Precipitin Antigen Research Articles

Discovery of a Unique Set of Dog-Seroreactive Coccidioides Proteins Using Nucleic Acid Programmable Protein Array.

Valley Fever (VF), caused by fungi in the genus Coccidioides, is a prevalent disease in southwestern and western parts of the United States that affects both humans and animals, such as dogs. Although the immune responses to infection with Coccidioides spp. are not fully characterized, antibody-detection assays are used in conjunction with clinical presentation and radiologic findings to aid in the diagnosis of VF. These assays often use Complement Fixation (CF) and Tube Precipitin (TP) antigens as the main targets of IgG and IgM reactivity, respectively. Our group previously reported evidence of over 800 genes expressed at the protein level in C. posadasii. However, antibody reactivity to the majority of these proteins has never been explored. Using a new, high-throughput screening technology, the Nucleic Acid Programmable Protein Array (NAPPA), we screened serum specimens from dogs against 708 of these previously identified proteins for IgG reactivity. Serum from three separate groups of dogs was analyzed and revealed a small panel of proteins to be further characterized for immuno-reactivity. In addition to CF/CTS1 antigen, sera from most infected dogs showed antibody reactivity to endo-1,3-betaglucanase, peroxisomal matrix protein, and another novel reactive protein, CPSG_05795. These antigens may provide additional targets to aid in antibody-based diagnostics.

An outbreak of infectious bursal disease in eight weeks old IBD vaccinated commercial poultry flock in Maiduguri, Nigeria

An outbreak of infectious bursal disease (IBD) on an IBD vaccinated commercial poultry farm was investigated using clinical and pathological findings as well as antigen and antibody detection by agar gel immunodiffusion test (AGDT). Mortality was observed 3 days after the onset of clinical signs and peaked on day 5 but declined significantly on day 6 after the onset of the clinical signs. The clinical and pathological lesions observed were those of hurdling anorexia, diarrhoea, hemorrhages on the thigh and breast muscles and on the gizzard-proventriculus junction as well as inflamed bursa. A morbidity rate of 90%, mortality rate of 53.3% and a case fatality rate of 59.3% were recorded. The sera collected on day 3 post infection were negative for IBD virus precipitin antibodies, while the sera obtained 15 days post-infection had IBD virus precipitin antibodies with titres ranging from 1:16 to 1:128. In addition, the tissue from infected bursae were all positive for IBD virus precipitin antigens with titres ranging from 1:2 to 1:32. The outbreak could have arisen from IBD vaccination failure. Keywords : infectious bursal disease outbreak, vaccination failure, agar gel immuno-diffusion test, Nigeria Tropical Veterinarian Vol. 22(3&4) 2004: 93-98

Cloning and expression of the gene which encodes a tube precipitin antigen and wall-associated beta-glucosidase of Coccidioides immitis.

We report the structure and expression of the Coccidioides immitis BGL2 gene which encodes a previously characterized 120-kDa glycoprotein of this fungal respiratory pathogen. The glycoprotein is recognized by immunoglobulin M tube precipitin (TP) antibody present in sera of patients with coccidioidomycosis, a reaction which has been used for serodiagnosis of early coccidioidal infection. The deduced amino acid sequence of BGL2 shows 12 potential N glycosylation sites and numerous serine-threonine-rich regions which could function as sites for O glycosylation. In addition, the protein sequence includes a domain which is characteristic of family 3 glycosyl hydrolases. Earlier biochemical studies of the purified 120-kDa TP antigen revealed that it functions as a beta-glucosidase (EC 3.2.1.21). Its amino acid sequence shows high homology to several other reported fungal beta-glucosidases which are members of the family 3 glycosyl hydrolases. Results of previous studies have also suggested that the 120-kDa beta-glucosidase participates in wall modification during differentiation of the parasitic cells (spherules) of C. immitis. In this study we showed that expression of the BGL2 gene is elevated during isotropic growth of spherules and the peak of wall-associated BGL2 enzyme activity correlates with this same phase of parasitic cell differentiation. These data support our hypothesis that the 120-kDa beta-glucosidase plays a morphogenetic role in the parasitic cycle of C. immitis.

Serological responses to various Coccidioides antigen preparations in a new enzyme immunoassay.

