Monoclonal Reagents Research Articles

A murine monoclonal anti-N, BIRMA-N, suitable for blood grouping in an automated system.

BIRMA-N, a murine monoclonal anti-N antibody of the IgG1 subclass, was assessed for suitability as a blood grouping reagent on the Olympus PK7100 automated blood grouping machine. At a selected dilution and over the pH range 5.0-8.0, the antibody performed accurately in this system as confirmed by parallel manual testing of donor blood samples with human anti-N and commercial monoclonal anti-N reagents. These findings show BIRMA-N to be extremely suitable for N typing blood samples in an automated system providing a convenient, objective and cost-effective method for large scale typing of the blood donor population.

Overview of the avian immune system

The avian immune system operates on the same general principles as the mammalian immune system. Antigenic stimulation initiates an immune response that involves cellular cooperation most notably between macrophages, B lymphocytes and T lymphocytes. Macrophages process the antigen and present the antigen to the lymphocytes. B lymphocytes, the principal cells that mediate humoral immunity, transform into plasma cells and produce antibodies. T lymphocytes, most important for cellular immunity, differentiate into functionally diverse subpopulations. The subpopulations of avian T cells have been identified with monoclonal reagents and appear to be similar to those of mammalian T cells. Lymphokines, the soluble products secreted by immune cells, mediate the functions of these cells. Studies on avian lymphokines have lagged behind those on mammalian lymphokines because the genes coding for avian lymphokines have not been cloned. The avian lymphokines studied thus far appear to function along the same lines as the mammalian lymphokines. The immune response in birds is highly regulated and breakdown in regulation often results in immunodepression.

Expression of HLA Class I and II (DR, DP and DQ) Determinants in Fetal and Postnatal Salivary Glands

Fetal (n = 20) and postnatal (n = 40) parotid glands were examined by two-colour immunohistochemistry combining monoclonal and polyclonal antibody reagents to study the expression of HLA class I and II (DR, DP, and DQ), CD45 and CD3. The epithelium lacked class II during fetal life, whereas class I determinants appeared in some acini and most major ducts. Fetal vessels were positive for both class I and class II (mainly DR), suggesting constitutive expression. Some class II-positive (DR greater than DP greater than DQ) histiocytic cells, scattered CD45+ leucocytes, and very few CD3+ T cells were present in the fetal stroma. The epithelium remained DR-negative the first few weeks after birth, but brisk expression was seen subsequently. DP and DQ remained virtually negative in the epithelium throughout the first year. A slight postnatal increase of class II expression (DR greater than DP greater than DQ), along with an apparent decrease in class I, was observed in the endothelium. The number of class II-positive histiocytic cells, CD45+ leucocytes and CD3+ T cells, as well as the proportion of presumably activated (DR+) T cells, increased a few weeks after birth. The local immune system hence seemed to be stimulated by extrinsic factors, but the overall number of T cells nevertheless remained small. Stimuli other than T cell-derived lymphokines, therefore, probably explained the brisk postnatal epithelial DR induction.

Expression of immunoglobulin genes in the avian embryo bone marrow revealed by retroviral transformation.

Analysis of the early stages of avian B lymphocyte differentiation has been hampered by the low frequency of extra-bursal B lineage cells in sites of hematopoiesis. Consequently, little is known about B lineage precursors prior to their migration into the bursa of Fabricius. Colonization of the bursa typically occurs between about days 8 and 14 of embryonic (e) development, although cells which can colonize the bursa, functionally defined as pre-bursal stem cells, can be demonstrated in embryo bone marrow up until about the time of hatch. As a novel approach to analyzing early stages of avian B lymphocyte development, we show here that transformed B lineage cells can be derived from chick embryo bone marrow after infection in vitro with the replication-defective retrovirus REV-T produced in the context of the non-cytopathic CSV helper virus. Thus, exposure of day 14e-15e chick embryo bone marrow cells to REV-T (CSV) results in the generation of transformed, polyclonal lines of cells. From these lines, cells expressing cell surface immunoglobulin were readily isolated by flow cytometric cell sorting and single cell cloning. Analysis of the phenotype of REV-T(CSV)-transformed clones with a panel of monoclonal antibody reagents demonstrated that transformation by v-rel likely leads to marked changes in cell surface antigen expression. Nonetheless, clones expressing cell surface immunoglobulin expressed apparently normal mRNA for immunoglobulin mu and light chain and contained apparently normal immunoglobulin heavy and light chain gene rearrangements. Furthermore, no evidence for chromosomal deletions or aberrations of the Ig loci was detected among either sIg+ or sIg- REV-T(CSV)-transformed clones.

