Characterization of monoclonal antibodies against ovine Sarcocystis spp. antigens by immunoblotting and immuno-electron microscopy

Six monoclonal antibodies were raised in mice against purified cytozoite extracts of Sarcocystis gigantea and S. tenella from sheep. Each monoclonal antibody was evaluated for specificity by enzyme immunoassay, immunoblotting and immuno-electron microscopy using homologous and heterologous antigenic preparations. All six monoclonal antibodies exhibited good species-specificity when reacted against crude soluble cystozoite antigens in enzyme immunoassays. However, only two monoclonal antibodies (IgM and IgG2a) exhibited reactivity in Western blots against specific protein bands. Both reacted against S. gigantea antigens of 100,000, 43,000 and 39,000 molecular weight. Neither monoclonal antibody reacted against the heterologous species S. tenella. Ultrastructural studies performed with colloidal-gold conjugated antisera revealed that both monoclonal antibodies reacted against antigens located around micronemes and amylopectin granules in S. gigantea cystozoites. Another monoclonal antibody (IgGI) reacted only against microneme determinants in S. tenella cystozoites. In contrast, polyclonal sheep and rabbit immune sera cross-reacted against a wide range of cystozoite antigens.     

Characterization of monoclonal antibodies to bovine IgG1

Monoclonal antibodies were developed to bovine IgG1. In addition, production of monoclonal antibodies to bovine light chain is also reported. Monoclonal antibody specificities were initially determined by solid-phase enzyme immunoassay. The monoclonal antibovine IgG1 was shown by a specificity-independent isotyping solid-phase enzyme immunoassay to be mouse IgG1 with kappa light chains. Ascites derived monoclonal antibovine IgG1 antibodies were linked to cyanogen bromide-activated Sepharose and used for affinity isolation of bovine IgG1. The bovine IgG1 eluted from the affinity column was characterized using immuno-electrophoresis, acrylamide gel electrophoresis, isoelectric focusing and solid-phase enzyme immunoassay. Affinity chromatography using monoclonal antibodies provided both a verification of monoclonal antibody specificity and a rapid technique for the isolation of bovine IgG1. This technique may also be employed to remove IgG1 contaminants during purification of bovine IgA.     

Characterization of monoclonal antibodies to avian leukosis viruses

Hybridoma cell lines secreting monoclonal antibody (MCA) to avian leukosis virus (ALV) structural proteins p27 and p19 have been established. In an indirect enzyme-linked immunosorbent assay (ELISA), MCA 6AL20 (IgG1 isotype) reacted with RPL-40 (ALV subgroup A), avian myeloblastosis virus (AMV) (a mixture of subgroups A and B), Rous-associated virus (RAV)-2 (subgroup B), and Carr-Zilber strain of Rous sarcoma virus (CZ-RSV) (subgroup D) but not with Prague strain of RSV (PrC-RSV) (subgroup C) or the endogenous virus RAV-0 (subgroup E). MCA 6AL22 reacted as above and also reacted marginally with PrC-RSV. Both MCAs immunoprecipitated p19 from 35S-methionine-labeled chicken embryo fibroblasts (CEFs) infected with RPL-40 or RAV-1, but not from CEFs infected with RAV-0, thus identifying the viral structural protein p19 as a polypeptide with subgroup-specific epitopes. Both MCAs can be used to differentiate RPL-40 from RAV-0 infection either in an indirect antibody ELISA or by immunoprecipitation. A third MCA, 6AL42 (IgG2a isotype), reacted with the above viruses of subgroups A, B, C, and D at an antibody titer up to 1000-fold higher than with subgroup E RAV-0 virus in indirect ELISAs. MCA 6AL42 immunoprecipitated p27 from cells infected with RPL-40, RAV-1, or RAV-0. These MCAs are potentially useful in developing immunological tests for differentiation of ALV strains.     

