Immunohistochemical localization of human and simian immunodeficiency viral antigens in fixed tissue sections.

Antigens of human (HIV) or simian immunodeficiency viruses (SIV) were identified with polyclonal or monoclonal antibodies and avidin-biotin complex (ABC) immunohistochemistry in fixed surgical pathology and autopsy specimens of humans or monkeys with the acquired immunodeficiency syndrome. With B-5 fixative, viral antigens were readily detected in lymph nodes of 8 of 13 patients with follicular hyperplasia, but in only 1 of 12 patients with follicular atrophy. Antigen was detected in follicular dendritic reticular cells and rare blastlike cells, extracellularly, and in postcapillary venules, medullary lymphocytes, sinus histiocytes, and macrophages in some lymph nodes. In the brain at autopsy, antigen could be found in gliomesenchymal-cell nodules, astrocytes, vascular endothelial cells, multinucleated cells, and astrocytes and macrophages associated with demyelination. In contrast, 4 rhesus monkeys with experimental SIV infection had abundant antigen in sinus histiocytes, macrophages, and multinucleated giant cells of lymph nodes and spleen and in thymic epithelial cells. Brain lesions of monkeys resembled those of humans, with antigen found in macrophages and multinucleated giant cells. Antibodies to HIV also were immunoreactive in formalin-fixed tissue sections of monkeys containing SIV antigens. The ABC technique provided a fast and efficient method for localizing HIV and SIV antigens in fixed surgical and autopsy specimens. These findings are consistent with those found with in situ hybridization, ultrastructural studies, frozen sections of lymph nodes, and permanent sections of brain.     

Monoclonal antibody localization of A and B isoantigens in normal and malignant fixed human tissues.

The expression of human blood group A and B isoantigens in normal and malignant tissues from stomach, colon, and pancreas was analyzed in an immunoperoxidase assay using monoclonal antibodies specific for these isoantigens. Appropriate isoantigen expression was demonstrated in the normal epithelium from the stomach, pancreas, and proximal but not distal colon of blood group A, AB, or B patients. Half of all gastric carcinomas and of proximal colon carcinomas showed complete loss of isoantigen, whereas the adjacent mucosa in these cases continued to express appropriate isoantigen. Isoantigen expression was completely lost in only 13% of pancreatic carcinomas tested. Neither A nor B isoantigen was detected in normal epithelium from the distal colon. By contrast, 85% of carcinomas derived from this site showed reexpression of isoantigen. Inappropriate expression of A isoantigen was detected in pancreatic carcinomas (2/5) but not in gastric or colon carcinomas (0/21). Inappropriate expression of B substance was not detected in any tissue (0/38). Interestingly, differential binding of antibodies to Type 1 versus Type 2 and/or difucosyl versus monofucosyl blood group B substances was manifested by differences in intensity of staining for endothelium and red blood cells.     

Monoclonal antibody to a highly glycosylated protein reacts in fixed tissue with melanoma and other tumors

A highly glycosylated protein with a molecular weight of 30,000 to 60,000 and a protein core of 20,000 daltons has been identified by antimelanoma monoclonal antibodies. The antigenicity of this melanoma-associated glycoprotein (MAG) was not destroyed in fixed paraffin-embedded melanoma tissue, and was present in malignant cells of cutaneous superficial spreading melanomas in skin (31/33) and in half of all metastatic melanomas examined (5/10). The antigen was not expressed by normal melanocytes. The strong reactivity of dysplastic nevi with the anti-MAG antibodies was comparable to that seen in radial growth phase melanoma. Antigen expression was much weaker in compound nevi where reactivity ranged from moderate in the junctional component and the upper dermis to absent at the base of the nevus.     

Monoclonal antibody against L-histidine decarboxylase for localization of histaminergic cells

The immunochemical and immunohistochemical properties of a monoclonal antibody (Mab HI 113-12) developed in mice against a partially purified preparation of rat gastric L-histidine decarboxylase (HD) were studied. The Mab recognised the HD activity from the antigen and from crude tissue extracts with a high histamine (HA)-synthesizing capacity (stomach, hypothalamus, striatum, mastocytoma). In contrast, neither a bacterial HD nor other decarboxylases (glutamic and DOPA decarboxylases) were immunoprecipitated. In preliminary immunohistochemical studies, staining of the cytoplasm of mastocytoma as well as of hypothalamic neurons, particularly magnocellular ones in the mamillary region, were observed. The latter presumably correspond to the cells of origin of a long ascending histaminergic pathway.     

