Workshop studies on monoclonal antibodies in the myeloid panel with CD11 specificity.

Several putative anti-human and swine CD11-specific monoclonal antibodies (mAbs) were included in the myeloid section of the Third International Swine CD Workshop. Failure of clustering analysis to group these mAbs together prompted additional analyses to define the specificities of these mAb. Combination of one and two-color flow cytometry (FCM) and immunoprecipitation (IP) allowed the definition of the mAb into three CD11 groups. Cellular distribution of the molecules recognized by anti-human CD11b and c mAbs on swine cells proved to be significantly different from that found in humans.     

Molecular cloning and expression analysis of interferon regulatory factor 8 (IRF8) in turbot, Scophthalmus maximus

Mucus immunoglobulin (Ig) of flounder (Paralichthys olivaceus) was purified by the combination of salting-out, Sephacryl S-300 gel filtration chromatography and DEAE Sepharose chromatography. According to the SDS-PAGE and native-PAGE, the purified mucus Ig showed apparent molecular weights of 72 kDa (heavy chain) and 26 kDa (light chain), and a total molecular weight of 798 kDa, which indicated mucus IgM was in tetrameric form. Purified mucus Ig was used to immunize the Balb/C mice, nineteen hybridomas secreting monoclonal antibodies (mAbs) against flounder mucus Ig were obtained by indirect enzyme-linked immunosorbent assay, and three of them designated as 1A-M2, 1C-M10 and 3F-M9 were cloned by limiting dilution. In Western blotting, the three mAbs specifically reacted to the heavy (H) chain of mucus Ig, but not reacted with serum Ig of flounder, whereas mAb 2D8 against serum Ig previously produced could react with the H chain of both mucus and serum Ig, indicating the composition of the mucus and serum Ig H chains was different. Meanwhile, surface Ig positive (sIg+) lymphocytes in the peripheral blood, spleen, skin and gills of healthy flounder, were analyzed by flow cytometry using mAb 1A-M2 and mAb 2D8, and the results revealed that both mAbs were reactive with the sIg+ lymphocytes. The positive reactivity rates for mAb 1A-M2 were 38.64% in the peripheral blood, 23.6% in the spleen, 16.56% in the skin and 6.26% in the gills, while the positive reactivity rates for mAb 2D8 were 48.89%, 33.7%, 15% and 6.02%, respectively, suggesting mucus Ig was similar, but not identical, to serum Ig. These results generated important mucosal immunological information and gave a valuable insight into understanding the mucosal immunity in flounder.     

Differentiation of porcine myeloid bone marrow haematopoietic cell populations.

The myeloid panel of monoclonal antibodies (mAbs) submitted to the Third Swine CD Workshop were analysed for reactivity with bone marrow haematopoietic cells (BMHC). Using single and triple immunofluorescence labelling by flow cytometry (FCM), the mAbs were grouped according to their capacity to recognise myeloid cell populations and/or maturation stages. Group 1 consisted of mAbs labelling the majority of myeloid BMHC, including neutrophilic, eosinophilic and monocytic cells. The ligands for SWC3 and CD11b-like mAbs of group 1 showed a maturation-dependent intensity of expression. The other antibodies of group 1 reacted with BMHC to give a sharp, single peak. Group 2 mAbs reacted only with monocytic cells. The anti-human CD49e mAb Sam-1 was the only mAb detecting the majority of monocytic cells, but not other BMHC. The mAbs in group 3 recognised antigens expressed on granulocytes, but not monocytes. The previously identified SWC8 in this group proved to be useful in differentiating major population of BMHC when cells were double labelled with the pan-myeloid SWC3. Other mAbs within group 3, such as MIL4 and TMG6-5 (an anti-human CD11b), only recognised subsets of neutrophils and eosinophils. Group 4 mAbs reacted with the more mature subpopulations of neutrophils and monocytes. Some of these antibodies might prove useful for assessment of cell maturity, such as anti-CD14 and the anti-human CD50 mAb HP2/19.     