A new enzyme immunoassay (EIA) was used to investigate immunoglobulin M (IgM) and IgG responses to various coccidioidal antigen preparations. Direct binding and inhibition assays both revealed that the IgG responses of many serum specimens were directed largely to the tube precipitin (TP) antigen even when the microwells were coated with a combination of coccidioidal antigens (CCAs). From a panel of 28 serum specimens containing antibodies to CCAs, 4 serum specimens yielded high IgG signals (absorbance, greater than 1.0) and were negative for IgM (absorbance, less than 0.2), yet all four IgG responses were inhibited by at least 90% with partially purified TP antigens (2.4 micrograms/ml). The IgM and IgG fractions of several serum specimens were separated by ion-exchange chromatography and assayed by using different antigen preparations adsorbed to the microwells. The binding of both IgM and IgG peaks to the microwells coated with the CCA preparation was inhibited significantly by preincubation with TP antigens. One serum specimen (specimen 26) yielded a large IgG response (absorbance, greater than 2.0) with the CCA preparation and also bound directly to microwells coated only with TP antigens. The IgG signal (absorbance) of serum specimen 26 was reduced by 98% when it was preincubated with TP antigens prior to the assay. Significant IgG signals from several other serum specimens were observed when microwells were coated with TP antigen preparations, but they were absent when periodate-treated preparations were used. Two cerebrospinal fluid specimens yielded IgG signals with CCA-coated microwells, which were not inhibited by TP antigens, and yielded no signal with microwells coated only with TP antigens. The results are consistent with the concept that the typical serologic response to TP antigens occurs early in disease progression, but they suggest that TP antigens stimulate both IgM and IgG responses.

Purification and Amino-Terminal Sequence Analysis of the Complement-Fixing and Precipitin Antigens from Coccidioides immitis

Purification and Amino-Terminal Sequence Analysis of the Complement-Fixing and Precipitin Antigens from <i>Coccidioides immitis</i>

Purification and amino-terminal sequence analysis of the complement-fixing and precipitin antigens from Coccidioides immitis

Two proteins (21 and 48 kilodaltons) purified from endospore-spherule culture filtrates of Coccidioides immitis are identified as precipitin and complement-fixing antigens, respectively. To allow specific structural comparison to antigens identified by other investigators and as a first step to eventual serodiagnostic antigen production by recombinant DNA technology, amino-terminal amino acid sequences were determined for these antigens.

Composition, serologic reactivity, and immunolocalization of a 120-kilodalton tube precipitin antigen of Coccidioides immitis

Diagnosis of coccidioidomycosis largely depends on serologic tests. In this investigation, the enzyme-linked immunosorbent assay (ELISA) was used to detect patient immunoglobulin M (IgM) precipitin antibody binding to a 120-kilodalton (kDa) fraction previously isolated from an alkali-soluble, water-soluble extract of the arthroconidial wall and mycelial culture filtrate plus toluene lysate of Coccidioides immitis. Results of the serologic response to this tube precipitin antigen (TP-Ag) in the ELISA correlated well with results of immunodiffusion assays of 30 serum samples from patients. Immunoelectron microscopic examinations of arthroconidia and spherules were performed with patient IgM precipitin antibodies isolated from sera eluted over a solid-phase immunosorbent column containing the purified 120-kDa TP-Ag. The antibody probe located the 120-kDa TP-Ag on the walls of in vitro-grown arthroconidia and spherules. Pronase digestion and heating (100 degrees C, 5 min) had no apparent effect on the activity of the 120-kDa TP-Ag, while periodate oxidation resulted in total loss of its immunodiffusion-TP activity. Analysis of the carbohydrate composition of the TP-Ag revealed xylose, 3-O-methylmannose (3-O-MM), mannose, galactose, and glucose. Competitive inhibition ELISAs were used to demonstrate that 3-O-MM is largely responsible for the reactivity of IgM precipitin antibodies with the 120-kDa TP-Ag. Synthetic 3-O-MM may be a useful probe for detection of anti-Coccidioides precipitin antibodies in the ELISA.

