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.     

小反刍兽疫病毒核蛋白单克隆抗体的制备与初步应用

针对小反刍兽疫病毒核蛋白制备特异性的单克隆抗体,并对其进行生物学特性鉴定和初步应用。以纯化的Bacmid-PPRV-N重组蛋白为抗原免疫BALB/c小鼠,取免疫小鼠的致敏脾细胞与SP2/0骨髓瘤细胞在PEG作用下融合,获得单克隆抗体,并通过染色体技术等方法研究其生物学特性,将其作为竞争单抗,Bacmid-PPRV-N重组蛋白作为检测抗原建立竞争ELISA检测方法。结果表明:经克隆和间接ELISA筛选,获得了2株能稳定分泌抗小反刍兽疫病毒N蛋白抗体的杂交瘤细胞株,分别命名为5B11和3H10-3B8。生物学特性鉴定试验表明:5B11和3H10-3B8抗体类型和亚类均为IgG2b;5B11单抗腹水的效价达1∶819 200,3H10-3B8达1∶12 800;血清学试验证明2株单抗均能与Bacmid-PPRV-N重组蛋白抗原结合,具有高度的特异性;相加ELISA试验结果显示,5B11和3H10-3B8 2株单克隆抗体分别识别N蛋白上不同的抗原位点;2株杂交瘤细胞的染色体均为99～104。应用建立的c-ELISA检测方法对222份血清样品进行PPRV抗体的检测,与参考试剂盒比较得到98.20%的符合率。本研究获得了2株能稳定分泌抗PPRV N蛋白单克隆抗体的杂交瘤细胞株,以单抗5B11作为竞争抗体建立了PPRV的c-ELISA检测方法。     

Competitive binding with putative Bo5 (CD5) cluster of monoclonal antibodies.

The relationship of seven monoclonal antibodies, putatively to the Bo5 (CD5) antigen, was tested. Five of the mAbs were confirmed to be directed against the Bo5 antigen. Three mAbs, CC29, BLT-1 and 8C11, effectively blocked binding to bovine PBM of mAb CC17, previously reported to be directed against this antigen. MAb 8-3F4 also blocked binding of mAb CC17, but less effectively than the others. MAbs IL-A67 and 79-5 did not inhibit binding of mAb CC17 because of antibody allelic specificity or technical reasons.     

Competitive enzyme-linked immunosorbent assay for heartwater using monoclonal antibodies to a Cowdria ruminantium-specific 32-kilodalton protein

Hybridomas producing monoclonal antibodies (mAb) to Cowdria ruminantium were raised. Four mAbs of the IgG isotype reacted in western blots with a 32-kilodalton Cowdria protein (Cr32), which had previously been shown to be conserved and immunodominant. A fifth mAb of the IgM isotype recognized a 40-kDa Cowdria protein. The latter mAb was negative in an indirect fluorescent antibody test (IFA), whereas the other four were positive. mAb No. 4F10B4 showed the strongest signal in western blots using three different stocks of Cowdria. Immuno-gold labeling of Cowdria organisms in vitro using 4F10B4 showed that Cr32 has surface-exposed antigenic determinants. Using mAb 4F10B4, a competitive ELISA was developed which detected specific Cowdria antibodies in goat, sheep and cattle sera. Antibodies in animal sera competed with binding of mAb 4F10B4 to a crude sonicated Cowdria antigen obtained from infected endothelial cell cultures. The competition ELISA (CELISA) detected antibodies in 55 out of 70 (79%) goats experimentally infected with one of eight different Cowdria stocks. Fourteen out of the 15 sera which were shown negative in the CELISA were also negative in the IFA. Nevertheless, all 15 sera recognized some epitopes of the immunodominant Cowdria-specific 32 kDa protein as judged from their reaction with this protein in western blots. Overall, there was 89% agreement between CELISA and IFA considering all 70 goat sera. Moreover, antibodies were detected in nine out of nine sheep infected with one of three different stocks of Cowdria and in sera from calves experimentally infected by two different strains of heartwater. There were no cross-reactions with Ehrlichia phagocytophila antibodies in goat sera, nor with Anaplasma marginale antibodies in bovine sera. Lack of cross-reactivity and detection of antibodies to eight geographically widely distributed stocks of Cowdria, makes the competition ELISA a promising test for use in heartwater endemic areas.     

