Keratins in malignant mesotheliomas and pleural adenocarcinomas: comparative immunohistochemical analysis with polyclonal and monoclonal antibodies

The distribution of intra-cellular keratins was studied in normal pleural mesothelium, malignant mesotheliomas and adenocarcinomas. This study was performed on deparaffinized sections of tissue fixed in Bouin's solution by indirect immunofluorescence with a monoclonal antibody (KL1) and a conventional keratin antiserum (AKS). Discrepancies were detected using one antibody or the other. Cells from normal mesothelium and 18 cases of malignant mesotheliomas (papillary, tubulary, solid epithelial type) were strongly labelled only by KL1. The 2 cases of sarcomatoid type were negative with both antibodies. In contrast 5 metastatic adenocarcinomas and 5 lung adenocarcinomas were weakly positive or negative with both antibodies. These data confirm the presence of cytokeratins in epithelial differentiation process. Although a clear-cut distinction between mesotheliomas and adenocarcinomas was not possible using these keratin antibodies. Our data point out the importance of reactivity pattern of the antibody used in such investigations.     

Improved detection of medically important fungi by immunoperoxidase staining with polyclonal antibodies

This study was performed to identify pathological fungi of eight species [Aspergillus fumigatus, Candida albicans, Torulopsis (Candida) glabrata, Cryptococcus neoformans, Fusarium anthophilum, Rhizopus oryzae, Sporothrix schenckii and Trichosporon beigelii] in formalin-fixed, paraffin-embedded tissue sections by indirect immunoperoxidase staining. Mature albino rabbits were immunized with formalin-killed organisms. Antibodies were prepared by precipitation. Immunoperoxidase staining was applied to the paraffin-embedded tissue sections of experimentally infected mice and human autopsy and surgical specimens. Although the cell walls of each fungus stained clearly, many cross-reactivities appeared. However, it was possible to obtain specificity for the eight species by absorption and dilution of the antisera.     

Identification of monoclonal antibodies to pancreatic islet cells by immunoperoxidase staining

Immunoperoxidase staining and enzyme-linked immunosorbent assay (ELISA) were used to identify monoclonal antibodies that reacted with pancreatic islet cells. All monoclonal antibodies produced against isolated human or rat pancreatic islets including one mouse autoantibody reacted with pancreatic islets in formalin-fixed pancreas sections, but not with rat kidney or thyroid. Reactivity was also found with suspensions of normal rat islet cells and rat insulinoma cells using a 3-stage immunoperoxidase procedure and an ELISA technique. Differences were observed in staining intensity between the various antigenic substrates tested suggesting variable cross-reactivity and/or number of epitopes. The sensitivity of the immunoperoxidase technique proved to be favourable for identification of monoclonal antibodies that recognize cellular constituents such as islet cell antigens present in low concentrations.     

Differential staining of cytoid bodies and skin-limited amyloids with monoclonal anti-keratin antibodies

The authors have used 5 different monoclonal antikeratin antibodies to study the antigenic profiles of cytoid bodies and skin-limited amyloids. Monoclonal antibodies AE1 (which stains the basal cell layer in normal human epidermis), AE2 (suprabasal layers), AE3 (whole epidermis), EKH4 (lower 2-3 layers), and EKH1 (recognizes all classes of intermediate filaments) were used to stain frozen skin sections by the indirect immunofluorescent or indirect immunoperoxidase technique. Cytoid bodies in lichen planus (LP) and discoid lupus erythematosus (DLE) were strongly stained with AE1, AE3, EKH4, and EKH1 antibodies but were negative with AE2. In contrast, amyloids in lichen amyloidosus and macular amyloidosis were stained strongly with EKH4 but only weakly or not at all with AE1, AE2, AE3, and EKH1. Amyloid associated with epithelial tumors showed closer immunologic profiles to cytoid body. These findings suggest that epidermal keratins are the major precursor substance of skin-limited amyloids as well as cytoid bodies in LP and DLE. Sequential changes in antigenic profiles from basal cells to amyloids through cytoid bodies further suggest that cytoid bodies may represent one of the precursor substances of skin-limited amyloids.     

