Detection of antibodies against African swine fever virus using infected monolayers and monoclonal antibodies

Three monoclonal antibodies, specific for porcine IgG, IgM and IgA, were used to develop isotype-specific immunoperoxidase monolayer assays for the detection of antibodies against African swine fever virus. A mixture of anti-IgM and anti-IgG monoclonal antibodies was used in an assay designed for screening sera. This test was compared with a commercially available ELISA by using experimental sera and field sera obtained after an outbreak of African swine fever on two farms in the Netherlands in 1986. Although the ELISA was less sensitive than the immunoperoxidase monolayer assay on sera taken early after infection, the tests were equally useful for screening purposes. The isotype-specific assays gave epizootiological information about the stage of infection on the two farms.     

A novel enhanced dot blot immunoassay using colorimetric biosensor for detection of Toxoplasma gondii infection

This study reports development of a novel point of care assay, namely an enhanced immuno-dot blot assay, for discrimination of anti-Toxoplasma IgG and anti-Toxoplasma IgM antibodies. This method has been designed based on formation of a sandwich complex between a gold nanoprobe (chitosan gold nanoparticle-anti-human IgG or anti-IgM) and anti- Toxoplasma lysate antigen (TLA) which holds anti-TLA antibodies, either IgG or IgM. Briefly, anti-human IgG or anti-IgM antibody was conjugated to chitosan gold nanoparticles via glutaraldehyde chemistry. Then, lysate antigen was immobilized on the surface of nitrocellulose membrane, which followed by addition of the sera sample and gold nanoprobes. The positive signals were readily detectable via observation with naked eye. This positive color change was further intensified via gold enhancement chemistry. The intensity of biosensor signal was proportional to the concentration of active antibodies on the surface of nanoparticles, titer of T. gondii antibodies in the sera samples and concentration of Toxoplasma lysate antigen coated on the nitrocellulose membrane. A minimum concentration to use the antibodies for conjugation, to detect titer of Toxoplasma IgG and IgM antibodies, and the concentration of TLA coated in nitrocellulose membrane were 0.5 mg/mL, 2 IU/mL, 10 IU/mL, and 20 μg/mL, respectively. This enhanced immuno-dot blot assay offers a simple diagnostic technique without expensive equipment requirement for distinguishing of anti- T. gondii IgM and IgG antibodies in field conditions, pregnant women, and immunocompromised patients.     

Monoclonal antibodies against sheep immunoglobulin light chain, IgM and IgA

Monoclonal antibodies specific for sheep immunoglobulin light chain, IgM and IgA were produced by conventional cell fusion technology. Purified light chain and IgM were used to verify the specificity of anti-light chain and anti-IgM hybridoma supernatants using passive haemagglutination assays, radioimmunoassays and immunoelectrophoresis. In the absence of pure IgA, verification of monoclonal anti-IgA was, based on paired staining of intestinal lymph smears and comparing the percentage of cells stained with hybridoma supernatant with the percentage of cells stained with polyclonal anti-alpha serum.     

Monoclonal antibodies to equine IgM improve the sensitivity of West Nile virus-specific IgM detection in horses

West Nile virus (WNV) is a zoonotic pathogen of global importance. In horses with neurological signs, detection of WNV-specific immunoglobulin M (IgM) in serum is widely used to identify clinical cases of WNV encephalitis. Here, we describe the development of two monoclonal antibodies (mAbs) to equine IgM which were used in a WNV IgM-specific enzyme-linked immunosorbent assay (ELISA). Their performance was compared to an established assay based on polyclonal anti-IgM. Check test serum samples from the National Veterinary Service Laboratory (NVSL) were used to evaluate the performance of the three anti-IgM antibodies. The anti-IgM 1-22 mAb correctly identified all NVSL samples. Both the polyclonal antibody and monoclonal anti-IgM 2B-63 identified eight out of ten samples correctly. The three assays were then compared using serum samples from clinically healthy animals (n=33) and horses with neurological signs (n=21). High Spearman rank correlations (0.76-0.86) were found among the ELISA results. Inter-test agreements (weighted kappa) for assay interpretation resulted in strong agreement (0.95) of the results obtained by the mAbs and moderate agreements when monoclonal and polyclonal anti-IgM-based assays were compared. To determine the analytical sensitivities of anti-WNV IgM detection, serial dilutions of WNV IgM-positive serum samples were analyzed. The highest sensitivity was obtained by using the anti-IgM 1-22 mAb to capture IgM from equine serum. In conclusion, the use of monoclonal anti-IgM antibodies can improve the sensitivity of IgM detection in the acute phase of WN disease.     

