Simultaneous blockade of Fc gamma receptors and indirect labeling of mouse lymphocytes by the selective detection of allotype-restricted epitopes on the kappa chain of rat monoclonal antibodies

BACKGROUND: Incubation of mouse hemopoietic cells with rat monoclonal antibodies (mAbs) of the IgG class sometimes results in Fc-region mediated binding of immunoglobulins by Fc-receptors. This unwanted binding can be prevented by preincubation of target cells with the rat anti-mouse anti-CD16/32 (2.4G2) mAb. METHODS: To avoid the cross-reactivity of fluorochrome-conjugated secondary anti-rat antibodies with the Fc-receptor blocking 2.4G2, direct fluorochrome-conjugated immunoglobulins need to be used. However, we report that a mouse mAb (MRC OX12) with a strong rat Igk(a) allotype preference can be used for flow cytometric measurements in conjunction with unlabeled rat mAbs with the simultaneous blockade of Fc gamma receptors by the 2.4G2 mAb. Results and Discussion This lack of reactivity of OX12 against the 2.4G2 mAb is remarkable, as it could efficiently detect another Sprague-Dawley-derived rat mAb. This staining procedure (unlabeled rat mAb of the appropriate strain detected by OX12 mAb in the presence of 2.4G2 IgG) is an attractive alternative to using direct antibody conjugates, while satisfying the need for an effective Fc gamma-receptor blockade.     

Immunogenic regions of the GA733-2 tumour-associated antigen recognised by autoantibodies of patients with colorectal carcinoma

The tumour-associated antigen (TAA) GA733-2 is overexpressed by >90% of human colorectal carcinomas (CRC). The antigen has previously been shown to be recognised by B and T cells. The aim of the present study was to define B cell epitopes of GA733-2. Fifteen percent of CRC patients with no previous immunotherapy have recently been shown to elicit an anti-GA733-2 IgG antibody response. Sera of these patients ( n=136) were analysed by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies against 23 partly overlapping synthetic peptides (18 amino acids: aa) derived from the extracellular domain of GA733-2. An 18-aa long sequence at the N-terminal region of the antigen (peptide 2) was found to be an immunodominant B cell epitope. Fifty percent of the patients had antibodies against peptide 2, while 8% to 9% had antibodies against peptides 1, 4, 7, 8 or 20. In healthy donors ( n=30) antibodies against peptides 2 and 8 were also detected in 13% and 3% of cases respectively, while no antibodies were found against the other peptides and the complete protein. Thirteen percent of CRC patients ( n=30) with no IgG antibodies against the GA733-2 antigen elicited antibodies against peptide 2. The specificity of peptide-reactive sera was verified by inhibition ELISA. The binding of sera to GA733-2 was significantly inhibited by peptides to which CRC sera bound, but not by control peptides. Binding to peptide 2 of sera showing both peptide 2 and GA733-2 reactivity was specifically inhibited by the complete GA733-2 antigen, while binding of peptide 2-reactive sera showing no GA733-2 reactivity was not inhibited. CRC sera interfered with the binding of monoclonal antibody (mAb) 17-1A and mAb C215 that recognise distinct epitopes of GA733-2. No significant correlation was found between the presence of anti-peptide antibodies in CRC patients and clinical stage or overall survival. The results provide additional evidence for immune recognition of CRC by the host.     

Differential Expression of a Mycobacterium tuberculosis Protein Recognized by a Mouse Monoclonal Antibody

Murine monoclonal antibodies were produced against Mycobacterium tuberculosis (Mtb) using standard hybridoma procedures. By a whole cell enzyme-linked immunosorbent assay (ELISA), one monoclonal antibody (mAb), HB28, demonstrated high level specific reactivity to Mtb. Western blot analysis demonstrated reactivity to a single 65 kDa Mtb protein in the cell wall extract and culture filtrate. HB28 mAb appears to be recognizing a 65 kDa Mtb protein that is over-expressed by Mtb but not other species under certain culture conditions. Differential expression and detection of this protein by HB28 mAb may have potential for diagnostic applications.     

