Development of immunoglobulin class-specific enzyme-linked immunosorbent assays for measuring antibodies against avian rotavirus

Immunoglobulin class-specific enzyme-linked immunosorbent assays were developed for detecting antibodies against avian rotavirus in serum, intestinal contents, and bile from experimentally infected specific-pathogen-free (SPF) chickens. Both indirect and antibody-capture (AbC) assays were developed based on monoclonal antibodies specific for chicken IgG, IgM, and IgA. Treatment of purified rotavirus with sodium thiocyanate before coating the plate improved the rotavirus-specific reading in the indirect assay. Use of Immunolon 2 plates facilitated attachment of monoclonal antibodies to the plate in the AbC assay. Addition of 5% powdered skim milk to the diluent buffer reduced nonspecific background readings. The indirect assay was superior for detecting rotavirus-specific IgG, whereas the AbC assay was better for detecting rotavirus-specific IgM and IgA. The presence of intestinal contents in the assay wells did not reduce the measurable titers of IgG, IgM, or IgA. These assays showed that SPF chickens produced systemic and mucosal antibodies against avian rotavirus.     

Measurement of Pasteurella haemolytica-specific lung and serum antibodies by ELISA

The modified enzyme-linked immunosorbent assay (ELISA) was used to determine the relative quantities of class-specific antibodies to Pasteurella haemolytica. IgG1, IgG2 and IgA were present in significantly higher quantities in bronchoalveolar washings (BAW), but in decreasing quantities, respectively; IgM was present in very low amounts. IgM, IgG1 and IgG2 were present in serum, again in decreasing quantities, respectively. IgA antibody quantities were lowest in serum. The indirect antibody ELISA was found to be superior to the indirect bacterial agglutination (IBA) technique for determining antibody titres against P. haemolytica.     

Immunoglobulin class distribution of systemic and mucosal antibody responses to Newcastle disease in chickens

In serum, tracheal wash fluid, and bile from chickens that were inoculated with live or inactivated Newcastle disease virus (NDV), the kinetics and immunoglobulin (Ig) class distribution of an antibody response were demonstrated. The Ig classes (IgM, IgG, and IgA) were captured using monoclonal antibodies (MAbs) in enzyme-linked immunosorbent assays (Ig-capture ELISA). The antibody specificity of the captured Ig was confirmed by binding of NDV. After inoculation with live virus, antibodies of the IgG and IgM classes were mainly found in serum. IgM was produced early from day 4 postexposure (PE) onward, IgG was detected later from day 7 PE onward, and in the tracheal wash fluid and bile, all three Ig classes were demonstrated. After inoculation of inactivated virus, a delayed response of all three classes was observed in serum, and only IgM and IgG were recognized in the tracheal fluid and bile. The type of vaccine and the mute of antigen entrance may have determined the immunoglobulin class produced. The Ig-capture ELISA assay developed in this study can be useful for evaluating various strategies to improve the efficacy of Newcastle disease vaccines and to study the evoked immune mechanisms.     

Distribution of antibodies to rotavirus in serum and lacteal secretions of naturally infected swine and their suckling pigs

Rotavirus antibodies were demonstrated in lacteal secretions and sera of 20 parturient sows and in sera of their newborn by an enzyme-linked immunosorbent assay blocking technique, using bovine rotavirus cell culture antigen and monospecific antibody to bovine rotavirus. Antibodies to rotavirus occur in the 3 immunoglobulin (Ig) classes IgM, IgA, and IgG in lacteal secretions. High and long-persisting antibody activity was mainly associated with the IgA class. The IgM and IgG decreased to undetectable concentrations in most sows during the 14-day investigation period. Serum antibodies of newborn pigs nursing their dams also decreased rapidly during this time. The heterologous enzyme-linked immunosorbent assay blocking technique was a reliable and rapid procedure for the demonstration of rotavirus antibodies.     