Preparation of heavy chain specific antisera to bovine IgA, IgM, IgG1 and IgG2

Goats, guinea pigs and rabbits were immunized with bovine IgM or with intact molecules, heavy chains, Fc portions or light chains of bovine IgG1 and IgG2. Rabbits and guinea pigs were immunized with bovine secretory IgA. Goats and guinea pigs produced heavy chain specific antisera to intact IgM whereas rabbits produced anti-light chain antibody and in one instance anti-alpha 2-macroglobulin antibody in addition to the anti-mu response. Goats and guinea pigs produced antisera to bovine IgG1 and IgG2 and their Fc portions that needed little absorption to render them monospecific for the heavy chain. In addition to antibody to the heavy chains, rabbits produced anti-light chain antibody when immunized with intact IgG1 or IgG2 molecules. These latter sera, and those produced by rabbits immunized with Fc portions of IgG1 or IgG2 required extensive absorption before they were monospecific for their respective heavy chains. Heavy chains were poor immunogens in all three species. Rabbits immunized with IgA produced both anti-alpha and anti-light chain antibodies while guinea pigs produced sera with antibody activity to the alpha chain only.     

Generation and characterisation of murine monoclonal antibodies specific for cervine immunoglobulin light chain, IgM and IgG.

Monoclonal antibodies (mAb) which react with cervine immunoglobulin (Ig) light chain, IgM and IgG were produced using conventional cell fusion technology. Hybridoma supernatants were initially screened for specificity against cervine Ig using an enzyme-linked immunosorbent assay (ELISA). The specificity of supernatants against size-fractionated cervine Ig was further determined. Supernatants were characterised using western blotting and autoradiographic techniques. The mAb OU1G, OU2G and OU3G were specific for cervine gamma-chain of IgG, whereas OU1L was specific for light chain of Ig. A further mAb (OU1M) bound IgM and not IgG. These mAb were found to have varying cross-reactivity against Ig from other species.     

Immunoglobulin genes and diversity: what we have learned from domestic animals

ABSTRACT: This review focuses on the diversity of immunoglobulin (Ig) genes and Ig isotypes that are expressed in domestic animals. Four livestock species--cattle, sheep, pigs, and horses--express a full range of Ig heavy chains (IgHs), including mu, delta, gamma, epsilon, and alpha. Two poultry species (chickens and ducks) express three IgH isotypes, mu, upsilon, and alpha, but not delta. The kappa and lambda light chains are both utilized in the four livestock species, but only the lambda chain is expressed in poultry. V(D)J recombination, somatic hypermutation (SHM) and gene conversion (GC) are three distinct mechanisms by which immunoglobulin variable region diversity is generated. Different domestic animals may use distinct means to diversify rearranged variable regions of Ig genes.     

Development of a monoclonal antibody-based sandwich ELISA for detection of guinea pig interleukin-2

Interleukin 2 (IL-2) is a T cell proliferation factor released by Th0- and Th1-type helper T cells and is an essential cytokine for immune responses. In the present study, recombinant glutathione S-transferase (GST)-guinea pig IL-2 (GPIL-2) fusion protein was prepared by Escherichia coli (E. coli) and by using this protein as an immunogen, monoclonal antibodies (mAbs) against GPIL-2 were produced to establish a basis for a research on immune responses in guinea pigs. Three stable hybridoma cell lines were established, and specific binding of each mAb to recombinant GPIL-2 produced by E. coli and insect cells infected with recombinant baculovirus was shown by enzyme linked immunosorbent assay (ELISA) and/or immunoblot analysis. Isotype analyses of these mAbs revealed that all three mAbs were IgG1 and had kappa chain. Furthermore, assessment of their epitopes by competition binding assay indicated that the mAbs obtained in this study bound to three different epitopes. Thus, a sandwich ELISA based on the two mAbs specific to different GPIL-2 epitopes was developed for detection of GPIL-2, which had a sensitivity threshold of about 0.3 ng/ml of GPIL-2.     