Production and characterization of four monoclonal antibodies against porcine pancreatic colipase

Four monoclonal antibodies directed against porcine colipase have been generated by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. Antibodies were screened by binding to immobilized colipase in a solid-phase assay. Monoclonal antibodies were purified by affinity chromatography on colipase coupled to Sepharose. All monoclonal antibodies are of the IgG1 class with high affinity for the antigen. The dissociation constant of the complex formed in solution between porcine colipase and antibody varied from 1.1 X 10(-10) M to 1.8 X 10(-8) M. Epitope specificity was studied for each antibody and in pairs with an enzyme-linked immunosorbent assay (ELISA). Results indicate that the four monoclonal antibodies react with at least three different antigenic regions of colipase. Finally, three monoclonal antibodies were found to be potent inhibitors of colipase activity. Antiporcine monoclonal antibodies appear to be suitable probes for studying the lipid affinity site of the protein cofactor of pancreatic lipase.     

Treatment of murine B cell lymphoma with bispecific monoclonal antibodies (anti-idiotype x anti-CD3).

It has previously been reported that T lymphocytes can be targeted by using bispecific antibodies consisting of anti-target antibody and anti-CD3. In the present study, a bispecific mAb was developed by somatic hybridization of mouse hybridomas, one producing a mAb against the Id determinant of the mouse B cell lymphoma 38C13 and the other a mAb against a polymorphic determinant on murine CD3. The bispecific antibody, anti-38C13 x anti-CD3, is bi-isotypic (IgG1 x IgG2a) and was purified by ion exchange and affinity chromatography. The dual specificity of the hybrid hybridoma-produced mAb could be demonstrated by flow cytometry, the induction of T cell proliferation, the induction of IL-2 secretion by polyclonal T cells, and redirected lysis of the relevant target cells. The hybrid (bi-isotypic) Fc part of the bispecific antibodies was nonfunctional in FcR-dependent redirected lysis. In vivo studies demonstrate that this bispecific mAb could efficiently target T cells towards the tumor cells, resulting in long term survival and cure of the lymphoma.     

乙氧基磷酸酯类有机磷农药单克隆抗体的制备与鉴定

制备针对乙氧基磷酸酯类有机磷农药的单克隆抗体,以此为基础建立该类农药的快速免疫筛选检测方法．以二乙基磷酸乙酸为通用结构半抗原,分别使之与牛血清白蛋白和鸡卵清蛋白共价偶联,合成免疫原和包被原并对其进行结构鉴定．偶联成功后的免疫原用于免疫Balb/c小鼠．将免疫成功小鼠的脾细胞与小鼠SP2/0骨髓瘤细胞融合,筛选能稳定分泌抗乙氧基磷酸酯类有机磷农药单克隆抗体的杂交瘤细胞株．获得的小鼠腹水用辛酸-硫酸铵法纯化,所得纯化抗体以琼脂双扩散法鉴定其免疫球蛋白类型,间接竞争ELISA方法测定其对半抗原的灵敏度、特异性和亲和性．结果表明,该抗体分泌IgG1亚类的单克隆抗体,且与二乙基磷酸乙酸的亲和性较高(1.4×10⁷ L/mol),所得抗体对毒死蜱、对硫磷、丙溴磷、氧化乐果、除线磷、二嗪农、溴硫磷、辛硫磷、喹硫磷、三唑磷等农药有特异性反应．该检测技术可用于上述农药的快速定性或定量检测．     

Generation of monoclonal antibodies to Macaca mulatta (rhesus) IgA

One IgG1 and five IgM murine monoclonal antibodies (mAb) specific for rhesus (Rh) IgA were generated. These mAbs bound to Rh IgA but not IgG or IgM when tested by enzyme-linked immunosorbent assay. Immunoblotting revealed that the mAbs reacted with the alpha heavy chain of Rh but not human IgA. The IgG1 anti-Rh IgA mAb detected IgA-producing cells in sections of monkey gut examined by immunofluorescent staining. These mAbs should be useful for characterizing IgA responses in the Rh monkey.     

Characterization of Mono- and Polyclonal Antibodies Against Highly Purified Choline Acetyltransferase: A Monoclonal Antibody Shows Reactivity in Human Brain

Choline acetyltransferase (ChAT) from porcine brain was purified by immunoaffinity chromatography, and the highly purified enzyme was subsequently used for immunization of mice and rabbits. After fusion of mouse spleen cells, 32 cultures producing monoclonal antibodies directed against ChAT were detected by an enzyme-linked immunosorbent assay (ELISA) with immunoaffinity-purified ChAT. Of these original 32, the most active 11 cultures were cloned and used for ascites production. The 11 clones generated monoclonal antibodies of the immunoglobulin (Ig) M class (three), the IgG1 subclass (seven), and the IgG2b subclass (one). The isoelectric points of the antibodies of the IgG class were different in each case. The monoclonal antibodies exhibited different binding characteristics in the above ELISA and on western blots. Two monoclonal antibodies demonstrated excellent immunohistological results with neurons of rat brain and spinal cord. One of them reacted well immunohistochemically with neurons of human brain and also recognized partially purified human placenta ChAT in the ELISA.     