Quantification of bovine IgG, IgM and IgA antibodies to Clostridium perfringens B-toxin by enzyme immunoassay I. Preparturient immunization for enhancement of passive transfer of immunity

A quantitative competitive binding "triple-sandwich" enzyme immunoassay was developed and used to evaluated pathogen/class-specific antibody responses in Holstein-Friesian cows vaccinated against Clostridium perfringens B-toxin. Vaccination of cows at six weeks and again at two weeks prepartum increased pathogen-specific IgG levels in each dam's colostrum and respective calf's serum. Pathogen-specific IgG and IgM concentrations in dams' sera and colostra were related to subsequent pathogen-specific IgG and IgM neonatal sera concentrations. Only pathogen-specific IgA in dams' colostra was correlated to neonatal levels, possibly owing to a different origin and role of this immunoglobulin class. All class-specific colostral immunoglobulin levels were related to subsequent neonatal concentrations. Isotypic antibody responses against C. perfringens B-toxin were found with pathogen-specific IgM predominant in dams' sera and pathogen-specific IgA predominant in colostra and neonatal sera.     

Immunoglobulin class response to canine distemper virus in gnotobiotic dogs

Serial serum samples from 27 gnotobiotic dogs infected with R252-canine distemper virus (CDV) were tested for anti-viral IgG, IgM and IgA immunoglobulins using an enzyme-linked immunosorbent assay (ELISA). The results were compared retrospectively to clinicopathological course of disease and to previously reported patterns of complement-fixing and virus neutralizing antibody titers determined in these same sera. Virus-specific IgA was never detected in the sera. High levels of IgG correlated with recovery from disease, whereas the antiviral IgM levels were equivalent in both persistently infected animals and those animals which recovered from disease. The inability to sustain a significant antiviral antibody response in either IgM or IgG classes was characteristic of dogs with fatal encephalitis. The data suggests that IgG is the most important Ig class for recovery from disease.     

雏鸡服用益生素后免疫器官指数及局部体液免疫球蛋白相对含量的动态变化

用商品益生素和自制益生素分别灌服1日龄雏鸡，灌服益生素后1、4、7、10、18d测定胸腺、脾脏、法氏囊的器官指数动态变化，用间接酶联免疫吸附试验(ELISA)方法测定泪液、气管液、胆汁和肠液的IgA、IgM、IgG相对含量的动态变化，结果发现服用商品益生素的雏鸡免疫器官指数在第7天高于未服用益生素的对照雏鸡，上述4种体液的IgA、IgM、IgG相对含量在服用益生素后7～10d高于未服用益生素的对照雏鸡，服用自制益生素的雏鸡上述4种体液的IgA、IgM、IgG相对含量在服用益生素后4～7d高于未服用益生素的对照雏鸡。表明益生素对雏鸡的免疫系统有一定的影响，能够促进免疫器官的生长发育，提高雏鸡呼吸道和消化道局部体液的免疫球蛋白相对含量。     

Isotype- and subclass-specific responses to infection and reinfection with parainfluenza-3 virus: comparison of the diagnostic potential of ELISAs detecting seroconversion and specific IgM and IgA.

Isotype- and subclass-specific indirect enzyme-linked immunosorbent assays were developed to detect parainfluenza-3 virus-specific IgG1, IgG2, IgM, and IgA responses. Sera were treated with protein G-agarose prior to testing for specific IgM and IgA to eliminate the possibility of false-positive results due to IgM-rheumatoid factor and to remove interisotypic competition due to specific IgG. IgM and IgA absorbance values were expressed as a percentage of the absorbance values of positive reference sera included on each plate (S/P%), and respective positive/negative threshold values of 15.0% and 28.0% were determined. The mean interval between experimental infection of 3 calves and initial detection of specific IgG1 and IgG2 responses was 8.0 and 9.3 days respectively, rising rapidly to an initial plateau 13.7 and 11.0 days postinfection (dpi). Reinfection of these calves at 30 dpi resulted in further rapid increases, with higher plateau values reached 13.0 (IgG1) and 13.7 (IgG2) days later. The mean interval between infection and the first positive IgM and IgA responses was 6.7 and 12.3 days, respectively. IgM S/P% values peaked at 13.0 dpi, with all 3 calves showing a secondary anamnestic response to reinfection, peaking 4.7 days later. The IgA response to initial infection was weak, with only 2 calves showing an obvious peak response at 15.0 dpi. A strong anamnestic IgA response to reinfection occurred in 2 calves, with a peak response 9.5 days later. Apparent biphasic and triphasic IgM and IgA responses were evident in some calves. Acute and convalescent serum samples from 80 calves involved in 17 outbreaks of respiratory disease were tested for specific IgM and IgA. Positive IgM results were detected in 15 outbreaks, with 71 sera from 44 calves testing positive. Although IgA-positive results were detected in the same 15 outbreaks, only 42 sera from 31 calves were positive. In a previous study, seroconversion was detected in 21 of these calves from 10 outbreaks. Thus the diagnostic potential of the assays was in the order IgM > IgA > seroconversion. The correlations between IgM and IgA, IgM and seroconversion, and IgA and seroconversion results for each calf were 73.8%, 58.8% and 62.5%, respectively.     