The serological response of calves to Leptospira interrogans serovar hardjo vaccines and infection as measured by the microscopic agglutination test and anti-IgM and anti-IgG enzyme-linked immunosorbent assay

The microscopic agglutination test (MAT) and the anti-IgM and anti-IgG enzyme-linked immunosorbent assays (ELISA) were used to examine sera taken over the course of 16 weeks from 35 calves vaccinated and/or infected with Leptospira interrogans serovar hardjo. The relationship between the IgM and IgG responses to vaccination and infection were determined. The rapid and high rise in IgM levels following challenge made the anti-IgM ELISA a potentially good indicator of recently established infection although some transitory high levels were seen where infection did not become established. The slow IgG response to infection made the anti-IgG ELISA of limited diagnostic use.     

The effect of including anti-Ig sera in the haemagglutination inhibition test forMycoplasma gallisepticum

Addition of anti-immunoglobulin M (anti-IgM), G (anti-IgG) and A (anti-IgA) sera to the haemagglutination-inhibition (HI) test (anti-Ig HI test) forMycoplasma gallisepticum resulted in 2- to 8-fold increases in the HI titres. On investigating the anti-Ig HI reaction using IgM and IgG antibodies separated by affinity chromatography, it was confirmed that, in the enhanced HI titres, specificity existed between the chicken Ig classes having antibody activity and the antisera used in the test. Four days after inoculation ofM. gallisepticum, the anti-Ig HI reaction was markedly enhanced by anti-IgM antiserum in the intravenously inoculated chickens and by anti-IgA serum in the nasally inoculated chickens. Ten days after inoculation ofM. gallisepticum marked enhancement of the reaction was produced by anti-IgG serum in both intravenously and nasally inoculated chickens, but the enhancement of the anti-Ig HI reaction diminished from the second week after inoculation.     

Monoclonal antibodies specific for pig serum and cell surface IgM

Two monoclonal antibodies were prepared which react specifically with pig serum immunoglobulin and with the population of B lymphocyte-bearing surface immunoglobulin. Comparison of our monoclonal antibodies with reagents specific for gamma, mu and alpha immunoglobulin chains in double immunodiffusion and immunoelectrophoresis revealed that the monoclonal antibodies recognise IgM in pig serum and mu chain or mu chain-like molecules on B lymphocytes. The monoclonal antibodies, designated LIG 2 and LIG 4, reacted positively with adult pig sera but not with fetal or precolostral sera or with sera from other animal species. LIG 2 and LIG 4 reacted with 15 per cent of cells from the peripheral blood lymphocyte population, 20.2 per cent of spleen cells and 20 per cent of lymph node cells, but did not react with pig erythrocytes, granulocytes or cells isolated from thymus, or with the lymphocytes of other species. Positive reactions were also found on lymphatic and intestinal tissue sections. No genetic polymorphism was found in the pig population revealed by the monoclonal antibodies. The monoclonal antibodies LIG 2 and LIG 4 may be useful for studying the pig immune system, especially as a standard reagent for measuring pig serum IgM and for the identification of positive B lymphocytes.     

