Recognition of two Dermatophagoides pteronyssinus-specific epitopes on antigen P1 by using monoclonal antibodies: binding to each epitope can be inhibited by serum from dust mite-allergic patients

Monoclonal antibodies were raised against Antigen P1, the major allergen of the house dust mite (Dermatophagoides pteronyssinus). The majority were Antigen P1 specific, isotype IgG1, and did not react with a comparable D. farinae allergen. These antibodies bound 38 to 50% of 125I Antigen P1 in antigen-binding assays (titer greater than or equal to 1/1,000,000), and the quantities of IgG antibody in ascites were 2 to 4 logs greater than those in polyclonal mouse antiserum or in serum from a mite-allergic patient. Two IgM antibodies showed weak binding to Antigen P1 but reacted strongly with D. pteronyssinus in enzyme immunoassay (titer greater than or equal to 1/100,000). Assessments of the specificity of the IgG antibodies by using two inhibition radioimmunoassays suggested that they were directed against two different epitopes. Antibodies 10B9 F6 and 5H8 C12 were purified by preparative isoelectric focusing (isoelectric points of pI 6.25 and 7.4, respectively) and radiolabeled with 125I. Cross-inhibition experiments, using ascites dilutions to inhibit binding of each radiolabeled antibody to Antigen P1, confirmed that these antibodies recognized two distinct epitopes. Analysis of antibodies from 39 clones/hybrids showed that the majority were directed against the same epitopes as either 10B9 F6 or 5H8 C12 (3 out of 39 [8%] and 29 out of 39 [74%], respectively). None of the monoclonal antibodies significantly inhibited (greater than 10%) human IgE binding to Antigen P1 in the radioallergosorbent test. However, 12 of 14 sera from mite allergic patients inhibited binding by the monoclonal antibodies. One serum from a mite-allergic patient inhibited binding of both 10B9 F6 and 5H8 C12 by greater than 85% and showed parallel inhibition curves. The results suggest that these monoclonal antibodies could be used to assay Antigen P1 in both D. pteronyssinus and house dust extracts. It should also be possible to use monoclonal antibodies in inhibition assays to define the antigenic/allergenic determinants recognized by human IgG and IgE antibodies on this mite allergen.     

Production of Monoclonal Antibodies to Guinea Pig Liver Transglutaminase

Monoclonal antibodies are now a powerful tool in biology and medicine. Transglutaminase has been implicated in diverse biological functions, and the characteristics of its catalytic action are suitable for applied enzymology. In this study, we produced hybridoma cells which synthesize monoclonal antibodies against guinea pig liver transglutaminase by fusing mouse myeloma cells with spleen cells of mouse immunized with the enzyme protein. Eight hybridoma clones (coded 2F, 4B, 7C, 8B, 8D, 8E, 9F and 11C) were selected to produce monoclonal antibodies. The subclass of IgG produced by clone 9F was IgG_2a and those from the seven other clones were all IgG₁ The 9F antibody inhibited transglutaminase activity, but the other antibodies did not. A solid-phase antibody-binding assay showed that of these antibodies, 8D antibody has the highest affinity to the antigen. Transglutaminase protein in crude liver extract was identified with Western blotting analysis using 8D antibody as the probe.     

抗人IgM单克隆抗体的研究—脾内免疫建立抗人IgM单克隆抗体杂交瘤细胞株

用多发性骨髓瘤病人血清中提纯的ＩｇＭ抗原,用脾内免疫ＢＡＬＢ／ｃ小鼠后与Ｓｐ２／Ｏ骨髓瘤细胞融合,获得了７株分泌人ＩｇＭ单克隆抗体的杂交瘤细胞株,分别命名为２５Ｇ１、２５Ｇ３、２５Ｇ４、２５Ｇ５、２５Ｄ２、２５Ｄ３和２５Ｄ５。注射同系小鼠后可诱生含较高效价抗人ＩｇＭ腹水（ＰＨＡ＝１２１２）。该杂交瘤细胞经组织培养传代半年,冻存１４个月后复苏,其分泌ＩｇＭ性能稳定。用ＰＨＡ、ＥＬＩＳＡ做人ＩｇＭ、ＩｇＧ、ＩｇＡ阻断试验,仅ＩｇＭ可阻断。用琼脂糖双扩散证明其与羊抗人ＩｇＭ有共同沉淀线     

Toxoid of Clostridium botulinum type F: purification and immunogenicity studies.