感染鸡贫血病毒雏鸡接种新城疫疫苗后局部粘膜免疫功能的变化

雏鸡1日龄感染鸡贫血病毒（CAV），8日龄接种Lasota疫苗，以未感染免疫雏鸡为对照，于免疫后7、14、28d检测其哈德尔腺和盲肠扁桃体T细胞及IgG^ 、IgM^ 、IgA^ 抗体生成细胞数量，泪液、气管液、肠液、胆汁中IgG、IgM、IgA含量以及泪液、胆汁HI抗体滴度的动态变化。揭示了感染CAV雏鸡接种ND疫苗免疫后哈德尔腺、盲肠扁桃体的T细胞和IgG^ 、IgM^ 、IgA^ 抗体生成细胞数量，泪液、气管液、肠液、胆汁中免疫球蛋白IgG、IgM、IgA含量以及泪液、胆汁HI抗体滴度，均较未感染免疫雏鸡明显减少。表明眼部、呼吸道和消化道局部粘膜免疫防御能力减弱。     

Quantitation of the immunoglobulins in reproductive tract secretions of the mare

IgG, IgA, IgM and albumin concentrations were measured in serum, follicular fluid and oviductal, uterine and intestinal secretions of the horse. Follicular protein concentrations were found to be dependent on serum concentration and molecular size. Of the immunoglobulins only IgG was detectable in oviductal secretions, but IgG:albumin ratios did not differ significantly from those in serum. IgG, IgA and IgM were measured in uterine secretions, with IgG predominant. Serum transudation into uterine secretions was minimal. In intestinal secretions, IgA levels were slightly higher than IgG, with albumin and IgM at low levels. In five mares with histories of chronic metritis, IgG, IgA and albumin concentrations were significantly elevated in uterine secretions.     

毒害艾美耳球虫初次感染对雏鸡外周血液免疫功能的影响

应用免疫SPA菌体花环、间接ELISA及细胞培养技术和四甲基偶氮唑盐(MTT)测定法对毒害艾美耳球虫(Eimerianecatrix)初次感染雏鸡外周血液的T、B细胞数量及其对ConA或PMA的增殖功能和IgG、IgM、IgA含量的动态变化进行了较全面系统的研究。结果发现,E.necatrix初次感染雏鸡外周血液的T、B细胞数量及其相应增殖功能、血清中上述三种免疫球蛋白含量均不同程度地高于未感染的对照雏鸡,表明E.necatrix初次感染雏鸡外周血液的细胞免疫和体液免疫功能均明显提高。     

Relative deficiency in IgA production by duodenal explants from German shepherd dogs with small intestinal disease