Monoclonal anti-equine IgE antibodies with specificity for different epitopes on the immunoglobulin heavy chain of native IgE

In this study we describe the generation of monoclonal antibodies (mAbs), which recognize different epitopes of the equine IgE constant heavy chain. Equi-murine recombinant IgE (rIgE), composed of the murine V(H)186.2 heavy chain variable region, linked to the equine IgE constant heavy chain and expressed together with the murine lambda(1) chain in J558L cells was used to immunize BALB/C mice. A total of 17 different mAbs were obtained, which recognized the rIgE heavy chain constant region. None of the mAbs reacted with monoclonal equine isotypes IgM, IgG1 (IgGa), IgG3 (IgG(T)), IgG4 (IgGb) or isolated equine light chains, IgGc and IgA from horse serum, or the native mAb B1-8delta, expressing the same heavy chain variable regions and light chains. One of the mAbs (alphaIgE-132) recognized the recombinant equine IgE, but did not recognize any protein in equine serum, i.e. native IgE. A total of 16 mAbs detected a serum protein of approximately 210,000Da on Western blots, corresponding to the expected MW of native IgE. In addition, one of the mAbs (alphaIgE-176) detected a protein of 76,000Da under reducing conditions, most likely the equine IgE heavy chain. According to binding inhibition studies, the equine IgE specific mAbs recognize at least two different epitopes of the equine IgE. In an ELISA using two anti-IgE mAbs which recognized different epitopes, no significant differences in the concentration of total serum IgE could be detected between adult Icelandic horses with IgE-mediated type I allergy (summer eczema) and healthy control animals. In Icelandic horse foals, no serum IgE could be measured 6 months post partum. All anti-IgE mAbs recognized a small population (1.3+/-0.5%) of leukocytes from adult Icelandic horses by surface immunofluorescence, but no cells could be detected in foal blood. The stained leukocytes from adult horses could be enriched by magnetic cell sorting and contained 32% basophils, 53% monocytes and/or large lymphocytes, 13% small lymphocytes and 2% eosinophils.     

Cross-reaction of anti-human CD monoclonal antibodies on guinea pig cells: a summary of the guinea pig section of the HLDA8 animal homologues data

A panel of 377 commercially available monoclonal antibodies (mAbs) specific for a total of 144 CD antigens was submitted to the animal homologue section of the Eighth International Workshop on Human Leukocyte Differentiation Antigens (HLDA8, Adelaide, Australia) for cross-reactivity studies in a range of vertebrate species. Each of the mAbs in this study was screened for positive reactivity with guinea pig splenocytes by flow cytometry. In the first phase of this study 36 of the total 367 mAbs (9.81%) cross-reacted with splenocyte surface molecules. The majority (26 of 36) of these cross-reactive mAbs were analysed further to confirm appropriate cell subset expression by two-color immunofluorescence. Our results indicate that 15 anti-human CD9, CD10, CD14, CD20 (two clones), CD22, CD25, CD29 (two clones), CD32, CD47 (two clones), CD49d, CD49e, and CD86 mAbs exhibit clear cross-reactivity with guinea pig splenocytes. These mAb can potentially be added to the limited repertoire of reagents available for studies in this model system. This data clearly indicates that mouse anti-human CD mAb guinea pig cross-reactions have been defined and that an aim of this HLDA8 section has been fulfilled, i.e., to identify mAbs which recognize conserved, species-independent CD epitopes. These results will contribute to the availability of mAbs and tools in veterinary medicine and immunology.     