Production and characterization of two serotype independent monoclonal antibodies against foot-and-mouth disease virus

Two foot-and-mouth disease virus (FMDV) monoclonal antibodies (mAbs) were produced from mice immunized with either FMDV serotype A, subunit (12S) or FMDV serotype O, whole virus (140S). Both mAbs (F1412SA and F21140SO) recognized all seven serotypes of FMDV in a double antibody sandwich (DAS) ELISA, suggesting that the binding epitopes of the two mAbs are conserved between serotypes. These mAbs are IgG1 isotype and contain kappa light chains. In order to define the mAb binding epitopes, the reactivity of these mAbs against trypsin-treated and denatured FMDV were examined using an indirect ELISA. The binding site of the mAb, F1412SA is trypsin sensitive and the epitope is linear. Both ELISA and Western blot results suggested that the polypeptide VP2 contributed to the immunodominant site. This mAb showed reactivity to VP2 peptide (DKKTEETTILEDRIL). The mAb, F21140SO, recognized an epitope which is trypsin resistant and discontinuous. This mAb binding to FMDV is dependent on conformational structures of intact viral (140S) or subunit (12S) particle, since it failed to recognize any viral protein in Western blot. This conformational and highly conserved epitope is the first identified epitope among all seven FMDV serotypes. Because the use of mAbs increases the specificity, accuracy and efficiency of diagnostic tests compared to polyclonal antisera, these two mAbs with different specificities are suitable for type-independent diagnosis of FMDV, such as DAS ELISA, or could be adapted to immuno-chromatographic or flow-through rapid test.     

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.     

Production and characterization of monoclonal antibodies detecting chicken interleukin-2 and the development of an antigen capture enzyme-linked immunosorbent assay

Eleven monoclonal antibodies (mAbs) which are specific for chicken interleukin-2 (chIL-2) were produced and characterized by enzyme-linked immunosorbent assay (ELISA), Western blotting and neutralizing assays. These mAbs were used to develop a mAb-based antigen capture ELISA specific for chicken IL-2 detection. Anti-IL-2 mAbs bound specifically to E. coli-derived rchIL-2 in ELISA and identified a 16 kDa IL-2 polypeptide band in Western blot. Several mAbs were shown to neutralize the biological activities of both rchIL-2 and native chicken IL-2 as measured by concanavalin A (ConA)-induced lymphocyte proliferation assay, IL-2 bioassay, and natural killer cell assay. Among the neutralizing mAbs, the mAb chIL-2/11 was most potent in neutralizing IL-2 activity. To develop a sensitive ELISA for the detection of chicken IL-2, an antigen capture ELISA was developed using the mAb chIL-2/16 as the antigen capture antibody and rabbit anti-IL-2 peptide antibody as the detection antibody. Using the mAb-based antigen capture ELISA, significant correlation between the level of IL-2 detected in bioassays and in ELISA was observed. These results showed that the mAb-based antigen capture ELISA is less time-consuming and more reliable compared to a conventional IL-2 bioassay for chicken IL-2. These neutralizing mAbs will facilitate basic immunobiological studies of the role of IL-2 in normal and disease states in chickens.     

应用改良阻断ELISA检测禽网状内皮组织增殖病血清抗体

应用禽网状内皮组织增殖病病毒纯化抗原和抗ＲＥＶ单克隆抗体建立了改良阻断ＥＬＩＳＡ用于鸡血清中ＲＥＶ抗体检测，并对北京地区鸡群中随机采样的３６份血清样本进行了检测，阳性率为５．６％。与间接ＥＬＳＩＡ的检测结果进行了统计学比较，两种方法的阳性率无显著差异。结果表明本试验所建立的改良阻断ＥＬＩＳＡ可以用于鸡群ＲＥＶ感染的血清学调查。     