Matched samples of serum, saliva and tears were collected from four groups of dogs; two of the groups were German shepherd dogs (GSDs) either with (Group 1) or without (Group 4) a variety of small intestinal disorders; the remaining two groups were dogs of other breeds, again with (Group 2) or without (Group 3) small intestinal disease. Capture ELISAs were used to measure IgG, IgM, IgA and albumin concentrations within these samples; intestinal humoral immune status of clinical cases was assessed by quantifying immunoglobulin production from duodenal explant cultures.There were no significant differences in IgG, IgM or IgA concentrations in serum, saliva or tears between the different groups of dog. Moreover, no significant differences were noted between groups for IgG, IgM and IgA salivary and tear secretory indices. IgA production by 24-h explant cultures was significantly lower in GSDs compared with non-GSDs with small intestinal disease (groups 1 and 2, respectively), but the numbers of lamina propria IgA(+) plasma cells in duodenal biopsies were not different between groups. These results suggest that there may be a relative deficiency in local IgA secretion in GSDs with small intestinal enteropathies, which is not reflected in either serum IgA concentrations, or in secretion at unaffected mucosal sites. It remains to be determined whether such a deficiency is a breed-related primary defect, or whether it arises secondary to the pathological processes within the intestinal mucosa.     

Monoclonal antibodies against porcine immunoglobulin isotypes

Monoclonal antibodies (MCAs) against porcine immunoglobulin isotypes* G, G1, G2, M and A have been produced and characterized in detail. Epitope analysis using a competitive direct enzyme-linked immunosorbent assay (ELISA) indicated that the MCAs recognized 3 class-specific epitopes of IgG, 4 epitopes specific for IgG1, 3 epitopes specific for IgG2, 2 epitopes of IgM and 2 epitopes of IgA. Two MCAs against IgG2 were shown to react with an allotypic determinant (B2) and one MCA against IgM is probably allotype specific. The production of MCAs specific for IgG and for its subclasses G1 and G2 and, in addition, the one-step isolation of nearly pure IgG1 and IgG2 preparations by immunoaffinity chromatography using MCA 34.1.1a (anti-IgG2) confirmed the existence of at least two subclasses of IgG. Preliminary results further suggested the existence of a subpopulation of IgG1 which could be eluted selectively from Protein A-Sepharose columns at pH 5.0. MCA 34.17.2a appeared to react preferentially with this IgG1 subpopulation and could be used to isolate a similar IgG1 subpopulation by immuno-affinity chromatography. Several of the MCAs have been successfully applied for the detection of porcine immunoglobulin isotypes by a double antibody sandwich ELISA and for the (isotype-specific) detection of antibodies against various porcine viruses. The availability of a full set of MCAs against porcine immunoglobulin isotypes will stimulate and facilitate the further study of the porcine immune system.     

Serum antibody response to soluble extract of the third-larval stage of Ostertagia ostertagi in cattle

Serum IgG, IgM, and IgA antibody responses against L₃ antigens of Ostertagia ostertagi were monitored by enzyme-linked immunosorbent assay (ELISA) after one, two or multiple sequential inoculations of this nematode in calves. Following the first infection, antibody levels did not change. After a second inoculation, IgG increased significantly (P < 0.05) after 2 months. IgG was not significantly increased 1 month after challenge inoculation. IgM and IgA antibody levels did not change following the first or second inoculations of L₃. IgG antibody levels rose only slightly following multiple sequential inoculations with infectious L₃.

Results indicate that calves with ostertagiasis have very weak serum antibody responses to L₃, and these appear to be of little value in detection of the infection in these animals.     

Class-specific circulating antibodies in infections with Eimeria tenella

Using a three-layer immunofluorescent test, class-specific, parasite-specific circulating antibody responses to Eimeria tenella were investigated following oocyst infection, drug-truncated oocyst infection and the injection of non-living antigens. Following all three means of antigenic stimulation, IgG, IgM and IgA antibodies were detected. The response to drug-truncated infections was dose-dependent. The sequence of appearance of antibody was IgM, IgA, IgG, whilst the relative quantities were IgG greater than or equal to IgM greater than IgA.     

IgG, IgM and IgA in the serum of cattle naturally infected with Mycobacterium paratuberculosis

Serum IgG, IgM and IgA antibody response in 20 cattle naturally infected with Mycobacterium paratuberculosis and in 15 non-infected cattle were measured by enzyme-linked immunosorbent assay. A strong IgG response was detected in 16 (80%) of the infected animals. Diagnostic levels of IgM were detectable in all of the infected animals as well as in 8 (53%) of the non-infected animals. Animals with paratuberculosis had a very weak specific serum IgA response and this appears to be of little value in detection of infection in these animals.     

