Use Of Antigen Research Articles

Development and application of an ELISA for detecting antibodies to Salmonella enteritidis in chicken flocks

Enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of IgG antibody to Salmonella enteritidis in poultry flocks. A lipopolysaccharide (LPS) and heat-extracted (HE) antigen for use in the ELISA were evaluated together with the rapid slide test (RST), microagglutination test (MT) and the microantiglobulin (MAG) test. In experimentally infected specific pathogen free chickens, good correlation was seen between all tests although, generally, the MT and MAG detected antibody earlier and titres peaked earlier than the ELISAs. The LPS antigen detected antibody earlier than the HE antigen but the latter gave higher titres in the later stages of infection. Cross reactions were seen between S enteritidis and S typhimurium in the ELISAs although homologous reactions were always much higher. Antisera to S montevideo or S senftenberg gave weak positive reactions in both S enteritidis ELISAs. Serological and bacteriological examinations of representative samples from two commercial chicken flocks were carried out. In flock A the HE-ELISA and MAG test detected antibody in nearly all birds. The LPS-ELISA detected antibody in over 60 per cent of birds, while the MT and RST detected few seropositive birds. The whole blood test using the stained S pullorum antigen on the farm detected antibody in just under 25 per cent of the birds. S enteritidis was isolated from the organs of 25 per cent of the birds. All birds in flock B were seronegative by all tests; no salmonellae were isolated from the organs of these birds.

Immunoprecipitation studies with biotinylated Entamoeba histolytica antigens

The biotinylation of protein antigens for use in immunoprecipitation studies with parasite antigens is described. This technique was standardized to produce maximal labelling without compromising either the viability of the parasite or the antigenicity of labelled proteins. Live axenic Entamoeba histolytica trophozoites were optimally labelled by incubation of 10(7) parasites in 1 ml phosphate-buffered saline for 20 min at 37 degrees C with a final concentration of 10 mM N-hydroxy succinimido-biotin (NHS-B). Examination of antigens recognized by immunoglobulin from convalescent amoebic liver abscess patients and precipitated by protein A-Sepharose showed a general increase in immune response to amoebic antigens with time following treatment. Mass ratio mol. wts of immunoprecipitated antigens concur with those presented by other authors using alternate technology, in addition to recognizing antigens previously not identified by human sera. We therefore recommend biotinylation as a viable alternative to radiolabelling techniques for the study of parasite antigens and the humoral immune responses raised against them.

Crossed immunoelectrophoretic analysis ofMycobacterium paratuberculosis

Antigenic analysis of M. paratuberculosis revealed extensive cross-reactivity with M. avium; however, the number of cross-reactive antigens found was dependent on the strain of M. avium tested. One antigen was shown to be the common antigen while another appeared to be iron-regulated in its production. A commercial polyclonal antibody to M. paratuberculosis produced a CIE precipitin pattern comparable to that of the antibody produced for the present study. An antigen designated no. 6 was consistently precipitated by sera from cattle infected with M. paratuberculosis. This antigen exhibited complete cross-reaction with M. avium and partial cross-reaction with M. phlei. Among three commercially available complement fixation (CF) antigen that could be precipitated by M. paratuberculosis antibodies. A commercial antigen for use in an agar gel immunodiffusion test for Johne's disease diagnosis produced 12 precipitins with the M. paratuberculosis antibody, one of which was identical with antigen 6.

Comparison of adult somatic and excretory-secretory antigens in enzyme-linked immunosorbent assay for serodiagnosis of human infection with Paragonimus heterotremus

Adult somatic antigen extract of Paragonimus heterotremus was compared with excretory-secretory (ES) antigen in an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of human paragonimiasis. The absorbence values in ELISA using the adult somatic antigen were not significantly different from the values obtained using ES antigens ( P>0·05). The sensitivity of the ELISA using either antigen was 100%, but the specificity was 96% with somatic and 98% with ES antigens due to a cross reaction with fascioliasis sera. It appears that both somatic and ES antigens are effective antigens for use in serodiagnosis of human paragonimiasis heterotremus.