Production of polyclonal and monoclonal antibodies against group A, B, and C capsular polysaccharides of Neisseria meningitidis and preparation of latex reagents

Polyclonal and monoclonal antibodies against capsular polysaccharides of Neisseria meningitidis serogroups A, B, and C were produced in order to develop immunological reagents allowing both the detection of soluble antigens during meningococcal meningitis and antigenic serogrouping of N. meningitidis cultures. The performance characteristics of monoclonal and polyclonal antibody latex reagents were compared. For the detection of soluble polysaccharide antigen, polyclonal antibody latex reagent was selected for N. meningitidis A and C. The latex reagent prepared with polyclonal antibodies against N. meningitidis B could not detect capsular polysaccharide even at 1 mg/ml. The monoclonal antibody B latex reagent which detected 100 ng of polysaccharide per ml was therefore chosen. For the serogroup identification of N. meningitidis, the use of a confirmatory test results in an overall specificity of 100% with polyclonal or monoclonal antibody latex reagents.

Fine‐needle aspiration biopsy of malignant melanoma: A cytologic and immunocytochemical analysis

The immunoreactivity of alcohol-fixed cell blocks from 15 fine-needle aspiration (FNA) specimens of malignant melanoma was investigated using monoclonal antibodies to keratin and vimentin intermediate filaments, melanoma cytoplasmic antigen (HMB-45), and S-100, as well as polyclonal antibodies to S-100. The results were compared with the immunoprofiles obtained using formalin-fixed surgical specimens from 10 of the same patients. In all cases, immunostaining for keratin was negative and immunostaining for vimentin was positive. Immunostaining for HMB-45 was positive in 13/15 aspirates and in 9/10 surgical specimens. Immunostaining for S-100 protein showed the greatest variability in staining, with 5/15 fine needle aspiration biopsies and 9/10 surgical specimens being positive using the polyclonal antibody and only 1/15 FNA specimens and 7/10 surgical specimens being positive using the monoclonal S-100 reagent. Our findings indicate that immunocytochemical studies can be very useful as an adjunct in the FNA diagnosis of melanoma. Also included in our series is an unusual variant of malignant melanoma, the so-called signet ring melanoma. Given the location of the anal verge, the use of immunocytochemical markers was essential in establishing the correct diagnosis in this case. While S-100 protein is of limited value as a marker of melanoma in alcohol-fixed FNA specimens, a definitive diagnosis of malignant melanoma can be made using a panel of antibodies including keratin, vimentin, and HMB-45.

The anti‐mitozantrone monoclonal antibody NO‐1, protects acute leukaemia cell lines from the cytotoxic effects of mitozantrone

The monoclonal antibody NO-1 was raised against the potent anti-cancer drug mitozantrone by immunization of a BALB/c mouse with a mitozantrone-keyhole limpet haemocyanin conjugate in Freund's complete adjuvant. This antibody was shown to be highly effective in vitro at neutralizing the cytotoxic effects of mitozantrone for the acute leukaemia cell lines ALL-1 and MOLT4. In order to achieve complete protection, a drug to antibody molar ratio of 1.5:1 was required. The neutralizing effect was specific for mitozantrone, as NO-1 antibody offered no protection of the MOLT4 cell line to the cytotoxic effects of the anthracycline drug daunorubicin when used at a near identical molar ratio. NO-1 antibody has already proven a highly successful monoclonal reagent for use in a competitive enzyme-linked immunosorbent assay for the accurate and sensitive quantitation of mitozantrone in serum. The neutralizing properties of NO-1 suggest other possible applications for this antibody. These could include a use in the rapid clearance of pharmacologically active mitozantrone from the circulation following very high dose administration prior to bone marrow transplantation and for the construction of bispecific antibodies for targeting mitozantrone to tumour cell populations.

