Detection of chromogranin A in the adrenal gland extracts of different animal species by an enzyme-linked immunosorbent assay using Thomsen-Friedenreich antigen-specific Amaranthus caudatus lectin

The reactivity of different lectins with crude chromogranin A (CgA) obtained from different animals, namely, cow, horse, dog, pig, and dolphin, was examined to identify lectin(s) that would be useful as coating reagent(s) in a sandwich enzyme-linked immunosorbent assay (ELISA). Of the different lectins studied, the Amaranthus caudatus lectin (ACA), which is specific for the Thomsen-Friedenreich (T)-antigen (Galβ1-3GalNAc), was found to react with the CgA from different animals by western blotting. Purified rabbit anti-bovine CgA antibody was also found to cross-react with the crude CgA preparations. On the basis of these findings, a sandwich ELISA was developed with ACA as the coating reagent and anti-bovine CgA antibody as the probing antibody. Using this method, concentration-dependent curves ranging from 0.003 μg/mL to 25 μg/mL and from 0.02 μg/mL to 25 μg/mL were obtained for bovine CgA and canine CgA, respectively. Similarly, concentration-dependent curves were obtained for the equine, swine, and dolphin crude CgA extracts. Thus, ACA is concluded to be a valuable reagent for CgA detection in crude extracts from different animal species, and for CgA isolation/purification.     

Measurement of plasma chromogranin A concentrations for assessment of stress responses in dogs with insulin-induced hypoglycemia

OBJECTIVE: To determine whether cross-reactivity exists between canine chromogranin A (CgA) and anti-human CgA antibody and investigate the usefulness of plasma CgA concentration measurements as an index of acute stress responses in dogs. ANIMALS: 12 healthy Beagles. PROCEDURE: Canine CgA was extracted and purified from canine adrenal glands of cadaver dogs for studying cross-reactivity with anti-human CgA antibody. Western blotting with anti-human CgA antibody was performed. Blood samples were collected from dogs at 0, 10, 20, 30, 40, 60, 120, and 180 minutes after IV administration of saline (0.9% NaCl) solution or insulin. Canine plasma CgA concentrations were determined by use of a CgA ELISA kit with rabbit antiserum against the carboxy-terminal fragment of human CgA. Plasma cortisol and catecholamine (ie, norepinephrine and epinephrine) concentrations were measured by use of an ELISA and a high-performance liquid chromatography method, respectively. RESULTS: Purified canine CgA was specifically detected by use of western blot analysis and an ELISA with anti-human CgA antibody. An increase in plasma CgA concentrations was observed in insulin-induced hypoglycemic dogs. Changes in plasma CgA concentration were correlated with changes in plasma cortisol or catecholamine concentrations of hypoglycemic dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Use of the CgA ELISA kit for determination of human plasma CgA concentrations is applicable to the measurement of canine plasma CgA concentrations. Canine plasma CgA concentrations, along with measurements of plasma cortisol and catecholamine concentrations, correctly reflect insulin-induced hypoglycemic stressed conditions in dogs. Measurement of canine plasma CgA concentrations may provide a useful index for evaluation of an acute stress response.     

Comparative evaluation of indirect and sandwich ELISA for the detection of antibodies to bovine respiratory syncytial virus (BRSV) in dairy cattle

Seroprevalence of bovine respiratory syncytial virus (BRSV) infection in both exotic and crossbred cattle were described. A baculovirus expressed recombinant purified nucleocapsid (N) protein was used in indirect and sandwich ELISA for screening of 499 bovine sera samples from all over the state for the presence of BRSV antibodies. The seroprevalence rate of BRSV was found to be 46.09% through indirect ELISA while it would found to be 65.33% by sandwich ELISA. The result also indicated that exotic breeds were more susceptible to BRSV infection compared to crossbred cattle. A comprehensive analysis on susceptibility to BRSV as regards to various factors like age and sex was also summarized.     

