Characterisation of the anti-bladder-cancer monoclonal antibody BLCA-8: identification of its antigen as a neutral glycolipid

 A monoclonal antibody, BLCA-8, was raised against the human bladder cancer cell line, UCRU-BL-17CL. By flow cytometry and immunoperoxidase staining, this antibody was found to possess high specificity for bladder tumours, some reactivity with fetal tissues, and no reactivity with normal bladder, or any normal or malignant tissue. This high specificity and the stability of the antigen to the urinary environment suggest that BLCA-8 may have potential for use as an anti-bladder-cancer therapeutic agent. By thin-layer chromatography and autoradiography, BLCA-8 was found to bind four components within the neutral lipid fraction of a bladder cancer cell line, UCRU-BL-17/23α. These components had R _F values of 0.22, 0.16/0.15 (doublet), 0.12 and 0.08, and migrated below globoside, indicating the presence of more than four sugars. By enzyme-linked immunosorbant assay and thin-layer chromatography it was found that the binding of BLCA-8 to the lipid extract was increased by both mild alkaline hydrolysis and enzymatic treatments, indicating that adjacent phospholipids and glycolipids interfere with the accessibility of the antibody-binding site. Full biochemical characterisation of the BLCA-8 antigen is currently underway. Received: 24 April 1995 / Accepted: 11 July 1995     

Characterisation of the anti-bladder-cancer monoclonal antibody BLCA-8: Identification of its antigen as a neutral glycolipid

A monoclonal antibody, BLCA-8, was raised against the human bladder cancer cell line, UCRU-BL-17CL. By flow cytometry and immunoperoxidase staining, this antibody was found to possess high specificity for bladder tumours, some reactivity with fetal tissues, and no reactivity with normal bladder, or any normal or malignant tissue. This high specificity and the stability of the antigen to the urinary environment suggest that BLCA-8 may have potential for use as an anti-bladder-cancer therapeutic agent. By thin-layer chromatography and autoradiography, BLCA-8 was found to bind four components within the neutral lipid fraction of a bladder cancer cell line, UCRU-BL-17/23α. These components hadR _F values of 0.22, 0.16/0.15 (doublet), 0.12 and 0.08, and migrated below globoside, indicating the presence of more than four sugars. By enzyme-linked immunosorbant assay and thin-layer chromatography it was found that the binding of BLCA-8 to the lipid extract was increased by both mild alkaline hydrolysis and enzymatic treatments, indicating that adjacent phospholipids and glycolipids interfere with the accessibility of the antibody-binding site. Full biochemical characterisation of the BLCA-8 antigen is currently underway.     

ELISA定量分析尿液BLCA-1/-4水平诊断膀胱癌的临床价值分析

目的:探讨膀胱癌患者尿液膀胱特异性核基质蛋白(bladder cancer specific nuclear matrix proteins,BLCA)-1/-4水平及其临床应用价值。方法:本研究纳入38例膀胱癌患者、40例正常对照组。采集受试者尿液样本,通过竞争性酶联免疫吸附法(enzymelinked immunosorbent assay,ELISA)定量分析尿液中BLCA-1和BLCA-4的水平,绘制受试者工作曲线,确定cut-off值。结果:膀胱癌患者尿液BLCA-1/-4水平均显著高于对照组(P0.001);当cut-off值取0.859 ng/mL时,BLCA-1诊断膀胱癌的敏感性和特异性分别为71%(27/38)、90%(36/40)。肌层浸润性膀胱癌患者尿液BLCA-1较非肌层浸润性膀胱癌患者水平显著升高(P0.001),但不同分级膀胱癌患者尿液BLCA-4水平无显著差异(P0.05)。高级别膀胱癌患者尿液BLCA-4水平较低级别膀胱癌患者显著升高(P0.05),但不同分期膀胱癌患者尿液BLCA-4水平无显著差异(P0.05)。以cut-off为0.859 ng/mL时,BLCA-1诊断膀胱癌的敏感性和特异性分别为71%(27/38)、90%(36/40)。以cut-off为0.620 ng/mL时,BLCA-4诊断膀胱癌的敏感性和特异性分别为76.3%(29/38)、97.5%(39/40)。联合检测尿液BLCA-1和BLCA-4诊断膀胱癌的敏感性和特异性分别为84.2%(32/38)和100%(40/40),准确度为0.923(77/78),阳性预测值为100%(32/32),阴性预测值为86.9%(40/46)以及YOUDEN指数分别为0.842。结论:膀胱癌患者尿液BLCA-1和BLCA-4水平显著升高,且敏感性和特异性均较高。联合检测尿液BLCA-1和BLCA-4较单一检测用于诊断膀胱癌的临床应用价值更高。     