Production and characterization of monoclonal antibodies to porcine immunoglobulin gamma, alpha, and light chains

Monoclonal antibodies (MAB) to porcine immunoglobulins were produced by fusion of SP2/0 cells with splenic lymphocytes of mice that had been immunized with porcine IgG or IgA. Of 16 MAB selected for detailed study, 13 reacted with heavy chains (7 anti-gamma, 6 anti-alpha) and 3 reacted with light chains of native immunoglobulin. Several of the same MAB (3 anti-gamma chain, 1 anti-alpha chain, 3 anti-light chain) also reacted with denatured immunoglobulin by use of immunoblotting analysis. Collective results of competitive ELISA, immunodiffusion, and immunoblotting analysis indicated that the 7 MAB of anti-gamma chain specificity were directed to 3 epitopes, the 6 MAB of anti-alpha chain specificity were directed to at least 2 epitopes, and the 3 MAB of anti-light chain specificity were directed to at least 2 epitopes that may have been located of different types of light chains. When tested by immunodiffusion with 5% polyethylene glycol incorporated in the agar matrix, all anti-gamma chain and antilight chain MAB, but no anti-alpha chain MAB, had precipitating activity. When polyethylene glycol was not used, only 4 MAB (all of the anti-gamma chain specificity and of IgM isotype) had precipitating activity. These MAB, with specificity for gamma and alpha chains and those reported earlier with mu-chain specificity, should be invaluable in the detection and quantification of porcine immunoglobulin isotypes. These MAB have potential applications in delineating the porcine immune response to selected immunogens.     

Monoclonal antibodies against chicken interleukin-6

Monoclonal antibodies (mAb) were produced against a recombinant (r) chicken interleukin-6 (IL-6). Eight mAbs produced were tested for isotype; ability to inhibit recombinant forms of chicken (ch), human (h) and murine (m) IL-6; and recognition of rchIL-6 by Western immunoblotting. The mAb isotypes were represented by IgG1 (one), IgG2a (six) and IgG2b (one). In a mouse B9 hybridoma cell bioassay with rmIL-6, four mAbs effectively inhibited activity of rmIL-6. Further bioassays with the four mAbs at varying concentrations showed that two of these mAbs (1.20.7 and 1.26.4) were quite effective at inhibiting rmIL-6. Recombinant forms of ch, h and mIL-6 were all tested in a bioassay with the most potent inhibiting mAb (1.26.4), and this mAb was effective in inhibiting all three recombinant IL-6 proteins. Western immunoblotting revealed identification of the original IL-6 immunogen used for mAb production. Based upon inhibition of IL-6 activity in a standard bioassay and IL-6 recognition by Western immunoblotting, mAb 1.26.4 was judged the most useful antibody for future studies and applications.     

Analysis of monoclonal antibodies that recognize γδ T/null cells

Thirty two monoclonal antibodies (mAbs) from the first round of analysis in the Second International Swine CD Workshop were placed together with additional mAb derived from the first workshop in the null cell panel for further evaluation. Preparations of peripheral blood leukocytes, concanavalin A stimulated peripheral blood mononuclear cells, and spleen cells were used in flow cytometric analyses. Nineteen mAbs identified molecules that were not expressed on null cells, not lineage specific, or recognized activation molecules. Sixteen mAbs including control mAbs were identified that were specific for null cells. One of the latter mAbs, 041 (PGBL22A), that recognizes a determinant on a constant region of porcine γδ TcR established the majority of null cells are γδ T cells. Use of this mAb in further comparisons demonstrated the γδ T cell population is comprised of two major subpopulations, one negative and one positive for CD2. Two color analyses demonstrated that 11 of the mAbs formed a broad cluster that included control mAbs 188 (MAC320) that defined the CD2 negative SWC6 cluster in the first workshop and mAb 122 (CC101) that might recognize an orthologue of bovine WC1 and nine mAbs that recognize determinants on one or more molecules with overlapping patterns of expression on subsets of CD2⁻ γδ T cells. Two groups of mAbs formed the previously identified subset clusters SWC4 and SWC5. Two new mAbs formed a third subcluster. Three mAbs did not form clusters. Three mAbs predicted to recognize TcR in the first workshop (020 [PT14A], 021 [PT79A], and 022 [MUC127A]) and mAb PGBL22A were shown to immunoprecipitate a 37, 40 kDa heterodimer.     