Toxin from Clostridium botulinum type F was recovered from dialysis cultures and partially purifed by: (i) ammonium sulfate and ethanol precipitation; (ii) O-(diethylaminoethyl)-cellulose chromatography; or (iii) diethylaminoethyl-cellulose chromatography followed by O-(carboxymethyl)-cellulose chromatography. Toxin purities as reflected by specific activity were 1.83 X 10(6), 9.8 X 10(6), and 2.0 X 10(7) mouse 50% lethal doses (LD50)/mg of N, respectively, for toxins purified by the three methods. The toxins were converted to toxoids by incubation at 35 C in the presence of 0.3 to 0.45% formalin for 21 to 35 days. Toxoids were immunogenic in guinea pigs, as demonstrated by serum antitoxin response and the immunized animals' resistance to challenge by type F botulinal toxin. The immune response to type F toxoids was lower when toxoids of serotypes A, B, C, D, and E were combined with the type F toxoid than when the type F toxoid only was administered. The toxoid prepared from the most highly purified toxin (method [iii]) conferred the highest immunity in guinea pigs at a given dose level. A relation between serum antitoxin level and resistance to challenge was observed. At least 50% of the groups of guinea pigs with 0.015 antitoxin units or more per ml survived challenge by 10(5) mouse LD50 of type F botulinal toxin. A dose of 3.75 mug of N of the most highly purified type F toxoid in combination with the other five serotypes of botulinal toxoid invoked an immune response in guinea pigs comparable to that considered adequate for the other toxoids.     

Serologic survey of select infectious diseases in coyotes and raccoons in Nebraska

To obtain data about select zoonotic and other infectious diseases in free-ranging predators in five ecoregions in Nebraska, sera were collected from 67 coyotes (Canis latrans) and 63 raccoons (Procyon lotor) from November 2002 through January 2003. For coyotes, antibodies were detected against canine distemper virus (CDV, 61%), Francisella tularensis (32%), Rickettsia rickettsi (13%), and flaviviruses (48%). None of the coyote sera had antibodies to Borrelia burgdorferi, Brucella canis, or six serovars of Leptospira interrogans. Because serologic cross-reactivity exists among flaviviruses, 14 sera from flavivirus-positive coyotes were also tested for St. Louis encephalitis virus (SLE) antibodies and two (14%) were positive, suggesting that up to 48% of coyotes tested had antibodies against West Nile virus (WNV). For raccoons, antibodies were detected against CDV (33%), F. tularensis (38%), and three serovars of L. interrogans (11%).     

Comparison of Clostridium botulinum toxins type D and C1 in molecular property, antigenicity and binding ability to rat-brain synaptosomes

Botulinum type D neurotoxin was purified 950-fold from the culture supernatant with an overall yield of 32%. The purified toxin had a specific toxicity of 5.8 X 10(7) mouse minimal lethal dose per mg of protein and a relative molecular mass of 140000. The purified toxin had a di-chain structure consisting of heavy and light chains with relative molecular masses of 85000 and 55000, respectively, linked by one disulfide bond. These subunits had different amino acid compositions and antigenicities. A similarity in molecular constructions and amino acid compositions was observed between type D and type C1 toxins as well as between their subunits. Among the seven kinds of monoclonal antibodies against type D toxin, six reacted with the heavy chain of type D toxin, while one of the six also reacted with the heavy chain of type C1 toxin and neutralized the toxicities of the two toxins. The other one of monoclonal antibodies reacted with the light chains of both toxins. This evidence indicates that both toxins have common antigenic sites on their heavy and light chains and that the antigenic site on the heavy chain may contribute to the neutralization of both toxins by antibody. The binding of type D toxin to rat brain synaptosomes was examined by use of 125I-labelled type D toxin. The binding was competitively inhibited not only by unlabelled type D and C1 toxins, but also by the heavy chains of both toxins, however, it was not inhibited by the light chain of type D toxin. These results suggest that the toxin receptors on synaptosomal membrane are common for type D and C1 toxins, and that the heavy chain contributes to the binding of toxin to synaptosomes and the structure of the binding sites on the heavy chains of both toxins is quite similar.     