Biodistributions of intact monoclonal antibodies and fragments of BLCA-38, a new prostate cancer directed antibody

Background: Monoclonal antibodies (MAbs) are used for targeting agents to tumours while minimizing normal tissue exposure. Methods: A new anti–prostate cancer MAb, BLCA-38, was radioiodinated (I¹²⁵) and assessed for its ability to target subcutaneous human prostate cancer (DU-145) xenografts after systemic intraperitoneal administration. For comparison, the profile of J591 MAb (now in clinical trial) against LNCaP-LN3 tumours was examined. Biodistribution profiles were obtained at various times, by assessing injected dose/gram (%ID/g) and xenograft to blood (X/B) ratios. Microautoradiography of xenografts was performed. After conjugation with a melittin peptide toxin, the profiles of BLCA-38 and J591 were compared with that of an irrelevant antibody, DS-1. Results: Xenograft localization by ¹²⁵I-labeled BLCA-38 and J591 MAbs to their relevant antigen-positive tumors was comparable, and there was no unusual localization in nontumour tissues. F(ab)₂ and Fab fragments gave improved X/B ratios, but the %ID/g xenograft was decreased and they accumulated in kidneys, bladder and stomach. In contrast, the conjugates of irrelevant antibody showed no tumour targeting. Microautoradiography showed more tumour accumulation of MAbs than F(ab)₂s or Fabs. Conclusions: BLCA-38 can target prostate cancer in vivo almost as effectively as J591. Given that J591 is used clinically, BLCA-38, which targets a different antigen, has potential for radioimmunoscintigraphy and for therapeutic targeting of prostate cancer.     

BLCA-4在膀胱癌中作用的研究进展

近年来,膀胱癌的发病率与死亡率呈逐年上升,已成为泌尿系统最常见的肿瘤。随着分子生物技术的不断发展,核基质蛋白（SMVs）、膀胱肿瘤抗原（BTA）、透明质酸和透明质酸酶（HA—Haase）、细胞角蛋O（CK）等诸多膀胱肿瘤标志物被发现。其中,膀胱癌特异性核基质蛋白-4（BLCA-4）是一种可溶性的复合物,在正常膀胱组织中不表达而存在于膀胱癌组织中。检测尿样中BLCA-4水平有望用于膀胱癌的筛查及诊断,具有较高的敏感性和特异性,也可用于普通人群与膀胱癌高危人群（脊髓损伤患者）的筛选与检测;而术后病理组织检测BLCA-4的表达也可为膀胱癌患者的预后预测提供参考。本文对近年来BLCA-4在膀胱癌中的作用进行了综述。     

BLCA-4 在膀胱癌中作用的研究进展

摘要：近年来,膀胱癌的发病率与死亡率呈逐年上升,已成为泌尿系统最常见的肿瘤。随着分子生物技术的不断发展,核基质蛋白 (NMPs)、膀胱肿瘤抗原(BTA)、透明质酸和透明质酸酶(HA-HAase)、细胞角蛋白(CK)等诸多膀胱肿瘤标志物被发现。其中,膀胱癌 特异性核基质蛋白-4(BLCA-4)是一种可溶性的复合物,在正常膀胱组织中不表达而存在于膀胱癌组织中。检测尿样中BLCA-4 水平有望用于膀胱癌的筛查及诊断,具有较高的敏感性和特异性,也可用于普通人群与膀胱癌高危人群(脊髓损伤患者)的筛选与 检测;而术后病理组织检测BLCA-4 的表达也可为膀胱癌患者的预后预测提供参考。本文对近年来BLCA-4 在膀胱癌中的作用进 行了综述。     

Immunohistochemical characterisation of the monoclonal antibody BLCA-38 for the detection of prostate cancer