Demonstration of anti-keratin antibodies by ELISA using keratin or thiol-containing compounds in urea as antigens

During the development of an enzyme-linked immunosorbent assay (ELISA) for the demonstration of anti-keratin antibodies (AKA) it was observed that rabbit anti-keratin antisera also reacted with polystyrene surfaces treated with beta-mercaptoethanol in 8 M urea (ME-urea). Sera from non-immunized rabbits or rabbits immunized with antigens unrelated to keratin failed to react. The specificity of the reaction was further assessed by absorption experiments and by testing affinity-purified AKA. IgM activity against ME-urea could be demonstrated in 62.5% of sera from patients with infectious mononucleosis and in 37.5% of sera from patients with rheumatoid arthritis, and there was a good correlation to the presence of AKA. Coating of the solid phase with compounds containing free SH groups in 4-8 M urea generated the antigen of this ELISA. The exact molecular configuration of this presumptive synthetic antigen is obscure, but the ME-urea ELISA seems to provide a simple way to detect anti-keratin antibodies of a certain specificity.     

Antigenic classification of Rickettsia felis by using monoclonal and polyclonal antibodies

Rickettsia felis is a flea-transmitted rickettsia. There is a discrepancy between its reported phylogenic and phenotypic identifications. Following the first report of R. felis, it was considered by tests with serologic reagents to be closely related to another recognized flea-transmitted rickettia, R. typhi. Subsequently, it appeared to be more closely related to spotted fever group (SFG) rickettsiae by genetic analysis. In the present work, R. felis was studied by microimmunofluorescence (MIF) serologic typing and with monoclonal antibodies (MAbs). Mouse polyclonal antisera to R. felis cross-reacted only with SFG rickettsiae. A neighbor-joining analysis based on MIF indicated that R. felis is actually related to SFG rickettsiae antigenically, clustering with R. australis, R. akari, and R. montanensis. A panel of 21 MAbs was raised against a 120-kDa protein antigen or a 17-kDa polypeptide of R. felis. They cross-reacted with most members of the SFG rickettsiae but not with R. prowazekii, R. typhi, or R. canadensis of the typhus group (TG) rickettsiae. Sixty-four MAbs previously generated to seven other ricketttsial species were tested with R. felis. Three MAbs reacted with the 120-kDa antigen and were generated by R. africae, R. conorii, and R. akari, respectively. They exhibited cross-reactivities with R. felis. All our data show that R. felis harbors the antigenic profile of an SFG rickettsia.     

Heterogeneity of rat thymic epithelium defined by monoclonal anti-keratin antibodies

Immunohistochemical characterization of rat TEC has been studied using a panel of monoclonal anti-keratin antibodies. These mAbs identified three distinct patterns of keratin subunit expression within thymic epithelium assessed by streptavidin-biotin immunoperoxidase staining and double immunofluorescence staining. K8 and KII mAbs labelled almost all epithelium, while K18 stained cortical epithelium and a subpopulation of medullary TEC. KI, K7 and K19 mAbs bound to the subcapsular/subtrabecular flat epithelial cell layer, TEC lining some perivascular spaces in the cortex, a subpopulation of medullary TEC and interstitial epithelial cystic structures. Double immunostaining revealed further heterogeneity of subunit keratin compositions in epithelial cells of particular thymic microenvironments suggesting their different origin or development stage.     

Use of monoclonal antibodies to keratin 7 in the differential diagnosis of adenocarcinomas.