Resolution of adhesion- and activation-associated components of monoclonal antibody-dependent human NK cell-mediated cytotoxicity.

Flow cytometry was used to investigate two functional parameters of human natural-killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC): (i) the frequency of NK cells which formed conjugates (NKC) with autologous monoclonal antibody (mAb)-coated lymphocyte target cells, a measure of the avidity of CD16-dependent cell-cell adhesion, and (ii) the rise in the intracellular concentration of ionized calcium ([Ca2+]i) elicited in NKC by contact with target cells, a measure of CD16-dependent NK cell activation. For each of four rat IgG2b mAb directed against target cell antigens CDw52, CD5, CD45, and class I HLA, there existed quantitatively similar relationships between ADCC and rise in NKC[Ca2+]i but significant inter-mAb differences with respect to the ADCC vs the NKC frequency relationship. Cytolytic efficiencies of mAb appeared to be determined at the level of the NK cell, dependent upon CD16 and LFA-1, but restricted with respect to quantitative levels of NKC[Ca2+]i. In concert with this notion, targets coated with an IgG1 isotype-switch variant alpha CDw52 mAb promoted significant conjugate formation but failed to elicit a rise in NKC[Ca2+]i or ADCC. Thus, Fc regions of antibodies make contacts with NK cell CD16 which may strengthen cell-cell adhesion without eliciting an activation stimulus, a finding which supports a complexity of CD16 functional regulation of probable significance in the clinical consequences of antibody responses or therapeutic mAb manipulations.     

Initial experience with treatment of human B cell lymphoma with anti-CD19 monoclonal antibody

Summary Six patients with progressive B cell non-Hodgkin's lymphoma have been treated with an IgG2a mouse monoclonal antibody (mAb) against the B cell differentiation antigen CD19, with total doses varying from 225 mg to 1000 mg. Free mAb was detected in the serum after doses of 15–30 mg. After the mAb infusions the number of circulating tumour cells was temporarily reduced, but in some cases antibody-coated cells remained in the circulation for several days. mAb penetrated to extravascular tumour sites; in general higher doses were required to saturate cells in the lymph nodes than to sensitize tumour cells in the bone marrow. mAb doses of up to 250 mg were given i.v. over 4 h without major toxicity. One patient twice achieved a partial remission after two periods of mAb treatment with an 8-month interval; the second remission lasted for 9 months. One patient showed a minor response. None of the patients made antibodies against the mouse immunoglobulin. Serum immunoglobulin levels were followed as a measure of the function of the normal B cell compartment; no significant changes were seen up to 6 months after mAb treatment.Supported by the Dutch Cancer Society (grant NKI 84-14)     

Monoclonal IgM antibody protection in mice against infection with an encapsulated strain of Staphylococcus epidermidis.

Passive protective activities of three different classes of monoclonal antibodies in mice against challenge with strain ATCC 31432 (capsular type I) of Staphylococcus epidermidis were examined. Monoclonal IgM antibody passively protected mice against challenge with the homologous strain, whereas monoclonal IgG1 and IgG2b antibodies did not. The protective activity of IgM was absorbed by the cell surface antigen extracted from the homologous strain but not by the antigen from heterologous strains. Rapid reduction of viable cells took place in the peritoneal cavity of mice immunized with monoclonal IgM as early as 6 h after the challenge with the homologous strain. An enzyme-linked immunosorbent inhibition assay showed there was remarkable inhibition with the homologous cell surface antigen but not with heterologous preparations from other strains. Results suggest that in the mouse the major passive protection against the S. epidermidis strain is provided by the IgM antibody to the cell surface antigen.     

