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A monoclonal antibody, designated NDOG1, has been used to stain a series of human and monkey placentae as well as several adult human tissues using immunoperoxidase techniques. In early placentae, NDOG1 was found to stain extracellular material associated with proliferating, extravillous cytotrophoblast cell columns and with the cytotrophoblast shell at the feto-maternal junction. The immunohistology suggests that NDOG1 antigen may be secreted by the anchoring cytotrophoblast into the immediately adjacent maternal tissues. NDOG1 antibody also shows extracellular staining in the stroma of early human placentae and reacted with the apical villous syncytiotrophoblast plasma membrane throughout pregnancy. Biochemical experiments demonstrated that extracts of this latter membrane contained NDOG1 antigenic activity which was susceptible to digestion with bovine testicular hyaluronidase. Hyaluronic acid was the only glycosaminoglycan found in this membrane, thereby implying a reaction between NDOG1 antibody and hyaluronic acid. Whilst no such direct interaction could be demonstrated in vitro, NDOG1 was shown to compete with two other antibodies which themselves demonstrated specificity for hyaluronic acid. The proposed identity between the NDOG1 antigen and hyaluronic acid is discussed particularly in terms of placentation where the distribution of NDOG1 staining may confirm the role of hyaluronic acid in providing an open matrix structure during stages of cell proliferation, migration and invasion.

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A monoclonal antibody (NDOG2) against placental alkaline phosphatase (PLAP) in ovarian cancer has been used in three ways by the Bristol University Department of Obstetrics & Gynaecology. First, in an indirect immunoperoxidase technique, NDOG2 demonstrated positive standing in 64% of 56 ovarian carcinomas as well as in 25% of 44 benign tumours. The majority of these positive tumours were serous cystadenocarcinomas or serous cystadenomas and there was considerable variation in the expression of this antigen from tumour to tumour. NDOG2 was also used as the basis of two serum assays and, when labelled with 123-iodine (123I), in radioimmunoscintigraphy (RIS) to monitor patients' response to therapy. The first serum assay measures the enzymic activity of PLAP and the second recognizes the antigenicity of the molecules. Assay 2 proved more useful in that it predicted the course of the disease in 45% of patients followed up, whereas Assay 1 was only of use in 25% of cases. RIS proved to be a useful imaging technique and was at least as sensitive as conventional imaging techniques. The common causes of false-positive and false-negative results are described.

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Two cases of gestational choriocarcinoma have been examined for the expression of HLA and trophoblast antigens using the indirect immunoperoxidase technique on frozen tissue sections. Approximately 40-70% of tumour cells express MHC Class I antigen as detected by a panel of antibodies to monomorphic, or framework, MHC Class I antigenic determinants. Evidence in one case suggests that most of these cells may not express the paternal, polymorphic HLA antigenic determinants but that a small subpopulation do carry the fully antigenically active molecule. These latter may give rise to patient anti-paternal HLA antibodies. Class II (HLA DR or DC) antigens are expressed by none, or very few, tumour cells.

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The presence of placental alkaline phosphatase has been demonstrated immunohistochemically using a monoclonal antibody, in frozen sections of human endometrium. The enzyme is present in glandular epithelium, but is found most commonly in the surface epithelial layer throughout the menstrual cycle. It has also been demonstrated in malignant endometrial epithelium in eight out of twelve patients.

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A radiolabelled monoclonal antibody (NDOG2) directed against placental alkaline phosphatase (PLAP) was used in the radio-immunodetection of ovarian carcinoma. Tumour deposits were successfully visualized in 11 of 15 patients and the abnormalities demonstrated were classified as focal or diffuse. Of the 11 patients, eight showed focal abnormalities alone and three had a diffuse abnormality, of which two also showed a focal abnormality. False-positive results may occur not only due to uptake of 123I by gut mucosa and an inadequately blocked thyroid gland but also from activity in an incompletely emptied bladder. A false-negative result occurred due to high background activity in the liver masking a known, discrete tumour deposit.