Monoclonal antibodies (MAbs) to specific keratin subtypes were prepared and characterized by immunoblotting and immunohistochemical assays on human cell cultures and normal and malignant human tissues. Chain-specific MAbs to keratin 7 (RCK 105, OV-TL 12/30) and keratin 18 (RGE 53, RCK 106, CK18-2), as well as broadly cross-reacting keratin MAbs (RCK 102, OV-TL 12/5) could be shown to react with different types of human epithelial tissues and were therefore tested for their usefulness in the differential diagnosis of carcinomas. The two broad-spectrum antibodies stained virtually all of the more than 350 carcinomas tested, especially when combined, and distinguished them from most nonepithelial tumors. The keratin 18 MAbs distinguished adenocarcinomas (which are keratin 18 positive) from most squamous cell carcinomas (which are generally keratin 18 negative). The MAbs to keratin 7 could be shown to recognize specific subtypes of adenocarcinoma and could, for example, distinguish between ovarian carcinomas (keratin 7 positive) and carcinomas of the gastrointestinal tract (keratin 7 negative), or between transitional cell carcinomas (keratin 7 positive) and prostate cancer (keratin 7 negative). In general, malignancies showed the expected keratin reactivity pattern as concluded from the keratin pattern of its cell of origin or its type of differentiation. The use of an extended series of malignancies did, however, also illustrate that exceptions to this rule exist. For example, certain antibodies to keratin 18 stained tumor areas in squamous cell carcinomas of the lung. Also a certain percentage of tumors, which generally showed no keratin 7 expression, were positive with RCK 105 or OV-TL 12/30. On the other hand, a certain percentage of tumors, which were generally positive for keratin 7, did not show a staining reaction with these MAbs. Furthermore subtle differences between reactivity patterns of different MAbs recognizing the same keratin protein were observed, both in the normal and malignant human tissues, indicating that specific keratin epitopes may be masked in certain tissues and that unmasking of such epitopes can occur with malignant progression. This phenomenon may be of some use in a further subtyping of carcinomas, especially those of the gastrointestinal tract. Despite these exceptional staining patterns, the keratin MAbs described above have proved to be useful tools in the characterization of epithelial tumors in routine histopathology and cytopathology, in which they add to a more refined diagnosis of (adeno)carcinomas.     

Immunohistochemical study of nasopharyngeal carcinoma using monoclonal keratin antibodies

Nasopharyngeal carcinoma (NPC) provides a unique opportunity to evaluate distinctive epidemiologic features and a possible etiologic relationship with Epstein-Barr virus (EBV) in human malignancy. The lack of a uniformly accepted pathologic classification for NPC has limited the application of this data, although the World Health Organization (WHO) developed a classification that may solve this problem. Monoclonal keratin antibodies were used for staining of NPC for evaluation of its assistance in diagnosis and classification. In the present immunohistochemical study, monoclonal keratin antibodies, designated AE1, AE2, and AE3, and a polyclonal keratin antibody (RAK) were used for study of the presence of keratin in 121 cases of NPC obtained from China and the United States. AE1 monoclonal antibody, which recognizes keratin protein classes 56.5K, 50K, and 40K, was shown to be the most sensitive and specific for NPC tumor cells among the keratin antibodies studied. In addition, some different keratin expression patterns could be identified between different kinds of epithelium and different tumor groups, with possible relevance to the histogenesis of the histologic subtypes of NPC.     

Application and staining patterns of commercial anti-Pneumocystis carinii monoclonal antibodies.

Commercially available monoclonal antibodies to Pneumocystis carinii were compared with respect to immunofluorescence staining patterns of human immunodeficiency virus-inactivated smears. Only the indirect staining kits were suitable for application to ethanol-inactivated samples. When antibodies from Dakopatts and Northumbria were compared, the staining of cysts and trophozoites showed different patterns.     

Malignant mesotheliomas. Improved differential diagnosis from lung adenocarcinomas using monoclonal antibodies 44-3A6 and 624A12.