Characterization and Distribution of B Cells in the Lymphoid Organs of Goats

The distribution of B cells in the lymphoid organs of the goat was studied using a panel of 13 monoclonal antibodies (mAbs) developed against different markers for bovine B cells. Samples of mesenteric lymph nodes, jejunal and ileal Peyer's patches, duodenum, jejunum, ileum, colon, caecum and rectum were taken from four 7-month-old male Murciano-granadina goats using the avidin-biotin-peroxidase (ABC) method on frozen sections as described by Hsu et al. (1981). The mAbs against immunoglobulins (Ig) recognized a large number of cells, particularly in the light zones of the germinative centres of the lymphoid follicles, regardless of the isotype against which they were directed. However, the greatest numbers of B cells in the germinative centres and outer coronas of the lymphoid follicles of the lymph node, spleen and Peyer's patches were recognized by mAbs against the L lambda chain of Ig and against IgM. This was also the case in other locations where B cells were abundant, such as the medulla of the lymph node and the dome of the Peyer's patches. These mAbs recognized not only B lymphocytes but also plasma cells, showing an intracytoplasmatic reaction (numerous in the spleen red pulp and the intestinal lamina propria when mAbs were used against the L lambda chain of the Ig, scarce in the intestinal lamina propria when used against IgM and scarce in spleen red pulp and numerous in the intestinal lamina propria when mAbs against IgA were used). The mAbs BAQ44 A, GC65 A and GB25 A are of interest because, besides marking cells in the B areas where lymphocytes show surface Ig, they give a positive reaction in areas where there are Ig-cells (the dark zone of the germinative centre) and do not immunostain plasma cells. Thus, these mAbs recognize a surface marker which is not an Ig.     

Production and characterization of monoclonal antibodies against porcine interleukin-4

Interleukin 4 (IL-4) is an important regulatory cytokine produced by activated T lymphocytes and mast cells, and regulates the growth and differentiation of cells such as B and T lymphocytes. In the present study, recombinant thioredoxin (Trx)-porcine IL-4 (pIL-4) fusion protein was prepared by Escherichia coli (E. coli), and by using this protein as an immunogen, monoclonal antibodies (mAbs) against pIL-4 were produced to establish a basis for a research on immune responses in pigs. Six stable hybridoma cell lines were successfully established and specific binding of each mAb to recombinant pIL-4 produced by E. coli and insect cells infected with recombinant baculovirus was shown by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Isotype analyses of these mAbs revealed that the subclass of 5 out of 6 mAbs was IgG1 and the rest was IgG2b. Further, assessment of their epitopes by competition binding assay indicated that the mAbs obtained in this study bound to 4 different epitopes. The recombinant proteins and mAbs produced in this study will be useful tools for the assessment of porcine immune system.     

Porcine IgE in the context of experimental food allergy: purification and isotype-specific antibodies

Measurement of allergen-specific immunoglobulin E (IgE) is a common practice in the investigation of allergy. It has not been possible to measure porcine IgE due to unavailability of anti-porcine IgE. This study was undertaken to purify and characterize porcine IgE from sera of allergic pigs, identify heterologous anti-IgE reactive with pig IgE and to use purified heavy (H) chain of porcine IgE to generate rabbit anti-IgE. A four-step process for the purification of porcine IgE is reported using ammonium sulphate precipitation, Protein G affinity chromatography, DEAE cellulose anion-exchange chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to obtain IgE H chain. The resultant IgE was evaluated for purity using SDS-PAGE and immunoreactivity was detected by Prausnitz-Küstner (PK) tests and passive cutaneous anaphylaxis with the allergen, crude peanut extract, used to induce experimental allergy. Cross-reactivity with anti-mouse and anti-human IgE antibodies were confirmed in western blot and enzyme-linked immunosorbent assays (ELISA). The H chain of IgE was excised from SDS-PAGE gels and used to develop rabbit anti-porcine IgE antisera. Antiserum obtained from rabbits immunized with porcine IgE, as well as heterologous murine and human-specific anti-IgE, induced reverse cutaneous anaphylaxis in pig skin and detected allergen-specific IgE in ELISA but did not react with IgG H chain in western blots. These results confirm allergy-associated bioactivity of porcine IgE and describe both homologous and heterologous anti-pig IgE suitable for use in allergen-specific and other assays. This will enhance utility of pig allergy models and provide an additional measure of type-2 immune response in pigs.     