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Frozen sections of three specimens of hydatidiform mole were stained with monoclonal antibodies to HLA Class I and Class II antigens by means of an indirect immunoperoxidase technique. Class I (HLA A, B, C) antigens were detected on proliferating extravillous trophoblast and on villous stromal cells but not on quiescent villous trophoblast. Trophoblast Class I antigen was detected with four different antibodies to monomorphic determinants but not with antibodies to the appropriate polymorphic HLA A or B type. Stromal cells were reactive with all Class I antibodies. Class II (HLA DR) antigens were not detected on any molar tissue. The expression of HLA antigens by molar tissue is similar to that of the normal first-trimester human placenta.

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The biodistribution of a radiolabelled monoclonal antibody (123I-NDOG2) was studied in patients with ovarian tumour. It was found that the uptake patterns in known tumour sites was variable and that the clearance of the agent from the vascular pool was due to renal excretion of the radionuclide and the redistribution of the radioactively labelled compound into other compartments. The mean (+/- SD) plasma clearance time was 20.8 (+/- 2.3) h and the ratios of target (tumour) to background (thigh) ranged between 1.4 and 4.8. The ratio between the plasma radioactivity at either 3.5 or 20 h after administration was calculated relative to the initial plasma radioactivity. These values (0.79 and 0.48, respectively) were incorporated into an image-subtraction technique that allowed for redistribution outside the vascular pool. A whole-body dose equivalent to 16.3 mu Sv MBq-1 (60.4 mrem.mCi-1) was calculated.

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The villous stroma and fetal endothelium in chorionic villi adjacent to maternal decidua in a placenta of a woman suffering from pemphigoid gestationis were found to have abnormal expression of HLA-DR antigen. This aberrant DR expression may be a reflection of an immune attack on the placenta.

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A monoclonal antibody, designated NDOG2, has been shown to react with placental alkaline phosphatase. It does not bind to liver, kidney, bone, or intestinal alkaline phosphatase enzymes and has shown a positive reaction with all of over 50 placentae. Immunohistological studies have shown (a) a reaction with respiratory bronchiolar epithelium of lung, endocervical gland epithelium, the epithelium of fallopian tube, and certain reticular cells in the thymic medulla; (b) no detectable staining with ten other normal tissues including testis and tonsil; and (c) a significant reaction with nine of 13 ovarian cystadenocarcinomas and little or no staining with four malignant ovarian tumors of other histopathological types. The NDOG2 antibody may be of value as a marker in patients with ovarian cystadenocarcinoma.

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Using immunohistological techniques, class 1 products of the major histocompatibility complex (MHC) have been demonstrated on various forms of human extra-villous trophoblast in placental tissues taken from first, second and third trimester pregnancies. This contrasts with the absence of class 1 MHC antigens on all forms of villous trophoblast. The extra-villous trophoblast which reacted with antibodies to monomorphic class 1 MHC antigens consistently failed to bind HLA-A or HLA-B antibodies specific for the foetal phenotype. This suggests, but does not prove, that the MHC antigen expression of trophoblast may be restricted to HLA-C or some other undefined class 1 antigen.

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The distribution of leukocytes in first trimester human decidual tissue has been studied by using a panel of monoclonal antibodies in an indirect immunoperoxidase technique on acetone-fixed cryostat sections. The results indicate that bone marrow-derived cells are abundant in the placental bed and a proportion of these are HLA-DR positive. However, a major leukocyte population in the decidua of early pregnancy is of cells which carry the E-rosette receptor but which do not express peripheral pan-T cell antigens nor HLA-DR. The distribution of these cells suggests that they are endometrial granulocytes. A similar large number of cells express OKT 10, a marker of immature or activated cells. The presence of this unusual T lineage cell raises the possibility that a form of lymphocyte processing is occurring in the decidua in early pregnancy, perhaps in response to foetal antigens presented on trophoblast.

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A large number of cells in the first trimester human placenta have been shown to express leucocyte-common antigen by the use of a monoclonal antibody in an immunoperoxidase technique. The distribution of endometrial granulocytes in the decidua of early pregnancy indicates that a large proportion of these cells bear the leucocyte-common antigen and are thus bone-marrow derived.