Tumor vaccines

Vaccination against tumor, either as a prophylactic procedure or as a mode of treatment, has been a distant goal of immunologists for many years. Ideally, the less specific therapies such as chemotherapy would be replaced by an anti-tumor immune response in the host that would be present on a continuing basis. However, progress has been hampered by a lack of understanding of the role of viruses in human tumor development and the molecular nature of tumor-associated antigens. Recent developments using the techniques of molecular biology and monoclonal antibody reagents are beginning to remedy this deficiency so that vaccination has become a real possibility for certain human cancers. The natural fluctuations in growth rates of some human tumors, and the observation that tumors can occasionally remain dormant for years, has led to the idea that the host has an intrinsic ability to control tumor growth, and that this ability is a property of the immune system. Attempts to enhance this putative control are being made by treating the host with defined biological modifiers that stimulate cells involved in immunity in vivo, and by seeking and expanding such cells in vitro before reinfusing them into the host. These attempts to harness the immune system to attack tumor cells that have evaded the host's defenses might be considered optimistic, but they will at least tell us a great deal about tumor cell behavior and the ability of the host to influence it.

Monoclonal antibody-based kits or reagents for HSV infection

Monoclonal antibody-based kits or reagents for HSV infection

A New Qualitative Variant of the RhE Antigen Revealed by Heterogeneity among Anti‐E Sera

We have recently detected a new qualitative variant of the RhE antigen characterised by negative reactions with a minority of anti-E sera, and an inability of the relevant cells to completely absorb most, if not all, anti-E samples that we tested. In tube tests, using a two-stage enzyme technique, cells from the proposita (V.R.) were completely agglutinated by 8 out of 12 undiluted polyclonal sera, weakly agglutinated (2+ and 3+ reactions) by 2 sera, but not agglutinated by the 2 remaining sera. Cross-absorption tests using 1 non-reacting, 1 weakly reacting and 4 strongly reacting polyclonal anti-E sera showed that V.R.'s cells only slightly reduced their anti-E titres versus r"r cells, whilst completely removing their reactivity against her own cells. Absorptions with R2R2 cells abolished activity against both cell types. The cells of V.R. failed to absorb or elute anti-Ew, and had normal expression of c. Three monoclonal human anti-E reagents were tested by fluorescence flow cytometry; one reagent failed to react with the cells of V.R., a second bound as strongly with V.R.'s cells as with R1R2 controls while the third bound much more weakly with V.R. than with any other E+ sample. The variant of RhE in the V.R. family is therefore distinct from Ew, and lacks an epitope recognised by a major constituent of most anti-E sera. This epitope is unlikely to be ET, since all of 91 samples from E-positive Australian aborigines (many of which are expected to lack ET) were agglutinated by a serum that failed to react with V.R.'s cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormalities associated with the bursal microenvironment in spontaneous dysgammaglobulinemia of UCD line 140 chickens

UCD line 140 chickens have been previously reported to develop a syndrome of spontaneous 7s immunoglobulin deficiency and the presence of autoantibodies. Earlier studies demonstrated that these inbred birds have normal peripheral blood T and B cell numbers; they also respond normally to allogeneic stimulation. Although the 7s immunodeficiency does not manifest itself until several months of age, line 140 birds have a premature degeneration of bursa. Because of the recent development of monoclonal reagents specific for bursal elements, including surface epithelium, basement membrane associated epithelium, follicle associated epithelium, and lymphoid subpopulations, we have examined line 140 and control birds for the expression of bursal epithelial cell antigens. Line 140 birds, in contrast to control chickens, have a dramatic early alteration in the expression of an epithelial cell marker in the bursa, thymus, and intestine. Moreover, to further address this issue, we transplanted bursa from 10-day embryos onto the chorioallantoic membrane, a privileged site. Bursae from control birds became abnormal when transplanted onto line 140 CAM; they remained normal when transplanted among several control chicken lines. In contrast, line 140 bursa remained abnormal independent of the transplant procedure. Due to the marked bursal abnormality observed specific to the dysgammaglobulinemia chicken line, we propose that the microenvironmental features of line 140 bursa may predispose these birds to the development of humoral immunodeficiency and autoantibodies.

Human anti-mouse antibody response induced by anti-CD4 monoclonal antibody therapy in patients with rheumatoid arthritis

