Monoclonal antibody-based capture enzyme immunoassays for specific serum immunoglobulin G (IgG), IgA, and IgM antibodies to respiratory syncytial virus

Monoclonal antibodies (MAbs) to the fusion protein (F), attachment protein (G), and nucleoprotein (N) of respiratory syncytial (RS) virus were evaluated for use as detector antibodies in immunoglobulin G (IgG), IgA, and IgM capture enzyme immunoassays. MAb assays were tested against assays using polyclonal antibodies (PAbs) with serum specimens from patients with and without evidence of recent RS virus infection. Assays developed with N MAbs were comparable to or better than PAb assays for detecting specific IgG and IgM antibodies but were somewhat less sensitive for IgA. F MAb assays were less sensitive for IgG and IgM antibodies but identified specific IgA in some specimens negative by N MAb assay. G MAb assays were insensitive for IgG and IgM antibodies but did detect about 50% of the IgA antibodies identified by the PAb assay. The basis for the low sensitivity of the G MAb assays is unclear, since many of these specimens were positive for IgG antibodies to G by Western immunoblot. The sensitivity of MAb assays varied with patient age: N MAb assays detected specific antibody responses to RS virus in all immunoglobulin classes in both adults and infants less than 1 year of age, F MAb assays detected specific IgG responses in adults and IgA responses in both adults and infants, and G MAb assays only detected IgA responses in adults. A mixture of N and F MAbs was complementary overall, identifying 54 of 55 (IgG), 51 of 52 (IgA), and 16 of 17 (IgM) serum specimens positive by PAb assay. These MAb assays were also specific with specimens tested from persons without a history of recent RS virus infection. The availability of these MAb-based assays offers other laboratories the opportunity to have long-term, standardized reagents and tests for serological diagnosis of RS virus infection.     

Characterization of rubella virus-specific antibody responses by using a new synthetic peptide-based enzyme-linked immunosorbent assay.

Rubella virus (RV)-specific immunoglobulin G antibodies were studied by enzyme-linked immunosorbent assay (ELISA) techniques in sera from RV (RA 27/3)-vaccinated individuals, patients experiencing natural RV infection, congenital rubella syndrome patients, and individuals failing to respond to repeated RV immunization. Results obtained by using whole-RV ELISAs (detergent-solubilized M33 strain or intact Gilchrist strain) and hemagglutination inhibition (HAI) and neutralization (NT) assays were compared with results obtained with the same sera by using ELISAs employing a synthetic peptide, BCH-178, representing a putative neutralization domain on the RV E1 protein. Murine RV E1-specific monoclonal antibodies with HAI and NT activities exhibited strong reactivity in ELISAs with BCH-178 peptide. In sera from RA 27/3-vaccinated individuals collected at 0 (prevaccine), 1, 2, 3, 4, 5, 6, 12, and 24 to 52 weeks postvaccine, the development of E1-peptide-reactive antibodies closely paralleled increases in RV-specific antibodies measured by whole-RV ELISAs and HAI and NT assays. Similarly, sequential serum samples obtained from patients during acute and convalescent phases of natural RV infection showed a coordinate increase in RV-specific antibodies as measured by whole-RV and peptide ELISAs. Conversely, congenital rubella syndrome patient sera, although exhibiting high levels of antibody in whole-RV ELISAs, had little or no antibody directed to the neutralization domain peptide. Sera from patients failing to respond to repeated RV immunization contained very low levels of RV-specific antibody in all ELISAs. Our results that the sequence represented by BCH-178 peptide may be a previously unidentified neutralization epitope for human antibodies on the RV E1 protein and may prove useful in determining effective RV immunity.     

Detection of IgM and IgA HIV antibodies after removal of IgG with recombinant protein G

Indirect assays for IgM and IgA antibodies often lack sensitivity and specificity due to interference from IgG antibodies. To overcome this problem we have developed a simple procedure using recombinant protein G coupled to agarose beads to remove the interfering IgG. A series of HIV seroconversion panels was tested by IgM and IgA immunoblot after protein G treatment in order to evaluate IgG removal and to study appearance of IgM and IgA antibodies in early HIV infection. Protein G treatment removed 99.9% of the IgG and reduced IgG anti-HIV titers of over 1/100,000 to undetectable levels. Both IgM and IgA HIV antibodies were detected as early in seroconversion as were IgG HIV antibodies. IgA HIV antibodies persisted for a longer period of time, reacted with more HIV proteins, and showed more intense staining than IgM HIV antibodies.     