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An immunoelectron microscopic technique was used to analyse the distribution in term human placentae at the ultrastructural level of three antigens recognized by recently developed monoclonal antibodies. Two of the antigens (recognized by NDOG-1 and NDOG-2) were present only on the outer villous membrane of the syncytiotrophoblast. The other (HLA-A, B, C) was absent from syncytiotrophoblast and present on some capillary endothelial cells and on Hofbauer cells in the villous stroma.

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This paper describes an immunoperoxidase technique for labelling cryostat tissue sections which is routinely used in the authors' laboratories both in the initial screening of hybridoma culture supernatants, and also during the subsequent cloning and growth of antibody-secreting cell lines. The technique can readily be performed on 100 samples in less than 3 h and is free of non-specific background labelling. The staining pattern of a monoclonal antibody on a single tissue section allows semiquantitative assessment of its reactivity against a wide variety of tissue constituents and is thus inherently much more informative than conventional screening techniques (such as binding assays) which yield only a single numerical value for each test performed. In consequence it is often possible to identify the probable specificity of a new monoclonal antibody at the primary screening stage. A further important advantage of immunohistological screening is that it detects antigens on cells or other tissue structures which do not readily enter suspension and also antibodies against nuclear and cytoplasmic antigens. Examples of monoclonal antibodies analysed by immunohistological screening include antibodies against C3b receptor, HLA-DR, factor VIII-related antigen, human syncytiotrophoblast, dendritic reticulum cells and a proliferation-associated cell surface glycoprotein.

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Frozen sections of human placentae taken at selected stages throughout gestation were stained with monoclonal antibodies to HLA-A, B, C and DR antigens, using an indirect immunoperoxidase technique. No staining of villous trophoblastic tissue was detected. Antibody to HLA-A, B, and C antigens clearly stained membranous elements of the villous stroma. Antibody to HLA-DR antigens stained rare, elongated cells of the villous stroma in the term placenta. Monoclonal antibodies to syncytiotrophoblast membrane were used as a control. This antigen distribution was confirmed by absorption of these monoclonal antibodies in an indirect radioimmunoassay. Adult spleen homogenate gave much stronger absorption than a trophoblast membrane preparation with antibodies to HLA-A, B, C or DR antigens. The opposite absorption pattern was observed with the anti-trophoblast monoclonal antibodies. Detergent solubilisation did not affect the absorption capacity of trophoblast membrane for HLA-A, B, C or DR antigens. Maternal antibody could be eluted from trophoblastic areas of term placental sections by washing at physiological pH, but this did not effect the binding of monoclonal antibodies. Thus the HLA antigens were not masked by preformed maternal alloantibody. These observations confirm that chorionic villous trophoblast does not express detectable HLA antigen. This may be the principal reason for the lack of rejection of the fetal allograft.

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Samples were taken from the human chorionic sac at 6 wk postconception and were stained with monoclonal antibodies to trophoblast and to HLA antigens by means of an indirect immunoperoxidase technique. Antibody to HLA A, B, C antigens did not stain chorionic villous trophoblast in accordance with previous studies, but did stain nonvillous trophoblast of the cytotrophoblast cell columns and cytotrophoblastic shell. Antibody to HLA DR antigens showed no staining of trophoblast. This is the first demonstration of HLA antigens on a human fetal tissue in direct maternal contact. We have discussed the immunologic implications of this finding.

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Two monoclonal antibodies, NDOG1 and NDOG2, have been produced which are directed towards human syncytiotrophoblast. The antigens detected by these antibodies are absent from human liver, heart, kidney, brain and from normal human sera. The NDOG1 antigen but not NDOG2 antigen is a component of pregnancy sera and falls to undetectable levels within 6 hours of placental delivery. This molecule is not human chorionic gonadotrophin, human placental lactogen, placental alkaline phosphatase. SP1 or PAPP-A. Immunoperoxidase staining shows that both antigens are on apical aspects of the syncytiotrophoblast and not in the villous stroma.

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