Forty-three malignant pleural mesotheliomas and 10 known metastatic pulmonary adenocarcinomas to the pleura were studied by immunohistochemistry using monoclonal antibodies 44-3A6 and 624A12. Monoclonal antibodies 44-3A6 and 624A12 were raised against human pulmonary carcinoma cell lines; they recognize a membrane-associated protein of 40,000 mol wt and a specific sugar sequence of lacto-N-fucopentose III, respectively. Samples were also studied with a broad-spectrum antikeratin antibody and a polyclonal antibody to carcinoembryonic antigen (CEA). These investigations were performed on formalin-fixed and paraffin-embedded tissues. The mesotheliomas comprised only grossly evident, pleurectomized, or pneumonectomized cases; they included 22 epithelial, 15 biphasic, and 6 spindle cell types. Electron-microscopic study was also done on 9 cases. None of the mesotheliomas was immunoreactive to 624A12, while 9/10 metastatic pulmonary adenocarcinomas were convincingly immunoreactive. Monoclonal antibody 44-3A6 immunostained all of the metastatic adenocarcinomas strongly, whereas only 10/43 mesotheliomas were focally and weakly immunoreactive. The latter included 5 epithelial and 4 biophasic mesotheliomas and 1 spindle cell mesotheliomas; the immunoreaction was confined to scattered single cells, and the staining pattern was readily discernible from that of adenocarcinomas. Forty of 43 mesotheliomas were strongly immunoreactive with the broad-spectrum anti-keratin antibody, whereas 8/10 metastatic pulmonary adenocarcinomas showed focal and rather weak staining. Seven of 10 metastatic adenocarcinomas were immunoreactive to anti-CEA antibody, while only 15/43 mesotheliomas displayed weak immunoreactivity. It is concluded that monoclonal antibodies 44-3A6 and 624A12 are excellent phenotypic markers of metastatic pulmonary adenocarcinomas to the pleura and thus are useful for the differential diagnosis of pleural mesotheliomas. Given conventionally fixed and processed tissues, it appears that the combined use of these monoclonal antibodies may be more effective for that differential diagnosis than anti-CEA and anti-keratin antibodies.     

Flow cytometric analysis of surface light chain expression patterns in B-cell lymphomas using monoclonal and polyclonal antibodies

Light chain (LC) expression by flow cytometry (FC) in B cell non-Hodgkin lymphomas (B-NHLs) can occasionally be detected with one anti-LC antibody but not with another. We retrospectively analyzed 564 four-color FC files from B-NHLs, assessing LC staining with monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs). Discrepancies in LC expression between mAbs and pAbs were present in 9.2% of cases, mainly in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL; 11.1%), diffuse large B-cell lymphoma (DLBCL; 10.2%), follicular lymphoma (9.5%), and mantle cell lymphoma (11.1%) and most frequently in body fluids. Equal proportions of cases were LC+ only with pAbs (4.8%) or mAbs (4.4%). Negative LC expression with both antibodies was present in 7.5% of cases, most frequently in DLBCL (21.6%) and body fluids (27.6%). Evaluation with both mAbs and pAbs increases the sensitivity for LC detection, with no single reagent outperforming the other, although CLL/SLL preferentially showed LC expression with pAbs.     

Detection of microsporidia by indirect immunofluorescence antibody test using polyclonal and monoclonal antibodies.

During a screening for monoclonal antibodies (MAbs) to the microsporidian Encephalitozoon hellem, three murine hybridoma cell lines producing strong enzyme-linked immunosorbent assay (ELISA) reactivities were cloned twice, were designated C12, E9, and E11, and were found to secrete MAbs to the immunoglobulin M isotype. On subsequent ELISAs, the three MAbs reacted most strongly to E. hellem, and they reacted somewhat less to Encephalitozoon cuniculi and least to Nosema corneum, two other microsporidian species. The MAbs produced values of absorbance against microsporidia that were at least three times greater than reactivities obtained with control hybridoma supernatants or with uninfected host cell proteins used as antigens. By Western blot immunodetection, the three MAbs detected three E. hellem antigens with relative molecular weights (M(r)s) of 62, 60, and 52 when assayed at the highest supernatant dilutions producing reactivity. At lower dilutions, the MAbs detected additional proteins with M(r)s of 55 and 53. By using indirect immunofluorescence antibody staining, the MAbs, as well as hyperimmune polyclonal murine antisera raised against E. cuniculi and E. hellem, were able to detect formalin-fixed, tissue culture-derived E. cuniculi and E. hellem and two other human microsporidia, Enterocytozoon bieneusi and Septata intestinalis, in formalin-fixed stool and urine, respectively. E. bieneusi, however, stained more intensely with the polyclonal antisera than with the MAbs. Neither the MAbs nor the hyperimmune murine polyclonal antibodies detected Cryptosporidium, Giardia, Trichomonas, or Isospora spp. At higher concentrations, the polyclonal antisera did stain N. corneum and yeast cells. The background staining could be absorbed with Candida albicans. These results demonstrate that polyclonal antisera to E. cuniculi and E. hellem, as well as MAbs raised against E. hellem, can be used for indirect immunofluorescence antibody staining to detect several species of microsporidia known to cause opportunistic infections in AIDS patients.     