Comparison of two antigens for use in an enzyme-linked immunosorbent assay to detect African swine fever antibody

SUMMARY Two African swine fever virus (asfv) antigens were tested for use in an elisa to detect antibody to asfv. Antigens used were the cytoplasmic soluble fraction (cs-p) of infected cells grown in the presence of porcine serum and the semipurified viral structural protein VP73 (svp73). Both antigens were tested by elisa against 72 sera obtained during several asf field episodes and from asfv- inapparent carriers. Of the 72 sera, only 2.8% had positive results by elisa against cs-p antigen; 60% of positive-reacting sera (to both antigens) had higher elisa values when the cs-p antigen was used. Samples (with positive results) that reacted only to cs-p antigen had results confirmed by immunoblot analysis. Such sera reacted against asfv-infection proteins IP25, IP25.5, and IP30, but not against IP73. In time-course experiments to detect appearance of asfv-antibodies in infected miniature pigs, antibodies were detected by immunoblot analysis on postinoculation day (pid) 8. At that time, only the polypeptides IP25, IP25.5, IP30, and IP31 were recognized; IP73 and IP12 were first detected 3 and 4 days later, respectively. In the same experiments, asfv antibodies were detected by elisa, using cs-p or svp73 antigens, on pid 7 and 9, respectively. These results could explain the percentage of sera not having positive results by elisa using svp73 antigen, if the sera were obtained from asfv-infected pigs during the first days of infection before induction of antibody response against the IP73 protein. This feature makes the use of cs-p antigen advantageous in early serologic detection of asfv-infected pigs.

A subgroup-specific antigenic site in the G protein of respiratory syncytial virus forms a disulfide-bonded loop

An antigenic site (represented by 15 amino acids, residues 174 to 188, designated peptide 12) of the large glycoprotein G of respiratory syncytial virus was demonstrated to be subgroup specific in peptide enzyme-linked immunosorbent assay tests with murine monoclonal antibodies and human postinfection sera. The role of individual amino acids in this subgroup-specific site was determined by use of single-amino-acid-deletion sets of peptides. When monoclonal antibodies were reacted with the deletion sets, a broad amino acid dependence of 11 or 12 residues, Cys-176 (Ile-175 in subgroup B) to Cys-186, was found. Human postinfection sera exhibited a narrower reaction profile (for subgroup A, Cys-182 to Trp-183; for subgroup B, Cys-176 to Lys-183). Reduction of peptides on microtiter plates by treatment with dithiothreitol completely destroyed their antigenic activity in tests with monoclonal antibodies and human postinfection sera of subgroup B. A variant of peptide 12 containing all four cysteines of the G protein (represented by 16 amino acids, residues 172 to 187, designated peptide 12var) also was subgroup specific. We concluded that the activity of the antigenic site in tests with monoclonal antibodies for subgroups A and B appears to depend on intrapeptide disulfide bonds. Reactions with postinfection sera of subgroup B also may depend on a disulfide bond. In contrast, postinfection sera of subgroup A appeared to have the capacity to identify a subgroup-specific site in a linear form of the selected 15-amino-acid-long peptide. Treatment of peptides with dithiothreitol had no effect on their antigenic activity in tests with human postinfection sera of subgroup A. These findings have relevance for molecular engineering of peptide antigens for use in respiratory syncytial virus subgroup-specific site-directed serology.

Identification of a Theileria mutans‐specific antigen for use in an antibody and antigen detection ELISA