Background: Monoclonal antibodies (MAbs) can be used to detect, image and treat cancers. This study aimed to characterise the binding of BLCA-38 MAbs to human prostate cancer cell lines, human prostate cancer biopsy samples and normal tissues to enable future targeted studies. Methods: BLCA-38 antigen expression on cancer lines was determined by flow cytometry; that on patient specimens from normal tissues and cancers was tested by immunohistochemistry using fresh frozen tissues or paraffin-embedded tissues that had undergone antigen retrieval. Results: Cell surface BLCA-38 antigen expression was seen on DU-145, PC-3, PC-3 M and PC-3 M-MM2 prostate cancer lines, but LNCaP, MDA PCa 2a or MDA PCa 2b lines were negative. Other human lines, including 8/12 bladder cancer and A431 vulval epidermoid cells, but not breast cancer lines, expressed BLCA-38 antigen. Staining occurred in glandular epithelial cells in the majority of frozen, and paraffin-embedded prostate cancer tissues and was occasionally seen in prostatic intraepithelial neoplasia (PIN). No staining was observed in normal cadaver tissues or in benign areas from various other cancer tissues. Conclusions: The BLCA-38 antibody binds to the majority of human prostate cancers but not to normal cells, and has potential for targeting novel therapies in patients with this disease.     

Antiproliferative effects of bacillus Calmette-Guérin and interferon α2b on human bladder cancer cells in vitro

Direct inhibitory effects of bacillus Calmette-Guérin (BCG) and interferon 2b (IFN2b) on six human bladder carcinoma cell lines, UCRU-BL-13, UCRU-BL-17, UCRU-BL-28, 5637, T24 and J82, were studied using an in vitro proliferation assay. Effects on proliferation following exposure to BCG or IFN2b were analysed by [³H]thymidine incorporation over 7 days. BCG had an antiproliferative effect on all bladder lines, while sensitivity to IFN2b varied greatly, being as remarkably low as 1 U/ml for some lines. The antiproliferative effect was greatest when cells were exposed continuously to either agent, but was still evident with a limited exposure. When clinical concentrations were simulated in vitro, BCG+IFN2b was more effective than BCG alone and as effective as a double BCG concentration. We conclude that, in addition to their immunomodulatory effects, BCG and IFN2b directly inhibit the proliferation of human bladder cancer cells, and often at extremely low concentrations.     

A phase II trial of murine monoclonal antibody 17-1A and interferon-γ: Clinical and immunological data

Summary A group of 15 patients with metastatic colorectal adenocarcinoma received a combination of interferon (0.1 mg/m², days 1–15) and the murine monoclonal antibody 17-1A (400 mg, days 5, 7, 9 and 12). The treatment was tolerated with minimal toxicity. Of the 14 evaluable patients, 13 developed human antibody to murine 17-1A, with 11 patients demonstrating antibody to the variable region of 17-1A (anti-idiotype). Antibody to the variable region was inhibited by 17-1A but not by mouse immunoglobulin. Sera from patients with substantial anti-idiotype reactivity were capable of inhibiting the binding of murine 17-1A to antigen expressing LS174-T cells thus indicating the presence of antibody directed against the 17-1A combining site (mirror-image anti-idiotype). The median survival of the whole group was 56 weeks and there was no correlation between clinical response/survival and the development of anti-idiotype antibody.Supported by the Veterans Administration Medical Center and by Public Health Services grant CA 45 232 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services     

In vivo cervical cancer growth inhibition by genetically engineered cytotoxic T cells

Purpose: The CD44 v7/8 splice variant that is frequently expressed in cervical carcinoma and rarely expressed in normal tissues displays promising properties as a target antigen for cancer immune therapy. In this study, cytotoxic T lymphocytes (CTLs) were genetically engineered to gain CD44v7/8 target specificity. Methods: Clone 96 (CI96), an established murine cytotoxic T-cell line, and naïve murine T cells were retrovirally transduced with a fusion gene construct encoding for the single chain fragment scFv of the monoclonal antibody VFF17 and for the chain of the T-cell receptor (TCR). The therapeutic potential of genetically engineered T cells was tested in vitro and in vivo. Results: Surface expression of the chimeric TCR on infected Cl96 and naïve T cells was shown by FACS analysis. CD44v7/8-positive target cells were efficiently lysed by transduced Cl96 and naïve T cells, demonstrating the functionality and specificity of the chimeric TCR. In a xenograft BALB/c mouse model, efficient growth retardation of CD44v7/8-positive tumours was mediated by genetically engineered Cl96(VFF17)cyYZ cells. Conclusions: We were able to reprogramme the target specificity of recombinant Cl96 and naïve CTLs resulting in efficient cytolysis of CD44v7/8-positive cervical cancer cells. High transduction rates and the specific cytolysis of CD44v7/8-redirected CTLs are promising tools for an immune gene therapy approach for advanced cervical cancer.Abbreviations Ab Antibody - CTL Cytolytic T lymphocyte - mAb Monoclonal antibody - TCR T-cell receptor     