Monoclonal antibody detection of carbohydrate-defined and protein-defined H-2Kk antigens

Three different monoclonal anti-H-2K^k antibodies, 27R9, 30R3 and 11-4 were examined for the biochemical nature of the antigenic determinants they recognize. When these were compared on the basis of their sensitivity to pronase and various glycosidases, 27R9 was shown to bind to protein-defined H-2K^k antigens, while 30R3 and 11-4 bound to H-2 antigens defined by carbohydrate. From sugar inhibition studies, and treatments with specific glycosidases, d-mannose appears to be the immunodominant sugar involved in the antigenic site recognized by 30R3, while several sugars, namely sialic acid, d-mannose and α-and β-linked d-galactose would appear to be components of the antigenic site bound by 11-4. The carbohydrate determinants appear to be present on glycolipid molecules, since both the 30R3 and 11-4 antibodies could be inhibited by glycolipid extracts from spleen cells of the appropriate H-2 haplotype, as well as from several other strains of mice previously shown to be cross-reactive targets for these antibodies. This finding is supported by evidence that the molecule carrying the protein-defined antigen is distinct from that carrying the carbohydrate-defined antigens. The results are discussed in the light of current information on the nature of glycolipid Ia antigens, as well as the role of H-2 antigens in T-cell interactions.     

Monoclonal antibodies to tissue-specific cell surface antigens. I. Characterization of an antibody to a prostate tissue antigen

Monoclonal antibodies were raised to PC-3 human prostate adenocarcinoma cells, and one hybridoma, designated F77-129, was extensively purified and used to characterize a PC-3 antigen. The F77-129 antibody also showed serological reactivity with the Du-145 prostate cancer line and with three of four breast carcinoma lines tested; it showed variable binding to a colon carcinoma line. Several other lines tested, including melanomas, fibrosarcomas, and leukemias, were completely negative. Immunoperoxidase staining of frozen surgical specimens showed binding to both normal and malignant prostate and breast tissue. Injection of radioiodinated F77-129 into tumor-bearing nude mice showed specific in vivo targeting to prostatic cancer implants. The antigen also showed surface modulation by bound antibody, suggesting possible clinical utility of this antibody in delivering immunotoxins to tumors.     

Monoclonal antibody defined-human melanoma-associated antigens: Molecular and phylogenetic studies in normal serum

Normal human sera were analyzed for the presence and molecular form of two human melanoma-associated antigens (MAAs), the 250 “melanoma-specific” glycoprotein and the 100 K “common tumor antigen”. The 250 K MAA was not synthesized by any cultures other than human melanoma and was not detectable in normal human serum. In contrast, the 100 K MAA, which is present in spent medium of cultured human melanoma, carcinoma and fetal melanocytes but not of adult normal cells, was found in normal human serum in nanogram quantities. This serum form of the 100 K MAA was also found in pooled sera of higher apes but not of lower species. The cell-derived form of the 100 K MAA, present in spent culture medium, had a similar phylogenetic distribution. The molecule was produced by cultured brain glia from gorilla, but not by melanoma cells from miniature swine or dog. The 100 K MAA from gorilla glia had a mol. wt identical to the molecule produced by human melanoma cells. Molecular characterization of this MAA in normal human serum showed that it was heterogeneous in size and was present in fractions> 100 kd after analytical HPLC gel sieving under non-denaturing conditions. In contrast, MAA from spent culture medium of melanoma cells was 100 kd or less in chemically defined medium (CDM) with no protein supplement, but had a higher mol. wt in CDM with BSA or fetal calf serum supplement, similar to the serum form of the molecule. An association of the 100 K MAA with albumin was demonstrated by analytical HPLC gel sieving and SDS-PAGE analysis of monoclonal antibody immunoprecipitates. The 100 K MAA was dissociated from albumin in normal human serum by treatment with SDS and fractionation by gel sieving. Under these conditions 100 K MAA from serum co-migrated with similarly treated 100 K from melanoma cells. These results indicate that the 100 K MAA is a normal serum constituent which forms a strong, non-covalent association with albumin and is evolutionarily restricted to higher apes or humans.     