Development and characterization of a monoclonal antibody against the tube precipitin antigen of Coccidioides immitis

Primary infection with Coccidioides immitis is commonly accompanied by the production of an immunoglobulin M precipitin antibody which is detected by the tube precipitin (TP) assay or by the immunodiffusion assay for TP antibody (IDTP assay). In the present investigation, spleen cells from spherulin-immunized BALB/c mice were fused with SP2/O Ag14 myeloma cells, and the resulting hybridomas were screened for antibody to the IDTP antigen by using an enzyme-linked immunosorbent assay. Positive hybridomas were cloned by limiting dilution and injected into pristane-primed mice for ascites production. Characterization of antibody reactivity was accomplished with the IDTP assay, two-dimensional immunoelectrophoresis, and immunoblotting. An immunoglobulin G1 monoclonal antibody which reacts with the IDTP antigen of C. immitis is described. The epitope that is recognized by the monoclonal antibody is also present, but to a lesser extent, on a second coccidioidal antigen which has been designated antigen 2. The monoclonal antibody was not reactive in immunoblots of histoplasmin or blastomycin, indicating that the epitope recognized by this antibody may be specific for C. immitis.

Detergent-Treated Newcastle Disease Virus as an Agar Gel Precipitin Test Antigen

A soluble Newcastle disease virus (NDV) agar gel precipitin (AGP) antigen was prepared by treating 100-fold concentrated NDV with a nonionic detergent. Virus concentration prior to detergent treatment was best accomplished by ultracentrifugation or by a simple, less expensive, and more practical method involving acid (HCl) precipitation of NDV. Virus concentrated by polyethylene glycol precipitation was found to have a low antigen titer and was not considered suitable as an AGP antigen.Antigens derived from the LaSota, Roakin, and Texas GB strains formed at least two lines of identity in the AGP test as early as 24 hr after inoculation of the agar gels. Virus used for AGP antigen production could be grown in chicken embryos from an NDV-immune as well as susceptible breeder flock. The NDV AGP antigen was found to be stable after 20 consecutive freezing and thawing cycles and storage at −20 C or 4 C for at least 6 months.Detergent-treated NDV was used as an AGP test antigen to determine serum antibody responses of chickens following infection and vaccination. Hemagglutination-inhibition, virus neutralization, and enzyme-linked immunosorbant assay antibody production was also evaluated for comparative purposes.The AGP test was found to be useful as an aid in diagnosing field infections and assessing inactivated virus vaccination responses. These purposes were achieved by demonstrating an increase in the number of AGP positive chickens between preinfection and postinfection or vaccination bleedings.The ease of performance and low cost of the AGP test favors its use for screening large numbers of serum samples, perhaps in conjunction with a quantitative serological test. Further, the AGP test may be useful in regions where limited facilities or technical capabilities preclude the use of other serological procedures.

Humoral antibody responses to specific antigens of Coccidioides immitis.

To identify antigens important to the diagnosis or pathogenesis of coccidioidomycosis, we analyzed three coccidioidal extracts by immunoblotting techniques with sera from patients infected with Coccidioides immitis and from immunized rabbits. When denatured fungal extracts were used, sera from at least 9 of 10 patients reacted with antigens of 100, 60, and 45 kilodaltons (kDa). By agar diffusion, the 100-kDa antigen appeared to be identical to the conventional tube precipitin antigen. Five of the six sera from patients with pulmonary syndromes reacted with another antigen of 70 kDa, but sera from patients with disseminated disease did not react with this antigen. Both the 100-kDa and the 70-kDa antigens were present in the 70-130-kDa region of the nondenatured fungal extracts. Our results suggest that the 100-kDa antigen may be useful in future diagnostic tests and that the 70-kDa antigen may identify patients whose infection is confined to the chest.

Preparation of hitherto undetected haemagglutinin and precipitin antigens of wanowrie (G-700) virus

Summary The presence of haemagglutinin and precipitin antigens in brain suspensions of mice inoculated with Wanowrie (G-700) virus, a tick-borne ungrouped arbovirus, is demonstrated. The method by which antigens for haemagglutination and agar gel precipitation were prepared is fully described and the significance of the findings discussed.

