BeWo human choriocarcinoma and two human embryonal carcinoma cell lines. The antibody did not react with 22 other cell lines or with hCG. The antibody was labeled with 131I and injected into nude mice bearing BeWo tumors and evaluated for tumor localization by performing whole body scans with a gamma camera 5 days later. Six mice injected with the antibody showed positive tumor localization without the need for background subtraction while six mice injected with MOPC-21, a murine myeloma immunoglobulin, demonstrated much less tumor localization. Tissue distribution studies performed after scanning showed specific tumor localization (8:1 tumor: muscle) for the monoclonal antibody and no specific localization for MOPC-21. This antibody thus has selective reactivity with the surface of tumor cells from embryonal carcinoma (testicle) and choriocarcinoma both in vitro and in vivo.     

A tumor-associated antigen in the scirrhous gastric carcinoma cell line MK-01 defined by monoclonal antibody S202

A monoclonal antibody (MAb) S202, with an IgG₁ isotype, that reacted strongly with the scirrhous gastric carcinoma cell line MK-01 was established. MAb S202 reacted with the colonic cancer cell line, SW116, and the pancreatic cancer cell line, PK-1, when tested by indirect immunofluorescence. The S202 reactive antigen was expressed in the majority of acetone-fixed fresh frozen cancer tissues. Eighty to 100 per cent of the paraffin-embedded sections of stomach, colon and pancreatic adenocarcinoma were positive for the S202 antigen, with diffuse cytoplasmic staining, whereas esophageal and breast cancers demonstrated markedly less immunostaining. Supplemented serum-free medium collected from 7 day old tumor cell cultures were assayed for the presence of antibody-defined antigens. Antigens detected by MAb S202 were released by the cell lines SW1116 and PK-1. The binding of MAb S202 to the colonic adenocarcinoma sections was reduced after treatment with sodium periodate which suggests that respective antigenic determinants are of carbohydrate nature without sialic acid residues.     

A tumor-associated antigen in the scirrhous gastric carcinoma cell line MK-01 defined by monoclonal antibody S202

A monoclonal antibody (MAb) S202, with an IgG1 isotype, that reacted strongly with the scirrhous gastric carcinoma cell line MK-01 was established. MAb S202 reacted with the colonic cancer cell line, SW1116, and the pancreatic cancer cell line, PK-1, when tested by indirect immunofluorescence. The S202 reactive antigen was expressed in the majority of acetone-fixed fresh frozen cancer tissues. Eighty to 100 per cent of the paraffin-embedded sections of stomach, colon and pancreatic adenocarcinoma were positive for the S202 antigen, with diffuse cytoplasmic staining, whereas esophageal and breast cancers demonstrated markedly less immunostaining. Supplemented serum-free medium collected from 7 day old tumor cell cultures were assayed for the presence of antibody-defined antigens. Antigens detected by MAb S202 were released by the cell lines SW1116 and PK-1. The binding of MAb S202 to the colonic adenocarcinoma sections was reduced after treatment with sodium periodate which suggests that respective antigenic determinants are of carbohydrate nature without sialic acid residues.     

Murine hybridoma antibodies against human transitional carcinoma-associated antigens

Spleen cells from mice immunized with the human urinary bladder transitional cell carcinoma cell line 647V have been fused with a syngeneic myeloma cell line to produce hybridomas. Screening of supernatants from 40 hybridomas which reacted with the immunizing cell line identified antibodies recognizing a variety of common, shared and tumor-associated antigens as well as newborn calf serum dependent antigens. Three hybridoma antibodies, 9A7 , 2E1 and 2A6 , recognize antigens found on all the human transitional cell carcinoma cell lines and tissue preparations tested, but the antigens were not found on normal human tissue (including urothelium), thus demonstrating the capability of the antibodies to distinguish normal from malignant bladder transitional epithelium. These antibodies, however, otherwise differ in their patterns of reactivity, with 1 recognizing an antigen which is also expressed on highly anaplastic malignant non-transitional cell carcinoma cell lines and tumors, while the other 2 demonstrate reactivities which are far more restricted to transitional cell carcinoma.     

