Evaluation of high-molecular weight adiponectin in horses

Objective-To characterize adiponectin protein complexes in lean and obese horses. Animals-26 lean horses and 18 obese horses. Procedures-Body condition score (BCS) and serum insulin activity were measured for each horse. Denaturing and native western blot analyses were used to evaluate adiponectin complexes in serum. A human ELISA kit was validated and used to quantify high-molecular weight (HMW) complexes. Correlations between variables were made, and HMW values were compared between groups. Results-Adiponectin was present as a multimer consisting of HMW (> 720-kDa), low-molecular weight (180-kDa), and trimeric (90-kDa) complexes in serum. All complexes were qualitatively reduced in obese horses versus lean horses, but the percentage of complexes < 250 kDa was higher in obese versus lean horses. High-molecular weight adiponectin concentration measured via ELISA was negatively correlated with serum insulin activity and BCS and was lower in obese horses (mean ± SD, 3.6 ± 3.9 μg/mL), compared with lean horses (8.0 ± 4.6 μg/mL). Conclusions and Clinical Relevance-HMW adiponectin is measurable via ELISA, and concentration is negatively correlated with BCS and serum insulin activity in horses. A greater understanding of the role of adiponectin in equine metabolism will provide insight into the pathophysiology of metabolic disease conditions.     

Canine adiponectin: cDNA structure, mRNA expression in adipose tissues and reduced plasma levels in obesity

Adiponectin is a protein synthesized and secreted by adipocytes. Decreased adiponectin is responsible for insulin resistance and atherosclerosis associated with human obesity. We obtained a cDNA clone corresponding to canine adiponectin, whose nucleotide and deduced amino acid sequences were highly identical to those of other species. Adiponectin mRNA was detected in adipose tissues, but not in other tissues, of dogs. When 22 adult beagles were given a high-energy diet for 14 weeks, they became obese, showing heavier body weights, higher plasma leptin concentrations, but lower plasma adiponectin concentrations. The adiponectin concentrations of plasma samples collected from 71 dogs visiting veterinary practices were negatively correlated to plasma leptin concentrations, being lower in obese than non-obese dogs. These results are compatible with those reported in other species, and suggest that adiponectin is an index of adiposity and a target molecule for studies on diseases associated with obesity in dogs.     

Development,validation, and pilot application of a semiquantitative Western blot analysis and an ELISA for bovine adiponectin

Adiponectin is an adipose tissue-derived glycoprotein circulating as highly abundant multimers. It regulates glucose metabolism and insulin sensitivity. In ruminants, valid data about serum concentrations and tissue-specific protein expression are lacking, and we, therefore, aimed to generate a polyclonal antibody against bovine adiponectin to apply it in immunodetection. The specificity of the purified anti-adiponectin antibody was established by Western blot analysis with the use of reducing and denaturing conditions applied to both the purified protein and the bovine serum samples. Besides bovine serum, the applicability of the antibody for immunodetection of adiponectin was confirmed for the supernatant fluid of in vitro–differentiated bovine adipocytes, for protein extracts from bovine adipose tissue, and also in a multispecies comparison: bands comparable in size with monomeric bovine adiponectin were obtained under denaturing conditions in serum of camel, horse, human, mouse, pig, roe deer, and sheep. In addition, when used in immunohistochemistry on bovine adipose tissue sections, a characteristic adipocyte-specific staining pattern was obtained with this antibody. The antibody was used for establishing a semiquantitative Western blot procedure and the development of an ELISA. Both methods were extensively validated and were first applied to characterize the serum adiponectin concentrations in multiparous dairy cows during the transition from pregnancy to lactation, that is, 3 wk before until 5 wk after calving. With both assays a time effect (P = 0.017, P = 0.026, respectively) with lowest values at the day of parturition was observed. We thus established 2 useful tools to validly assess bovine adiponectin at the protein level.     