The development of human anti-mouse monoclonal antibodies (HAMAs) was investigated ib 10 patients with rheumatoid arthritis (RA) who had undergone an experimental therapeutic trial with an anti-CD4 monoclonal antibody. In this patient group, the antibody 16H5 of the IgG1 isotype had been administered in a median total dosage of 140 mg per treatment cycle. Four patients took part in a second treatment regimen 6–8 weeks later. After the first treatment cycle, detectable HAMAs developed in 5 out of 10 patients. In 4 individuals undergoing a second course of therapy, increases of HAMAs were evident only in the 3 patients with previous HAMA responses. HAMAs were primarily of the IgG isotype, while the presence of rheumatoid factors usually interfered with the detectability of IgM HAMAs. However, using isolated F(ab) 2 fragments of the monoclonal reagent used for therapy, HAMAs of the IgM isotype were also detectable. HAMAs of the IgG isotype did not exceed levels of 2.0 mg/liter after a single treatment cycle and 2.2 mg/liter after a repeated cycle. No IgE responses were detectable. Absorption experiments indicated that approximately 25% of the HAMA activity was directed against specific determinants of the 16H5 monoclonal antibody, presumably including anti-idiotypic reactivities. These data demonstrate that HAMAs developed only in a proportion of RA patients treated with the anti-CD4 monoclonal antibody 16H5. However, the amounts were rather low compared to other monoclonal reagents used in cancer patients and were therefore allowed for repeated applications without an apparent loss of efficacy.

Subclass and molecular form of immunoglobulin A antibodies to Actinobacillus actinomycetemcomitans in juvenile periodontitis

Patients with juvenile periodontitis frequently have elevated levels of serum immunoglobulin A (IgA) antibodies to antigens of Actinobacillus actinomycetemcomitans. IgA occurs in two subclasses, IgA1 and IgA2, and in monomeric and polymeric forms. Because IgA1 is susceptible to cleavage by IgA1 proteases produced by microorganisms found at mucosal sites and in the gingival crevice, we wished to determine the IgA subclass distribution of antibodies to antigens of A. actinomycetemcomitans. The molecular form was examined because it may indicate the origin of the IgA and because the form differs in acute and chronic infections. There is also evidence that monomeric and polymeric IgA have different biological functions. Serum was taken from patients with juvenile periodontitis before and at intervals during and after initiation of therapy. IgA subclass distribution was determined against a sonic extracts of A. actinomycetemcomitans ATCC 2952a (serotype b) by using monoclonal anti-subclass reagents in an enzyme-linked immunosorbent assay. To determine the molecular form of the antibodies, sera were separated by high-performance liquid chromatography on a size-exclusion column. Fractions were assayed for antibody activity by the enzyme-linked immunosorbent assay, and described above. The results of the subclass analysis of the sera indicated that while both IgA1 and IgA2 antibodies to A. actinomycetemcomitans sonic extract are often found before, during, and after treatment, IgA1 antibodies dominated the response. There was a predominance of monomeric IgA1 antibodies to A. actinomycetemcomitans sonic extracts in most samples before, during, and after treatment. The monomeric form is consistent with what is seen in other chronic infections. The predominance of IgA1 antibodies implies that any protective effects of the IgA response to A. actinomycetemcomitans could be compromised by microbial IgA1 proteases.

Typing of normal and variant red cells with ABO, Rh, and Kell typing reagents using a gel typing system

Abstract In 1989 Lapierre et al. described a novel method of detecting agglutination reactions by the use of a Sephadex (DiaMed ID Typing System) gel held in a microtube. This report examines the use of gels containing ABO, Rh, and Kell system specific antibodies. The anti-A and -B were monoclonal reagents; anti-A,B, and those for the Rh and Kell systems were polyclonal. Five hundred and fifty-one tests performed for the ABO system detected all but the most weakly reacting variants, a detection rate superior to most commercially available reagents. Five hundred and thirty samples were typed for Rh antigens. One hundred and twenty-seven of these were of various D category III through VII types (Dcats) and 154 were DUs. The gel system detected all but seven DVI variants and seven DUs. The seven DVI variants, from individuals with no anti-D in their sera, gave reactions identical to the seven DUs when tested against a panel of over 50 monoclonal IgG and IgM anti-Ds. The 554 samples tested for the K1 antigen gave correct results. Immunohematology 1991;7:94–97.

High concentrations of a cGMP-stimulated phosphodiesterase mediate ANP-induced decreases in cAMP and steroidogenesis in adrenal glomerulosa cells.