Detection of antibodies to ovine lentivirus using recombinant capsid and transmembrane proteins

The coding sequences of the capsid protein p25 and transmembrane protein of Maedi-Visna virus were amplified using polymerase chain reaction and cloned into the plasmid expression vector pRSET-B. Both DNA constructs expressed proteins tagged with polyhistidine. The recombinant proteins were purified using Ni-NTA agarose and used in immunoblot to detect antibodies against Maedi-Visna virus. A total of 260 ovine serum specimens was analysed. The total number of p25-positive sera was 111 (42.7%). Higher sensitivity was achieved with rTM antigen, which detected antibodies in 118 (45.4%) sera. The combination of both recombinant proteins as antigens resulted in higher sensitivity of serological detection compared to whole virus antigen.     

Serological reactions of leptospirosis-positive (MAR and CFT) bovine sera in ELISA

The collection of test sera for measuring ELISA results was composed of bovine sera with MAT titres of greater than or equal to 1:200 in the leptospirosis MAT and of greater than or equal to 1:5 in the CFT together with sera from a serologically negative and clinically non-suspicious cattle herd. To establish cut-off ODs, the geometric mean net-extinction of the negative serum collection plus 1, 2, and 3 standard deviations were calculated. By comparison of 3 different conjugates from rabbits, it was demonstrated that results from anti-total bovine Ig were superior to anti-IgG and anti-IgM conjugates. Considerations regarding sensitivity and specificity led to the recommendation to use a test serum dilution of 1:160, to apply anti-total bovine Ig conjugates, and to establish the cut-off OD at the geometric mean net-extinction of negative sera plus 3 standard deviations. Under such conditions, agreement between leptospirosis MAT/CFT positivity on the one side and ELISA positivity on the other was reached in 74%. This recommendation is made for cross-sectional studies but not for examinations of clinically suspicious cattle herds.     

Japanese encephalitis virus immunoglobulin M antibodies in porcine sera

A solid-phase enzyme-linked immunosorbent assay (ELISA) was developed for detection of porcine immunoglobulin (Ig)M antibodies to Japanese encephalitis virus (JEV). Antibodies in sera were captured onto the solid phase of Microtiter plates sensitized with mouse monoclonal antibodies to porcine mu heavy chain. Virus antigen binding to the lawn of IgM was quantitated by subsequent binding of peroxidase-labeled human hyperimmune anti-JEV IgG, which in the final step, catalyzed a substrate color change. In sucrose density-gradient fractionated sera from recently infected pigs, the peak of ELISA JEV IgM activity corresponded to the peak of 18-S, 2-mercaptoethanol-sensitive hemagglutination-inhibiting (HAI) antibody activity. Within 2 to 3 days, JEV-infected sentinel pigs developed high JEV IgM activity; this activity decreased within 2 weeks. Among specimens collected from 99 random swine at abattoirs in Thailand during a period of low JEV transmission, none of 25 JEV HAI-negative sera had JEV IgM activity, 7 of 74 JEV HAI-positive sera did have JEV IgM activity, and the remaining 67 sera had readily detectable JEV HAI antibodies, but lacked JEV IgM. The JEV IgM solid-phase ELISA was useful for rapidly diagnosing active or recent JEV infections in swine.     

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.     

Isolation and purification of a diagnostic antigen for bovine cysticercosis by hydrophobic chromatography

An ammonium sulfate fraction of Taenia hydatigena cyst fluid (ThFAS) was further fractionated by hydrophobic interaction chromatography, using alkylagarose and omega-amino alkylagarose columns, in an effort to isolate and purify a specific diagnostic antigen in the ThFAS preparation. The less than 12 kDa antigen was found to have an affinity for immobilized alkanes with chain length of six carbons or greater. The antigen was recovered in an ethylene glycol eluate from a hexylagarose column then analyzed by Western blot; it reacted with bovine and human cysticercosis infection sera and with specific monoclonal antibodies but not with control sera or fascioliasis infection sera. When the eluate was used as coating antigen in a plate ELISA assay no false positive reactions were seen in sera from cattle infected with Fasciola hepatica; false positive reactions were observed for the unfractionated ThFAS antigen preparation.     