酶联免疫吸附试剂盒与免疫金标快速检测试纸检测 PRRS 抗体的比较

应用美国IDEXX公司生产的PRRS抗体ELISA试剂盒和PRRS抗体免疫金标试纸同时检测12份猪血清，阳性率均为41．67％、(5／12)。二种试剂都具有微量、特异、准确的优点。前者需要有酶标仪等仪器，试验时间长，成本高，但能用准确的数字表达抗体水平；后者不需要特殊仪器设备，试验时间短，成本低，适用于基层兽医站、养猪场使用。     

Canine heterophilic antibodies as a source of false-positive B-type natriuretic peptide sandwich ELISA results

BACKGROUND: Interference by heterophilic antibodies is a well-known cause of false-positive sandwich ELISA results in human medicine. They are considered rarely in veterinary species and have not been characterized but could become important as newer, highly sensitive sandwich immunoassay technologies are developed. OBJECTIVES: The goals of this study were to use a B-type natriuretic peptide (BNP-32) sandwich ELISA to determine the effect of heterophilic antibodies on test performance; to characterize canine heterophilic antibodies; and to develop and test a method for heterophilic antibody removal. METHODS: A sandwich ELISA was developed using a mouse IgG(1)K monoclonal and a rabbit polyclonal antibody to two synthetic peptides of canine BNP-32. The effects on false-positive results of heterophilic antibody depletion and blocking by various techniques were compared. The titers of canine heterophilic antibodies were compared with various blood antigens from other species and the relative amount of canine IgG was compared with that of IgM heterophilic antibody. RESULTS: Heterophilic antibodies in dog plasma were shown to be capable of causing false-positive ELISA results. They reacted with blood proteins from a variety of animal species at relatively low titers and consisted of both IgG and IgM. Protein A agarose antibody precipitation, in conjunction with mouse IgG(1)K blocking antibody, was effective in eliminating false-positive sandwich ELISA results while retaining adequate test performance. CONCLUSIONS: Canine heterophilic antibodies can interfere with sandwich ELISA assays and cause false-positive test results. An effective technique for their removal that has a potentially broad application was developed, and allows measurement of canine blood constituents at low picomolar concentrations.     

Insulin regulates the expression of adiponectin and adiponectin receptors in porcine adipocytes

Adiponectin is an adipocyte-derived hormone that can improve insulin sensitivity. Its functions in regulating glucose utilization and fatty acid metabolism in mammals are mediated by two subtypes of adiponectin receptors (AdipoR1 and AdipoR2). This study was conducted to determine the effect of insulin on the expression of adiponectin and its receptors. We demonstrated that in the presence of 10 nM insulin, addition of 1 μM of insulin or rosiglitazone (a peroxisome proliferator-activated receptor γ (PPARγ) agonist) had no effect on the expression of adiponectin and AdipoR genes in differentiated porcine adipocytes. However, the addition of 1 μM insulin plus 1 μM rosiglitazone significantly increased the AdipoR2 mRNA in differentiated porcine adipocytes. Using the phosphatidylinositol 3-kinase inhibitor (PI3K inhibitor, LY 294002), we found that insulin inhibited the expression of AdipoR2 through the PI3K pathway and this inhibition was blocked by addition of rosiglitazone. When porcine adipocytes were cultured without insulin, supplementation with 10 nM insulin inhibited the expression of AdipoR2 and this inhibition effect was also blocked by addition of rosiglitazone. Therefore, these data suggest that a PPARγ agonist increases expression of AdipoR2 and that insulin inhibits the expression of AdipoR2 through the PI3K pathway.     

蛋白芯片在兽药残留检测中的应用

通过对猪和鸡组织样品中的克仑特罗、链霉素等兽药的检测,比较了蛋白芯片检测试剂盒、ELISA检测试剂盒和确证方法在兽药残留检测中的应用.结果表明:蛋白芯片检测试剂盒与ELISA试剂盒性能相当,与确证方法的结果有很好的一致性,且具有同时检测多种兽药,前处理方法简单、速度快等优点.     