Presence of antibotulinum neurotoxin antibodies in selected wild canids in Israel

Serum samples from 35 golden jackals (Canis aureus syriacus), eight wolves (Canis lupus), and four red foxes (Vulpes vulpes) from various regions of Israel were collected during the years 2001-04 and tested for antibodies to Clostridium botulinum neurotoxin (BoNT) types C and D. Antibodies against BoNT types C and D were detected in 10 (29%) and in 3 (9%) of 35 golden jackals, respectively, using enzyme-linked immunosorbent assay. This report describes detection of anti BoNT antibodies in wild canids other than coyotes (Canis latrans) for the first time and demonstrates that C. botulinum type C is prevalent in Israel.     

Serologic and molecular evidence of Ehrlichia spp. in coyotes in California

In order to determine the role of coyotes in the epidemiology of granulocytic and monocytic ehrlichial agents in California (USA), we tested 149 serum samples for antibodies against Ehrlichia equi, E. risticii, and E. canis, using an indirect immunofluorescent antibody test. Polymerase chain reaction (PCR) assay was used to survey for the presence of members of the E. phagocytophila genogroup, E. risticii and E. canis in blood samples of 95 coyotes. Sixty-eight (46%) samples were seropositive for E. equi, two (1%) for E. risticii and none of the samples had antibodies reactive to E. canis. Two and one coyote were positive for E. risticii and members of the E. phagocytophila genogroup by PCR assay, respectively. In contrast, the 95 samples were negative for E. canis by PCR. Ninety-five percent of the 68 E. equi seropositive coyotes and the one coyote PCR positive for members of the E. phagocytophila genogroup originated from a coastal area. However, the two E. risticii seropositive coyotes and the two coyotes PCR positive for E. risticii were from northern California. Sequence analysis of the three amplified PCR products revealed the agent to be similar in two coyotes to the sequences of E. risticii from horses originating from northern California and identical in one coyote to the agent of human granulocytic ehrlichiosis and E. equi from California. Thus, coyotes are exposed to granulocytic ehrlichiae and E. risticii and may play a role in the epidemiology of these ehrlichial agents in California.     

Brucella abortus in coyotes. I. A serologic and bacteriologic survey in eastern Texas.

Prevalence of Brucella abortus serum antibodies in coyotes from east central Texas was determined by the buffered Brucella antigen (card test), rivanol, standard agglutination tube, and cold complement fixation tube tests. Eighteen percent (9 of 51) of the coyotes were positive serologically. B. abortus biotype 1 was isolated from various tissues from 7 of 43 coyotes by bacteriologic culture. Congenital transmission was found.     

Role of Salmonella and Yersinia in the pathogenesis of spondyloarthropathies and rheumatoid arthritis. II. Antibodies to Salmonella and Yersinia in sera and synovial fluids

IgA, IgG and IgM antibodies against Yersinia Yop proteins, Yersinia LPS and Salmonella LPS from different serogroups were determined by enzyme-linked immunosorbent assay (ELISA) in a 885 serum samples and 92 synovial fluids. The control group consisted of 200 healthy blood donors. Compared with control subjects, patients with arthritis showed significantly increased titres of antibodies against Yersinia Yop, Yersinia LPS and Salmonella LPS appropriately in 21.7%, 44.0% and 56.0% serum samples. The prevalence of positive antibody levels was highest in Yersinia serogroup O3 and Salmonella serogroup B and D antibodies. The IgA titres were found to be much higher in adults than in children and youngsters but IgM titres consequently decreased with age. Investigation of synovial fluids obtained from patients with arthritis showed that Yersinia and Salmonella antibodies in synovial fluid mirror those in serum by concentration, by specificity and by distribution in classes.     

免疫磁珠技术分离唾液A/B血型抗原并用于吸附血清A/B抗体

目的:应用免疫磁珠分离技术获得具有良好抗原性的A/B血型抗原,并探究其作为ABO血型抗体吸附剂去除A/B抗体的可行性。方法:将含有血型物质的唾液进行预处理,再与包被了抗体的磁珠混合,分离出纯度较高的A/B抗原,运用酶联免疫及凝集抑制试验验证所得抗原的抗原性及是否存在交叉反应。用未纯化A/B抗原和纯化A/B抗原包被磁珠,对含有抗A/B IgM、IgG的血清进行抗体吸附,用纯化A/B抗原对100份来自O型血孕妇的临床血清样本进行抗体吸附,分别评价其吸附效果。结果:纯化抗原与对应抗体反应后,其吸光度显著高于对照组(A抗原与A抗体0.85±0.12 vs.0.27±0.03,P0.01;B抗原与B抗体0.86±0.09 vs.0.24±0.06,P0.01),与其它类型抗体反应后的吸光度值与对照组比较差异无统计学意义(P0.05)。进行红细胞凝集抑制试验时,纯化抗原可显著抑制相应抗体与红细胞的凝集反应,对其它类型抗体与红细胞的凝集没有抑制作用。血清抗体吸附实验表明纯化抗原的吸附效率比未纯化抗原的高(97.00%vs.88.00%,P0.001)。临床样本抗体吸附实验显示,纯化A抗原对抗A IgM/IgG的吸附效率分别为96.88%、98.44%;纯化B抗原对抗B IgM/IgG的吸附效率分别为96.88%、98.44%。结论:磁珠纯化抗原能特异性地与对应抗体结合,有效吸附血清中的血型抗体,有望作为合成A/B抗原的替代品。     