Immune reactions in patients with superficial bladder cancer after intradermal and intravesical treatment with bacillus Calmette-Guérin

Summary The immune reactivity of patients with strongly recurrent superficial bladder cancer was followed after combined intravesical and intradermal bacillus Calmette-Guérin (BCG) immunotherapy. All patients in this study were previously treated without success with intravesical chemotherapy. The BCG treatment regimen consisted of weekly administrations with BCG (RIVM) for six consecutive weeks, both intravesically and intradermally. In this study, sera and peripheral blood leukocytes (PBL) of patients were tested serially. Besides BCG-antigen-specific reactions, e.g. skin reactivity to purified protein derivatives of Mycobacterium tuberculosis (PPD), antibody formation and antigen stimulation of PBL in vitro, non-antigen-specific immune reactivities were also measured, e.g. mitogen response and spontaneous cytotoxic activity of PBL. In addition the antibody response to bladder carcinoma antigens and the cytotoxic activity of PBL for the bladder carcinoma cell line T24 and the natural-killer-sensitive K562 cell line were investigated. The results obtained from the various assays were evaluated for their prognostic value in relation to the length of the tumor-free interval after the BCG treatment. Because sera and PBL were only obtained during the first 6 months after the BCG treatment, the immune reactivity was compared to the clinical results at that same time. At 6 months after therapy 12 out of 40 BCG-treated patients were tumor-free whereas 28 out of 40 showed a recurrence. Skin reactivity to tuberculin PPD was measured in 40 patients during a period of 3–6 months after therapy. Of patients who showed a recurrence of the tumor within 6 months, 48% of them showed a transient response or developed no response at all to PPD. In the group of patients with a longer tumor-free period (n=10), only one patient lost the response to tuberculin PPD. Although PBL of a limited number of patients were tested, it was observed that the cytotoxicity to the bladder carcinoma cell line T24, and the natural-killer-sensitive K562 cell line increased in a number of the patients (7 out of 14, and 9 out of 14 respectively). Reactivity of PBL to mitogens and subset distribution (ratio T-helper: T-suppressor/cytotoxic) were not influenced by the BCG treatment. Antibody response to mycobacterial antigen was detected in 9 out of 23 patients investigated. Of these 9 patients, 8 belonged to the group with a recurrence of the tumor within 6 months (n=17). There was no correlation between the skin reactivity and the antibody response to tuberculin PPD. Furthermore, none of the 25 patients showed an antibody response to bladder carcinoma antigens. Sera of bladder carcinoma patients (n=19) reduced the mitogen-induced proliferation of lymphocytes, compared to sera of healthy controls (n=13), indicating the presence of circulating suppressor factor(s). Our results indicate that the absence of a Mantoux conversion or the presence of transient reaction to tuberculin PPD were highly related (91%) to a relapse of the disease. On the other hand, the cytotoxic activity of PBL to T24 and K562 cell lines, or their reactivity to tuberculin PPD or mitogens, gives no predictive information about the clinical results (tumor-free interval) of the BCG therapy. Abbreviations used: BCG, bacillus Calmette-Guérin; NK, natural killer; PBL, peripheral blood leukocytes; PPD, purified protein derivative of Mycobacterium tuberculosis; ELISA, enzyme-linked immunosorbent assay     

Production and characterization of a monoclonal antibody (BBH5) directed to ganglioside lactone