Preparation of monospecific antiserums against porcine immunoglobulins, using agarose-linked immunosorbents.

Immunoglobulins IgG, IgA, and IgM were isolated from porcine serum and milk, and antiserums against the 3 immunoglobulin classes were prepared. Monospecificity of the antiserums for the gamma-, alpha-, and mu-chains was obtained by absorbing them in agarose-linked immunosorbent columns. These immunosorbents were prepared by linking IgG or IgA-IgM to CNBr-activated agarose. Contaminating anti-alpha2-macroglobulin antibodies in the anti-IgA and anti-IgM serums were removed with agarose-linked fetal globulins.     

Seroprevalence of bovine immunodeficiency virus in dairy and beef cattle herds in Korea.

Infection of bovine immunodeficiency virus (BIV), a lentivirus, is thought to sporadically occur throughout the world, but seroepidemiological surveys concerning the incidence of BIV are limited and have not been undertaken in Korea. A total of 266 sera from different twenty dairy (Holstein) and twenty-six Korean native beef (Hanwoo) farms of the south-western part of Korea was analyzed for the presence of anti-BIV antibodies by Western blotting. Thirty five percent and 33% of dairy and beef cattle, respectively, were BIV-seropositive. By nested polymerase chain reaction, it was confirmed that these seropositive cows had provirus in the peripheral blood mononuclear cells. To demonstrate the correlation with BIV and bovine leukemia virus (BLV) infection, these sera were also analyzed for anti-BLV antibodies by immunodiffusion test, resulting in high prevalence of BLV infection but relatively a few dual infections. We report herein the first serological detection of antibodies to BIV in Korea.     

李斯特氏菌因子血清的制备及食品检测的免疫磁性分离-PCR(MIPA)方法的建立

首先进行了李斯特氏菌因子血清的研制,制备出了可对所有李斯特氏菌分型的 １５ 个 Ｏ 抗原因子和４ 个 Ｈ 抗原因子的因子血清。利用复合因子血清的多克隆抗体包被磁性球,对食品中的单核细胞增多性李斯特氏菌进行免疫磁性分离,并与 Ｐ Ｃ Ｒ 方法相结合,建立了检测食品中单核细胞增多性李斯特氏菌的 Ｍ Ｉ Ｐ Ａ方法（免疫磁性分离—聚合酶链反应方法,ｍ ａｇｎ ｅｔｉｃ ｉｍ ｍ ｕｎｏｐｏｌｙｍ ｅｒａｓｅ ｃｈａｉｎ ｒｅａｃｔｉｏｎ ａｓｓａｙ）。对菌液、模拟样品的检测表明,本方法能够有效地克服食品基质、培养基成分和杂菌对 Ｐ Ｃ Ｒ 检验的干扰作用。食品样品在 Ｅ Ｂ增菌液中增菌 １２ ｈ 后,检测的敏感度达 ５ Ｃ Ｆ Ｕ／ｍ Ｌ,可以在 ２０ ｈ 内完成检测。本方法对实际食品样品的检测结果,与国家标准检验方法检测的结果一致。     

Pitfalls in immunofluorescence testing in dermatology. II. Pemphigus-like antibodies in the cat, and direct immunofluorescence testing of normal dog nose and lip

Indirect immunofluorescence testing for pemphigus-like antibodies was performed on 75 cats: 25 cats with various nonpemphigus dermatologic diseases, 25 cats with various nondermatologic diseases, and 25 normal cats. Pemphigus-like antibodies were not detected. It was concluded that neither true pemphigus antibodies, nor pemphigus-like antibodies are commonly detected by indirect immunofluorescence testing in the cat. Direct immunofluorescence testing for IgG, IgA, IgM, and C3 was performed on the nose and lip of 15 normal dogs. Granular deposition of IgM at the basement membrane zone of the nose was demonstrated in 11 of the 15 dogs. Lip was consistently negative. It was concluded that direct immunofluorescence testing of canine nose by only polyvalent immunoglobulin antisera or anti-IgM antisera may lead to misinterpretation and misdiagnosis in up to 73% of all dogs tested.     