Evaluation of serological tests for detecting tick-borne encephalitis virus (TBEV) antibodies in animals

Tick-borne encephalitis (TBE) in animals is not well understood yet. TBE virus (TBEV) serology in several host species could be valuable for epidemiological analyses in the field as well as for the detection of clinical cases. However, performance and suitability of the available test systems are not well assessed. Therefore, we evaluated two commercial TBEV-ELISA kits in a pilot study and compared them for their suitability in veterinary applications. For this purpose, we tested 163 field collected goat sera and evaluated the results by serum neutralization test (SNT) as "gold standard". Twenty-eight SNT positive sera (17.2%) were detected. The best suited ELISA kit was used for determination of a species-specific cutoff for horses, cattle, sheep, goats, pigs, mice, dogs, rabbits and monkeys with defined sera from animals without known or with improbable contact to TBEV. The level of non-specific ELISA results does not only differ between animal species but may also be influenced by the age of the tested animals. The number of sera which tested false positive by ELISA was higher in older than in young sheep. In order to obtain defined polyclonal sera as references, two dogs, cattle, goats, sheep, rabbits and pigs each, as well as one horse and 90 mice were immunized four times with a commercially available TBEV vaccine. In conclusion, our results demonstrated that commercial TBEV-ELISA kits are suitable for application in veterinary medicine for both, verification of clinical TBE cases and epidemiological screening. However, positive ELISA results should be verified by SNT. Only a very low number of false negative ELISA-results were found.     

Validation of a rapid solid-phase radioimmunoassay for canine, bovine, and equine insulin

A rapid and convenient commercial radioimmunoassay kit, developed for quantifying hormones in specimens from human beings, was validated for use in measuring insulin in serum of dogs, cattle, and horses. The procedure uses polypropylene assay tubes treated with rabbit anti-porcine insulin serum and porcine [125I]iodoinsulin. Specificity was proven by demonstrating that standard solutions of porcine insulin and serial dilutions of canine, bovine, and equine sera and pancreatic extracts inhibited binding of [125I]iodoinsulin to the antibody in a parallel manner. Gel-filtration chromatography of pancreatic extracts yielded a major peak of immunoreactive material that eluted identically with [125I]iodoinsulin. Immunoreactivity was not associated with fractions that contain larger and smaller molecular weight peptides (eg, proinsulin and C-peptide, respectively). Biological specificity of the assay was shown by demonstrating increased insulin in serum after injection of glucose into heifers and glucagon into dogs and horses. Purified insulin and insulin in pancreatic extracts could be quantitatively recovered from serum, thereby demonstrating accuracy of the assay. Interassay precision of 5 control specimens run in 20 consecutive assays ranged from 6.7% to 20.1% (coefficient of variation) and intra-assay precision of 6 control specimens each assayed 10 times ranged from 4.4% to 10.7% (coefficient of variation). Sensitivity of the assay was 3.2 microIU/ml. This radioimmunoassay for insulin is ideal for veterinary research and diagnosis, because a single set of reagents and procedures can be used for at least 3 species.     

赤羽病病毒的纯化及其单克隆抗体的制备

将人工繁殖、纯化的赤羽病病毒作为免疫原,通过常规杂交瘤技术,制备亲和性强、特异性好的小鼠抗赤羽病病毒单克隆抗体,并进行相应的纯化和标记工作。以澳大利亚进口的试剂盒进行验证,结果证明:进口单抗与我们制备的AAK纯品及AAK-HRP显示同样结果;且质控结果表明,AAK纯品及其衍生物AAK-HRP的特异性和亲和性均基本满足进行封闭ELISA检测方法的要求,为制备国产化ELISA试剂盒提供了基础。     

动物源性食品中莫能菌素残留ELISA试剂盒的研制

利用碳化二亚胺法合成抗原,进行动物免疫,制备特异性强的单克隆抗体。在筛选莫能菌素单克隆抗体的基础上,建立了莫能菌素ELISA检测方法,并研制出利用单克隆抗体对动物源性食品中残留的莫能菌素进行检测的试剂盒。本试剂盒的最低检测限为1μg/kg,试剂盒标准品变异系数为5.4%～13.1%,肌肉、肝脏样本的变异系数为5.1%～14.2%,肌肉和肝脏样本添加回收率分别为67.5%～87.4%和64.7%～86.7%。该方法的建立为莫能菌素的残留检测提供了可靠的分析检测手段。     

两种不同ELISA法在检测猪繁殖与呼吸综合征抗体中的选择与应用

猪繁殖与呼吸综合征（简称猪蓝耳病）是引起母猪繁殖障碍和仔猪、育成猪呼吸道症状的一种高度接触性传染病。本文比较了两种常用的检测猪繁殖与呼吸综合征抗体的ELISA方法，确定IDEXX猪蓝耳病抗体ELISA检测试剂盒适合蓝耳病感染抗体的检测和早期诊断；LSI猪蓝耳病抗体ELISA检测试剂盒适合蓝耳病免疫抗体的监测。两种ELISA方法在应用范围和诊断意义方面完全不同，因此在日常监测中，应根据监测目的合理选择猪蓝耳病抗体检测试剂盒。     