Antibody response to rabies virus in Syrian hamsters.

Syrian hamsters were injected with inactivated, attenuated, and virulent rabies virus (RV), and the antibody response was quantified by a neutralization test and the immunoglobulin class of the virus antibody was characterized by indirect fluorescent microscopy. Serum antibodies to RV were found to be predominantly of the immunoglobulin G2 (IgG2) class, although IgG1 anti-RV also were detected in high-titered sera obtained after secondary challenge. Brain extracts of hamsters inoculated intracerebrally with RV contained only IgG2 anti-RV. IgA and IgM anti-RV were not detected. The preferential IgG2 response to RV is in marked contrast to the isolated IgG1 response detected after inoculation of hamsters with soluble purified protein antigens.     

Diagnosis of recent primary rubella virus infections: significance of glycoprotein-based IgM serology, IgG avidity and immunoblot analysis

Reliable serodiagnosis of rubella virus (RV) infections requires discrimination of specific IgM induced by primary rubella from persistent, reactivated or non-specific IgM reactivity. Sera from 130 pregnant women with recent or past RV infection/vaccination, persistent IgM or negative rubella serology, 26 patients with other acute infections and 5 patients with rheumatoid factor-positivity were analyzed for RV-specific IgM by ELISA coated with whole-virus lysate or native glycoprotein, followed by determination of IgG avidity and E2-specific IgG using lysate-coated ELISA and non-reducing immunoblot. Compared to a reference μ-capture IgM ELISA, the sensitivity for diagnosing recent rubella infection/vaccination was 90.0% and 100% for the lysate-based and glycoprotein-based IgM ELISA, respectively. With respect to women with past RV infections or negative histories of RV infection/vaccination, both assays were 97.5-100% specific, whereas for patients with other acute infections the glycoprotein substrate provided a specificity of 92.3% compared to only 80.8% using whole-virus antigen. Analyzing anti-RV IgG avidity and anti-E2 IgG reactivity allowed the time point of primary infection to be determined unambiguously in >86% of samples. In conclusion, using RV glycoprotein antigen improves the specificity of indirect IgM ELISA. In cases of RV-specific IgM reactivity, recent primary rubella infection can be confirmed or excluded efficiently by specific IgG avidity and immunoblot analysis.     

Enzyme-linked immunosorbent assays for measurement of reovirus immunoglobulin G, A, and M levels in serum.

Enzyme-linked immunosorbent assays were developed for the quantitation of anti-reovirus immunoglobulin G (IgG), IgA, and IgM in serum. The assays were specific, sensitive, and reproducible and measured antibodies to the reovirus group antigen. They should permit diagnosis of recent reovirus infection and longitudinal study of the development of reovirus group antibodies.     

Use of antibody avidity assays for diagnosis of severe acute respiratory syndrome coronavirus infection

An indirect immunofluorescent assay (Euroimmun AG, Luebeck, Germany) was used to investigate the avidity of immunoglobulin G (IgG), IgM, IgA, and total Ig (IgGAM) antibody responses to severe acute respiratory syndrome coronavirus (SARS CoV) infections. Serial serum samples from eight patients collected during the first, third, and ninth months after the onset of infection were evaluated. It was found that low-avidity IgG antibodies were detected in 15/15 (100%), 1/5 (20%), and 0/8 (0%) serum samples collected during the first, third, and ninth months after the onset of symptoms, respectively. Low-avidity antibodies of IgA and IgM subclasses were detected in 14/14 (100%) and 3/14 (21%) serum samples, respectively, collected in the first month after the onset of infection. However, IgA antibodies remained low in avidity in a proportion of patients even during late convalescence. As a consequence, IgG antibody avidity assays gave better discrimination between acute-phase and late-convalescent-phase serum samples than IgM, IgA, or IgGAM assays. In two of these patients, sequential serum samples were also tested for IgG avidity against human CoV strains OC43 and 229E in parallel. While SARS CoV infections induced an anamnestic IgG antibody response to the 229E and OC43 viruses, these cross-reactive antibodies remained of high avidity from early (the first month) postinfection. The results showed that assays to detect low-avidity antibody may be useful for discriminating early from late antibody responses and also for distinguishing anamnestic cross-reactive antibody responses from primary specific responses. This may be useful in some clinical situations.     

Laboratory diagnosis of congenital syphilis by immunoglobulin M (IgM) and IgA immunoblotting.