Anti-insulin antibodies in dogs with naturally occurring diabetes mellitus

The presence of anti-insulin antibodies was determined by ELISA in serum samples from 30 diabetic dogs receiving bovine insulin therapy and 30 normoglycaemic dogs. Twenty of the diabetic dogs had significant reactivity to both bovine (heterologous) and porcine (homologous) insulin compared to control dogs. In contrast there was no significant difference between the two populations in reactivity to canine distemper virus (CDV) or canine thyroglobulin. The high degree of correlation between anti-bovine insulin and anti-porcine insulin antibodies suggested cross-reactivity which was confirmed by performing a competition ELISA, with antibody binding to bovine insulin inhibited by pre-incubating serum with porcine insulin. The insulin B-chain, rather than the A-chain was the most reactive component of the insulin molecule although in some cases, diabetics with antibody reactivity to whole insulin protein showed minimal reactivity to the individual subunits. The data suggest that treatment of diabetic dogs with bovine insulin can lead to anti-insulin antibody production. These antibodies cross-react with homologous insulin and recognise conformational as well as linear epitopes.     

Detection and characterization of leptospiral antigens using a biotin/avidin double-antibody sandwich enzyme-linked immunosorbent assay and immunoblot.

A biotin/avidin double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of antigens of Leptospira interrogans serovars in experimentally inoculated bovine urine samples was evaluated. Immunoglobulin G (IgG) from rabbits immunized with L. interrogans serovar hardjo type hardjobovis sonicated, whole cell, and formalinized-heated antigen preparations were purified by a protein A-superose column coupled to fast protein liquid chromatography, and evaluated for species specificity in the ELISA. The ELISA using each specific IgG detected as few as 10(4) leptospires of the homologous serovar hardjo diluted in phosphate-buffered saline solution with Tween 20 (PBSS-Tween 20). On immunoblot analysis of proteinase-K-digested whole cell leptospiral preparations, each IgG revealed the presence of bands specific to serovar hardjo, suggesting the presence of serovar-specific epitopes on the lipopolysaccharide molecules. The minimum number of cells of heterologous serovars pomona, grippotyphosa, bratislava, icterohaemorrhagiae and copenhageni detected by each ELISA was greater, ranging from 10(6) to 10(7). The common antigenic determinants observed on immunoblot analysis were different for each specific IgG, except for a major cross-reacting, possibly flagellar, protein doublet at approximately 36-36.5 kDa. Leptospires were equally well detected by the ELISA in both bovine urine and PBSS-Tween 20.     

Seminal plasma lactoferrin but not transferrin reflects gonadal function in dogs

Lactoferrin purified from canine seminal plasma by a three-step chromatography procedure had a molecular mass of 75.2 kDa and cross-reacted with antiserum to equine seminal plasma lactoferrin. Seminal plasma lactoferrin concentrations were determined by a competitive enzyme-linked immunosorbent assay (ELISA) by using rabbit anti-equine lactoferrin antibody and alkaline phosphatase-labeled goat anti-rabbit IgG antibody in 14 normal dogs and found to range from 12 to 197 micro g/ml, with a mean value of 77 +/- 59 micro g/ml (the mean +/- SD). Seminal plasma transferrin concentrations were determined by a sandwich ELISA with goat antibody to canine serum transferrin and alkaline phosphatase-conjugated goat anti-canine transferrin antibody and found to range from 0.32 to 12.6 micro g/m l, with a mean value of 2.44 +/- 3.25 micro g/m l. The lactoferrin concentration significantly correlated with the sperm concentration (r=0.7025, P<0.01), but there was no significant correlation between the seminal plasma transferrin concentration and sperm density. These results indicate that seminal plasma lactoferrin, but not transferrin, reflects gonadal function.     

ELISA法检测犬腹泻粪样中的犬冠状病毒

用ＦＥ细胞增殖犬冠状病毒（ＣＣＶ）参考株，分别免疫家兔和ＢＡＬＢ／ｃ小鼠制备ＣＣＶ多抗和单抗，建立了夹心ＥＬＩＳＡ及Ｄｏｔ－ＥＬＩＳＡ诊断方法。在检测的８４例犬腹泻粪样中，多抗、单抗夹心法显示ＣＣＶ阳性１６例，Ｄｏｔ－ＥＬＩＳＡ阳性１３例，后１３例包括在前１６例中。从８４例腹泻犬粪样中随机取３８例作ＣＣＶ、犬细小病毒（ＣＰＶ）双项检测，ＣＣＶ阳性１６例，ＣＰＶ阳性６例，ＣＣＶ、ＣＰＶ混合感染４例。结果显示，在南京地区流行的犬腹泻中，ＣＣＶ感染比例有超过ＣＰＶ的趋势。     