Drug resistance in rat colon cancer cell lines is associated with minor changes in susceptibility to cytotoxic cells

The development of resistance to anticancer drugs urges the search for different treatment modalities. Several investigators have reported the concomitant development of drug resistance and resistance to natural killer (NK), lymphokine-activated killer (LAK) or monocyte/macrophage cell lysis, while others described unchanged or even increased susceptibility. We investigated this subject in the rat colon carcinoma cell line, CC531-PAR, which is intrinsically multidrug-resistant (MDR), and in three sublines derived from this parental cell line: a cell line with an increased MDR phenotype (CC531-COL), a revertant line from CC531-COL (CC531-REV), which demonstrates enhanced sensitivity to anticancer drugs of the MDR phenotype, and an independently developed cisplatin-resistant line (CC531-CIS). In a 4-h⁵¹Cr-release assay we found no difference in susceptibility to NK cell lysis. No significant differences in lysability by adherent LAK (aLAK) cells were observed in a 4-h assay. In a prolonged 20-h⁵¹Cr-release assay an enhanced sensitivity to aLAK-cell-mediated lysis was observed in the revertant, P-glycoprotein-negative cell line and in the cisplatin-resistant cell line (CC531-CIS). None of the cell lines was completely resistant to lysis by aLAK cells. Therefore, a role for immunotherapy in the treatment of drug-resistant tumors remains a realistic option.     

Construction and characterization of the chimeric antibody 8C11 to the hepatitis E virus

Homodimers of the truncated hepatitis E virus (HEV) capsid proteins, E2 and p239, were conformed to model the dominant antigenic determinants of HEV. Using E2 as an immunogen, two neutralizing monoclonal antibodies (mAbs), namely 8C11 and 8H3, were produced. We constructed a mouse-human chimeric antibody derived from 8C11 and its expression in Chinese hamster ovary (CHO) cells. cDNAs encoding variable regions of heavy and light chains were isolated from hybridoma cells and inserted into mammalian expression vectors containing cDNA of human gamma-1 and kappa constant regions, respectively. The vectors were then cotransfected into CHO cells, and a stable cell line was established. Results from indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showed that the chimeric antibody was assembled correctly to the native IgG molecule and could be secreted from the cells. Similar to the original mAb, the expressed chimeric antibody displayed HEV antigen-binding activity and an enhancement effect on 8H3 binding to HEV antigen. The chimeric antibody could specifically inhibit the binding of p239 to HepG2 cells and compete with HEV IgG in positive serum by antibody-competitive ELISA. The chimeric antibody is expected to be less immunogenic in human and more suitable for antibody therapy of hepatitis E.     

Shared idiotope on monoclonal anti-Ia.7 antibodies reactive with determinants in a structural domain of the I-E molecules

In previous studies, heterologous anti-idiotypic (anti-Id) antisera against the C3H.SW 14-4-4S or the A.TH 41.A anti-Ia.7 monoclonal antibodies (mAb) were shown to identify an interstrain cross-reactive idiotypic specificity (IdX.Ia.7) expressed on monoclonal or conventional anti-Ia.7 alloantibodies. The objective of the present investigation was to characterize further this IdX at the idiotopic level. To this end, 11 hybridomas producing IgG1, IgG2a, or IgM anti-Id mAb were derived from a rat immunized with a mixture of 10 A.TH or A.BY anti-Ia.7 mAb. The specificity of the latter anti-Id mAb was determined by direct Id binding radioimmunoassay (RIA) with the use of a panel of 52 anti-Ia mAb derived from hybridomas produced in various inbred mouse strains. These rat anti-Id mAb recognized idiotopes expressed on i) all anti-Ia.7 mAb against determinants in the topographic domain I of the I-Ek molecule but not on 18 other anti-I-Ek mAb directed at epitopes in domains II or III; ii) three of 19 anti-I-Ak mAb; and iii) one A.TL-derived anti-I-As mAb. Competitive Id binding assays revealed that among the 14 IdX+ anti-Ia.7 mAb, one (81.B) was bound to a lesser extent by various rat anti-Id mAb, suggesting that heterogeneity probably exists in this antibody family. By contrast, two isologous (B10.S(7R)) anti-Id mAb to the IdX.Ia.7+ mAb 41.A displayed a specificity restricted to 41.A individual idiotopes (IdI). Rat anti-IdX.Ia.7 and mouse anti-41.A IdI mAb inhibited the binding of 125I-labeled mAb 41.A to CBA spleen cells. These two sets of mAb bound in a noncompetitive fashion to mAb 41.A-coated plates, indicating that their corresponding public or private idiotopes were spatially distinct. These data may have implications for in vivo manipulations of anti-Ia immune responses.     