An adrenal cGMP-stimulated phosphodiesterase (cGS-PDE) has been shown to mediate atrial natriuretic peptide (ANP)-induced reductions in aldosterone secretion and cAMP levels in primary bovine glomerulosa cells. High concentrations of cGS-PDE have been localized to the zona glomerulosa cell layer of the adrenal cortex using biochemical and immunological techniques. Immunoblot analysis using an affinity-purified, isozyme-specific antiserum revealed a single band that comigrated with a purified cGS-PDE (105 kDa) (1) and that was most highly concentrated in the outermost 1-2 mm of the cortex, representing the capsule and zona glomerulosa regions. Greater than 90% of the overall phosphodiesterase activity present in tissue extracts prepared from these regions was immunoprecipitated using a solid-phase monoclonal antibody reagent, indicating the cGS-PDE as the predominant phosphodiesterase isozyme. Immunohistochemical staining experiments of frozen thin sections of intact adrenal tissue revealed that the cGS-PDE present in this region was localized in the glomerulosa cells themselves. The role of this isozyme as a mediator of ANP-induced decreases in intracellular cAMP concentrations and aldosterone production was tested in primary cultures of bovine adrenal glomerulosa cells. In cells stimulated by ACTH, ANP treatment produced dose-dependent reductions in aldosterone secretion and cellular cAMP content over the same concentration range. Increases in aldosterone production elicited by three cell-permeable cAMP derivatives (8-bromo-cAMP, 8-p-chlorophenylthio-cAMP, and N6-2'-O-dibutyryl-cAMP) were antagonized by ANP, indicating a site of action distal to adenylate cyclase for this hormone. Because the relative magnitude of the ANP effect differed depending upon the derivative used, the three derivatives were compared with respect to their relative rates of in vitro hydrolysis by adrenal cGS-PDE. A positive correlation between their rates of hydrolysis and the degree to which the steroidogenic response produced by these derivatives was antagonized by ANP was demonstrated, further suggesting an ANP-induced activation of the cGS-PDE as being responsible for this effect. The possible contribution of an additional pathway mediated by an inhibitory guanine nucleotide binding regulatory protein (Gi) acting on adenylate cyclase was tested by pretreatment of primary glomerulosa cells with pertussis toxin. Levels of pertussis toxin sufficient to inhibit subsequent in vitro ribosylation did not significantly alter the ANP effect on aldosterone production, although a partial reduction in the ANP effect on cAMP levels was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

CD4 rat × rat and mouse × rat T cell hybridomas produced by fusion of established T cell lines and clones to W/Fu (C58NT)D

Previously, fusion of established T cell lines or clones has been claimed to be difficult. We now report our experiences in the fusion of both long term cultures of rat T cell clones and mouse T cell lines to rat W/Fu (C58NT)D. Upon fusion of rat T cell clones the hybrids obtained expressed antigen specificities identical to those of the parent clones. In addition, C58 was used for interspecies hybridisation of murine T cell lines. The specificity of intra- and inter-species hybrids was maintained by subcloning. We conclude that the C58 cell line can be used to generate continuously growing monoclonal T-cell reagents of sufficient stability using both intra- and inter-species hybri disation.

Microsporidia and Isospora Belli infections in humans

Microsporidial infections in humans are generally caused by four genera, Nosema, Enterocytozoon, Pleistophora , and EncephalHozoon . Infection is through ingestion of the infective spores, either from stool or urine or in tissues. These organisms have been associated with diarrhea and malabsorption, blindness, muscle weakness and contractures, and hepatitis in the liver. The host cells are damaged and often rupture with granulomatous foci in the tissues. In the intestine, there may be flattening of the villi, elongation of the crypts, and an increase in inflammatory cells in the lamina propria. Diagnosis is usually based on demonstration of the spores in tissue biopsy specimens (PAS positive granule, methenamine silver positive, Gram and acid fast variable) or the presence of polar tubules in transmission electron micrographs. Spores have not yet been demonstrated in stool; the development and use of monoclonal reagents are still experimental and not commercially available. Treatment is not well defined and very little has been done on prevention and/or control. With documented diagnosis of these infections in patients with AIDS and other immune deficiencies, the clinician needs to consider microsporidia as possible etiologtc agents of disease in all compromised patients, particularly with symptoms of diarrhea or myositis. Isospora belli is transmitted through ingestion of the infective oocysts. Symptoms may range from mild gastrointestinal symptoms to severe diarrhea. Immunosuppressed patients often present with profuse diarrhea, weakness, anorexia, and weight loss. The disease is usually sell-limiting in the immunocompetent patient. Changes range from broadening of the villus tips to a flat mucosal surface and crypt elongation. There may also be inflammatory infiltrates in the lamina propria. Diagnosis is usually made by finding the oocysts in concentrated stool material (wet preparation examination). Multiple stools are recommended and biopsy material may be positive while the stool examinations are negative. These organisms are found worldwide and are probably more common in the tropics. The drug of choice is trimethoprim-sulfamethoxazole; however, there may be side effects, particularly in patients with AIDS. Although deaths have been reported, the prognosis is usually good with early diagnosis and treatment. Good personal hygiene and proper disposal of human fecal material should help decrease this infection.