Characterization of monoclonal antibodies to avian Escherichia coli Iss

Colibacillosis accounts for annual multimillion dollar losses in the poultry industry, and control of this disease is hampered by limited understanding of the virulence mechanisms used by avian pathogenic Escherichia coli (APEC). Previous work in our laboratory has found that the presence of the increased serum survival gene (iss) is strongly associated with APEC but not commensal E. coli, making iss and the protein it encodes (Iss) candidate targets of colibacillosis-control procedures. Previously, we produced monoclonal antibodies (MAbs) against Iss to be used as a reagent in studies of APEC virulence and colibacillosis pathogenesis. Unfortunately, the utility of these MAbs was limited because these MAbs exhibited nonspecific binding. It was thought that the lack of specificity might be related to the fact that these MAbs were of the immunoglobulin M (IgM) isotype. In the present study, new MAbs were produced using a different immunization strategy in an effort to generate MAbs of a different isotype. Also, because Iss bears strong similarity to Bor, a lambda-derived protein that occurs commonly among E. coli, MAbs were assessed for their ability to distinguish Iss and Bor. For these studies, the bor gene from an APEC isolate was cloned into an expression vector. The fusion protein expressed from this construct was used to assess the potential of the anti-Iss MAbs produced in the past and present studies to distinguish Bor and Iss. The MAbs produced in this study were of the IgG1 isotype, which appeared to bind more specifically to Iss than previously generated antibodies in certain immunologic procedures. These results suggested that the MAbs generated in this study might prove superior to the previous MAbs as a reagent for study of APEC. However, both MAbs recognized recombinant Iss and Bor, suggesting that any results obtained using anti-Iss MAbs would need to be interpreted with this cross-reactivity in mind.     

Characterization of monoclonal antibodies against fowl poxvirus

Vaccines for the prevention of fowl pox in chickens and turkeys have been available for more than five decades. However, in recent years outbreaks have occurred in several previously vaccinated chicken flocks. Presumably, fowl poxviruses (FPVs) antigenically different from the attenuated vaccine strains are responsible for such occurrences. In support of this concept, we previously detected minor antigenic changes in field isolates based on comparative immunoblotting with polyclonal anti-FPV serum. Realizing the need for antibodies specific against the dominant antigens of FPV, monoclonal antibodies (MAbs) were produced by immunizing mice with either a field strain of FPV or a pigeon poxvirus, currently used for vaccination. Three hybridoma clones producing MAbs reacting with a specific FPV protein were selected from a total of 83 clones. In immunoblots, two of the MAbs, P1D9 and P2H10, recognized an antigen with an apparent molecular weight varying from 39 to 46 kD, depending on the FPV strain. The third MAb, P2D4, reacted with an approximately 80-kD protein, regardless of which FPV isolate was tested. Immunofluorescent staining with P1D9 and P2D4 revealed that these MAbs react with intracytoplasmic antigens in FPV-infected cells.     

Characterization and use of monoclonal antibodies to identify Haemophilus paragallinarum serovars

Two serovar-specific monoclonal antibodies (MAbs) to Haemophilus paragallinarum serovars A/1 and C/2 strains, respectively, were developed and characterized by hemagglutination-inhibition (HI) and dot-blotting tests using representative H. paragallinarum serovars A/1, B, and C/2 strains. In both the HI and dot-blotting tests, one MAb (E5C12D10), raised against strain 221, serovar A/1, reacted only with serovar A/1 strains, while the other MAb (F2E6), raised against strain S1 of serovar C/2, reacted with only serovar C/2 strains examined. In both tests, the two MAbs did not react with two serovar B strains. These results indicated that the two MAbs recognize serovar-specific hemagglutinating (HA) antigens of H. paragallinarum serovars A/1 and C/2 strains, respectively, and that a dot-blotting test using these MAbs is a practical alternative to the HI test for serotyping H. paragallinarum. Strains 0222 and Spross of serovar B, which did not react with these two MAbs, were found to possess serovar-specific HA antigen in cross-HI tests.     