The detection of transmissible gastroenteritis viral antibodies by immunodiffusion.

Precipitating antibodies against transmissible gastroenteritis viral antigens were detected by the immunodiffusion test in two transmissible gastroenteritis viral hyperimmune antisera and in antiserum prepared against haemagglutinating encephalomyelitis virus but not in sera from several species of normal animals, in antisera prepared against a variety of othet viruses and bacteria or sera from swine with bacterial enteritis. When the immunodiffusion test was compared with the virus neutralization test for the detection of transmissible gastroeneritis viral antibodies in 20 swine sera certain samples which contained high titres of virus neutralizing antibodies failed to produce precipitation while other sera were positive in the immunodiffusion test although their virus neutralizing antibody titres were relatively low. Precipitating antibodies were also detected by immunodiffusion in several samples of milk whey from a sow which had been vaccinated with inactivated transmissible gastroenteritis virus.     

Bovine babesiosis: anti-erythrocyte antibodies purification from the sera of naturally infected cattle

Babesia bigemina and Babesia bovis are intra-erythrocytic protozoan parasites transmitted by ticks to cattle in which they induce babesiosis, a disease that resembles human malaria. Anemia, caused by the destruction of non-infected erythrocytes, is a critical feature of the disease. Anti-erythrocyte antibodies could be one of the explanations for such destruction. These antibodies are found in the sera of dogs and mice respectively infected with B. gibsoni and B. rodhaini. However, data concerning the presence of anti-erythrocyte antibodies in the sera of infected cattle are not conclusive. In the present study, we made an attempt to detect anti-erythrocyte antibodies from the sera of cattle naturally infected with B. bigemina. Erythrocytes from a non-infected calf were used in ELISA reaction for the detection of antibodies from samples. Results confirmed the presence of anti-erythrocytes antibodies in higher amounts in the serum of infected cattle. In order to correlate this increment with the parasite, anti-erythrocyte antibodies from the sera from infected calves were purified, coupled to a Sepharose-4B column and than used for anti-idiotypic antibodies purification. These antibodies were found to react with the parasites, suggesting a correlation between both anti-parasite and anti-erythrocyte antibodies.     

Change of antibody levels to ferritin in the sera of foals after birth: Possible passive transfer of maternal anti‐ferritin autoantibody via colostrum and age‐related anti‐ferritin autoantibody production

Antibody (immunoglobulin G (IgG), IgM or IgA) levels relative to ferritin in six foal sera (three male and three female) after birth (day 0 and 2, 6, 10, 20, 28, 36, 40, 52 and 56 weeks of age) were semi‐quantitatively measured with normalization with antibody activity to ferritin in one adult horse serum. After addition of horse spleen ferritin to the serum sample, the complex formed between antibodies to ferritin in the serum and ferritin was co‐immunoprecipitated using antibody to horse spleen ferritin. Antibody classes of the co‐immnoprecipitate were detected with antibodies specific for horse IgG, IgM or IgA heavy chain. Six adult horse serum samples were found to have ferritin‐binding activities in all immunoglobulin classes examined. Although ferritin antibody activities (IgG, IgM and IgA) were scant in the foal sera before sucking colostrum (day 0), their activities increased at 2 weeks of age. IgG antibodies showed a biphasic response and IgM antibody activity increased up to 40 weeks of age. Antibody (IgG, IgM and IgA) activities to ferritin in three colostrum samples were significantly higher than in adult horse serum samples. These results demonstrate that antibody to ferritin in foal serum is derived from colostrum after birth and is produced thereafter.