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.     

Toxoplasmosis II. Studies of Animal Sera Using Complement-Fixation Tests

Serological studies were performed in guinea pigs, a sheep, calf, goat and two pigs experimentally infected with toxoplasmosis. The direct complement-fixation method was effective in detecting antibodies in guinea-pig, goat and sheep sera. The modified complement-fixation technique supplementing complement with normal bovine serum fraction, was required when testing bovine serum. With swine sera best reactions occurred in the indirect complement-fixation test and definite but low grade reactions were produced in the direct test after pro-complementary activity was removed by pH treatment of the sera.

Allergic skin reactions were produced in the experimental animals but improvement in the antigen is necessary before the test could be used generally in the field as a diagnostic method for animal toxoplasmosis.

     

Species specificity in thermostable and ethanol insoluble tissue antigens. I. Immunization of rabbits and goats with bovine antigen.

Kidney and spleen antigens from cow, sheep, goat, European moose, reindeer, deer and roe deer were prepared by boiling and ethanol precipitation and tested against rabbit and caprine anti-bovine kidney sera. Two different levels of antigen concentration were used for immunizing animals in the various groups. In the group using high antigen concentrations, the precipitation reaction obtained initially disappeared after further inoculations, probably due to immunological tolerance. Sera tested from animals inoculated with low level antigen concentration showed a variation in the immunological response. The caprine antibovine sera showed distinct species specific reaction, while rabbit antisera showed either species specific or organ specific reaction, with a limited degree of cross reaction. In the production of species specific antisera against ruminant tissue antigens, goat seems to be preferable to rabbit.     

Bovine IgM: does it fix guinea pig complement in the absence of bovine complement components?

Purified bovine isotypes IgM, IgG1, IgG2, and IgA (secretory), affinity purified with Brucella abortus, were tested in a complement fixation test (CFT) for their ability to activate guinea pig complement directly or in the presence of 'normal' bovine serum. Only IgG1 fixed guinea pig complement in the direct test and approximately 250 ng of antibody was required to activate 50% of 3 CH50 units with a standard amount of antigen. Addition of 'normal' bovine serum as an additional source of complement resulted in activation of guinea pig complement by IgM, IgG2 and secretory IgA at levels of approximately 1200, 700 and 2250 ng, respectively for 50% of 3 CH50 units. Addition of 'normal' bovine serum did not enhance complement activation by bovine IgG1.     

Requirement for host Fc receptors and IgG antibodies in host immune responses against Rhipicephalus appendiculatus

Guinea pigs sensitized by prior feeding of larval Rhipicephalus appendiculatus ticks expressed complete immunity to challenge feeding resulting in 100% tick rejection. Passive transfer of 1 ml of serum from animals expressing resistance into naive animals conferred recipients with significant protection (88% tick rejection). Successful transfer of resistance was blocked by pretreatment of recipients with rabbit IgG but not sheep IgG1. Passive transfer of IgG1 or IgG2 purified from tick-sensitized guinea pig serum by ion-exchange chromatography failed to confer resistance to naive guinea pigs. Furthermore, IgG1 from guinea pigs expressing resistance obtained from serum by passage through a heavy chain specific rabbit anti-guinea pig IgG1 column failed to confer resistance to naive guinea pigs, as did the eluate. These results suggest that both IgG subclasses are needed for the expression of resistance, or IgG1 in conjunction with IgE.     

Effects of metyrapone and ACTH on intestinal absorption of immunoreactive bovine IgG in cesarean-derived pigs.

Newborn cesarean-derived pigs were injected with 5.0 mg of metyrapone/kg, or 1 USP U of ACTH/kg, or the vehicle 1.0 ml of 44 mM sodium tartrate and 88 mM NaCl soon after delivery (0 hour) and again 4, 8, and 12 hours later. Beginning at 2 hours, each pig in the 3 groups was given 40 ml of pooled bovine colostrum/kg by stomach tube every 8 hours for the duration of the experiment. Four hours after each feeding, pigs were killed; plasma and serum were collected and assayed for cortisol and bovine immunoglobulin (IgG), respectively. Some nonfed, nontreated pigs were killed at 0 hour also. Metyrapone significantly decreased plasma cortisol (hydrocortisone) concentrations at all times tested, whereas ACTH-treated pigs demonstrated a biphasic increase of plasma cortisol. Immunoreactive serum bovine IgG was not detected in nonfed, nontreated pigs. In vehicle-injected control pigs, bovine IgG was present in the serum at 6 hours; the concentration increases consistently to 22 hours, but not significantly thereafter. The concentration of bovine IgG in the serum of metyrapone-treated pigs also increased steadily before plateauing at 22 hours, but the values were significantly less than those of the controls at 14, 22, 30, and 38 hours. The concentration of bovine IgG in the serum of ACTH-treated pigs did not differ significantly from the control values at any of the times tested.     