Development and characterization of a monoclonal antibody which recognizes a new prostate-organ specific antigen]

PURPOSE: Development and characterization of monoclonal antibodies which recognizes a new prostate-organ specific antigen. METHOD: For development of monoclonal antibodies, hybrid cells were prepared by fusion of spleen cells of BALB/c mice immunized with the homogenates of surgically resected prostatic tissue and P 3 x Ag 8 U 1 (P 3 U 1) murine myeloma cells. Supernatants of hybrid clones were primarily screened using an ELISA on human prostatic cancer cell line PC-3 and human bladder cancer cell line T-24. In the secondary screening, they were tested on normal tissues by immunohistochemical staining. To characterize the antigens, biochemical analyses were performed using seminal plasma as an antigen by western blotting and gel filtration, and the reactivity of antibodies were compared with that of antibodies against prostatic acid phosphatase (PAP), prostate-specific antigen (PSA) and gamma-seminoprotein (gamma-Sm). RESULTS: A monoclonal antibody termed KP-9 was obtained and it only reacted with PC-3 and prostate tissues, but did not react with other cell lines and normal tissues. Immunohistochemical staining of prostate tissue revealed that KP-9 stained grandular epithelium and grandular exudate of normal and malignant prostatic tissues, and especially, strongly stained the apical site of grandular epithelium. Western blotting and gel filtration of seminal plasma suggested that the molecular weight of the KP-9 antigen was more than 300,000 and was different from PAP, PSA and gamma-Sm. CONCLUSION: We have developed a monoclonal antibody, KP-9 which specifically reacts with prostatic cancer as well as benign prostatic tissues. The antigen recognized by KP-9 appeared to be a new prostate-organ specific antigen and may be a useful marker for prostatic cancer such as PAP, PSA and gamma-Sm.     

Antibodies to human squamous cell carcinoma

We are studying membrane antigens of human squamous cell cancer with the use of naturally occurring autologous antibodies from patients' sera, along with a set of other serologic reagents and monoclonal antibodies raised against cultured squamous cell lines. Twenty-eight squamous cell carcinoma cell lines have been established in our laboratory from tissues obtained from 23 patients. Antibody reactivity has been found against the autologous tumor cell line in 13 of 23 patients. One of these is of sufficient titer for detailed analysis. Four cell lines are available from this patient. UM-SCC-17A is derived from the primary laryngeal carcinoma, and UM-SCC-17B is derived from a lymph node metastasis removed during the same surgical procedure. Fibroblasts have been cultured from normal mucosa, and a B-lymphoblastoid line has been developed by Epstein-Barr virus transformation of the patient's peripheral blood lymphocytes. Antibody from this patient reacts with the UM-SCC-17A and -17B tumor cell lines but does not react with the normal fibroblasts (UM-NF-17).     

Increased expression of the epidermal growth factor receptor on human colon carcinoma cells

Epidermal growth factor (EGF) is a small polypeptide hormone that promotes the growth of cells in culture and elicits the differentiation of epithelial tissues in vivo. The effect of EGF is mediated by a transmembrane receptor that is expressed in increased amounts on some tumor cells. We have used a monoclonal antibody to the EGF receptor to detect increased expression of the receptor on human colon carcinoma cells. All eight of the moderately well-differentiated colon carcinoma cell lines tested and several frozen colon carcinoma tissue sections showed increased expression of the EGF receptor, while five poorly differentiated colon carcinoma cell lines and normal colon tissue sections did not. Increased expression of the EGF receptor on moderately well-differentiated colon carcinoma cells but not on poorly differentiated colon carcinoma cells was also demonstrated by western transfer and iodine 125-labeled EGF binding assays. Increased expression of the EGF receptor on moderately well-differentiated colon carcinoma cells seems to be a useful marker for the differentiation of human colon carcinoma cells. In addition, it might provide a site for adjuvant hormonal therapy or immunotherapy.     

Induction of cytotoxicity from human lymphocytes coated with bispecific antibody against human glioma cells

A bifunctional hetero-F (ab') 2 fragment containing the Fab portions from anti-CD3 and anti-glioma monoclonal antibodies was prepared. The antibody simultaneously recognized two different molecules, the CD3 complex on effector T cells and human glioma-associated antigens on target glioma cells. This bispecific F (ab') 2 fragment induced peripheral blood lymphocytes (PBLs) from healthy donors to lyse cells of the human glioma cell line, U251MG, that is resistant to natural killer cell-mediated cytolysis. Compared with lymphokine-activated killer (LAK) activity which is obtained by exposure to interleukin (IL)-2 for more than 3 days, the maximum bispecific antibody-dependent cytotoxicity can be generated only after 24 hour exposure to IL-2. And cytotoxicity of lymphocytes triggered by the bispecific antibody was dependent upon the concentration of IL-2 in the culture medium. The effect of the bispecific antibody on LAK cells was tested in patients suffering from malignant glioma. One patient who received specific targeting therapy (LAK plus bispecific antibody) showed the disappearance of high density tumor mass from CT scan. But the patient who received only LAK therapy showed the recurrence of tumor one year after LAK treatment. These are preliminary data, but may be a promising approach in cancer immunotherapy.     