牛IRF7蛋白的原核表达及多克隆抗体的制备

本研究旨在通过原核表达系统表达牛干扰素调节因子7（interferon regulatory factor 7,IRF7）蛋白,并对其进行纯化,进而制备高纯度的牛IRF7兔多克隆抗体。使用生物信息学软件预测并分析牛IRF7潜在的生物学功能,参考GenBank已公布的牛IRF7基因序列（登录号：NM_001105040.1）设计引物,利用PCR技术从牛病毒性腹泻病毒（Bovine viral diarrhea virus,BVDV）感染的MDBK细胞中扩增牛IRF7基因,连接至pMD19-T克隆载体,提取质粒连接至原核表达载体pET-28a （+）,转化大肠杆菌DH5α感受态细胞,构建重组质粒pET-28a-IRF7。将重组质粒pET-28a-IRF7转化大肠杆菌BL21（DE3）感受态细胞,经IPTG诱导后,通过SDS-PAGE进行表达产物的分析与鉴定。通过Western blotting鉴定多克隆抗体的特异性,间接ELISA测定兔抗牛IRF7多克隆抗体效价,经BVDV感染细胞后,实时荧光定量PCR检测抗病毒因子IRF7的表达。生物信息学分析发现,IRF7蛋白不存在跨膜结构域,无信号肽,存在较多磷酸化位点,二级结构主要以无规则卷曲为主,与三级结构的三维模型一致,说明该蛋白可能存在很好的抗原潜力。PCR成功扩增出大小为1 497 bp的IRF7基因片段,成功表达牛IRF7蛋白,分子质量约为60 ku,间接ELISA测得兔抗牛IRF7多克隆抗体效价为1∶128 000,并可与牛IRF7蛋白发生免疫反应,感染BVDV毒株的MDBK细胞中IRF7表达量下降;BVDV感染过表达IRF7后的细胞发现,IRF7能显著促进干扰素-β（IFN-β）的表达（P<0.05）。综上,本研究成功表达纯化了牛IRF7蛋白,并制备兔抗牛IRF7多克隆抗体,为阐明牛IRF7在先天免疫抗病毒应答的分子机制提供了材料。     

Quantitation of canine distemper virus and antibodies by enzyme-linked immunosorbent assays using protein A and monoclonal antibody capture

Monoclonal antibodies produced from 19 cloned hybridomas were selected for this study. Specific canine distemper virus (CDV) antibodies in medium from cloned hybridomas were detected by direct enzyme-linked immunosorbent assays (ELISA) and by indirect immunofluorescence. Three different sandwich ELISA systems were developed either to detect CDV in cell cultures and clinical specimens or to detect specific antibody in canine sera. Protein A and monoclonal antibodies attached in sequence to a solid phase constituted the capture system in the assays. Viral antigens were detected by sandwiching extracts of clinical specimens (or infected cell cultures), monoclonal antibody, and peroxidase-labeled protein A in sequence onto the capture layer. In 1 procedure, biotin-labeled antibody and peroxidase-labeled avidin were used as the last 2 layers in the assay. The CDV antibodies in dog sera were quantitated in a similar manner, but the sequential sandwiching levels consisted of partially purified CDV, serum specimen, and peroxidase-labeled protein A, respectively. The procedures were specific and highly sensitive.     

Expression of Recombinant bovine L-selectin in Escherichia coli and insect cells.

Bovine L-selectin was expressed in bacteria using pGEX vector and in insect cells infected with recombinant baculovirus in order to obtain recombinant protein for preparation of specific antiserum and its functional studies. In bacterial expression, L-selectin fusion protein with glutathione S-transferase was detected in the insoluble fraction with the expected molecular weight of 60 kDa by SDS-PAGE and reacted with anti-bovine CD62L monoclonal antibody in immunoblot analysis. In insect cells infected with recombinant baculovirus, a band corresponding to L-selectin was not observed in SDS-PAGE with protein staining, but they apparently reacted with anti-bovine CD62L monoclonal antibody in immunoblot analysis. Furthermore, the indirect immunofluorescence test revealed that bovine L-selectin was efficiently expressed on the surface of Sf9 cells infected with recombinant baculovirus, and flow cytometric analysis showed that the percentage of CD62L positive cells in bovine PBMC was about 66% and that most Sf9 cells infected with recombinant baculovirus had specific immunofluorescence.     