Monoclonal antibody to type IV collagen with selective basement membrane localization

A monoclonal antibody (MCA IV-1) has been developed to a determinant of the high molecular weight fractions of human placental collagen, present also in bovine lens capsule and glomerular basement membrane type IV collagens. This unique determinant is pepsin and collagenase resistant and is apparently distinct from the alpha 1(IV) and alpha 2(IV) helical peptides. As part of the high molecular weight molecules, the determinant is located in a region additively deformable by reduction and sodium dodecyl sulfate denaturation. However, when a 20-kilodalton, largely collageneous, fragment containing this determinant is separated from the larger fraction by 37 degrees C collagenase treatment, the determinant is insensitive to reduction or sodium dodecyl sulfate denaturation. Immunohistologic analysis and comparison with a polyclonal antibody to type IV collagen shows a marked selectivity of localization in the glomerular basement membrane. MCA IV-1 reacts in the inner aspect of the glomerular basement membrane but primarily in the mesangium, where it selectively expands in diabetic nephropathy. Tissue selectivity is also evident in lens and corneal basement membrane.     

Expression of ABH, Lewis and related tissue antigens in the human thymus

Expression of ABH, Lewis and related antigens was studied in the thymus of children of known ABO, Lewis and secretor status using a panel of specific reagents. ABH and Lewis antigens partly under control of the secretor status were expressed on the Hassals' bodies and a large fraction of the medullary epithelial cells. The sialyl-Lea antigen was only present on some Hassals' bodies of Lewis-positive individuals. ABH but not Lewis antigens were also present on cortical epithelial cells but this was independent of the secretor status. The X, sialyl-X and Y antigens were only expressed on Hassals' bodies irrespective of the ABO, Lewis or secretor phenotype. Furthermore, the anti-X and sialyl-X antibodies labelled a subset of leucocytes of all the individuals tested. These results show that the genetic control of the expression of ABH and Lewis glycosidic tissue alloantigens in the thymus is different on cortical and medullary epithelial cells and stress the heterogeneity of the thymus epithelial cells.     

Monoclonal antibody Leu-22 (L60) permits the demonstration of some neoplastic T cells in routinely fixed and paraffin-embedded tissue sections

Monoclonal antibody Leu-22 (L60) detects a T cell-associated antigen which is stably expressed in routinely fixed and paraffin-embedded tissue sections. We investigated the utility of monoclonal antibody Leu-22 to immunophenotype routinely processed lymphoid neoplasms by determining its reactivity in 105 archival pathologic specimens of lymphoid neoplasia that had been previously immunophenotyped by standard cell suspension and frozen tissue section techniques. Monoclonal antibody Leu-22 reacted with 69% of T cell non-Hodgkin's lymphomas (NHLs), including cases belonging to each of the major clinicopathologic categories, and with 22% of B cell NHLs, but did not react with the Reed-Sternberg (RS) cells of Hodgkin's disease (HD). We concluded that monoclonal antibody Leu-22 reacts preferentially but not exclusively with T cell NHLs. Therefore, we performed parallel analyses of the same 105 cases with monoclonal antibodies leukocyte common antigen (LCA), Leu-M1, LN1, and LN2, which detect various paraffin-resistant antigens, and of 80 of these cases with monoclonal antibody UCHL1, which detects a paraffin-resistant T cell-associated antigen. UCHL1 reacted with 61% of the T cell NHLs studied. Sixty-nine percent of T cell NHLs expressed the LCA+, Leu-22+ or Leu-M1+, LN1- phenotype and 47% of B cell NHLs expressed the LCA+, Leu-22-, Leu-M1-, LN1+ phenotype. These phenotypes had a false-positive rate of only 7%. The substitution of UCHL1 for Leu-22 or the combined use of UCHL1 and Leu-22 in this panel did not improve our ability to correctly predict the T cell phenotype of these lymphoid neoplasms. LN1 and LN2 reacted with 13% and 56% of T cell NHLs, respectively, and LN2 reacted with RS cells in 85% of cases of HD. In summary, our results demonstrate that the judicious use of monoclonal antibody Leu-22 in combination with other selected commercially available monoclonal antibodies permits the determination of the B cell or T cell origin of a high proportion of NHLs, and is helpful in the differential diagnosis between HD and NHL among cases that have been routinely fixed and paraffin-embedded.     