非洲猪瘟病毒p54蛋白单克隆抗体制备及其抗原表位鉴定

旨在制备非洲猪瘟病毒(ASFV)p54蛋白的特异性单克隆抗体。本研究利用大肠杆菌表达系统表达p54蛋白,免疫BALB/c小鼠,取其脾细胞与SP2/0细胞进行细胞融合。利用纯化的p54蛋白作为包被抗原,采用间接ELISA方法筛选获得阳性杂交瘤细胞。经4次亚克隆后,取杂交瘤细胞上清进行单克隆抗体亚型鉴定,利用体内诱生法制备单克隆抗体并进行纯化。间接ELISA方法检测单克隆抗体的效价,利用交叉反应性试验、间接免疫荧光试验和蛋白印迹对所获单克隆抗体的特异性进行鉴定。根据预测的p54蛋白二级结构,采用逐步截短法分析鉴定单克隆抗体识别的抗原表位区域,并在p54的三级结构中进行标注。结果显示：成功筛选了6株分泌p54单克隆抗体的杂交瘤细胞,分别命名为28G12-1、31G7-1、31G7-2、35F10-1、35F10-2、38D3-1。其中28G12-1、31G7-1、31G7-2重链为IgG2a型,35F10-1、35F10-2、38D3-1重链为IgG1型;轻链均为κ链。单克隆抗体的最低效价为1∶25 600,与猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪流行性腹泻病毒、猪细小病毒、猪急性腹泻综...     

Production of antibodies against chicken interferon-gamma: demonstration of neutralizing activity and development of a quantitative ELISA

Four monoclonal antibodies (mAbs) specific for chicken interferon-gamma (ChIFN-gamma) were generated by gene gun immunization and were utilized to develop a mAb-based capture ELISA specific for ChIFN-gamma. Each mAb reacted specifically with both baculovirus and Escherichia coli-derived recombinant ChIFN-gamma in ELISA and Western Blot analysis or natural ChIFN-gamma in immunofluorescence experiments. As determined by competition ELISAs, mAbs 3D5, 4C6 and 3A3 recognized the same or adjacent epitopes on the ChIFN-gamma molecule, whereas mAb 1E12 recognized a distant epitope. Moreover, this latter mAb was able to highly neutralize the biological activities of both recombinant and natural ChIFN-gamma as measured by inhibition of viral replication and macrophage activation. To improve the detection of ChIFN-gamma, a capture ELISA was developed using mAb 1E12 as capture antibody and biotinylated mAb 4C6 as detection antibody. In addition to being more rapid and easier to perform than classical cell-mediated immunity tests, this ELISA has excellent sensitivity and improved specificity. The use of a specific rabbit polyclonal serum as revealing antibody further increased the sensitivity of the detection down to 0.5ng/ml of ChIFN-gamma. This ELISA would provide a sensitive tool to measure the in vitro release of ChIFN-gamma by T-cells in response to specific recall antigen.     

A monoclonal blocking ELISA detecting serum antibodies to Mycoplasma hyopneumoniae.

A monoclonal blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Mycoplasma hyopneumoniae in porcine serum has been developed. The monoclonal antibody (mAb) reacts with an M. hyopneumoniae specific epitope on a molecule of approximately 74 kDa. Only sera from M. hyopneumoniae infected pigs were able to block the binding of the mAb although antibodies from M. flocculare infected pigs also recognized a 74 kDa molecule. Sera from experimentally infected pigs as well as field samples were compared by the ELISA and by an indirect hemagglutination assay (IHA). In experimental pigs, the earliest detectable antibody response was found to be almost identical for both assays, but for some of the pigs the time of detection was significantly earlier by blocking ELISA than by IHA. In naturally infected herds more samples were found to be positive by ELISA than by IHA. Furthermore, the results indicate that sera from naturally M. flocculare infected pigs may give rise to cross-reactions in the IHA. The blocking ELISA appears to be a valuable and reproducible tool in the surveillance and serodiagnosis of M. hyopneumoniae infections in pigs.     

传染性法氏囊病病毒VP2单克隆抗体夹心ELISA检测试剂盒的研制

为研制传染性法氏囊病病毒（IBDV）快速检测试剂盒,用重组IBDV-VP2蛋白免疫BALB/c小鼠,制备免疫脾细胞,与SP2/0骨髓瘤细胞触合,获得3株稳定分泌抗VP2蛋白单克隆抗体（mAb）的杂交瘤细胞株,分别命名为1D11、2G8和2E5,抗体亚类分别为IgG1κ、IgG2bκ和IgG1κ。间接免疫荧光试验（IFA）证明,3株单抗均与VP2发生特异性反应。相加ELISA证明3株mAb识别VP2不同的抗原表位。在病毒中和试验中,1D11和2G8腹水对IBDV的中和效价分别为104和103,而2E5无中和活性。用亲和层析方法纯化1D11和2E5,分别作为包被抗体和标记抗体,建立了IBDV夹心ELISA检测方法,优化了试验条件,测定了其主要性能指标,对IBDV的最低检出量为102 TCID50/mL。用夹心ELISA、AGP和RT-PCR 3种方法同步检测8种试验样品,夹心ELISA与RT-PCR的检测结果一致,显著高于AGP方法。组装成的试剂盒,置于37℃保存7d、4℃保存6个月和-20℃保存24个月,其检测结果没有显著差异（P〉0.05）。     