Detection of circulating 54 kDa antigen in sera of bovine calves experimentally infected with F. gigantica

The antibody response and circulating antigen levels in bovine calves, infected experimentally with Fasciola gigantica, were monitored using enzyme-linked immunoelectrotransfer blot (EITB) and sandwich ELISA, respectively. By EITB, the infected calves' sera recognized the polypeptides in the range of 54-58 kDa as early as 2 weeks post-infection. By 12th week post-infection, the lower two polypeptides of 12 and 8 kDa had disappeared. In sandwich ELISA, the circulating 54 kDa and whole worm antigen of F. gigantica were detected in the sera samples of infected calves as early as 2 weeks post-infection and persisted until the end of experiment (26th week PI). The 54 kDa antigen of F. gigantica appears to be specific and possesses promising immunodiagnostic potential for early prepatent diagnosis of bovine fasciolosis.     

A blocking ELISA for the detection of specific antibodies to bovine respiratory syncytial virus

A blocking enzyme-linked immunosorbent assay (ELISA) has been adapted to detect specific antibodies in bovine sera to respiratory syncytial virus using a horseradish peroxidase-labeled monoclonal antibody to the fusion protein of the virus. This assay plus an indirect blocking ELISA and indirect ELISA were used to detect antibodies to the bovine respiratory syncytial virus (BRSV) in 159 field-origin bovine sera. Results of these assays were compared with serum antibody titers measured by the serum neutralization (SN) test. Over a 56-day period, the mean neutralization titers and the mean delta absorbance values for the blocking ELISA, on the same sera, showed similar declines. However, the calculated correlation coefficients between mean SN titer and mean absorbance value for the blocking ELISA of the individual sera ranged from -0.2 to -0.5 depending on the source of sera. Similar values were obtained whether using crude or purified viral antigen in the assays. Corresponding calculated correlation coefficients were generally higher for the indirect blocking ELISA or indirect ELISA than for the blocking ELISA. The blocking ELISA was between 70 and 64% as sensitive as the serum neutralization test with a specificity of 100 or 90% using the crude and purified viral antigen, respectively. The indirect blocking ELISA and indirect ELISA had similar calculated sensitivities and specificities. The blocking ELISA was faster to run than either of the other ELISA's or the neutralization test. Further, nonspecific background absorbance was obviated because the blocking ELISA detects antibodies to 1 specific viral protein, the fusion protein. These studies suggest that the blocking ELISA should be useful as a serological test for BRSV antibodies.     

牛白血病病毒GP51蛋白在大肠杆菌中表达及胶体金免疫层析试纸条检测方法的建立

为了建立一种快速诊断牛白血病(BLV)的检测方法,本研究从BLV中扩增出其囊膜糖蛋白gp51基因,经测序后克隆于表达载体pET32a(+)中,构建了重组表达质粒pET32a-gp51。将其转化受体菌BL21,用IPTG诱导后表达出约42ku的目的蛋白,经western blot检测表明目的蛋白具良好的反应原性。以胶体金标记的羊抗牛IgG(Fc)抗体作为标记抗体,将纯化的His-gp51重组蛋白和羊抗牛IgG分别标记于硝酸纤维素膜上作为检测线和质控线,各部件按序装配形成快速诊断试纸条。对22份样本分别用试纸条和ELISA试验进行检测,2种方法的阳性符合率为88.9%(8/9),表明本研究建立的胶体金免疫层析方法简便、快捷,具有较好的特异性和一定的敏感性,适宜基层初筛诊断和现场应用。     