The occurrence of lactones in various ganglioside preparations has been clearly demonstrated, yet the natural occurrence of ganglioside lactones in cells and tissues has been the subject of long debate, since lactones can be formed readily during preparation of gangliosides. We now report the generation of a monoclonal antibody (BBH5) that reacts specifically with lactones of disialogangliosides having the NeuAc2-8NeuAc2-3Gal sequence, but does not crossreact with the parent ganglioside. The specificity of the antibody resides on the first lactone ring between two sialic acid residues but not on the second lactone ring between sialic acid and galactose, as evidenced by reactivity with lactonized GD_1b having the first lactone ring (L1), and by reactivity with lactonized polysialic acid homo-oligomers ([NeuAc2-8] _n NeuAc). The sialic acid carboxyl involved in the lactone ring was unequivocally determined after ammonolysis followed by methylation and fast atom bombardment mass spectrometry. The antibody BBH5 thus provides a novel tool for studies of the natural occurrence of lactones in cells and tissues.Abbreviations BSA bovine serum albumin - CM chloroform-methanol - CMW chloroform-methanol-water - FAB-MS fast atom bombardment mass spectrometry - IHW isopropanol-hexane-water - MAb monoclonal antibody - PBS phosphate-buffered saline - TLC thin layer chromatography     

A new lymphocyte alloantigen (Ly-10) controlled by a gene linked to theLyt-1 locus

Spleen cells from a (BALB/cxC57BL/6)F₁ mouse immunized with CBA/J spleen cells were fused with the myeloma cell line NS-1. One of the six established hybrid cell lines continuously secreted antibody that recognized a new antigenic specificity, tentatively called Ly-10.1. This newly found antigen is expressed on thymocytes, on splenic T and B cells, on bone-marrow cells, and on the cells derived from brain, kidney and liver. It is also expressed on a continuous cell line, 416B, with stem-cell characteristics. The unique tissue distribution and, furthermore, a distinct strain distribution pattern distinguishes Ly-10.1 from any known murine lymphocyte alloantigen. On the basis of reactivity with cells of the C57BL/6-Lyt-1^a congenic strain, one gene governing Ly-10 expression is assigned to the Lyt-1 region of chromosome 19.We chose the notation Ly-10 rather than Ly-9 to allow for a future decision that Lgp 100 (Ledbetter et al. 1979) should be renamed Ly-9.     

Human tumor and normal tissue reactivity of the anti-(breast cancer) monoclonal antibody BA-Br-3 and its similarity to the anti-(epithelial membrane antigen) monoclonal antibody E29

Summary A mouse monoclonal antibody (BA-Br-3) raised against the breast carcinoma cell line CAMA-1 was previously shown to react with a 300-kDa globule-like glycoprotein from human milk fat also expressed in the cytoplasm and on the surface of human carcinoma cells of different histological types. In this report the reactivity of this mAb with a large number of normal and malignant human tissues was analyzed using immunoperoxidase techniques. When tested on sections of both fresh-frozen tissues and formalin-fixed, paraffin-embedded tissues, BA-Br-3 reacted with a formalin-resistant antigenic determinant expressed by normal and malignant epithelial cells. Preferential reactivity was observed at the apical portion of ductal epithelial cells in normal breast and in glandular epithelia distributed in several other organs. Reactivity with mucin-like secretions in the lumina of ducts was also found. BA-Br-3 reacted mostly in heterogenous staining patterns with 88% of 49 breast carcinoma specimens tested, regardless of their histological type or whether they were primary or secondary neoplasms. Testing of epithelial malignant tumors other than breast carcinomas with this antibody showed that 127 of 151 (84%) were also reactive. mAb BA-Br-3 and E29 (a commercially available anti-(epithelial membrane antigen) shared very similar staining patterns and distributions of reactivity with breast and other epithelial tumors. However, BA-Br-3 showed a significantly higher percentage of reactivity with melanoma (33% versus 6%,P = 0.003) and a trend toward a higher percentage of reactivity with sarcoma (55% versus 27%,P >0.05). This antibody, therefore, defines a molecule that is a member of the mucin-like epithelial membrane antigen family. Further studies are warranted to determine its usefulness in antibody-directed cancer diagnosis, prognosis, and immunotherapy.     