Sandwich ELISA system for cartilage oligomeric matrix protein in equine synovial fluid and serum

Reasons for performing study: Measurement of cartilage oligomeric matrix protein (COMP) in serum has potential for diagnosis of equine osteoarthritis (OA), but clinical use is currently limited by the lack of specificity of an inhibition ELISA as well as by baseline increases due to exercise. Improved methods for ELISA with increased antigen specificity and sensitivity are therefore required for reliable measurement. Hypothesis: Measurement of the serum level of COMP by sandwich ELISA allows identification of horses with OA. Methods: New monoclonal antibodies (mAbs) were elicited against equine cartilage COMP, their epitopes were determined and a sandwich ELISA was developed. The concentrations of COMP in synovial fluid (SF; n = 100) and sera (n = 100) from OA cases were measured by sandwich ELISA as well as by inhibition ELISA and compared with concentrations in normal joints (n = 95) and horses (n = 50). Results: Immunoblots of enzymatically cleaved COMP showed that the new mAbs recognised different epitopes located on a 20 kDa fragment between K⁶³ and K²³⁸ of the EGF‐like repeats. Inhibition ELISA with any mAb detected significantly increased levels of COMP in OA SF compared with normal SF, whereas no significant difference was detected between serum levels of COMP in OA and normal horses. Conversely, sandwich ELISA with the combination of unlabelled 2A11 × biotinylated 11F10 mAbs detected a significant increase in COMP levels in both serum and SF from OA cases compared with levels in normal animals. Conclusions and potential relevance: Measurement of serum COMP with sandwich ELISA may be useful in identifying horses with OA.     

H5 antibody detection by blocking enzyme-linked immunosorbent assay using a monoclonal antibody

Many commercial enzyme-linked immunosorbent assays (ELISAs) are unable to differentiate antibody responses to different avian influenza virus (AIV) subtypes. Developing an ELISA for specifically detecting the H5 antibody is the purpose of this study. Four monoclonal antibodies (Mabs) were raised using A/duck/Yunlin/04 (H5N2). They were confirmed as being specific to H5. Two of these antibodies showed hemagglutination inhibition (HI) activity using the HI test. Using immunodot blot assays, three Mabs recognized both Eurasian and American H5, whereas the other Mab recognized only the tested Eurasian H5 virus. When testing denatured H5 antigen, one of the Mabs lost its antigen binding activity using Western blotting. For detecting the H5 humoral response in serum, one monoclonal antibody was purified and labeled with horseradish peroxidase to set up a blocking ELISA. Chicken sera that blocked H5 Mab binding by > 29% were considered H5 antibody positive. Inhibition percentages for sera from chickens infected with other AIV subtypes, H1 to H15, were < 29%. This blocking ELISA was used for 478 field chicken serum samples. The results showed that the sensitivity and specificity of this ELISA were 98.3% (232/236) and 95.9% (232/242), respectively. This blocking ELISA could be used specifically for detecting the H5 humoral responses in chickens.     

An international comparison of different laboratory tests for the diagnosis of bovine leukosis: suggestions for international standardization

A collaborative study was conducted to compare the detection limit of different laboratory tests for antibodies against bovine leukemia virus (BLV). Serum and milk samples were tested in agar gel immunodiffusion (AGID), different modifications of indirect ELISA, blocking ELISA and ELISA procedures using monoclonal antibodies to BLV gp51 or BLV p24. The detection limit of reference serum E4 diluted 2-fold in negative serum gave a median value of 1:16 in AGID, indirect ELISA, and monoclonal ELISA p24, 1:128 in monoclonal ELISA gp51, and 1:1024 in blocking ELISA. The detection limit of a 4% immunoglobulin preparation of E4 diluted in negative milk showed median values of 1:800 in indirect ELISA, 1:1000 in monoclonal ELISA, and 1:2400 in blocking ELISA. None of the ELISA procedures could detect all the positive individual milk samples diluted 1:50. The AGID test is the official reference test for detection of antibodies against BLV. Reference serum E4 diluted 1:10 in negative serum must be scored positive in the AGID test. It is suggested that an international reference serum standard be established rather than an official recommendation of a particular ELISA test.     