The production and characterization of anti-bovine CD14 monoclonal antibodies

To characterize further the chemical and biological properties of bovine soluble (bos) CD14, a panel of ten murine monoclonal antibodies (mAb) reactive with recombinant (r) bosCD14 were produced. A sandwich ELISA, using murine mAb and rabbit polyclonal antibodies reactive with rbosCD14 was developed. All the mAb were reactive by ELISA with baculovirus-derived rbosCD14 and they recognized rbosCD14 (40 kDa) by western blot analysis. The mAb also identified by western blot sCD14 (53 and 58 kDa) in milk and blood and sCD14 (47 kDa) in a lysate of macrophages obtained from involuted bovine mammary gland secretions. Analysis by ELISA of whey samples after intramammary injection of lipopolysaccharide (LPS) (10 micro g) revealed increased sCD14 levels between 8 to 48 h after injection. Flow cytometric analysis showed that the mAb bound to macrophages isolated from involuted mammary gland secretions and mouse macrophages but not to swine or horse monocytes. Addition of anti-rbosCD14 mAb to monocytes stimulated with LPS reduced in vitro production of TNF-alpha. The anti-rbosCD14 antibodies generated in this study will be useful in studying CD14, an accessory molecule that contributes to host innate recognition of bacterial cell wall components in mammary secretions produced during mastitis.     

Purification and Characterization of Liver Ferritins from Different Animal Species

The ferritins were purified from liver homogenates of buffalo, camel, cattle, sheep and shark by thermal denaturation, ammonium sulphate fractionation, Sephacryl S-300 gel filtration and DEAE-blue gel affinity chromatography. The yield and iron content of affinity-purified liver ferritins ranged from 0.008 to 0.052 mg/g and 3.17% to 11.4% respectively. As they are glycoproteins, the ferritins contained variable amounts of neutral carbohydrates. Except for shark ferritin, the ferritins all exhibited immunological cross-reactivity with anti-buffalo liver ferritin and anti-horse spleen ferritin by immunodiffusion and immunoelectrophoresis. Gel electrophoresis, gel filtration and ultracentrifugal analysis indicated the presence of a monomeric ferritin in all cases. SDS-gel electrophoresis of shark ferritin gave a protein band of 18 kDa. Ovine, buffalo and bovine ferritin comprised two protein subunits, the H (20 and 21 kDa) and the L types (18 and 19 kDa). Oligomeric ferritin subunits with molecular weights of 27, 37 and 55 kDa were also found for bovine and buffalo ferritin. SDS-PAGE of camel ferritin revealed a complex pattern with four prominent bands of 61, 51, 44 and 39 kDa. Two fast-migrating components of 15 and 16 kDa were also found in the purified liver ferritins, including reference preparations. The PO₄ ^3–/Fe ratios of purified shark (0.10) and bovine ferritin (0.12) were similar to that of standard equine spleen ferritin (0.11). However, the ratio was higher in ovine (0.17), camel (0.22) and bovine (0.26) ferritins. The amino acid compositions, molecular weights and sedimentation coefficients of the different liver ferritins were similar.     

Immunohistochemical demonstration of chromogranin A in endocrine organs of the rat and horse by use of region-specific antibodies

Chromogranin A (CgA) is an acidic glycoprotein that is co-stored with hormones or neurotransmitters in granular components of endocrine cells and neurons, and released together with them in response to adequate stimulation. In addition to acting as a packaging protein, CgA functions as a precursor molecule that yields several bioactive peptides by proteolytic cleavage. The purpose of this study is to elucidate how different the processing of CgA is among endocrine tissues by immunostaining using multiple region-specific antisera, and to evaluate the availability of region-specific antisera. When various endocrine organs of rats were immunostained with four region-specific antisera against rat CgA (CgA 1-28, 94-130, 296-314, and 359-389), all amine/peptide-secreting endocrine tissues except the pineal body were stained positively. The adrenal medulla and gastric endocrine cells were equally intensely immunoreactive to all four antisera, while the other endocrine tissues, represented by pancreatic islets, showed different staining patterns depending on the antiserum. These results suggest that the processing of CgA differs from tissue to tissue. An antiserum against horse CgA 335-365, corresponding to rat CgA 359-389 which shows the highest concentration in the plasma and urine of the rat, again stained all endocrine tissues of the horse except the pineal body. Therefore, the anti-horse CgA 335-365 serum is useful for immunohistochemical survey of horse CgA, and may make possible the establishment of a CgA assay system for the measurement of CgA in the plasma, urine and saliva.     