Counterimmunoelectrophoresis for Identification of Infectious Pancreatic Necrosis Virus After Isolation in Cell Culture

An isolate of IPNV identified by seroneutralization test as serotype VR-299 was produced in RTG-2 cells, purified by density gradient centrifugation, and used to prepare the immune serum. Parameters of the CIE technique were adapted for the IPNV and one precipitin line was observed either with the purified antigen or with known positive specimens. A greater number of fish organ homogenates were positive after multiple passages in cell cultures than by direct CIE examination. However, the precipitin antigen was detected in cell culture fluid at early passages. The use of CIE following virus isolation in cell culture is recommended for routine identification of IPNV.

Characterization of an Isolate of Mycoplasma WVU 907 Which Possesses Common Antigens to Mycoplasma gallisepticum

Antigenic characteristics of an isolate of mycoplasma WVU 907 were compared with Mycoplasma gallisepticum (MG) and Mycoplasma synoviae. Mycoplasma WVU 907 and MG share common agglutinating and precipitin antigens. Although hemagglutinin is an integral part of WVU 907, hemagglutinating-inhibited antibody was not detected in sera of chickens inoculated with WVU 907. The clinical symptoms observed in chickens inoculated with WVU 907 were mild. Viral infections of chickens helped spread WVU 907 to contact controls. WVU 907 was more resistant to tylosin than MG was.

Circumoval precipitin antigens for the diagnosis of schistosomiasis. I. Development of an antiserum reactive with Schistosoma mansoni eggs by the circumoval precipitin test.

A serum was developed which reacts with Schistosoma mansoni eggs by the circumoval precipitin (COP) test. This antiserum to COP antigens was prepared by coupling the COP immune complexes to protein A-Sepharose, isolating the complex with acid buffer, and immunizing rabbits with the eluate. The antiserum reacted with S. mansoni eggs, forming globular bleb and septate precipitates. It also reacted in gel diffusion with a soluble egg antigen preparation of S. mansoni (SEA). Of four SEA antigenic components precipitated one was clearly MSA1. The antiserum to the COP antigens had anti-parasite antibodies absorbable with lyophilized cercariae or eggs. Three human immunoglobulins (IgG, IgM, and IgA) were involved in the COP reaction.

Antigenic difference between intracellular and membrane antigens induced by herpesvirus of turkeys.

The difference between intracellular antigen (IA) and membrane antigen (MA) induced by herpesvirus of turkeys (HVT) was examined by immunofluorescence analysis. Convalescent sera from chickens infected with HVT (HVT convalescent serum) and hyperimmune sera from chickens immunized with partially pruified virus (anti-HVT particle serum) or with a major HVT soluble precipitin antigen (anti-common Ag serum) were used as the source of antisera to specify both antigens in these experiments. Absorption of a convalescent serum from HVT-inoculated chicken with IA positive cells, resulted in reduction of their reactivity to IA but not to MA. Differential absorption of the hyperimmune sera by IA positive cells and MA positive cells resulted in reduction of their reactivity to homologous cells but not to heterlogous cells which were used in the absorptions. When the relationship between the levels of anti-IA and anti-MA antibodies was examined by a blocking immunofluorescence test using 38 sera from chickens inoculated with HVT, no correlation was observed (p greater than 0.25). These results provide evidence that MA and IA are antigenically different from each other.

Use of the Agar-Gel Precipitin Test to Evaluate Broiler Breeder and Commercial Layer Flocks for Mycoplasma gallisepticum Infection

In the absence of Mycoplasma gallisepticum (MG) isolation, the importance of agar-gel precipitin (AGP) tests along with microhemagglutination-inhibition (mHI) and serum plate agglutination (SPA) tests for the evaluation of poultry flocks for MG infection was demonstrated. A good correlation between AGP, mHI, and SPA tests for the early confirmation of MG in a flock was observed, except for the breeder flocks from which WVU 907 was isolated. The serums of such breeder flocks were positive for SPA and AGP test but negative for the mHI test. Isolation of MG for confirmation of the status of the flock was unsatisfactory in the later stages of infection or when nonpathogenic organisms were present in appreciable numbers. In the absence of MG or M. synoviae (MS) isolation, demonstration of agglutinin, precipitin, and/or mHI antibodies in the serums of such flocks were confirmatory for MG or MS infection. The new isolate WVU 907 gave positive reactions on the SPA and AGP tests with MG antiserum. The isolate hemagglutinated chicken red blood cells (RBC) and induced low HI antibody levels (less than or equal to 1:20) when inoculated into chickens. Like MG, WVU 907 precipitin antigen was partially denatured by urea, Triton x-100, and sodium dodecyl sulfate treatment.