Interaction of bovine immunoglobulins with complement

Whereas complement (C) in rabbit serum (CR) was bound by bovine antibodies in seven different IgG1 preparations, only two IgG1 preparations could bind the C in guinea pig serum (CGP). Addition of the Clq component of CR to CGP was alone sufficient to render the C-cascade in CGP activable in the presence of bovine erythrocytes sensitized with specific antisera, i.e. reagents. Normal bovine serum was also capable of restoring the haemolytic activity of CGP. However, the bovine serum was much more temperature sensitive than was CR and, as was observed in the sera from MZ twins, it showed considerable variation both in titre values and in prozones when added to CGP.     

Characterization of monoclonal antibodies to bovine IgG1

Monoclonal antibodies were developed to bovine IgG1. In addition, production of monoclonal antibodies to bovine light chain is also reported. Monoclonal antibody specificities were initially determined by solid-phase enzyme immunoassay. The monoclonal antibovine IgG1 was shown by a specificity-independent isotyping solid-phase enzyme immunoassay to be mouse IgG1 with kappa light chains. Ascites derived monoclonal antibovine IgG1 antibodies were linked to cyanogen bromide-activated Sepharose and used for affinity isolation of bovine IgG1. The bovine IgG1 eluted from the affinity column was characterized using immuno-electrophoresis, acrylamide gel electrophoresis, isoelectric focusing and solid-phase enzyme immunoassay. Affinity chromatography using monoclonal antibodies provided both a verification of monoclonal antibody specificity and a rapid technique for the isolation of bovine IgG1. This technique may also be employed to remove IgG1 contaminants during purification of bovine IgA.     

Studies on the efficiency of absorbed bovine PPD in tuberculin and serological tests

The passive haemagglutination, enzyme-linked immunosorbent assay and indirect fluorescent antibody tests were applied to study the non-specific reactions in experimentally infected guinea pigs and tuberculin positive bovines. These cross-reactions were greatly decreased after absorption of either sera with avian PPD or bovine PPD antigen with anti-avian PPD serum. The use of both absorbed sera and antigen raised the specificity of PHA and ELISA to 100%. The use of absorbed sera rendered the IFA specific in 95%. The absorption has reduced the sensitivity of ELISA, IFA and PHA by 14, 27 and 29%, respectively.     

A class capture enzyme immunoassay for immunoglobulin level determinations in bovine sera.

An enzyme immunoassay for determination of individual isotype concentrations in bovine serum was developed. Polystyrene tubes were coated with affinity purified goat antibovine IgA, IgG1, IgG2 or IgM, washed and then incubated with purified isotypes to ascertain crossreactivity and sensitivity limits. Bound isotype was detected using the homologous affinity purified antibody conjugated to horseradish peroxidase and hydrogen peroxide-2,2-azino-di-(3-ethylbenzthiazoline sulfonic acid), as the substrate/chromogen. A standardized serum was diluted and used as a control for comparison. Several dilutions were used initially, however, determinations may be made with a single dilution, 1:200, for all isotypes. Results for 100 sera were compared to data obtained with the same samples using a radial immunodiffusion technique. A low correlation coefficient was noted between results from the two assays. Day to day variation and within test repeatability were determined for both assays using ten samples. For the enzyme immunoassay method, day to day variation for IgA, IgM, IgG1 and IgG2 determinations was 17.5, 19.3, 7.6 and 7.3% while variation in repeatability (within a test) was 6.2, 5.9, 3.3 and 4.5%, respectively. Day to day variation for the single radial immunodiffusion test for IgA, IgM, IgG1 and IgG2 was 15.4, 26.0, 11.5 and 18.3% and variation repeatability (within a test) was 11.6, 13.9, 5.9 and 8.3%, respectively. The procedures consistently detected 0.1 micrograms of immunoglobulin whereas the radial diffusion sensitivity limit was approximately 500 micrograms.     