Development of protein A-gold immunoelectron microscopy for detection of bovine coronavirus in calves: comparison with ELISA and direct immunofluorescence of nasal epithelial cells

A protein A-colloidal gold immunoelectron microscopy (PAG-IEM) technique was developed for the detection of bovine coronavirus (BCV) in the feces and nasal secretions of infected calves. Feces or nasal swab fluids were incubated sequentially with hyperimmune bovine anti-bovine coronavirus serum and protein A-gold, negatively stained, applied to formvar-coated copper grids and viewed using an electron microscope. The PAG-IEM method specifically identified BCV particles and possible subviral particles in feces and nasal-swab fluids from infected calves. The PAG-IEM method did not label other enveloped enteric viruses or morphologically similar fringed particles commonly found in feces. Detection of BCV using PAG-IEM was compared with ELISA and direct immunofluorescence (IF) of nasal epithelial cells by monitoring fecal and respiratory tract shedding of BCV from two experimentally infected and two naturally infected calves from birth to 3 weeks of age. PAG-IEM and ELISA detected shedding of BCV in fecal (4/4 animals) and nasal (3/4 animals) samples for an average of 5.25 days each. The observed agreement of BCV detection by PAG-IEM and ELISA was 85%. PAG-IEM may be a more sensitive immunoassay for the detection of BCV in diagnostic specimens from infected neonatal calves than ELISA. BCV infection of nasal epithelial cells was detected by immunofluorescence in 4/4 calves, persisted for the duration of the study in 2/4 calves and was sporadic in the other two animals. The observed agreement of BCV detection by PAG-IEM and IF was 57%.     

Evaluation of ELISA based on the conserved and functional middle region of nucleocapsid protein to detect distemper infection in dogs

A 287 bp fragment from the middle region of the nucleocapsid protein of canine distemper virus (CDV) was amplified from the conjunctival samples of distemper-infected dogs and was cloned into pRSET B vector. The recombinant protein was expressed as a 16-kDa-fusion protein with histidine tag in E. coli. Sera of distemper-infected and vaccinated dogs contained IgG antibodies against the purified recombinant protein as observed by enzyme linked immunosorbent assays (ELISA) and showed a strong correlation (r = 0.882, p < 0.0001 at 95% CI) and good agreement (kappa = 0.718) with the conventional tissue culture viral antigen based ELISA. Further, the results of recombinant protein based ELISA and Western blotting with the sera from the infected and vaccinated dogs correlated well (kappa = 0.8226). These findings recommend the use of the recombinant protein in the serodiagnosis of canine distemper virus infection in dogs.     

Serological evidence of an Australian bovine lentivirus.

Recombinant 26 kDa capsid (CA) proteins of bovine lentiviruses, bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV), were expressed in Escherichia coli and utilised as antigens for an enzyme-linked immunosorbent assay (ELISA) and a western immunoblot (WIB) procedure for the detection of antibody in dairy cattle in Western Australia. A total of 690 serum samples, 30 from each of 23 farms, were tested by ELISA with a JDV CA protein antigen, and antibody was detected in 3.8% (p<0.05) of the sera. Nine sera from each farm were also tested by WIB with JDV CA protein antigens and antibody was detected in 15.9% of these samples. All ELISA-positive results were also WIB-positive, and all sera antibody-positive by WIB with JDV CA protein antigens were also antibody-positive by the WIB using recombinant BIV CA antigens. This study showed that recombinant protein antigens can be used for serological tests to detect bovine lentivirus infection in Australia.     

Characterisation and quantitation of pilus antigens of Moraxella bovis by ELISA

Hyperimmune serums raised in rabbits to purified pili from 9 Australian and 2 American strains of Moraxella bovis from infectious bovine keratoconjunctivitis (IBK) affected herds were used to study the degree of binding between combinations of antigen and antiserum in a conventional enzyme linked immunosorbent assay (ELISA). With the aid of appropriate absorption tests major antigenic differences among pili were found permitting 6 distinct serogroups to be recognised. Further, production of specific antiserums to representative strains of each serogroup in goats facilitated the development of a double antibody sandwich ELISA which could be used to quantitate pilus expression of a given strain of M. bovis, or to differentiate pilus serogroups of 22 strains of M. bovis obtained from a total of 12 Australian herds. Most isolates were found to belong to serogroups designated IV and V. One strain from the United States of America showed total homology with Australian serogroup IV while the other showed some cross-reactivity with serogroups V and VI.