IgG Subclasses in the Serum and Skin in Subacute Cutaneous Lupus Erythematosus and Neonatal Lupus Erythematosus

IgG subclasses differ in their biologic and chemical properties, such as complement fixation, protein and cellular binding, and placental transfer. In this study, IgG subclasses of anti-Ro/SSA antibodies in subacute cutaneous lupus (SCLE) and neonatal lupus (NLE) are examined in the serum and in the skin. IgG subclasses in NLE beginning in utero (NLE-heart disease) are compared to subclasses in NLE beginning after birth (NLE-skin disease). Human skin was grafted onto athymic mice, mice were injected with one of eight anti-Ro/SSA maternal NLE sera (four heart block, four skin disease) or seven anti-Ro/SSA SCLE sera, and grafts were examined for IgG subclasses using monoclonal anti-human IgG subclass reagents in an immunofluorescent technique. Lesional skin was examined from four SCLE patients. IgG1 was the only IgG subclass detected in the grafts and skin lesions. IgG1 was the predominant anti-Ro/SSA IgG subclass detected in SCLE and NLE sera in an ELISA using a synthetic Ro/SSA polypeptide. These studies show that the maternal anti-Ro/SSA autoantibodies in NLE-heart disease sera are predominantly IgG1 and are therefore likely to be present in the fetus at the time of gestation, when heart block usually develops. Second, differences in the clinical presentations of NLE (in utero vs. postnatal disease) cannot be attributed to differences in anti-Ro/SSA IgG subclasses. Finally, the subclass bound in the skin in SCLE is IgG1, a subclass capable of mediating tissue injury via complement or cellular effectors.

Percutaneous Umbilical Blood Sampling and Umbilical Vein Transfusions

Percutaneous umbilical blood samples (PUBS), obtained under ultrasound guidance, are used for prenatal diagnosis and management of hemolytic disease of the newborn (HDN) and other fetal disorders. Rapid testing at the time of sampling is vital to distinguish fetal from maternal blood. Blood typing was performed by slide technique in the treatment room during 38 procedures on 25 patients. Anti-I was used to test 50 presumed PUBS; venous I-positive maternal blood was tested in parallel. Because anti-I cannot detect fetal blood after umbilical vein transfusion (UVT) of I-positive donor blood, ABO and Rh blood typing reagents were used to test 29 samples when maternal and fetal or donor blood groups differed. Monoclonal reagents were used for optimal detection of weak AB antigens in fetal blood. Avid, chemically modified anti-D was used for Rh typing. Blood typing showed 27 (34%) of 79 samples to be maternal blood. Fetal blood was obtained in 8 of 10 cases investigated for fetal disorder and in 16 cases of potential HDN (anti-D, 5; -CD, 5; -cE, 2; -K, 2; -c; -E). The absence of HDN (antigen-negative fetus) was determined in 4 cases. UVT afforded live birth of 9 of 10 infants with HDN and was not indicated in two cases.

The Application of Immunocytochemical Techniques to Routinely-Fixed and Stained Cytologic Specimens: An Aid in the Differential Diagnosis of Undifferentiated Malignant Neoplasms

Routinely-fixed and Papanicolaou stained smears with the cytologic diagnosis of undifferentiated malignant neoplasm that had been prepared with cells obtained by fine needle aspiration biopsy (n = 7), pulmonary lavage (n = 5), or thoracentesis (n = 3) from 15 unselected patients were stained by an immunocytochemical technique to evaluate the presence of keratin proteins and the leukocyte common antigen (LCA). Commercially available, well-characterized monoclonal antibodies with specificities for keratin proteins and the leukocyte common antigen, and a streptavidin-biotin-horseradish peroxidase labelling method were used. Evaluation of the stained smears revealed the presence of one of the two antigens in material obtained from each patient, thus indicating the probable cell-lineage of the neoplastic cells. The specificity of the monoclonal antibody reagents used was further evaluated in routinely-fixed and stained cytologic material from 24 histologically confirmed carcinomas and 12 lymphomas. In conclusion, immunocytochemical techniques may be successfully applied to routinely processed archival cytologic smears to determine the antigenic profile of morphologically undifferentiated cells and therefore aid in the differential diagnosis of undifferentiated malignant neoplasms.