Natural antibody to a human bladder carcinoma cell line

Summary Sera from 144 healthy blood donors were screened for the presence of antibody against the bladder carcinoma cell line RT4 by means of an immune adherence assay. Only one serum (9241) demonstrated high activity with a titer of 1/124. Qualitative and quantitative absorption analyses were carried out with a wide variety of cells to characterize the specificity of the reaction. Cells used for absorption were both cultured and non-cultured and normal and malignant. The reactivity was absorbed by the bladder cancer cell lines RT4 and 5637 and the lung cancer cell line SK-LC-LL. In addition, surgically obtained urothelium from a patient with transitional cell carcinoma abrogated reactivity. No other absorbing cells removed the reactivity of serum 9241 for RT4. The antigen defined by this serologic reaction is heat-stable and trypsin-resistant, as demonstrated by using heated or trypsinized RT4 cells for absorption.This study shows that naturally occurring antibody directed toward cell surface antigens of tumor cells is present in some normal individuals. This finding is important in the evaluation of a possible causal relationship between the presence of antibody specific for tumor cell antigens and the development of neoplasia.     

Diagnosis of bladder cancer with urinary cytology, immunocytology and DNA-image-cytometry.

DNA-image-cytometry and antibodies directed against the Lewis X- and the 486p 3/12 antigen were applied to improve diagnostic accuracy of urinary cytology for the detection of bladder cancer. Cytology, immunocytology and DNA-image-cytometry were performed in spontaneously voided urine samples and barbotage bladder washings from 71 patients. The DNA content was determined using the CM-1 Cytometer according to the recommendation of the ESCAP Consensus Report on Standardization of DNA-image-cytometry (1995). For immunocytological examination we used the monoclonal anti Lewis X antibody P-12 and antibody 486p 3/12. All patients underwent subsequent cystoscopy and for any suspicious lesion biopsy or transurethral resection was done. Histological findings revealed 31 patients with transitional cell carcinomas of different stages and grades of malignancy. 40 patients had various benign diseases of the urinary bladder. Cytology yielded a sensitivity of 68% and a specificity of 100%. DNA aneuploidy was detected in 81% of cancer patients with a specificity of 100%. By combination of these two methods the overall sensitivity increased to 87%. Immunocytology with Lewis X and 486p 3/12 antibodies showed reactivity in 84% and 87% in combination with a specificity of 80% and 70%, respectively. By combining urinary cytology, immunocytology and/or DNA-image-cytometry the overall sensitivity increased to 94% with no change in specificity. DNA-image-cytometry should be used to evaluate particularly urothelial cells suspicious for malignancy in urinary specimens. Because of low specificity the monoclonal antibodies against Lewis X- and 486p 3/12 antigens are not helpful in screening for bladder cancer. Nevertheless, their high sensitivity may justify their use in case DNA image cytometry is not available and in the follow up of patients with transitional cell carcinoma.     

Epitope masking of rat esophageal carcinoma tumor-associated antigen by certain coexisting glycolipid and phospholipid molecules: a potential mechanism for tumor cell escape from the host immune responses

A monoclonal antibody (mAb-5G) produced against a tumorigenic rat esophageal cell line, B2T, was shown to react specifically with a unique glycolipid antigen expressed on the cell surface of tumorigenic and certain non-tumorigenic, immortalized rat esophageal cell lines [Cancer Immunol Immunother 36: 94 (1993)]. In enzyme-linked immunosorbent assay experiments, mAb-5G reacted with crude lipid extracts prepared from B2T cells cultured in vitro, but showed very little reactivity with crude lipid extracts prepared from the same cell line passaged once in vivo, unless the antigen was separated from other lipid components by column or thin-layer chromatography (TLC). When a secondary tissue-culture cell line was established from the above B2T tumor tissues and serially subcultured in vitro, the percentage of positively stained cells was increased significantly in immunofluorescence assay. It was also demonstrated that the amount of extractable antigen was increased as the cells were subcultured in vitro up to passage 15, and stabilized thereafter. These results indicate the presence of certain lipid components in crude lipid extracts from B2T cells grown in vivo that are capable of interfering with antigen-antibody binding. On TLC plates, these interfering lipids were identified as phosphatidylcholine, phosphatidylserine, sphingomyelin and gangliosides. The interfering lipids did not bind the antibody, rather they appeared to interfere with antigen accessibility. These lipid substances may modify tumor cell surface antigen(s), thus protecting the tumor cells from host immune destruction.     