牛γ-干扰素ELISA检测方法的建立与初步应用

以纯化的rHis-BoIFN-γ制备兔多抗血清,辛酸—硫酸铵两步法对体内诱生的抗rBoIFN-γ单抗腹水进行纯化,用美洲商陆素（Pokeweed mitogen,PWM）刺激奶牛全血产生的分泌性天然BoIFN-γ筛选出与之反应最佳的单抗5E11。将单抗5E11以40μg/mL浓度包被,与1∶3 000倍稀释的兔抗rHis-BoIFN-γ多抗血清（13.8μg/mL）配对,以1∶6 000稀释的商品化酶标羊抗兔IgG为指示抗体,建立了BoIFN-γ抗原捕获ELISA检测方法,该方法可以检出2.56 U/100μL（30.5 pg/100μL）的rHis-BoIFN-γ、8U/100μL的rBac-BoIFN-γ和1U/100μL的分泌性天然BoIFN-γ。以商品化试剂盒作为平行对照,将获得的奶牛临床检测血浆样品使用BoIFN-γ抗原捕获ELISA法进行检测,结果显示两种方法检测符合率达到83.9%。本研究建立的BoIFN-γ抗原捕获ELISA检测方法可有效检测分泌性IFN-γ,为进一步开发BoIFN-γELISA检测试剂盒奠定了基础。     

非洲猪瘟病毒p54蛋白单克隆抗体的制备及阻断ELISA方法的建立

旨在建立检测非洲猪瘟病毒(ASFV)血清抗体的阻断ELISA方法.采用原核表达的ASFVp54蛋白作为包被抗原,并制备了针对p54蛋白的单克隆抗体,采用方阵滴定法确定了阻断ELISA方法的最佳反应条件,并对建立的方法进行了敏感性、特异性、重复性和符合性评价.结果显示,抗原最佳包被浓度为2.0 μg·mL-1,抗原包被温...     

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.     

鸡毒支原体烯醇化酶单克隆抗体研制及黏附阻断

通过对鸡毒支原体烯醇化酶的克隆和原核表达,利用表达产物免疫小鼠,制备单克隆抗体,以超声波裂解的MG全菌作为筛选抗原,结果获得了6株特异性单抗,分别命名为：1A4、2D7、2E4、2F4、384和3D8。经间接ELISA、免疫荧光试验及Western blot检测发现：1A4具有荧光效价和高滴度的ELISA效价,但无免疫印迹效价;而其他5株单抗,免疫印迹呈阳性,但无表面染色的活细胞免疫荧光。在所有6株单抗中,仅1A4单抗有MG细胞黏附抑制作用,可导致MG的黏附率降低34．40％,而且1A4与其他5种单抗混合后,则黏附率可下降57．69％。这些抗体的制备将为烯醇化酶生物学活性研究奠定基础。     

Analysis of various antigens in golden hamster testis by monoclonal antibodies.

A total of 38 hybridomas producing monoclonal antibodies (mAbs) was established by immunizing BALB/c mice with extracts of the golden hamster testis. Six mAbs stained the acrosome of developing spermatids by immunofluorescence. Two mAbs (1A11 and 4D8) reacted with spermatid components other than acrosome. The mAbs 1C9 and 4D3 recognized a 103 kilodalton (kDa) protein on immunoblots, and were reactive to spermatocytes and early spermatids, but not to late spermatids and spermatozoa. This finding suggests that the protein functions for meiosis or early spermiogenesis. Four mAbs (3G2, 2E5, 2G3, and 3F10) stained all stages of spermatogenic cells. The remaining 24 mAbs showed a positive reaction to the basement membrane of the seminiferous tubule. Two of them, 3D6 and 3E5, recognized approximately 150 kDa major proteins, indicating that the antigen is an extracellular matrix.