Antigenic profiling of yersinia pestis infection in the Wyoming coyote (Canis latrans)

Although Yersinia pestis is classified as a "high-virulence" pathogen, some host species are variably susceptible to disease. Coyotes (Canis latrans) exhibit mild, if any, symptoms during infection, but antibody production occurs postinfection. This immune response has been reported to be against the F1 capsule, although little subsequent characterization has been conducted. To further define the nature of coyote humoral immunity to plague, qualitative serology was conducted to assess the antiplague antibody repertoire. Humoral responses to six plasmid-encoded Y. pestis virulence factors were first examined. Of 20 individual immune coyotes, 90% were reactive to at least one other antigen in the panel other than F1. The frequency of reactivity to low calcium response plasmid (pLcr)-encoded Yersinia protein kinase A (YpkA) and Yersinia outer protein D (YopD) was significantly greater than that previously observed in a murine model for plague. Additionally, both V antigen and plasminogen activator were reactive with over half of the serum samples tested. Reactivity to F1 was markedly less frequent in coyotes (35%). Twenty previously tested antibody-negative samples were also examined. While the majority were negative across the panel, 15% were positive for 1-3 non-F1 antigens. In vivo-induced antigen technology employed to identify novel chromosomal genes of Y. pestis that are up-regulated during infection resulted in the identification of five proteins, including a flagellar component (FliP) that was uniquely reactive with the coyote serum compared with immune serum from two other host species. Collectively, these data suggest that humoral immunity to pLcr-encoded antigens and the pesticin plasmid (pPst)-encoded Pla antigen may be relevant to plague resistance in coyotes. The serologic profile of Y. pestis chromosomal antigens up-regulated in vivo specific to C. latrans may provide insight into the differences in the pathogen-host responses during Y. pestis infection.     

Incidence of antibodies against human immunodeficiency virus, human T-cell lymphotropic virus type 1, hepatitis B virus, hemorrhagic fever with renal syndrome virus and Chlamydia in Tonga and western Samoa

Among the populations of Tonga and Western Samoa, serum antibodies against human immunodeficiency virus or hemorrhagic fever with renal syndrome virus were not detected (0/904 and 0/192). No serum samples were considered to be positive for antibody against human T-cell lymphotropic virus type 1 (0/527). Hepatitis B antigen and antibody were found in 4% (8/192) and 47% (90/192), respectively. Chlamydia trachomatis IgG and C. psittaci IgG antibodies were detected in 39% (75/192) and 47% (91/192), respectively. The possibilities of the spread of human immunodeficiency virus and hemorrhagic fever with renal syndrome virus on the islands when the viruses invade from abroad were discussed.     

Monoclonal antibodies against baboon endogenous virus and against host cell antigens.

Monoclonal antibodies were produced by murine hybridomas after immunization with semipurified baboon endogenous virus. In a solid-phase radioimmunoassay, two antibodies (F12-9 and B9-18) reacted with viral antigen only. The antibodies A6-8 and C9-12 also reacted with virus-producing cells but not with control cells, whereas antibodies E4-6 and D12-2 bound to virus-free cells as well. The cytofluorometry technique confirmed these results and showed a competition between antibodies A6-8 and C9-12 for binding to virus-producing cells as well as a competition between antibodies D12-2 and E4-6 for binding to virus-free human cells. An immune precipitation assay with disrupted virions indicated that antibodies A6-8, B9-18, and C9-12 were directed against the gp70 glycoprotein, and that antibody F12-9 reacted with a viral antigen with a molecular weight of 18,000. The syncytia induced in RSa cells by baboon molecular weight of 18,000. The syncytia induced in RSa cells by baboon endogenous virus could be inhibited either when antibody A6-8 or C9-12 was combined to the virus or when the RSa cells were treated with the anticellular antibody D12-2 or E4-6. These two effects were not observed with Mason-Pfizer virus. Thus, of three antibodies with specificities for viral gp70, two (A6-8 and C9-12) were directed at viral sites responsible for syncytium formation. Another antiviral antibody (F12-9) reacted with a protein of unknown function with a molecular weight of 18,000. The two anticellular antibodies were directed at similar or neighboring epitopes, which may be situated within the receptor to the virus.     