We screened cord blood or serum samples from 101 infants at risk for congenital syphilis and serum samples from their mothers for immunoglobulin G (IgG), IgM, and IgA antibodies to Treponema pallidum by western blotting (immunoblotting). Clinical evaluation showed that six infants had signs and/or symptoms consistent with congenital syphilis. The sera from five of these infants were IgM blot positive, and four were IgA blot positive. Four asymptomatic infants had serologic evidence of congenital syphilis. The sera from three of these infants were IgM blot positive, and two were IgA blot positive. However, the IgM reactivity of the serum from one asymptomatic infant, which was also IgA positive, was abolished by protein G treatment. An IgM capture enzyme-linked immunosorbent assay corroborated the presence of IgM antibodies in six of seven IgM blot-reactive sera. Overall, for detection of symptomatic congenital syphilis, a sensitivity of 83% for IgM blotting and 67% for IgA blotting was obtained. The significance of positive IgM or IgA Western blots for asymptomatic infants requires further study to confirm infection in these infants.     

Enzyme immunoassay for detection of immunoglobulin G (IgG), IgM, and IgA antibodies against type 6B pneumococcal capsular polysaccharide and cell wall C polysaccharide in chinchilla serum.

Conjugation of the capsular polysaccharides of Streptococcus pneumoniae to protein carriers has introduced a new generation of pneumococcal vaccines which may be efficacious in preventing pneumococcal otitis media during infancy. The chinchilla model has been used extensively for studying the pathogenesis of pneumococcal otitis media and for testing the efficacy of early pneumococcal capsular polysaccharide (PCP) vaccines, but immunologic studies in the chinchilla have been limited by the lack of antibodies against specific immunoglobulin isotypes. By using affinity-purified rabbit immunoglobulin G (IgG) anti-chinchilla IgG, IgM, and IgA, we developed a sensitive enzyme immunoassay that is highly specific for IgG, IgM, and IgA antibodies against type 6B PCP (anti-6B) and against C polysaccharide in chinchilla serum. Antibody titers increased in serum from five chinchillas immunized with a type 6B outer membrane protein complex vaccine. Increases of anti-6B IgG and IgM antibody titers were more striking than increases of anti-6B IgA or anti-C polysaccharide IgG, IgM, or IgA titers were.     

Evaluation of rubella virus immunoglobulin G (IgG) and IgM assays with the new Vidia instrument

For rubella virus immunoglobulin G (IgG) and IgM detection, Vidia assays were compared to Vidas, AxSYM, and Liaison assays with 419 serum samples. Only Vidia produced a sensitivity of 100% for IgG and IgM. Vidia specificities were 98.4% for IgG and 99.8% for IgM, versus Vidas specificities of 100 and 99.3%. Vidia IgG and IgM assays performed equally well.     

Rubella reimmunization: comparative analysis of the immunoglobulin G response to rubella virus vaccine in previously seronegative and seropositive individuals.

Rubella virus (RV)-specific immunoglobulin G (IgG) antibodies were studied in military recruits undergoing unselected immunization with live attenuated measles, mumps, and rubella virus (MMR) vaccine. Three different whole-RV enzyme immunoassays (EIAs) and an epitope-specific EIA with a synthetic peptide (BCH-178c) representing a heutralization domain on the RV E1 envelope protein were used. Before vaccination, 84.2, 87.7, and 84.5% of the subjects tested (n = 399) were found to be seropositive (> 10 IU/ml or assay equivalent) by the three whole-RV EIAs, respectively, while only 82.5% were seropositive by the BCH-178c EIA. Although prevaccination seropositivity rates were similar for the whole-RV EIAs (sensitivity, 94 to 100%), many sera considered seropositive by the whole-RV EIAs had E1 peptide EIA antibody levels of < 10 IU/ml (sensitivity, 77.4 to 80.7%). One month after vaccination, 97.8, 97.2, and 93.5% of the subjects who were followed (n = 356) were seropositive by the three whole-RV EIAs, respectively, while 89% had BCH-178c peptide-specific IgG titers of > 10 IU/ml. After vaccination, depending on the assay used, up to 20.6% of initially seropositive individuals exhibited a greater than fourfold increase in RV-specific IgG, while up to 47.3% showed a greater than twofold increase. Increased antibody titers after vaccination (seroboosting) were most frequently associated with low levels of BCH-178c peptide-specific IgG before vaccination. RV protein-specific IgG was also studied by immunoblot assays in a subset (n = 56) of individuals receiving the MMR vaccine. Of these, 89.4 and 91.1% exhibited RV protein (E1, E2, and C protein)-specific IgG before and after vaccination, respectively. Seroboosting (two- to fourfold increase in EIA titers of individuals seropositive by the whole-RV EIA before vaccination) was usually accompanied by a shift in the IgG immunoblot pattern from a single (E1) to multiple (E1-E1, E1-C, or E1-E2-C) specificities, suggesting exposure of new epitopes as a result of viral replication.     