Maternal-neonatal immunoregulation: suppression of de novo synthesis of IgG and IgA, but not IgM, in neonatal pigs by bovine colostrum, is lost upon storage

Fifty-four neonatal pigs were allotted to 4 groups and reared in an electrically controlled automatic feeding device (autosow). Each group was reared on a different pool of bovine colostrum: fresh, stored 1 month, stored 6 months, and stored 8 years. Bovine and porcine immunoglobulins in the sera of these pigs, and in a group of conventionally reared pigs, were measured periodically during the first 42 days after birth. The maximal concentration of absorbed bovine immunoglobulin was reached between 12 and 18 hours and equaled or exceeded the amount of porcine immunoglobulin absorbed by the conventionally reared pigs. Large differences in the concentrations of the bovine immunoglobulin isotypes among the various pools of colostrum were positively correlated with concentration of these isotypes in the sera of the neonatal pigs fed these pools. Relative to their concentrations in colostrum, approximately 41% of the IgG1, 55% of the IgG2, 29% of the IgM, and 67% of the IgA was absorbed. The IgA was absorbed the best and IgM was least absorbed. Significant trends or differences in absorption were not observed among groups. Neonatal pigs given fresh colostrum, which had a higher fat content, had significantly more weight gain (P less than 0.05). This occurred, despite the fact that the fresh colostrum had the lowest concentration of bovine immunoglobulin. Serum half-lives for bovine IgG1 and IgG2 were significantly less than for porcine IgG (P less than 0.05), whereas the half-lives for bovine and porcine IgM and IgA were similar. De novo-synthesized immunoglobulins were detectable in serum after 6 days; IgM concentrations reached a maximum at 15 days in neonatal pigs given stored, but not fresh, colostrum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophoretic and immunoelectrophoretic analysis of feline serum proteins.

Serum from 28 clinically healthy cats was subjected to agarose gel electrophoresis and the migration distance relative to albumin was determined. The reference values for the relative and absolute concentrations of each protein fraction were determined and compared to previous reports. The immunoelectrophoretic, crossed immunoelectrophoretic and crossed line immunoelectrophoretic pattern, of a pooled sample of serum from clinically normal cats was determined. The cross-reactivity between goat and/or rabbit monospecific antisera to human proteins and feline serum was determined using immunoelectrophoresis and crossed-line absorption immunoelectrophoresis. Feline alpha-2-macroglobulin, haptoglobin, B1C-globulin, IgG, albumin and ceruloplasmin cross reacted strongly with the monospecific antisera. Alpha-2-macroglobulin migrated anodal to haptoglobin. Lipoproteins and ceruloplasmin were studied using staining procedures described in man. Feline transferrin was precipitated with Rivanol.     

Bovine reaginic antibody III. Cross-reaction of antihuman IgE and antibovine reaginic immunoglobulin antisera with sera from several species of mammals.

Using antisera specific for the heavy chain of human IgE and bovine reaginic immunoglobulin, the degree of cross-reaction amongst sera from pig, rat, rabbit, guinea pig, goat, cow, horse, dog, cat and human was tested. Antihuman IgE antiserum gave strong reactions with pig, rabbit, cow, goat and human sera (100% to 15.1%) and weak reactions with rat, guinea pig, horse, dog and cat sera (10.1% to 3.22%). Antibovine reagin antiserum produced a considerable amount of cross-reaction with sera from pig, rat, rabbit, goat, horse and human (43.6% to 20.1%) with limited reactions with guinea pig, dog and cat sera (13.9% to 9.3%).     

Further evidence that bovine IgM does not fix guinea pig complement

In a study of sera from cattle vaccinated with 3 X 10(10) cfu of Brucella abortus strain 19, it was found that IgG1 antibody measured by an indirect ELISA was the only isotype to correlate with standard complement fixing antibody titers using heated serum samples and guinea pig serum as a source of complement. A supplement of normal unheated bovine serum resulted in IgM fixing guinea pig complement, giving data similar to those obtained with unheated serum in the complement fixation test.     