Solid-phase adsorption of antigens for efficient production of antibodies reactive with native and fixed tissue antigens

A study has been made of the efficacy of different immunization protocols using low antigen levels for the generation of monoclonal antibodies capable of detecting antigens (ADCP) in processed tissues. Protocols using unprocessed native antigen immobilized on nitrocellulose were more efficient than soluble antigen in generating serum antibodies reactive with both native antigen and processed tissues. The derived monoclonal antibodies reacted with native but not processed antigen. The use of antigen immobilized on polyvinylidene (PVDF) and subsequently processed as for histochemistry was successful in generating monoclonal antibodies reactive with processed antigen.     

Hepatitis B core and surface antigens in liver tissue. Light and electron microscopic localization by the peroxidase-labeled antibody method.

Hepatitis B core antigen (HBcAg) and hepatitis B surface antigen (HBsAg) were localized in human liver tissues by the peroxidase-labeled antibody method at the light and electron microscopic levels. Several methods of fixation, staining, and inhibition of endogenous peroxidase activity were studied. The periodate-lysine-paraformaldehyde fixative effectively preserved the tissue structure and the antigenicity of both antigens, and the peroxidase-labeled Fab' fraction of IgG penetrated well into hepatocytes. HBcAg was present in nuclei, or cytoplasm of hepatic cells, or both. In nuclei, the antigen was found both in virus-like particles of approximately 20 nm. diameter and in nuclear ground substance. In the cytoplasm, the antigen was found on membrane-bound ribosomes and free polysomes, and also in the ground substance of the cytosol near ribosomes and around nuclear membranes, especially near nuclear pores. HBcAg-positive virus-like particles were also demonstrated sparsely or in clusters in the cytoplasm. HBsAg was not present in nuclei but was found in the perinuclear space and in cisternae of endoplasmic reticulum, and on nuclear, endoplasmic reticulum, and cell membranes of hepatic cells. HBsAg-positive 25- to 30-nm. wide tubular forms, round particles (probably cross-sections of tubular forms), and a few large particles of 40 to 50 nm. diameter were seen in cisternae. Such HBsAg-positive particles were also present in the intercellular space and in Disse's space. These findings suggest that HBcAg produced on the cytoplasmic ribosomes migrates through nuclear pores to the nucleus and is assembled into core particles there. These particles may then move through nuclear pores to the cytoplasm where they are invested with HBsAg-positive membrane in cisternae of endoplasmic reticulum or as they enter the endoplasmic reticulum. These virus particles are then released together with other HBsAg-positive forms into the intercellular space by reversed phagocytosis.     

Monoclonal antibody that detects human type I muscle fibres in routinely fixed wax embedded sections.

A murine monoclonal antibody, F7, which selectively shows type I fibres in human skeletal muscle is reported. The antibody reacts with frozen sections and with formalin fixed wax embedded material. It should prove useful in the retrospective and prospective study of muscle pathology.     

Monoclonal antibody specific to virulence-associated 15- to 17-kilodalton antigens of Rhodococcus equi.

Virulent Rhodococcus equi produces 15- to 17-kDa surface protein antigens. These antigens are used as markers to identify virulent R. equi isolates from foals and their environment by Western blot (immunoblot) analysis with naturally infected foal serum. In the present study, a monoclonal antibody (MAb; 10G5) was generated against the 15- to 17-kDa antigens excised from sodium dodecyl sulfate-polyacrylamide gels to develop sensitive and specific immunoblot assays for the identification of virulent R. equi. MAb 10G5 strongly reacted with R. equi ATCC 33701 and L1, which expressed 15- to 17-kDa antigens by Western blot, colony blot, and dot immunobinding assays, but it did not react with strains ATCC 33701P- and L1P-, which lacked the antigens. For identification of virulent R. equi, clinical and environmental isolates were tested by these assays with the MAb, and all virulent strains were successfully identified; these strains possessed virulence plasmids. These results suggest that the MAb is a useful reagent for the identification of virulent R. equi.     

Monoclonal antibodies to HLA-DR antigens

Monoclonal antibodies (Mabs) have been invaluable in the study of human MHC class-11 (or la-like) antigens. For this to continue some pooling of resources was needed and in 1982 the British Medical Research Council's Clinical and Population Cytogenetics Unit in Edinburgh initiated an information exchange scheme on anti-class-11 Mabs in an attempt to classify and group at least some of these reagents. Over 100 Mabs from 25 laboratories were screened by several centres in various serological, biochemical and functional assays and the data were discussed in Edinburgh on 2–6 September 1983.