Transfer of antirotaviral antibodies from mothers to their infants

Levels of rotavirus-specific immunoglobulin G (IgG), IgA, IgM, and secretory immunoglobulin in maternal and cord serum, colostrum and milk, and infants' stools were measured by enzyme-linked immunosorbent assay in 92 mothers and their infants. Although antirotaviral IgG, IgA, and secretory immunoglobulin were present in most maternal sera, only IgG crossed the placenta. All samples of colostrum and milk tested contained antirotaviral secretory immunoglobulin and IgA except those of two women in whom IgA deficiency was subsequently described. Specific IgM and IgG were also detected in many colostral samples. Antirotaviral IgA was detected in many colostral samples. Antirotaviral IgA was detected in stools of breast-fed but not bottle-fed neonates. Apparently the human infant receives rotaviral antibodies both transplacentally and via maternal colostrum and milk.     

Selective reactivity of antibodies to human immunoglobulins G, M, and A with rubella virus proteins

Proteins of purified rubella virus were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted with human sera and immunoglobulin class heavy-chain-specific peroxidase conjugates. The levels of rubella antibodies in these sera were predetermined by the radial hemolysis test, the density gradient centrifugation method for immunoglobulin M (IgM) antibodies, and IgG-, IgM-, and IgA-specific enzyme immunoassays. In immunoblotting, rubella-specific IgG antibodies reacted with both envelope glycoproteins (E1 and E2) and the capsid protein (C). In contrast, rubella IgM antibodies reacted predominantly with E1, whereas the specific reactivity of IgA antibodies was directed mainly to the capsid protein. Purified IgM rheumatoid factor added to IgG-positive, IgM-negative serum did not give false-positive reactivity in the immunoblotting test as it did in solid-phase enzyme immunoassays. The immunoglobulin class-specific reactivities with the different viral proteins are expected to have diagnostic applications.     

Comparative immunoglobulin G antibody profiles between mother and child (CGMC test) for early diagnosis of congenital toxoplasmosis

Early diagnosis of congenital toxoplasmosis is rendered difficult when specific immunoglobulin M (IgM) and/or IgA antibodies are absent in the blood of the newborn infant. Since maternal IgG antibodies can cross the placenta, determination of IgG antibodies in newborn infants has hitherto not been used routinely for the diagnosis of congenital infection. The aim of this study was to assess the diagnostic usefulness of an immunoblot assay which compares the early IgG profiles between the mother and her child (comparative IgG profile between mother and child; CGMC test) directed against a total cell lysate of Toxoplasma gondii tachyzoites. Serum samples from 97 newborn infants at risk of toxoplasma infection were obtained from umbilical cord blood at birth or postnatally until 3 months of life and were directly compared with serum samples from the respective mothers. Congenital toxoplasmosis was diagnosed only when IgG-reactive protein bands that were present in any newborn serum samples were absent in the corresponding maternal serum sample. Congenital infection was defined by conventional serological assays when IgM and/or IgA antibodies were present in newborn infant blood or when IgG titers rose within the first 12 months or were persistently stable for more than 8 months. Using these criteria, congenital infection was definitely confirmed in 11 cases. Three additional cases were diagnosed based on indicative data. The CGMC test, which was performed without knowledge of the results of conventional serologal assays, had sensitivity and specificity of 82.4 and 93.0%, respectively, and positive and negative predictive values of 73.7 and 95.7%, respectively. When true positives and true negatives were considered, the comparative IgG profile had a ratio of 90.9% true results. The CGMC test thus is useful as an additional assay for the rapid diagnosis of congenital toxoplasmosis when paired serum samples from mother and child are available.     