The immune response to Schistosoma mansoni infections in inbred rats. VI. Regulation by T cell subpopulations

These studies assess the roles of subpopulations of T lymphocytes in inducing and modulating resistance to Schistosoma mansoni. CDF rats were depleted of RT 7.1+ (anti-Pan-T), W3/25+ (anti-T helper/inducer), or OX8+ (anti-T suppressor) cells by the in vivo administration of monoclonal antibodies (mAb). The development of parasites and immunity to challenge by S. mansoni were compared with results in undepleted normal and congenitally athymic rats. Discrete subpopulations of T lymphocytes were adoptively transferred to ascertain effects upon parasite development and the protective immune response. In vitro studies, involving utilizing cocultivation of cell subpopulations +/- cyclosporin A, were utilized to dissect mechanisms. Depletion of T lymphocytes by anti-RT7.1 mAb and anti-W3/25 mAb resulted in augmented initial worm development, suboptimal resistance, and decreased antibody and delayed-type hypersensitive reactivity directed against schistosome antigens. Depletion with OX8 mAb produced opposite effects. The adoptive transfer of T cell subpopulations produced concordant results with T cell regulation expressed B cell-dependent effector mechanisms. The coadoptive transfer of cells resulted in the suppression of resistance afforded by the W3/25+ cells by OX8+ cells, which could be augmented in vitro by cyclosporin A. Thus, protective immunity to S. mansoni in rats is regulated by discrete subpopulations of T lymphocytes. The findings suggest the possibility of selective immune regulation of resistance based on the manipulation of specific T cell subpopulation.     

利用hTM表达细胞株制备抗人血栓调节蛋白单克隆抗体

为了研制抗人血栓调节蛋白(hTM)的单克隆抗体(mAb),给经CHO细胞免疫的BALB/c小鼠腹腔注射环磷酰胺诱导其对CHO和CHO-TM5细胞的共同抗原表位产生免疫耐受,再用高效表达hTM的CHO-TM5常规免疫小鼠．细胞融合后,ELISA筛选分泌抗hTM的特异性mAb阳性克隆,并将其腹腔注射BALB/c小鼠诱生腹水．腹水中的mAb经亲和层析纯化后,采用ELISA、流式细胞术、免疫组织化学染色法及Western blotting对其进行特异性鉴定．结果显示,共获得了5株阳性克隆,其中2F7可稳定分泌IgG1亚型的mAb,腹水抗体效价为1×10_-6,含量为19.56 g/L．2F7在体内与正常组织的交叉反应极少,对HUVEC、CHO-TM5有特异性结合,并可识别细胞裂解液还原条件下分子质量为105 ku左右的蛋白质多肽．2F7的解离常数K_d约为1.22×10_-9 mol/L．实验结果表明,通过采用新的技术路线,直接应用hTM表达细胞株免疫小鼠,成功制备了具有高度特异性与高亲和力的抗hTM mAb,为其他来源困难的蛋白质mAb制备提供了可借鉴的技术方案,同时2F7的研究鉴定为进一步应用抗hTM mAb进行hTM生物学功能及临床意义研究提供了新的物质基础．     

Effective suppression of tumour cells by oligoclonal HER2-targeted delivery of liposomal doxorubicin

Synergistic effect of combined antibodies targeting distinct epitopes of a particular tumour antigen has encouraged some clinical trial studies and is now considered as an effective platform for cancer therapy. Providing several advantages over conventional antibodies, variable domain of heavy chain of heavy chain antibodies (V_HH) is now major tools in diagnostic and therapeutic applications. Active targeting of liposomal drugs is a promising strategy, resulting in enhanced binding and improved cytotoxicity of tumour cells. In the present study, we produced four anti-HER2 recombinant VHHs and purified them via native and refolding method. ELISA and flow cytometry analysis confirmed almost identical function of VHHs in refolded and native states. Using a mixture of four purified VHHs, PEGylated liposomal doxorubicin was targeted against HER2-overexpressing cells. The drug release was analyzed at pH 7.4, 6.4 and 5.5 and dynamic light-scattering detector and TEM micrograph was applied to characterize the produced nanoparticles. The binding efficiency of these nanoparticles to BT474 and SKBR3 as HER2-positive and MCF10A as HER2-negative cell line was examined by flow cytometry. Our results indicated effective encapsulation of about 94% of the total drug in immunoliposomes. Flow cytometry results verified receptor-specific binding of targeted liposomes to SKBR3 and BT474 cell lines and more efficient binding was observed for liposomes conjugated with oligoclonal VHHs mixture compared with monoclonal VHH-targeted liposomes. Oligoclonal nanoparticles also showed more cytotoxicity compared with non-targeted liposomes against HER2-positive tumour cells. Oligoclonal targeting of liposomes was represented as a promising strategy for the treatment of HER2-overexpressing breast cancers.     