分泌抗犬细小病毒单克隆抗体杂交瘤细胞株的建立

为制备犬细小病毒(CPV)单克隆抗体,用F81细胞繁殖CPV,病毒液经二乙烯亚胺(BEI)灭活、纯化后免疫BALB/c小鼠,将小鼠脾细胞与SP2/0骨髓瘤细胞进行融合,经筛选获得2株分泌抗CPV单克隆抗体的杂交瘤细胞株,命名为F1和B6.F1和B6分泌的单克隆抗体Ig亚类均为IgG1;以F1和B6制备的腹水,经检测,间接ELISA效价均为1×105,免疫荧光效价分别为1:3200和1∶6400;F1和B6培养上清的抗体中和效价分别为1∶1024和1∶2048,血凝抑制效价分别为1∶2048和1∶4096;免疫印迹(Western-blotting)和抗原表位分析结果显示,F1和B6分泌的单克隆抗体可能分别针对CPV的空间表位和线性表位.2株分泌抗CPV单克隆抗体的杂交瘤细胞株的建立可进一步用于开发CPV特异性诊断制剂和治疗制剂.     

Production and characterization of monoclonal bovine immunoglobulins G1, G2 and M from bovine × murine hybridomas

Three bovine × murine hybridomas, which secrete bovine IgG₁, IgG₂ and IgM, respectively, were derived by the fusion of normal bovine B lymphocytes to the murine cell line SP-2/0. The hybridomas were initially selected by testing the culture supernatant of individual clones with rabbit anti-bovine light chain antibody. The bovine nature of Ig secreted by the bovine × murine hybridomas was confirmed by the ability of bovine serum but not murine serum to inhibit specific adsorption by murine anti-bovine Ig coupled to Sepharose 4B and the inability of sheep anti-mouse Ig (all isotypes) to bind biosynthetically labelled Ig from the respective bovine × murine hybridomas or to react with bovine × murine hybridoma Ig in Ouchterlony analysis. The isotype of the bovine Ig produced by each hybridoma was determined by cRIA using bovine Ig isotype-specific murine mcab, Ouchterlony analysis with guinea pig anti-bovine Ig isotypes and molecular weight analysis of unreduced and reduced affinity purified Ig from the respective bovine × murine hybridomas. Even though the hybridomas in this study showed chromosome loss, they have continued to secrete bovine Ig in culture for over 16 months, indicating that it is possible to isolate and stabilize interspecific hybridomas retaining the chromosome, or at least the genes, encoding an Ig.These hybridomas will provide monoclonal bovine Ig for; 1) serological standards, 2) production of polyclonal and monoclonal antisera to bovine Ig isotypes, 3) sequencing studies, 4) serological and structural studies of bovine Ig classes and subclasses, and messenger RNA for the production of cDNA probes for the cloning of bovine Ig genes and for determining the organization of Ig genes in the bovine genome.     

Antigenic subtyping and epitopes' competition analysis of porcine circovirus type 2 using monoclonal antibodies