Purified piroplasms of Theileria mutans were used to immunize BALB/c mice to generate monoclonal antibodies (MoAbs). The MoAbs recognized an antigen of a relative molecular mass of 32 kDa in Western blots. This antigen was also recognized by sera from cattle which had recovered naturally from experimental tick-transmission or infections induced by the blood stages of T. mutans. The MoAbs did not react, in indirect immunofluorescence or enzyme-linked immunosorbent assays (ELISA), with the common haemoparasites of cattle, namely, T. parva, T. annulata, Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma congolense, T. vivax or T. brucei. An antigen capture ELISA was established with two of the MoAbs which recognized different epitopes on the 32 kDa molecule. Using this test it was possible to detect circulating antigens or immune complexes in sera collected from cattle during the acute or chronic phases of infection. When the purified 32 kDa protein was used as antigen in a micro-ELISA to detect circulating antibodies in both experimental and field cattle sera, it was found that the titres of antibodies ranged between 1:20 and 1:10,240. Results of this study indicate that the antigen and immune complex capture assays and the antibody detection ELISA can be complementary in the immunodiagnosis of acute and chronic T. mutans infections. Moreover, the tests are useful in the differential diagnosis of the disease and for epidemiological studies.

Detection of autoantibodies to ribosomal P protein using recombinant autoantigen in a quantitative immunoassay

Antibodies to ribosomal P proteins are markers for systemic lupus erythematosus (SLE) but are often missed by assays utilizing routine immunofluorescence or immunoprecipitation. Expression of autoantigenic sites encoded by complementary DNA (cDNA) clones offers an inexpensive source of antigen for use in quantitative immunoassays. Using a monospecific ribosomal P positive serum to screen a human placental lambda gt11 expression library, a cDNA clone was isolated which reacted with all anti-P human sera tested. Autoantibodies which were affinity purified from the expressed cDNA reacted with the 38 kDa ribosomal Po protein and reactivity was blocked by absorption with recombinant fusion protein. A stable beta-galactosidase fusion protein of 150 kDa was partially purified by a simple differential centrifugation method and used in an enzyme-linked immunoabsorbent assay to reliably quantitate anti-P responses, with a sensitivity and specificity equal to Western blotting. A survey of 84 normals and 151 patients with autoimmune diseases confirmed the high specificity of anti-P antibodies for SLE. The availability of cloned autoantigen will facilitate more detailed clinico-serologic correlations for anti-P antibodies.

Peptides containing epitopes of Plasmodium falciparum merozoite antigen for use in malaria vaccines, their derivatives and DNA sequences encoding them

Peptides containing epitopes of Plasmodium falciparum merozoite antigen for use in malaria vaccines, their derivatives and DNA sequences encoding them

Autoepitopes on La(SS-B): [formula omitted], L.J. McNeilage, A.D. Sturgess and G. Naselli, Walter and Eliza Hall Institute, Melbourne, (Australia)

Background: The detection of antibodies to La/SS-B, a nuclear RNA-binding protein in mammalian cells, aids in the diagnosis of Sjogren's syndrome and systemic lupus erythematosus (SLE). This is performed conventionally by immunoprecipitation using a crude splenic extract and more recently, by the more sensitive and rapid enzyme-linked immunosorbent assay (ELISA) which uses a purified La/SS-B antigen. The latter antigen is obtained from cellular extracts of the antigen or from bacterial cell lysates containing the recombinant antigen usually by affinity chromatographic method. Objective: To produce a La/SS-B antigen for use in ELISA that can be obtained easily and inexpensively without the need for extensive purification (including affinity chromatography). Study design: The antigen was produced as a fusion protein of the minor coat protein of M13 bacteriophage and used in this phage-associated form in an ELISA. La/SS-B cDNA derived from Hep-2 cells was cloned into the phagemid, pCANTAB-5E, and transfected to Escherichia coli. Phage clones selected for the presence of insert both by gene and antigenic analyses were used in the ELISA to detect anti-La/SS-B antibodies from patients with Sjogren's syndrome and SLE. Results: A phage clone was obtained which contained a La/SS-B cDNA fragment truncated at the C-terminal end (after base-pair 631). The phage-displayed antigen derived from this clone was obtained by precipitation of the phage particles from the bacterial culture supernatant with polyethylene glycoI. Used in the ELISA, this antigen detected 27 of 28 precipitin-positive sera and was negative for 50 control sera. The soluble (phage-free) form of the antigen was obtained from a nonsuppressor host as a cell lysate which could not be used in this form in an ELISA for antibody detection. It was useable, however, in Western blot analysis which confirmed the reactivity of the recombinant antigen. Conclusion: Phage-displayed antigens may be used in place of soluble forms of these antigens in detection assays which have the advantage that they are easy and inexpensive to produce.