Cross-reactivities with Cryptosporidium spp. by chicken monoclonal antibodies that recognize avian Eimeria spp

In a previous study, we have developed several chicken monoclonal antibodies (mAbs) against Eimeria acervulina (EA) in order to identify potential ligand molecules of Eimeria. One of these mAbs, 6D-12-G10, was found to recognize a conoid antigen of EA sporozoites and significantly inhibited the sporozoite invasions of host T lymphocytes in vitro. Furthermore, some of these chicken mAbs showed cross-reactivities with several different avian Eimeria spp. and the mAb 6D-12-G10 also demonstrated cross-reactivities with the tachyzoites of Neospora caninum and Toxoplasma gondii. Cryptosporidium spp. are coccidian parasites closely related to Eimeria spp., and especially C. parvum is an important cause of diarrhea in human and mammals. In the present study, to assess that the epitopes recognized by these chicken mAbs could exist on Cryptosporidium parasites, we examined the cross-reactivity of these mAbs with Cryptosporidium spp. using an indirect immunofluorescent assay (IFA) and Western blotting analyses. In IFA by chicken mAbs, the mAb 6D-12-G10 only showed a immunofluorescence staining at the apical end of sporozoites of C. parvum and C. muris, and merozoites of C. parvum. Western blotting analyses revealed that the mAb 6D-12-G10 reacted with the 48-kDa molecular weight band of C. parvum and C. muris oocyst antigens, 5D-11 reacted the 155 kDa of C. muris. Furthermore, these epitopes appeared to be periodate insensitive. These results indicate that the target antigen recognized by these chicken mAbs might have a shared epitope, which is present on the apical complex of apicomplexan parasites.     

Characterization of monoclonal antibodies directed against swine leukocytes

Hybridomas were produced from fusions of the SP2/0 mouse myeloma with splenic cells from: 1) an outbred Sprague Dawley rat immunized with swine peripheral blood mononuclear (PBM) cells; 2) a (CBA/NDub X BALB/c Dub) F1 mouse immunized with concanavalin A (Con A) activated swine PBM cells and 3) a (BALB/c Dub X C3H/He Dub) F1 mouse immunized with swine thymocytes. The resulting supernatants were screened by a microcytotoxicity assay for activity against swine PBM cells. Four hybridomas (MSA1, MSA2, MSA3 and MSA4) were selected, cloned and characterized by their cell reactivity and effect on mitogenic assays. MSA1 and MSA2 belong to the rat IgG2b subclass. MSA3 and MSA4 are of the mouse IgG2a subclass. These monoclonal antibodies reacted in the following manner: MSA1 with monocytes, granulocytes, red blood cells and bone marrow cells; MSA2 with subset of T cells; MSA3 with B cells and subsets of T cells and monocytes (class II molecule) and MSA4, a pan-T cell reagent (E-rosette receptor). The involvement of the various cell types reactive to the different monoclonal antibodies in the mitogenic response of swine PBM cells to Con A, phytohemagglutinin (PHA) or pokeweed mitogen (PWM) was investigated by cellular depletion with monoclonal antibody plus complement. Cellular depletion of PBM cells with the following monoclonal antibodies plus complement treatment resulted in: MSA1, almost total reduction in the mitogenic response to low doses of Con A or PWM; MSA2, partial reduction in the proliferative responses to any concentration of Con A, PHA or PWM; MSA3, partial reduction in proliferative responses to low concentrations of Con A or PWM and 4) MSA4, total elimination of any proliferative response to Con A, PHA or PWM.     

Characterization of infectious bronchitis virus using monoclonal antibodies

Three monoclonal antibodies (MABs) reactive against two structural proteins--the nucleoprotein (NP) or the surface (S) protein--of avian infectious bronchitis virus (IBV) were produced and characterized. The MABs did not neutralize virus infectivity or inhibit hemagglutination. Their reactivity patterns with the homologous strain and eight heterologous strains of IBV were determined using the indirect immunoperoxidase test, the indirect immunofluorescent test, transfer-immunoblotting of separated proteins, and a dot-immunoblotting assay (DIA). Two MABs, NP- or S-protein-specific, reacted with all nine strains; one (NP-specific) reacted with only two strains. The two MABs reacting with all nine strains of IBV also detected 18 IBV field isolates of unknown serotype in the DIA. The MAB detecting only two strains did not react in the DIA. The diagnostic application of these MABs appears promising.     