Porcine circovirus type 2 (PCV2) strains have been classified into two major genotypes (PCV2a and PCV2b) and 8 genetic clusters: PCV2b-1A to PCV2b-1C and PCV2a-2A to PCV2a-2E. To date, no studies have been performed to antigenically subtype PCV2 strains enclosing eight PCV2 clusters. The present study aimed to antigenically subtype PCV2 and perform epitopes' competition analysis using monoclonal antibodies (mAbs). Fourteen PCV2 strains representative for eight clusters were tested with 20 mAbs (fifteen of them were generated against PCV2a strain Stoon-1010 and 5 of them against PCV2b strain 1147) in immunoperoxidase monolayer assays. Four mAbs reacted to all 14 PCV2 strains and one mAb reacted with all strains except for a PCV2a-2C strain. One mAb reacted with all PCV2a strains, except for a PCV2a-2C strain and one mAb reacted with all PCV2b strains, except for a PCV2b-1C strain. Nine mAbs reacted with the strains of PCV2b-1A/1B, PCV2a-2A and PCV2a-2E. Three mAbs only reacted with the strains of PCV2a-2A and PCV2a-2E. One mAb reacted specifically with the strains of PCV2b-1A/1B. This suggests that discrete antigenic differences exist between different PCV2 genetic clusters and that these clusters can be discriminated by the use of a panel of universal and cluster-specific mAbs. Six mAbs were selected for cross-competition analysis by a competitive ELISA using PCV2 strain Stoon-1010. Six overlapping epitopes were identified on the PCV2 capsid protein. The universal mAbs recognized larger epitopes than the cluster-specific mAbs. These findings are helpful in the development of diagnostic tests and new generation vaccines against PCV2.     

Characterization of stage-specific and cross-reactive antigens from Eimeria acervulina by chicken monoclonal antibodies

The characterization of five chicken monoclonal antibodies (mAbs) that were developed against apical complex antigens of Eimeria acervulina sporozoites is realized and the mAbs reactivity to merozoites belonging to this species is tested. Using immuno-fluorescence assay (IFA), one mAb (HE-4) that recognized apical antigens common to sporozoites of E. acervulina and E. brunetti bound antigens localized on the apical tip of merozoites from all stages of development examined. The mAb 8E-1, reactive with antigens found on the apical tip of all chicken Eimeria sporozoites, also showed binding to antigens common to merozoites from all generations. Another mAb, 8C-3, which identified an antigen shared by sporozoites apical tip and sporocysts wall of E. acervulina reacted very weak and inconstantly with the merozoites from all generations whereas the mAbs 5D-11 and 8D-2 that recognized antigens shared by the sporozoites of E. acervulina and E. maxima (mAb 5D-11) and E. acervulina and E. brunetti (mAb 8D-2) did not react with the merozoites from any generation. Collectively, these results showed that the invasive stages of chicken Eimeria share cross reactive apical complex antigens which are inter-species and inter-generation-specific that might be components of a potential recombinant vaccine.     

Screening of anti-human leukocyte monoclonal antibodies for reactivity with equine leukocytes

Three hundred and seventy-nine monoclonal antibodies (mAbs) against various human CD molecules supplied to the HLDA8 animal homologues section (including four isotype controls) were analysed for cross-reactivity with equine leukocytes. First, flow cytometric identification of positively reacting mAbs was performed in one laboratory. Thereafter, a second round of flow cytometric evaluation was performed, involving three laboratories participating in the study. The first test-round indicated 17 mAbs as potentially positive. After the second round of flow cytometric analysis, 14 mAbs remained (directed against CD2, CD11a, CD18, CD44, CD45, CD49d, CD91, CD163 and CD172) where cross-reactivity was anticipated based on similarities between the human and equine staining pattern. Additionally, there was 1 mAb with weak likely positive reactivity, 12 mAbs with positive staining, which likely do not reflect valuable data, 5 mAbs with clear alternate expression pattern from that expected from humans, 5 mAbs with a questionable staining pattern itself, i.e. that was variable between the three labs, 32 mAbs with weak-positive expression and alternate staining pattern, and 279 negative mAbs (including the four isotype controls) were detected. In 31 cases, more appropriate target cells, such as thymocytes or stem cells, were not available for the screening. The results underline the value of this "cross-reactivity" approach for equine immunology. However, as only a few mAbs against leukocyte surface antigens reacted positively (approximately 4% of the mAbs submitted), the analysis of further anti-human mAbs and directed efforts to develop species-specific anti-CD mAb are still required.