抗PSA/抗CD3两种双特异性单链抗体的活性研究

目的:研究并比较已成功构建的抗PSA/抗CD3两种双特异性单链抗体的生物学活性。方法:利用流式细胞仪和51Cr释放试验评价抗PSA/抗CD3双特异性单链抗体的抗原亲和活性和体外介导杀伤靶细胞的效果。建立前列腺癌裸鼠模型,分为非治疗组、对照组、双特异性单链抗体(BsAb)组和多价双特异性单链抗体(mBsAb)组,分析两种双特异性单链抗体在体内介导细胞毒T细胞对肿瘤细胞杀伤的能力。结果:流式细胞仪结果显示:BsAb与LNCaP细胞和Jurkat细胞的阳性结合率分别为56.3%和55.4%;mBsAb与LNCaP细胞和Jurkat细胞的阳性结合率分别为74.0%和83.0%。在体外,有细胞毒T细胞存在时两种抗体均可引起前列腺癌细胞的裂解,裂解效率分别与抗体浓度和T细胞与靶细胞比例呈正相关。与对照组比较,接种前列腺癌细胞的裸鼠在体内注射激活的细胞毒T细胞的同时分别接受两种抗体的治疗后,肿瘤生长均明显受到抑制(P<0.05)。在亲和力、体外杀伤和体内抑制肿瘤生长等方面,mBsAb的活性明显优于BsAb。结论:抗PSA/抗人CD3 BsAb及其四聚体均具有良好的生物学活性,单链抗体四聚体的形成可以明显改善抗体的生物学活性。     

Monoclonal antibody Due ABC 3 directed against transitional cell carcinoma. I. Production, specificity analysis, and preliminary characterization of the antigen.

The development of the hybridoma technology allows the identification of tumor associated antigens with monoclonal antibodies (mAbs). Employing this technology mAb Due ABC 3 was obtained by immunization of a BALB/c mouse with bladder tumor cell line SW 1710 and subsequent cell fusion of spleen cells with P3. X63.Ag8.653 mouse myeloma cells. MAb Due ABC 3, an IgM antibody, was found to recognize an antigen present in the membrane of tumor cells in 25 out of 28 (89%) transitional cell carcinoma specimens but rarely (three out of 25 specimens, 12%) on normal urothelial cells. Cross reactions were seen with proximal tubular epithelium of the kidney and seven out of 12 renal cell carcinomas examined. Furthermore, the antigen was expressed by granulocytes, some gastrointestinal epithelia, ovarian and breast carcinoma. The antigen recognized by mAb Due ABC 3 was stable to fixation with formaldehyde and paraffin emmbedding, different proteases, alkaline treatment and heat exposure up to 70C. Antigenicity was abandoned by incubation with periodate but not with neuraminidase treatment. The antigen could be extracted with chloroform/methanol suggesting the involvement of a glycolipid. Immuno-thin layer chromatography revealed a single lipid band reacting with mAb Due ABC 3 but not with anti-CD15, directed against the Lewis X antigen. Although not tumor-specific, mAbs directed against differentiation antigens may be of value for the investigation of cell transformations as well as for diagnostic use.     

Human glioma-specific antigens detected by monoclonal antibodies.

Three murine monoclonal antibodies, designated GA-17, GB-4, and GC-3, were prepared by the hybridization of murine myeloma cells (NS-1) and spleen cells of BALB/c mice immunized with the crude membrane fraction of cultured human gliosarcoma cells (GI-1). Two of them (GA-17 and GB-4) reacted exclusively with the membrane of glioma cells, and the other (GC-3) reacted with the membrane of glioma cells and a T cell line (MOLT-4). Although these antibodies reacted with almost all of the gliomas, the reactions differed. GA-17 reacted equally well with all glioblastoma (17 cases) and low-grade astrocytoma (10 cases), whereas GB-4 reacted poorly with 7 cases of glioblastoma and GC-3 did not react with 7 cases of low-grade astrocytoma. The antigens, exclusively expressed on the cell surface, were analyzed by surface labeling with 125I followed by a cell lysis and immunoprecipitation with these antibodies. The findings obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that GA-17, GB-4, and GC-3 reacted with Mr 140,000-145,000, Mr 160,000, and Mr 145,000-150,000 proteins, respectively. Some evidence has been obtained indicating that these antigens are composed of the same polypeptide chain (Mr 120,000) with the carbohydrate chains being different.     