Serologic survey for canine infectious diseases among sympatric swift foxes (Vulpes velox) and coyotes (Canis latrans) in southeastern Colorado

Swift foxes (Vulpes velox) and coyotes (Canis latrans) are sympatric canids distributed throughout many regions of the Great Plains of North America. The prevalence of canid diseases among these two species where they occur sympatrically is presently unknown. From January 1997 to January 2001, we collected blood samples from 89 swift foxes and 122 coyotes on the US Army Pi?on Canyon Maneuver Site, Las Animas County, SE Colorado (USA). Seroprevalence of antibodies against canine parvovirus (CPV) was 71% for adult (> 9 mo old) and 38% for juvenile (< or = 9 mo old) swift foxes. Adult (<1 yr old) and juvenile (<1 yr old) coyotes had a seroprevalence for CPV of 96% and 78%, respectively. Presence of antibodies against canine distemper virus (CDV) was 5% for adult foxes and 0% for juvenile foxes. Seroprevalence of CDV was 46% for adult coyotes and 18% for juvenile coyotes. No swift foxes had canine adenovirus (CAV) antibodies, whereas 81% and 63% of adult and juvenile coyotes, respectively, had antibodies for CAV. Seroprevalence of antibodies against Yersinia pestis was 68% among adult foxes and 34% among juvenile swift foxes. Seroprevalence of Y. pestis antibodies was 90% and 70% for adult and juvenile coyotes, respectively. No swift foxes had antibodies against Francisella tularensis, whereas seroprevalence was 4% among both adult and juvenile coyotes. Antibodies against CPV and plague were common in both species, whereas antibodies against CDV and CAV were more prevalent in coyotes compared to swift foxes.     

Monoclonal antibodies to type A, B, E and F botulinum neurotoxins

Mouse monoclonal antibodies against the most acutely toxic substances, botulinum neurotoxins (BoNTs) of types A, B, E, and F, was generated and characterized, that recognize their respective toxins in natural toxin complex. Based on these antibodies, we developed sandwich-ELISA for quantitative detection of these toxins. For each respective toxin the detection limit of the assay was: BoNT/A - 0.4 ng/ml, BoNT/B - 0.5 ng/ml; BoNT/E - 0.1 ng/ml; and for BoNT/F - 2.4 ng/ml. The developed assays permitted quantitative identification of the BoNTs in canned meat and vegetables. The BNTA-4.1 and BNTA-9.1 antibodies possessed neutralizing activity against natural complex of the botulinium toxin type A in vivo, both individually and in mixture, the mixture of the antibodies neutralized the higher dose of the toxin. The BNTA-4.1 antibody binds specifically the light chain (the chain with protease activity) of the toxin, whereas BNTA-9.1 interacts with the heavy chain. We believe that the BNTA-4.1 and BNTA-9.1 monoclonal antibodies are prospective candidates for development of humanized therapeutic antibodies for treatment of BoNT/A-caused botulism.     

Antibodies to Borrelia sp. in wild foxes and coyotes from Wisconsin and Minnesota

Serum samples from 93 red foxes (Vulpes vulpes) and nine gray foxes (Urocyon cinereoargenteus) trapped in Wisconsin and 23 coyotes (Canis latrans) trapped in Wisconsin and Minnesota were tested for antibodies to Borrelia sp. with an indirect fluorescent antibody test which used Borrelia burgdorferi as the whole-cell antigen. Seven red foxes (8%) and two coyotes (9%) had antibody titers greater than or equal to 1:64. All the positive samples were from areas known to be endemic for human Lyme disease. Implications for the epizootiology of Lyme borreliosis in wild canids are not well understood, but even if these species are not actual reservoirs of B. burgdorferi they could serve to increase the range of the vector and establish new endemic foci of the spirochete.     

Inhibition of stimulation in murine mixed lymphocyte cultures with an alloantiserum directed against a shared Ia determinant.