Osteopontin in Seminal Plasma and Sperm Membrane of Dogs

The objective of this work was to describe the presence of osteopontin (OPN) in canine seminal plasma and sperm membranes. A pool of seminal plasma and sperm membrane extract from 30 dogs was used. Polyacrylamide electrophoresis gels were performed and the bands were transferred to nitrocellulose paper and Western blot was undertaken using an antibody anti-OPN. Two and 12 bands were marked in the seminal plasma (77.2 and 15.6 kDa) and sperm membrane extracts (70.6–26.6 kDa), respectively. However, from 12 marked bands in the sperm membrane extract, only three (46.4, 37.7 and 36.5 kDa) were strongly marked. We conclude that, seminal plasma and sperm membranes from dogs contain different isoforms of OPN; yet, further studies will be necessary to determine their function in this species.     

Monoclonal antibody based sandwich enzyme-linked immunosorbent assay for chicken growth hormone.

1. Monoclonal antibodies which bind to different epitopes of chicken growth hormone (cGH) were used to develop a homologous sandwich enzyme-linked immunosorbent assay (ELISA). 2. The first antibody, which is species specific, was immobilised on microtitre plates and concentrations of cGH in biological fluids were estimated by revealing bound hormone using a second, biotinylated monoclonal antibody. 3. The sensitivity was 0.024 ng/ml, which is at least ten-fold greater than current radioimmunoassays (RIA) and there was no cross-reactivity to other chicken pituitary hormones or to growth hormone from other species. 4. The accuracy and precision of the assay were similar to RIA, and the growth hormone concentrations measured in plasma samples by both RIA and this new ELISA showed a high degree of correlation. 5. The assay takes only 4 h using pre-coated plates which can be stored at 4 degrees C in sucrose. The advantages of being rapid and non-isotopic make this method attractive to both research and industrial laboratories.     

Comparison of a commercial H1N1 enzyme-linked immunosorbent assay and hemagglutination inhibition test in detecting serum antibody against swine influenza viruses.

Recently a commercial enzyme-linked immunosorbent assay (ELISA) kit for detecting antibody against H1N1 swine influenza virus (SIV) has been made available to diagnosticians and veterinary practitioners. Because the hemagglutination inhibition (HI) test has been considered the standard test for SIV serology, diagnostic performance of the new ELISA was evaluated using positive (n = 60) and negative (n = 188) serum samples from young pigs with known status of SIV infection and compared with that of the HI test. Both ELISA and HI test identified all negative animals correctly. None of the serum samples (n = 64) from pigs inoculated with H3N2 SIV was positive by ELISA for SIV antibody. The H1N1 SIV antibody detectable by ELISA appears to develop more slowly in comparison with antibody detectable by HI test. Although antibody was detected by HI test in all inoculated animals (n = 20) by day 7 postinoculation (PI), antibody was detected by ELISA in 0%, 75%, and 100% of the inoculated animals on days 7, 14, and 28 PI, respectively. Discrepancy in test results between the 2 serologic tests appeared to be because of differences in antibody isotypes detected by each test. Enzyme-linked immunosorbent assay mainly detected IgG antibody, whereas the HI test detects IgM antibody very efficiently as well as IgG antibody. Collectively, the commercial ELISA is highly specific for antibody to H1N1 SIV but may not identify positive animals at the early stage of infection as effectively as the HI test, particularly when SIV is introduced to a na?ve swine population.     

Comparative study of diagnostic testing for feline leukemia virus infection.