Tumor-specific monoclonal antibodies for renal cell carcinoma

Monoclonal antibodies, tumor-specific for renal cell carcinoma (RCC), were produced in Balb/C mice, hyperimmunized with tumor cell suspensions from a histological grade II tumor. Boosting with lectin-immobilized tumor-antigen rendered high yields of specific antibody-producing hybrids. Hybridoma supernatants were screened for specificity using a solid-phase enzyme-linked immunoassay. Testing in parallel for reactivity with tumor tissue and corresponding autologous normal kidney material, only those hybrids producing antibodies exclusively reactive with RCC were propagated, resulting in 4 stable, highly productive subclones. Using the immunoperoxidase staining technique, tissue sections from 97 different RCC specimens and corresponding normal kidneys were evaluated for reactivity with the monoclonal antibodies. Over 90% of RCC were strongly positive, whereas normal kidney tissue did not react. Other normal human organ sections, including pancreas, liver, lung, stomach, small intestine, spleen, lymph node, arteries, veins, skeletal muscle, heart, skin and fetal tissues were negative for tumor-associated antigens. Mucous substances and Panet's granular cells in colon mucosa showed nonspecific binding suppressible by addition of normal human serum. Adenocarcinoma of the stomach, colon, pancreas or breast did not demonstrate cross-reactivity with the antibodies to RCC. These monoclonal antibodies apparently recognize a tumor-associated antigen possibly specific for RCC. They could prove to be potent tools in the search for specific tumor markers applicable in the early diagnosis of the disease and immunotargeting cancer therapy.     

A human monoclonal antibody to insulin

To elucidate the immune aspects of insulin-dependent diabetes mellitus (IDDM), we attempted to generate human monoclonal anti-insulin antibodies by fusing peripheral blood lymphocytes obtained from 10 insulin-treated IDDM patients with cells from a human lymphoblastoid cell line. Hybridomas that secreted immunoglobulins appeared in 9 of 400 wells. One of these hybridomas secreted anti-insulin antibody of the IgM class. The lymphocytic partner of this hybridoma was obtained from an IDDM patient who had undetectable levels of antibodies to insulin in his serum. Thus, by employing the hybridoma technique, it was possible to reveal the presence of insulin-sensitized B-lymphocytes in a patient who was serologically negative for anti-insulin antibodies. The monoclonal antibody recognized intact human insulin and insulins of other species, but not isolated A- and B-chains. This indicates that the antibody was functionally an autoantibody directed to an epitope formed by the native conformation of a highly conserved portion of the insulin molecule. This is the first report of a human hybridoma antibody to insulin.     

Characterization of neuroectodermal antigen by a monoclonal antibody and its application in CSF diagnosis of human glioma

Monoclonal antibodies were produced by immunization of the human glioma cell line SK-MG-4. One of the antibodies, designated G-22, reacted with 18 of 20 glioma cell lines, two melanoma cell lines, and three lung cancer cell lines, but not with 39 cell lines derived from sarcoma, carcinoma, or hematopoietic tumors. The antigen was expressed in the brain of human fetuses in early gestation (9 weeks) but not in late gestation (8 months) or in normal adult brain, suggesting that the antibody recognizes neural differentiation antigens expressed by neuroectodermal origin. A high incidence of positive antigens has been observed in gliomas but not in the other neural tumors, such as ependymomas, meningiomas, and neuroblastomas. Thus, the antigen defined by the G-22 monoclonal antibody could be defined as glioma-associated antigen. Pulse-labeling with tritiated leucine and subsequent immunoprecipitation of the solubilized cell membrane revealed that the antigen recognized by this antibody had a molecular weight of 67 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It was shown by dot-blot enzyme-linked immunospecific assay (ELISA) that the antigen could be detected in the cerebrospinal fluid (CSF) from patients with gliomas. From analysis of affinity chromatography and SDS-PAGE, the antigen present in the CSF had a molecular weight similar to that of a 1% Nonidet P-40 (NP-40) extract from a glioma cell line. When the antigen in CSF was quantitatively assayed by ELISA, the mean antigen level (expressed as optical density at 450 nm) in the CSF of seven patients was 0.8 +/- 0.28 (mean +/- standard deviation), which was significantly higher than the 0.38 +/- 0.14 level observed in the CSF of 15 patients with nonglioma brain tumors and the 0.23 +/- 0.09 level in the CSF of four patients without brain tumors. These results indicate that the monoclonal antibody G-22 is useful for the diagnosis of glioma.