Characterization of rabbit cells by monoclonal and polyclonal antibodies.

Reagents for the identification of rabbit cell markers have been developed at a relatively slow rate. In this paper, rabbit cells are being characterized by polyclonal antibodies against a T-cell antigen (RTLA), a B-cell antigen (RABELA) and an analogue of murine Ia antigen. A number of monoclonal antibodies, specific for lymphocytes and/or bone marrow and/or polymorphonuclear leucocytes, have been used for the analysis of cells with identifiable membrane antigens. Populations that have cells with two of the above antigens in the membranes were identified. To these ends, complement-mediated cell kill by antisera alone and in mixtures was employed.     

Rapid detection of respiratory syncytial virus and influenza A virus in cell cultures by immunoperoxidase staining with monoclonal antibodies.

Peroxidase-labeled monoclonal antibodies against respiratory syncytial virus (RSV) and influenza A virus were used for immunoperoxidase staining (IPS) of cell cultures inoculated with nasopharyngeal aspirates. Cells were grown in 24-well plates, and specimens were inoculated by low-speed centrifugation. Cultures were incubated for 2 days at 37 degrees C and then fixed, stained, and observed by light microscopy. IPS was compared with standard virus isolation by using cultures of human diploid fibroblasts and Vero, HEp-2, and HeLa cell lines for RSV and Madin-Darby canine kidney cells for influenza A virus; these cultures were inoculated with specimens that were previously stored at -70 degrees C. Of 40 known RSV-positive specimens, 30 were found to be positive on reinoculation by both methods, and an additional 5 specimens were found to be positive by IPS only. Of 190 specimens tested for influenza A virus, 14 were positive by IPS and in tubes, and a further 8 specimens were positive by IPS only. IPS was also compared with direct detection of viral antigens in nasopharyngeal aspirates by a time-resolved fluoroimmunoassay (TR-FIA). Fresh nasopharyngeal aspirates were inoculated into human diploid fibroblasts and Madin-Darby canine kidney cells and tested for RSV and influenza A virus, respectively, by IPS. Of 110 specimens tested for RSV, 37 were positive in total, 32 were positive by IPS, and 33 were positive by TR-FIA. Of 150 specimens tested for influenza A virus, 39 were positive in total, 35 were positive by IPS, and 34 were positive by TR-FIA. IPS of cultures inoculated by centrifugation and incubated for 2 days is a sensitive method for the diagnosis of respiratory virus infections, and 24-well plates allow for the easy processing of a large number of specimens.     

Differential diagnosis of gastrointestinal carcinomas by using monoclonal antibodies specific for individual keratin polypeptides

Monoclonal antibodies which recognize particular keratin polypeptides have been used to analyze normal human tissues including pancreas, stomach, colon, gall bladder, and liver as well as tumors of the gastrointestinal tract by immunohistological techniques. Broad specificity (lu5), ker 8 (Troma 1) and ker 18 (CK2) antibodies were positive while a ker 14 specific antibody (CKB1) was negative on all specimens tested. Differential staining patterns were seen with a ker 7 (CK7) and a ker 19 (KA4) antibody. Both antibodies stained gall bladder epithelium, pancreatic ducts but not acinar cells, as well as pancreatic ductal adenocarcinomas. KA4 but not the CK7 antibody stained adenocarcinomas of the stomach and large bowel. Both antibodies stained bile ducts and cholangiocellular carcinoma of the liver but did not stain hepatocytes or hepatocellular carcinomas. The results with keratin monoclonal antibodies compare well with those obtained by others using two dimensional gel electrophoresis and they further support the idea that monoclonal antibodies specific for particular keratin polypeptides will find applications in routine pathological diagnosis.     