Inhibition of Human Cytomegalovirus by Rifampin

Replication of human cytomegalovirus (CMV) was inhibited by 50 mug/ml of rifampin. Nevertheless, a number of functions of CMV were still expressed in the presence of rifampin, including early cell rounding, and the development of immuno-fluorescent antigen, haemadsorption antigen, complement-fixing antigen and precipitin antigens. If rifampin was kept in the culture medium for longer than 48 h, infectious virus was not synthesized, but removal of rifampin resulted in restoration of virus titre within 24 h. In parallel with the restoration of infectivity, removal of the drug resulted in a sharp increase in macromolecular synthesis, first RNA and then virus DNA. The results suggest that rifampin blocks a stage in the production of m-RNA species.

Experimental reproduction and antibody inhibition of marble spleen disease of pheasants.

An extract of spleens from three pheasants affected with marble spleen disease was used as an intravenous inoculum to transmit the disease to pen reared pheasants (Phasianus colchicus x Phasianus torquatus). The disease was prevented by specific convalescent pheasant antiserum and by antiserum from turkeys that had recovered from hemorrhagic enteritis of turkeys. The causative agent of the disease passed through 0.22 mu filters, resisted chloroform and retained its precipitin antigen quality after propagation by bird passage. Filterability, chloroform resistance, antigenic characteristics and in vivo response to antibody strongly indicate that the causative agents of marble spleen disease is a virus very similar to the virus which causes hemorrhagic enteritis of turkeys.

Further studies on the antigenic properties and distribution of the putative bovine leukemia virus.

The C-type viruses found in long-term cultures. New Bolton Center (NBC) cell lines, of peripheral lymphocytes from leukemic cattle and in short-term cultures of bovine buffy coat(BC) cells share an immunofluorescent(IF)antigen detected in the cytoplasm of infected cells as well as an antigen demonstrable in gel diffusion experiments. Therefore the viruses from these cultures most likely represent different isolates of the putative bovine leukemia virus (BLV). The BLV precipitin antigen is analogous to the group specific (gs) antigens of the leukemia viruses of other species in that it is soluble, ether resistant, and apparently located within the virion. These observations, together with results showing that the specificity of the BLV precipitin antigen differs from that of the gs antigen of other mammalian leukemia viruses, indicate that the former antigen represents the intraspecies (gs-1) determinant of BLV. Antibodies to the precipitin viral antigen were found in 82% of cattle with leukemia and in 40% of clinically normal adult cattle in multiple-case herds. These groups of animals also had fluorescent antibodies to the virus, but with significantly higher frequencies (100% and 76%, respectively). On the other hand, in leukemia-free herds, precipitating antibodies were not found and the incidence of fluorescent antibodies was only 3%.

Effect of Passively Transferred Immunoglobulins on Marek's Disease

Early events in the pathogenesis of Marek's disease (MD) were studied in chicks with no passive MD antibodies, with congenitally derived parental antibodies, and with injected antibodies (immunoglobulins). MD responses in chicks inoculated with immunoglobulins at hatching were virtually identical to responses in chicks with parental antibodies. In vivo neutralization indices of 1.0 log1l lesionproducing-50% dose (LPD50) were obtained for cell-associated MD virus inocula in chicks with passive antibodies directed against either MD virus or an antigenically related herpesvirus of turkeys (HVT). Parallel studies with cell-free MD virus gave neutralization indices of 2.1 log1o LPD50. Latent periods (50% response times) for both lympholiferative lesions and for degenerative skin and bursal lesions were approximately 50-70% greater in antibody-free chicks. Also noted in MD-infected chicks with passive antibody were absence of bursal atrophy, delayed latent periods for precipitin antigen and cell-free virus production in the skin, and lower frequency of virus isolation from buffy coat cells. These data suggest possible mechanisms for the inhibitory role of passive antibody on the in vivo dissemination and primary oncogenic functions of MD virus.