Monoclonal antibody against the glutaraldehyde-conjugated polyamine,spermine

We developed a mouse monoclonal antibody (ASPM-29, mAb) against spermine (Spm) conjugated to human serum albumin (HSA) using glutaraldehyde-sodium borohydride, for applications in immunocytochemistry (ICC). The antibody specificity was evaluated by an enzyme-linked immunosorbent assay (ELISA) binding test, simulating the ICC of tissue sections. ASPM-29 showed an almost equal immunoreactivity to Spm and spermidine (Spd) but no reactivity to any of the other polyamine (PA)-related compounds tested. By use of this antibody, indirect immunoperoxidase staining was observed in different tissues fixed with glutaraldehyde in combination with borohydride reduction. In contrast, immunoreactivity was quite low in tissues fixed only with glutaraldehyde. Absorption controls indicated that the immunostaining could be completely inhibited by 50 g/ml of Spm or Spd and partially inhibited by N-acetylspermine (Ac-Spm), N¹-acetylspermidine (N¹-Ac-Spd), or N⁸-acetylspermidine (N⁸-Ac-Spd), but was hardly inhibited at all by other PA-related compounds or amino acids. The reactivity of the antibody with Spm conjugated on wells in an ELISA plate was inhibited by micromolar concentrations of Spm, Spd, Ac-Spm, N¹-Ac-Spd, or N⁸-Ac-Spd, in decreasing order, but not by other small molecules. Dense ICC staining was observed in the paranuclear and basal cytoplasm of acinar cells of rat pancreas, submandibular gland and paratid gland, these results being in complete agreement with our recent ICC methods using other mAbs produced against N-(-male-imidobutyryloxy) succinimide-conjugated Spm.     

Cytochemical observations on the development of antibody-forming cells in popliteal lymph nodes by an improved immunoperoxidase method

Summary Precursors of cells forming antibody to horseradish peroxidase (HRP) became detectable in lymph nodes of rats and rabbits after the sensitivity and the specificity of the previously used immunocytochemical procedure were increased. The sensitivity of the method was increased by shortening the time of fixation of the tissue from 24 h to 5 h, and by exposing the antibody in the tissue section to a higher concentration of the antigen for a longer time period than previously. The specificity of the method was enhanced by inhibiting the endogenous peroxidase activity with phenylhydrazine. Five to 8 days after the injection of the antigen, many precursors of plasma cells showed the specific reaction for the antibody in the medulla of the lymph node. Most of the precursor cells were smaller than mature plasma cells and had small, eccentrically located nuclei. The smallest of the precursor cells had the appearance of lymphocytes. From 8 days to several weeks after the injection of the antigen, mature plasma cells characterized by an intense antibody reaction coexisted in the medulla with more weakly stained precursor cells. Lymphocytes, blast cells, and transitional cells containing the specific antibody appeared in germinal centers of the cortex not earlier than 2–3 weeks after the injection of the antigen. From 2–3 weeks to several months after the injection of the antigen, a considerable number of small and medium-sized lymphocytes in the diffuse cortex and in the sinuses contained the anti-HRP antibody. In some animals, a strong antibody reaction occurred in the cytoplasm of these lymphocytes. In other animals, a relatively weak reaction was observed in the perinuclear areas and on the cell surfaces. It is suggested that these lymphocytes may be memory cells.This work was supported by General Research Support Grant BR-5366 from the General Research Support Branch, Division of Research Facilities and Resources, National Institutes of Health     

Predominant expression of the β subunit of prolyl 4-hydroxylase (disulfide isomerase) in human extravillous trophoblasts

Prolyl 4-hydroxylase is a heterodimeric enzyme that is crucial in the biosynthesis of collagen. The subunit of this enzyme is a multifunctional protein which is also known as protein-disulfide isomerase. Immunofluorescence and monoclonal antibody (Mab) 5B5 were used to localize the subunit in human extraembryonic tissues. The strongest sites of 5B5 reactivity were extravillous cytotrophoblasts in the basal plate, uteroplacental arteries and amniochorion, syncytiotrophoblast displayed variable weaker reactivity. Only a small fraction of placental 5B5 antigen was detected as a component of prolyl-4-hydroxylase by affinity chromatography on immobilized polyproline. The results indicate a difference in the expression of an endoplasmic reticulum marker between villous and extravillous trophoblast. The predominance of 5B5 antigen in extravillous trophoblast could be associated with an increased ability to synthesize collagen or other enzymatic reactions associated with prolyl 4-hydroxylase subunit.