The separation of immunoglobulin M from human serum by fast protein liquid chromatography

A fast protein liquid chromatography (FPLC) system was evaluated as a method for rapid separation of serum immunoglobulin M (IgM) from immunoglobulin G (IgG) and immunoglobulin A (IgA). The system incorporates the use of a strong anion exchanger. Evaluation was carried out in 3 ways. The effect of increasing the serum percentage in the 500 microliters volumes loaded on to the column was tested. Samples containing up to 60% serum resulted in only small concentrations of contaminating IgG and IgA in the IgM fraction. Reproducibility was tested by fractionating the same serum sample several times; the coefficient of variation (CV) of the IgM concentration in the IgM fraction was 6%. A number of sera which varied considerably in immunoglobulin concentration were fractionated without any significant adverse effects on the immunoglobulin ratios in the IgM fraction. One serum sample containing a high concentration of IgG and IgA was included. In contrast to gel filtration chromatography, FPLC can separate IgM from IgG and IgA within 6 min. On loading 500 microliters samples containing from 20 to 60% serum, less than 0.01 g/l IgG was detected in the IgM fractions when tested by the radial immunodiffusion method.     

Detection of Immunoglobulin G and A Antibodies to Rubella Virus in Urine and Antibody Responses to Vaccine-Induced Infection

Urine and serum samples from 89 healthy volunteers and three healthy individuals who underwent rubella vaccination were tested for immunoglobulin G (IgG), IgA, and IgM to rubella virus (RV) by enzyme-linked immunosorbent assay methods. Subjects with positive (n = 68) or negative (n = 21) results for serum IgG were exactly the same as those with the corresponding results for urinary IgG. Both urinary and serum IgG levels remained elevated from the 3rd or 4th week after vaccination until the end of the study. Both urinary IgA and serum IgM levels tended to increase rapidly between the 3rd and 5th week and then gradually decrease until the end of the study, but the levels of both remained positive except for one sample each at the end (26th week). On the other hand, the ratio of anti-RV IgA titer to anti-RV IgG titer in urine (urinary anti-RV IgA/IgG ratio) increased rapidly between the 3rd and 4th week after vaccination and then rapidly returned to the ratio levels of the subjects positive for serum IgG from among the healthy volunteers. In summary, detection of urinary anti-RV IgG should be useful for screening for previous RV infection, and measurement of urinary anti-RV IgA/IgG ratio might be useful for diagnosing recent infection.     

Intranasal Immunization with Pneumococcal Polysaccharide Conjugate Vaccines Protects Mice against Invasive Pneumococcal Infections

Host defenses against Streptococcus pneumoniae depend largely on opsonophagocytosis mediated by antibodies and complement. Since pneumococcus is a respiratory pathogen, mucosal immune responses may play a significant role in the defense against pneumococcal infections. Thus, mucosal vaccination may be an alternative approach to current immunization strategies, but effective adjuvants are required. Protein antigens induce significant mucosal immunoglobulin A (IgA) and systemic IgG responses when administered intranasally (i. n.) with the glyceride-polysorbate based adjuvant RhinoVax (RV) both in experimental animals and humans. The immunogenicity and efficacy of pneumococcal polysaccharide conjugate vaccines (PNC) of serotypes 1 and 3 was studied in mice after i.n. immunization with RV. Antibodies were measured in serum (IgM, IgG, and IgA) and saliva (IgA) and compared to antibody titers induced by parenteral immunization. The PNCs induced significant systemic IgG and IgA antibodies after i.n. immunization only when given with RV and, for serotype 1, serum IgG titers were comparable to titers induced by subcutaneous immunization. In addition, i.n. immunization with PNC-1 in RV elicited detectable mucosal IgA. These results demonstrate that RV is a potent mucosal adjuvant for polysaccharides conjugated to proteins. A majority of the PNC-1-immunized mice were protected against both bacteremia and pneumonia after i.n. challenge with a lethal dose of serotype 1 pneumococci, and protection correlated significantly with the serum IgG titers. Similarly, the survival of mice immunized i.n. with PNC-3 in RV was significantly prolonged. These results indicate that mucosal vaccination with PNC and adjuvants may be an alternative strategy for prevention against pneumococcal infections.     

Antilipopolysaccharide antibodies and differential diagnosis of chronic Pseudomonas aeruginosa lung infection in cystic fibrosis