Characterization of monoclonal antibodies to channel catfish, Ictalurus punctatus, leucocytes

Four monoclonal antibodies (MAbs) were evaluated for specific reactions to various catfish peripheral blood leucocytes and anterior kidney cells. The MAb 9E1 served as a standard and positive control for all test reactions because of its defined reactivity with channel catfish immunoglobulin and immunoglobulin bearing cells. Of the four MAbs, two have been characterized as being specific for a non-immunoglobulin marker on lymphocytes, thus marking T lymphocytes and two were specific for catfish neutrophils. Morphological, flow cytometric and functional analysis of the reactive cells verified these findings.     

Characterization of anti-channel catfish MHC class IIbeta monoclonal antibodies

This study characterizes four monoclonal antibodies (mAb) developed against the major histocompatibility complex (MHC) class II beta chain of the channel catfish, Ictalurus punctatus. Immunoprecipitations using catfish clonal B cells revealed that each of these mAbs immunoselected proteins of approximately 32 and 36 kD, which are of the appropriate sizes for MHC class II alpha and beta chains, respectively. Cell distribution studies using a fluorescence-activated cell sorter (FACS) combined with RT-PCR analyses demonstrated that MHC class II beta is expressed at a high density on catfish clonal macrophage, B and T cell lines, on alloantigen stimulated leukocytes, and on lipopolysaccharide-induced B-cell blasts. Collectively, these results demonstrate the potential importance of these antibodies as reagents in future studies dealing with the functional role of MHC class II molecules in immune recognition of self from non-self.     

重组牛朊蛋白特异性抗体的制备及鉴定

以原核表达后经纯化的重组牛朊蛋白为免疫原,免疫prnp-/-基因敲除鼠。4次免疫后,利用淋巴细胞杂交瘤技术,取脾细胞和SP2/0骨髓瘤细胞进行细胞融合。间接ELISA方法筛选出阳性杂交瘤细胞,采用有限稀释法对阳性杂交瘤细胞进行3次克隆,用间接ELISA筛选出了稳定分泌针对牛重组朊蛋白特异性单克隆抗体的杂交瘤细胞株,命名为5C9D6。Western blotting鉴定结果表明,5C9D6均能特异性识别重组牛朊蛋白、健康牛、BALB/c脑组织匀浆中的PrPc,不识别prnp-/-基因敲除鼠脑组织匀浆液。本试验制备了可与牛、BALB/c鼠反应的单克隆抗体,同时也为牛海绵状脑病的研究及其诊断方法的建立奠定了基础。     

Prevalence of Sarcocystis spp. in Argentinean cattle

Sarcocystis cruzi, S. hirsuta and S. hominis are apicomplexan parasites that affect cattle worldwide with variable prevalence. The aim of the present study was to evaluate the prevalence of Sarcocystis spp. in Argentinean cattle comparing microscopic fresh examination and molecular methods. Blood, myocardium and loin samples were collected in five slaughterhouses from a total of 380 bovines. Origin of animals was representative of the major beef cattle production area of Argentina. Samples were analyzed by fresh microscopical examination, transmission electron microscopy (TEM), IFAT and PCR-RFLP. Thin walled sarcocysts corresponding with S. cruzi were found in 99.5% of heart samples. Sarcocysts were detected in 73.1% of loin samples; 71.5% had S. cruzi cysts and 23.1% had thick walled sarcocysts (S. hirsuta or S. hominis). TEM observation revealed the presence of characteristic S. hominis and S. hirsuta cyst walls in 7 and 1 loin samples respectively. Using IFAT, 379/380 animals had titers 25 or higher, showing a full agreement with fresh examination. Amplification products were detected in 35.5% (135/380) of loin samples; however Sarcocystis species could only be determined by RFLP in 29 samples. Agreement between fresh examination and PCR was low (Kappa value=0.262). This is the first report of S. hominis and S. hirsuta in Argentina. Further studies are needed to improve the sensitivity of molecular methods for species identification, especially for differentiation of S. cruzi and S. hirsuta from the zoonotic species S. hominis. The results of the present study and others focusing on sensitivity and specificity of Sarcocystis spp. diagnostic methods should contribute to improve food safety.     