Evaluation of a dot ELISA kit for measuring immunoglobulin M antibodies to canine parvovirus and distemper virus

A dot ELISA for the detection of immunoglobulin M (IgM) antibodies to canine distemper virus (CDC) and canine parvovirus (CPV) was assessed. The titres of IgM antibodies to CDV and CPV in 100 dogs were measured by the Immunocomb ELISA kit and compared with the results derived from the immunofluorescence assay (IFA). There was a strong correlation between the results of the dot ELISA technique and the IFA (P < 0.001). The dot ELISA kit was also used to assess the changes in the levels of immunoglobulin G (IgG) and IgM antibodies to CPV and CDV in 10 puppies vaccinated with a polyvalent vaccine. High levels of IgM antibodies to CPV were first detected seven days after they were vaccinated, and after nine days all the pups had high titres of IgG antibodies to CPV. High levels of IgM antibodies to CDV were detected after nine days and the highest average titres were recorded after 12 days. IgG antibodies to CDV were present from nine days after vaccination.     

Immunological parameters in the intestinal lymph of pigs including changes during experimentally induced diarrhoea

Experiments were undertaken to provide basic immunological data on the intestinal lymph of young pigs. For this purpose indwelling cannulae were established in the main efferent intestinal lymphatic ducts of 12 animals and measurements were made of lymph flow rate, and concentrations of IgG, IgA and IgM. Measurements were also made of cell numbers, differential counts and immunoglobulin specificity of lymphoid cells in lymph. Similar measurements were also made on six pigs in which experimental diarrhoea was induced. The mean number of leucocytes in intestinal lymph was extremely low (0.66 X 10(5)/ml). However a high proportion of lymphocytes contained cytoplasmic IgA (19.65 per cent) and IgM (12.53 per cent), with few containing IgG (1.35 per cent). The concentrations of IgM and IgA in intestinal lymph were 0.51 mg/ml and 1.64 mg/ml respectively, values which suggest that the intestine is an important organ for synthesis of these two classes of immunoglobulin in young pigs. Following induction of diarrhoea and consequent dehydration of the intestine, the lymph: serum concentration ratios for immunoglobulins increased but subsequently declined when the water balance in the intestine returned to normal.     

Demonstration and quantitation of immunoglobulins in bovine serum, follicular fluid, and uterine and vaginal secretions with reference to bovine viral diarrhea and infectious bovine rhinotracheitis.

Immunoglobulin concentrations (IgG, IgM, and IgA) in bovine serum, follicular fluid, and uterine and vaginal secretions were determined. The specificities of IgG, IgM, and IgA for virus-neutralizing antibody against bovine viral diarrhea (BVD) and infectious bovine rhinotracheitis (IBR) viruses were also examined. High concentrations of IgG were present in both serum and follicular fluid. The IgG, IgM, and IgA concentrations were low in uterine and vaginal secretions. There was more IgG in the uterus during estrus than at any other time. Virus-neutralizing antibodies against BVD and IBR in serum of cows were mainly the IgG class. There was positive correlation between serum and follicular fluid virus-neutralizing antibody titers fro BVD and IBR. These antibodies may provide some protection for recently ovulated ova.     

IgG, IgM and IgA in the serum of cattle naturally infected with Mycobacterium paratuberculosis

Serum IgG, IgM and IgA antibody response in 20 cattle naturally infected with Mycobacterium paratuberculosis and in 15 non-infected cattle were measured by enzyme-linked immunosorbent assay. A strong IgG response was detected in 16 (80%) of the infected animals. Diagnostic levels of IgM were detectable in all of the infected animals as well as in 8 (53%) of the non-infected animals. Animals with paratuberculosis had a very weak specific serum IgA response and this appears to be of little value in detection of infection in these animals.