Pools of high titered alloantisera were raised by immunizing (B10.A/SgSn X A/WySn)F1 mice with C57BL/10Sn(B10) spleen cells. This serum (F1 anti-B10), when added to one-way mixed lymphocyte cultures (MLC), inhibited stimulation of B10.A splenic responders by both B10 and B10.D2/nSn irradiated, splenic stimulators. The B10 stimulation was suppressed approximately 85% whereas the mean suppression of B10.D2 stimulation was approximately 60%. In the ofrmer case, the serum contained antibodies reactive with multiple major histocompatibility complex determinants on the stimulator cells. In the latter case, the cytoxic reactivity of the serum was directed principally against an I region-associated determinant Ia.8) shared by B10 and B10.D2 and coded for by a gene(s) in the I-A subregion. The magnitude of the suppression of the response to B10.D2 cells (60%) was similar to the reduction in stimulation observed when the Ia.8 difference was eliminated genetically by using (B10 X B10.A)F1 responder cells against irradiated B10.D2 stimulators. Ihhibition of MLC by this antiserum was a function of reactivity with stimulator and not responder cells. Although some pools of F1 anti-B10 antiserum produced partial inhibition of the responder cell in a B10.D2 vs B10.Ax MLC combination, the results were inconsistent and not correlated with the anti-Ia.8 cytotoxicity titers. In addition, an F1 anti-B10 antiserum pool, which consistently failed to inhibit the responder cell, nevertheless inhibited both irradiated B10.D2 and (B10.A X B10.D2)F1 cells from stimulating B10.A responder cells. However, this same antiserum did not inhibit stimulation of B10.D2 responder cells by the (B10.A X B10.D2)F1 stimulators. Thus, the binding of antibodies to the non-stimulating antigens on the F1 stimulator cell did not interfere with the capacity of the appropriate stimulating antigens to cause stimulation. All of these results are consistent with the hypothesis that Ia allo-antigens are the major stimulating determinants of I region-associated MLC reactions.     

The characteristics of the antibody response of animals immunized with components of the surface structures of Francisella tularensis

Antibody formation in animals immunized with one of the components of F. tularensis surface structures was studied. The time course of antibody formation in 20 hamadryas baboons was studied in the passive hemagglutination (PHA) test, microagglutination (MA) test, and indirect enzyme immunoassay, used for the determination of IgG, IgA and IgM antibodies. The character of antibody response in the animals immunized with components of F. tularensis surface structures (S-complex) and with live tularemia vaccine was compared. The study revealed that immunization with the S-complex induced the formation of antibodies detected by all three methods. Antibody formation to the S-complex was found to be dose-dependent. With the increase of the injected dose of the S-complex, antibody titers determined in the PHA test decreased and those determined in the MA test increased, which was seemingly due to the induction of antibodies differing in their isotypes. After immunization with the S-complex the levels of IgG antibodies were lower and the levels of IgM antibodies by day 28 after immunization higher than after the injection of live tularemia vaccine.     

Development of monoclonal antibodies against different protein and carbohydrate epitopes of dipeptidyl peptidase IV from rat liver plasma membranes.

Dipeptidyl peptidase IV (DPP IV) is a serine exopeptidase expressed at high levels in rat kidney, liver and lung. We established eight monoclonal antibodies against partially purified DPP IV from rat liver plasma membranes. By means of a competitive dot blot assay with purified DPP IV, these antibodies were shown to recognize four different epitopes of the glycoprotein, designated A - D. The epitopes are located on the extracellular domain of DPP IV, as shown by papain digestion of liver plasma membranes. Treatment of DPP IV with neuraminidase and glycopeptide N-glycosidase F, as well as incubation of hepatocytes with the alpha-mannosidase I inhibitor deoxymannojirimycin, revealed that epitope A may be formed by a mannose-rich sugar chain and epitope D might represent a complex carbohydrate structure in the mature glycoprotein, while the epitopes B and C are formed by the protein moiety. Concanavalin A reduced the binding of monoclonal antibody to epitope A by 78%. Binding to epitope D was blocked by 73% with wheat germ lectin, and by more than 99% with sialic acid; epitopes B and C were unaffected by any of the lectins or sugars tested. The immunological cross-reactivity with DPP IV from Morris hepatoma 7777 was demonstrated with monoclonal antibodies against epitopes A-C. Epitope D was not recognized on hepatoma DPP IV. However, in addition to DPP IV, four hepatoma plasma membrane glycoproteins were precipitated by the monoclonal antibody against the epitope D, indicating that this epitope is not uniquely restricted to DPP IV.