Results of commercially available diagnostic test kits and commercial laboratory test results were compared for ability to detect FeLV antigen. Results of the immunofluorescent antibody (IFA) test were compared with test kit ELISA results and with results of a system in which samples were applied to an absorbent material, dried, sent to a laboratory, eluted, and assayed by a plate ELISA. Test kits were generally highly sensitive and specific, compared with the IFA test performed at a commercial laboratory. Feline heterophile antibody, specific for mouse immunoglobulin, was detected in approximately 0.14 to 0.57% of the cat population. Test kits B, E, and D contain reagents that correct for antimouse antibodies. During 1989 and 1990, 2,229 feline serum samples were tested for FeLV antigen (gsa p27); positive ELISA results were obtained for 204 (9%) of the samples. Results for 32 (1.4%) samples were interpreted as equivocal (color development slightly exceeded that of the negative control, but was much less than that of the positive control). Collectively, the data indicate that when testing serum or saliva, a negative test result may be a good predictor that a cat is not infected. In populations of cats in which FeLV prevalence is low, a positive test result may not be reliable and thus, a confirmatory test should be performed.     

呋喃妥因残留代谢物人工抗原的合成与鉴定

利用对醛基苯甲酸对呋喃妥因代谢物1-氨基乙内酰脲(1-Aminohydantoin hydrochloride,AHD)进行衍生化,衍生物1-氨基乙内酰脲-4-羧苯基肟(CPAHD)通过混合酸酐法与牛血清蛋白(bovine serum albumin,BSA)偶联,用紫外扫描(UV)和变性凝胶电泳(SDS-PAGE)对偶联物进行验证。将偶联成功的人工全抗原(CPAHD-BSA)免疫BALB/c小鼠,通过间接酶联免疫吸附法(ELISA)测定小鼠血清抗体效价为1:32 000。用间接竞争ELISA鉴定抗体特性,结果显示:抗体对AHD与对硝基苯甲醛的衍生物(NPAHD)的灵敏度高,IC50为30.63μg/L,特异性好,与CPAHD有部分交叉反应外,与其他3类硝基呋喃类药物及代谢产物无交叉反应(交叉反应率<0.01%)。结果表明,用CPAHD-BSA作免疫原免疫小鼠可诱导产生针对AHD衍生物(NPAHD)的特异性抗体,为进一步制备AHD单克隆抗体和研制快速筛选检测试剂盒奠定了基础。     

Serodiagnosis of goat warble fly infestation by Przhevalskiana silenus with a commercial ELISA kit.

Cross-reactivity between Hypoderma lineatum antigen and anti-Przhevalskiana silenus antibodies has been demonstrated by an ELISA technique. To evaluate the applicability of a commercial ELISA kit for the immunodiagnosis of goat warble fly infestation, different dilutions of serum and conjugate were tested, the development of antibody to P silenus in naturally infested goats was studied, and the results were compared with an ELISA technique using an antigen extracted from the first instar larvae of H lineatum. The best results were obtained with a serum dilution of 1:50; with both techniques the highest antibody concentration were recorded in October, November and December. In view of the confirmed cross-reactivity between H lineatum antigen and anti-P silenus antibodies, and the simplicity and rapidity of the assay, the commercial ELISA kit can be considered as a useful tool for the diagnosis of goat warble fly infestation.     

Binding of mouse mannan-binding lectins to different bacterial pathogens of mice

Humans have one mannan-binding lectin (MBL) in circulation but rodents, pigs, rabbits and rhesus monkeys have two, MBL-A and MBL-C. Plasma forms of these proteins have similar mannan-binding activity in vitro, but might differ in their ability to bind other microbial targets. In these studies, we compared carbohydrate-dependent binding of mouse plasma MBL-A and MBL-C to mannan-sepharose beads and to intact bacteria isolated as pathogens from mice. After incubation of mouse plasma with intact bacteria, MBL-A and MBL-C were eluted with N-acetylglucosamine (GlcNAc) and identified in nonreducing SDS-PAGE using Western blot analysis and MBL-A or MBL-C specific monoclonal antibodies. GlcNAc eluates of plasma incubated with mannan-sepharose beads, Klebsiella oxytoca and Staphylococcus aureus contained similar bands (mainly approximately 50kDa) that were immunoreactive with MBL-C antibody. Furthermore, a smaller form of MBL-C (approximately 45kDa) was detected bound to Pseudomonas aeruginosa. By comparison, immunoreactive MBL-A (a ladder of approximately 175kDa and larger bands) was identified in these GlcNAc eluates from mannan-sepharose beads, S. aureus and K. oxytoca but not P. aeruginosa. These studies demonstrate that mouse MBL-A and MBL-C in plasma are not equivalent in their ability to recognize bacteria that are pathogens for mice.