Detection of group B streptococcal antigen in necropsy specimens using monoclonal antibody and immunoperoxidase staining.

A murine monoclonal antibody, which recognises various serotypes of group B Streptococcus in formalin fixed, paraffin embedded tissue, was used to show the organism in necropsy specimens of newborn infant lung by an indirect immunoperoxidase technique. The method seemed to be complementary to that of Gram staining, and may be successfully used to identify group B Streptococcus antigen in histopathological material.     

The production of polyclonal and monoclonal antibodies in mice using novel immunization methods

Two novel immunization methods (intrasplenic and intra-inguinal lymph node) have been developed for the production of polyclonal and monoclonal antibodies in mice. Freund's complete adjuvant and antigen were mixed in the ratio of 1 : 2 (v/v). Various concentrations of human serum albumin (HSA) were used as antigen. No primary immune response was induced with 0.1 μg of HSA in either of the methods studied. Intrasplenic immunization resulted in the strongest primary immune responses using all other doses of HSA. The primary immune response induced by intrasplenic immunization with 0.5 μg of HSA was higher than any response induced by subcutaneous immunization with various doses of HSA. Inguinal lymph node immunization was less effective than intrasplenic immunization but better than subcutaneous immunization with 1–50 μg of HSA. Comparisons were also made of the efficacy of different adjuvants when inducing primary immune responses with 1 μg of HSA. Freund's complete adjuvant resulted in a much stronger response than Freund's incomplete adjuvant and alum. Both intrasplenic and inguinal lymph node immunization using 1–5 μg of HSA were able to induce strong primary immune responses. Secondary immunization with either method or intravenous injection 3 days before fusion resulted in a higher frequency of specific monoclonal antibodies.     

Antigenic differentiation of avian pneumovirus isolates using polyclonal antisera and mouse monoclonal antibodies

Avian pneumovirus (AVP) isolates F83, CC220 and 1260 from Great Britain and 1556, 657/4, 2119 and 872/S from France, Hungary, Italy and Spain, respectively, were compared in ELISA and virus neutralization (VN) tests for reactions with chicken polyclonal sera against each of the viruses and monoclonal antibodies (MAbs) against two British isolates. ELISA test results using the polyclonal antisera indicated that all seven viruses were antigenically related, but some variation between strains could be detected, especially when antigens were prepared from infected cells using Nonidet P40 (NP40) rather than freezing and thawing. In VN tests results also showed that all viruses tested were related but there was evidence that the three British isolates showed closer relationships with each other than with the viruses from Italy, Hungary and Spain. In ELJSA tests, isolates F83 and 1556 bound all 11 MAbs and 1260 reacted with 10/11 MAbs. Isolate CC220 showed reaction with all the MAbs but for 8/11 MAbs the optical density differences were low. Isolates 2119 and 872/S both reacted only with MAb 4 and none of the MAbs reacted with 657/4.     

Identification of T-lymphocytes and T-subsets in human tonsil using monoclonal antibodies and the immunoperoxidase technic

A study was performed using monoclonal antibodies and the immunoperoxidase technic to identify T-cells and T-subsets in human tonsil sections. The in situ topographic identification of T-cells, helper cells, and suppressor cells was achieved. This model may therefore be applied to other normal lymphoid tissues. Its potential pathologic importance lies in the fact that certain disorders may be associated with depletion of lymphocyte subsets from their normal areas of "homing," and that lymphoma cells may mirror their putative normal counterparts in their selective metastatic migration pattern. The improved knowledge of normal lymphocyte subset microenvironment afforded by this technic may therefore be of value in applied pathology.