Chronic lung infection in cystic fibrosis is characteristically associated with polyagglutinable, serum-sensitive, mucoid strains of Pseudomonas aeruginosa. Enzyme-linked immunosorbent assay (ELISA) methods for standard-free quantitation of immunoglobulin G (IgG) and IgM antibodies to P. aeruginosa lipopolysaccharides (LPSs) have been developed. We now report the development of assays for quantitation of monomer and dimer total IgA and IgA anti-LPS antibodies. Use of these methods in diagnosis of early chronic P. aeruginosa lung infection was assessed. IgG and IgA anti-LPS levels increased significantly at the onset of chronic infection and continued to increase to very high levels in the later stages of infection. IgM anti-LPS levels also rose at the onset of chronic infection but did not increase further. The function of true- and false-positive rates was illustrated by using various concentrations of IgG, IgA, and IgM anti-LPS for discrimination of patients. Values that gave optimum separations were used for statistical evaluation of the diagnostic sensitivities and specificities of anti-LPS antibody concentrations. The results obtained in these assays were compared with a diagnosis, based on the number of precipitins in crossed immunoelectrophoresis, of serum samples from cystic fibrosis patients. In 64 paired serum samples taken before and immediately after the onset of chronic infection, as defined by crossed immunoelectrophoresis precipitins, the predictive values of a positive ELISA were 86% for IgG and 89% for IgA. The predictive values for a negative ELISA were 98% for IgG and 97% for IgA. Results of the IgM anti-LPS ELISA had a lower predictive value. Immunoblotting and absorption studies showed that IgG anti-LPS antibodies were directed specifically against LPS of P. aeuruginosa. ELISAs were developed to determine the specific IgG sublclasses involved. The increase in IgG anti-LPS involved all four subclasses. Highest anti-LPS titers were seen with IgG1 and IgG4, but the largest relative increases were seen with IgG2 and IgG3.     

Evaluation of monoclonal antibody-based capture enzyme immunoassays for detection of specific antibodies to measles virus.

Monoclonal antibodies to the hemagglutinin protein, fusion protein, phosphoprotein, matrix protein, and nucleoprotein of measles virus were evaluated as detector antibodies in capture enzyme immunoassays (EIAs) for the detection of specific serum immunoglobulin G (IgG), IgA, and IgM antibodies to measles virus. A pool of monoclonal antibodies to hemagglutinin protein and nucleoprotein proved optimal and was further evaluated. Specific IgM was detected in 97% of adolescents with clinical measles, 97% of infants 3 weeks postvaccination, and less than 1% of normal serum specimens. Specific IgA antibodies were found in 97% of adolescents with clinical measles, 97% of infants 3 weeks postvaccination, and less than 1% of normal serum specimens. Specific IgA antibodies were found in 97% of clinical measles cases and vaccinees, in 26% of healthy persons, and in 36% of infants 8 months postvaccination; consequently, IgA antibodies were not a useful indicator of recent measles infection. A significant increase in IgG antibodies between paired specimens was detected in 92% of clinical cases and all vaccinees. Only 59% of infant specimens had persistent IgG antibodies as detected by capture EIA at 8 months postvaccination, whereas all specimens had antibodies as detected by hemagglutination inhibition and plaque neutralization. An alternative indirect EIA, in which antigen was directly absorbed to the solid phase, was more sensitive than the capture design, detecting IgG antibodies in all infants postvaccination. When standardized with a microneutralization assay for the detection of persistent antibodies, the indirect IgG EIA gave predictive values for positive and negative tests exceeding 90%. Our capture IgM and indirect IgG EIAs provide a practical combination of serologic tests for the determination of acute measles virus infection and past exposure to measles virus or vaccine, respectively.     

Isolation of porcine immunoglobulins and determination of the immunoglobulin classes of transmissible gastroenteritis viral antibodies

The porcine immunoglobulins M (IgM), A (IgA), and G (IgG) were isolated and purified and some of the properties of the porcine milk IgA were examined. Monospecific antisera which were prepared against these immunoglobulins in rabbits were then used to absorb a particular class of immunoglobulin from sow serum, colostrum, and milk in an attempt to identify the immunoglobulin classes of neutralizing antibodies to the porcine enteric virus, transmissible gastroenteritis (TGE). The results of these absorption studies suggest that in colostrum and milk from sows experimentally (orally) or naturally infected with live virulent TGE virus, IgA is the predominant immunoglobulin class of TGE antibodies. Both IgA and IgG TGE antibodies appeared to be present in the serum from these sows, but with IgG TGE antibodies predominating. In contrast, in the serum, colostrum and milk from sows vaccinated intramuscularly or intramammarily with live attenuated TGE virus, the TGE antibody activity was associated mainly with the IgG class of immunoglobulins. These results provide additional data indicating that the route of infection or vaccination markedly influences the immunoglobulin class of antibodies in colostrum and milk. Secondly, IgA antibodies in mammary secretions are probably essential for providing optimal passive immunity of nursing pigs against infection with TGE virus.