Characterization of monoclonal and polyclonal antibodies to bovine enteric coronavirus: establishment of an efficient ELISA for antigen detection in feces

Monoclonal antibodies to bovine enteric coronavirus (BEC) were produced. Additionally, polyclonal antibodies were made in rabbits and guinea pigs and extracted from the yolk of immunized hens. The antibodies were characterized by neutralization test, hemagglutination inhibition test, enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Neutralizing antibody titers of polyclonal antisera ranged from 1:1280 to 1:40,000. Only one out of 908 hybridoma colonies tested secreted antibodies with neutralizing activity. By ELISA, polyclonal sera exhibited high background reactions that could be significantly reduced by treatment with kaolin in the case of rabbit sera. Attempts to establish an ELISA for BEC antigen detection based on polyclonal sera failed due to low sensitivity and specificity. Optimal results were achieved when a mixture of two monoclonal antibodies was coated onto microplates for antigen capture, while rabbit hyperimmune serum served as detecting antibodies in an indirect assay. The combination of the two monoclonal antibodies did not increase sensitivity synergistically, but in a compensatory fashion, probably because of epitope differences between BEC field strains.     

Effect of addition of soybean trypsin inhibitor to colostrum on immunological status in goat kids

The aim of the study was to evaluate the influence of soybean trypsin inhibitor (TI) on immunoglobulin G (IgG) serum levels and growth in neonatal goat kids. Twenty‐four newborn kids were fed with natural colostrum (group A), and 24 kids received the same colostrum with 1 g of TI per litre (group B). Blood samples were obtained at birth and on days 1, 2 and 4 of life to analyze serum proteins, IgG and haematological parameters. There were no clinical signs of disease and no significant differences in body weight between the groups. Haematological parameters were not affected by treatment. The peak of serum IgG was reached at 24 h of life, but no effects of soybean TI was observed on serum IgG levels. The apparent efficiency of absorption of IgG was similar in both groups (group A 24.5% vs. group B 25.2%, p > 0.05). The addition of TI to colostrum did not change the concentration of serum proteins and their fractions in goat kids. The correlation between serum IgG and γ‐globulin was positive and significant (p < 0.01, r = 0.64) in group A, but not in group B (p > 0.05, r = 0.08), suggesting a negative influence of soybean TI on γ‐globulin absorption. These results show that addition of soybean TI to colostrum did not improve the performance or immunological status in goat kids.     

The effect of parenteral immunisation on antibody production in the pig colon

Local and systemic antibody production was studied in pigs to compare responses to live and killed bacterial antigen and purified protein antigen, with and without prior mucosal stimulation. Recovery from challenge with live bacteria and intramuscular injection with killed bacteria gave rise to similar high levels of serum IgG antibody, but the ratio of specific IgA to IgG in the colon was significantly higher after infection than following vaccination with killed bacteria. Vaccination with a protein antigen gave rise to serum and local antibody production. Prior feeding of the antigen had a tolerising effect on the serum antibody response, but production of IgG and IgA antibody by the colon was not suppressed.     

Alternative pathway fof bovine complement Immunochemical studies on factor B-like serum protein and its conversion product B gamma 2.

Rabbits produced antibodies to a factor B-like serum protein (factor Bbov), its conversion product B gamma 2 and some other bovine serum proteins after repeated immunization with zymosan which previously had been incubated with fresh bovine serum. Such antisera were used to monitor purification of B gamma 2 from fresh bovine sera incubated with zxymosan. Subsequently, antisera specific for factor Bbov and B gamma 2 were produced. Antiserum produced against B gamma 2 cross-reacted with factor Bbov. Functional assays for factor Bbov were carried out in a hemolytic system with guinea pig erythrocytes in EGTA buffer. Heat inactivation (56 degrees C/5 min) of bovine serum destroyed the antigenicity of factor Bbov but not that of B gamma 2. Factor Bbov had an apparent molecular weight of 95,000 and B gamma 2 a molecular weight of 40,000 daltons. Conversion of factor Bbov to B gamma 2 was determined qualitatively by immunoelectrophoresis and quantitatively by radial immunodiffusion. Conversion of factor Bbov to B gamma 2 in bovine serum, in the presence of zymosan or cobra venom factor (CoVF) required Mg++ but not Ca++, did not occur in heat inactivated (56 degrees C/5 min) serum and was maximal, but not complete, when fresh bovine serum was incubated with zymosan (20 mg/mL) at 37 for two hours.     

Interaction of certain bovine immunoglobulins with complement in a single radial haemolysis system

Bovine IgG1, IgG2, and IgM initiated haemolysis by bovine complement. With guinea pig complement bovine IgG1 and IgM appeared effective, but bovine IgG2 was much less effective. Single radial haemolysis systems using guinea pig complement to measure bovine antibody are likely to detect predominantly IgG1 and IgM. However, in vivo IgG2 should activate bovine complement.     

豚鼠动物模型评价牛口蹄疫Asia-1型灭活疫苗效力的研究

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