Characterization of monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 5.

Two monoclonal antibodies against Actinobacillus pleuropneumoniae serotype 5, designated as 5MAb-1 and 5MAb-6, were characterized. Enzyme-linked immunosorbent assay-inhibition tests with whole-cell antigens obtained from strains of serotype 1 through 12 of A pleuropneumoniae revealed that 5 MAb-1 bound to only serotype-5 strains. The epitope recognized by 5MAb-1 was a carbohydrate that was sensitive to periodate oxidation and resided on the structure of beta-1,6-linked D-galactose in an O-antigen polysaccharide of serotype-5 lipopolysaccharide. Analysis of these results revealed that the O-antigen polysaccharide of lipopolysaccharide was 1 of the antigenic determinants responsible for the serotype specificity of A pleuropneumoniae. On the other hand, 5MAb-6 reacted with strains of serotype 1 through 10 in varying degrees and its epitope was located on polypeptides sensitive to proteinase K. In an immunoblotting analysis, 5MAb-6 reacted with 2 polypeptide bands, with molecular weights of approximately 41,500 and 28,000, in the outer membrane protein-rich fraction obtained from strains of serotype 1 through 10. These results indicated that outer membrane proteins from several serotype strains of A pleuropneumoniae possessed common antigenic determinants.     

Characterization of monoclonal antibodies to bovine herpesvirus type I, Los Angeles strain.

We produced monoclonal antibodies (mAbs) against bovine herpesvirus type 1 (BHV-1), Los Angeles strain, and then evaluated them as potential candidates for preparing diagnostic reagents. Of the 318 cell lines expressing antibodies to the virus, 60% (192) secreted IgG and 40% (126) secreted IgM. Twenty-six mAbs were selected based on enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) titers and characterized by immunoprecipitation, immunofluorescence and immunoblots. The selected mAbs were assigned to one of four groups based on their immunoprecipitation patterns. Group A (4 mAbs) precipitated a complex of three glycoproteins with molecular weight (MW) 130 kDa, 72 kDa and 55 kDa, which presumably represented gI of BHV-1. Monoclonal antibodies of this group were highly reactive in ELISA but had low VN titers. Group B (4 mAbs) precipitated a glycoprotein with MW of 71 kDa (gIV). This group of mAbs had high VN titers. Group C (16 mAbs) precipitated a 97 kDa glycoprotein (gIII). Monoclonal antibodies of this group had high ELISA but low VN titers. Group D (2 mAbs) precipitated a double band of non-glycosylated proteins with MW of 37/32 kDa; these proteins could not be assigned to any of the antigens of BHV-1 previously described. ELISA and VN titers of this group of mAbs were low. To test the antigenic variability of the antigenic determinants which were recognized by these 4 groups of mAbs, we adapted Madin Darby bovine kidney cell-propagated BHV-1 Los Angeles strain to Crandell's feline kidney cell line. After the tenth passage in feline kidney cells, the epitopes on the 37/32 kDa peptide recognized by the mAbs group D were no longer detectable. Additional changes were noted in the electrophoretic mobility of the 130 kDa and 71 kDa glycoproteins (gI) identified by mAb of group A shifted downward. The 71 kDa glycoprotein (gIV) reactive with mAb group B and the 97 kDa (gIII) reactive with mAb group C remained stable. Since clone No. 191 of group B mAb was potent in ELISA, VN, immunoblots and immunofluorescence, and recognized an epitope which did not change under selective pressure, we feel that the mAb produced by this clone are a good candidate for the production of diagnostic reagents.