Pharmacokinetic studies of radiolabelled rat monoclonal antibodies recognising syngeneic sarcoma antigens

Summary The object of our current investigations is to explore the potential of antibodies for localisation and treatment of disseminated disease, using as a model rat monoclonal antibodies (mAbs) raised against syngeneic tumourspecific antigens. As part of this study, antibodies of differing isotypes with specificity for either HSN or MC24 sarcoma were labelled with¹²⁵iodine and injected intravenously into normal rats or those bearing paired tumours in contralateral flanks. The blood clearance rates of the radiolabelled antibodies were found to be influenced by immunoglobulin subclass (IgG2b > IgG2a > IgG1) and to be increased non-specifically by the presence of growing tumours. The tumour and normal tissue distributions of the antibodies tested were also found to vary according to their apparent degree of interaction with host Fc-receptor-bearing cells, to the extent that tumour specificity in vitro was not necessarily reflected in selectivity of localisation in vivo. Three IgG2b monoclonal antibodies showed preferential uptake in the spleens of syngeneic rats and non-specific accumulation in tumours. This effect was not observed with antibodies of IgG2a or IgG1 subclass, and was abolished by the use of IgG2b F(ab)₂ preparations. In spite of the use of immunoglobulin fragments, varying the assay time and testing tumours of different sizes, specific tumour localisation was low with all seven monoclonal antibodies tested. The maximum uptake achieved was less than 1% of the injected dose of antibody per gram of tumour. Much higher levels of antibody localisation have been reported for human tumour xenografts growing in nude mice, but these are rarely achieved in other systems. We propose that the use of autologous monoclonal antibodies recognising tumour-associated antigens of relatively low epitope density in syngeneic hosts provides a valid alternative model in which to investigate the factors limiting more effective, specific immunolocalisation of malignant disease.     

Determination of critical epitope of PcMab-47 against human podocalyxin

Podocalyxin (PODXL) is a type I transmembrane protein, which is highly glycosylated. PODXL is expressed in some types of human cancer tissues including oral, breast, and lung cancer tissues and may promote tumor growth, invasion, and metastasis. We previously produced PcMab-47, a novel anti-PODXL monoclonal antibody (mAb) which reacts with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in Western blot, flow cytometry, and immunohistochemical analysis. In this study, we used enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of PcMab-47. The minimum epitope of PcMab-47 was found to be Asp207, His208, Leu209, and Met210. A blocking peptide containing this minimum epitope completely neutralized PcMab-47 reaction against oral cancer cells by flow cytometry and immunohistochemical analysis. These findings could lead to the production of more functional anti-PODXL mAbs, which are advantageous for antitumor activities.     

Monoclonal antibodies against human mast cell tryptase demonstrate shared antigenic sites on subunits of tryptase and selective localization of the enzyme to mast cells

Two murine monoclonal antibodies were prepared against tryptase, the major neutral protease and protein component of human mast cells. The antibodies were termed G5 (IgG2B-kappa) and H4 (IgG1-kappa). They were specific for tryptase by an enzyme-linked immunosorbent assay and an immunotransblot technique. The latter procedure showed that H4 and G5 each bind to the 35,000 and 37,000 m.w. subunits of tryptase, indicating immunologic cross-reactivity between the subunits. The monoclonal antibodies reacted only with tryptase subunits in an extract of dispersed lung cells. By immunofluorescence microscopy, tryptase was further identified to be present only in cytoplasmic granules of Alcian Blue-stained mast cells in dispersed pulmonary cell preparations. No evidence for a mast cell subtype lacking tryptase was detected. In addition, a procedure for the purification of tryptase to homogeneity from dispersed pulmonary cells containing less than 10% mast cells was developed; this procedure involved high salt extraction, ammonium sulfate precipitation, and sequential chromatography with decyl-agarose, DEAE-agarose, and heparin-agarose. The procedure resulted in a higher yield even with less pure starting material than reported previously. Tryptase is a selective marker for mast cells in dispersed pulmonary cells, and can be detected with specific anti-tryptase antibodies.