Vector containing yeast arg3 regulatory region used for production of hepatitis B virus surface antigen for use in vaccine

Vector containing yeast arg3 regulatory region used for production of hepatitis B virus surface antigen for use in vaccine

Glutathione S-transferases — potential components of anti-schistosome vaccines?

Graham Mitchell discusses how work in his and other laboratories has suggested that glutathione S-transferases may be potential candidate antigens for use in multicomponent anti- Schistosoma vaccines.

Production of Eimeria tenella antigens for use as coccidiosis vaccine by irradiating cultured oocysts in potassium bichromate solution

Production of Eimeria tenella antigens for use as coccidiosis vaccine by irradiating cultured oocysts in potassium bichromate solution

Monoclonal antibodies that distinguish Trypanosoma congolense, T. vivax and T. brucei.

Monoclonal antibodies (MoAbs) were derived against in-vitro-propagated procyclic forms of Trypanosoma congolense, T. vivax, T. brucei brucei and T.b. rhodesiense in order to identify antigens for use in immunodiagnosis of African trypanosomiasis. The antibodies have been tested against procyclic and bloodstream form trypanosomes of 13 T. congolense, six T. vivax six T.b. brucei, four T.b. rhodesiense, five T.b. gambiense and three T. simiae isolates from different geographical areas by indirect immunofluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). The MoAbs raised against T.b. brucei reacted with all the brucei group of trypanosomes but not with T. congolense, T. simiae or T. vivax. Likewise, MoAbs against T. congolense reacted with T. congolense and T. simiae but not with any of the other species, while those against T. vivax reacted with T. vivax only. The antigens recognized by these MoAbs were present in lysates of bloodstream trypanosomes as well as midgut (for T. congolense and T. brucei) and epimastigote forms from infected Glossina morsitans centralis. There was no reactivity of the MoAbs with Theileria parva, Anaplasma marginale, Babesia bigemina or Plasmodium falciparum. These antibodies and the antigens they recognize should, therefore, prove useful in the development of assay systems for immunodiagnosis of African trypanosomiasis.

Caprine arthritis-encephalitis virus: an efficient method for the large scale production of serological antigens

Caprine arthritis-encephalitis virus (CAEV) was grown on a large scale in lamb corneal cell cultures. Antigens for use in two different CAEV agar gel immuno-diffusion tests were prepared from the same culture fluid. These antigens were separated on the basis of size by membrane filtration.

Immune response toTrichinellaepitopes: the antiphosphorylcholine plaque-forming cell response during the biological cycle

Phosphorylcholine (PC), an immunodominant component of the cell wall of certain bacteria, fungi and nematodes, is known to induce low anti-PC antibody levels during natural infection by Trichinella spiralis. This article reports a study in which spleen cells from BCF1 mice infected with Trichinella sp. larvae were found to produce large numbers of direct haemolytic plaques in response to PC conjugated to sheep red blood cells (SRBC) after muscle-encysted larvae had been killed by treatment with mebendazole. Inhibition of the response by PC-chloride, immunodiffusion and immunoelectrophoretic studies with the anti-PC IgA (TEPC-15) and anti-idiotype T15 serum assays showed the plaque-forming cell (PFC) response to be specific for PC. The absence of haemolytic plaques when unconjugated SRBC or TNP-SRBC were used as indicator cells ruled out involvement of a polyclonal response. Greatest anti-PC PFC response was found to be associated with a microsomal fraction designated FCp1, a particulate fraction behaving as a thymus-dependent antigen. The FCp1 fractions from all four strains of Trichinella employed induced anti-PC PFC responses when injected into mice. These results suggest that FCp1 is a suitable antigen for use in detailed studies of immune responses to Trichinella and related parasites.

The present status of serodiagnosis and seroepidemiology of schistosomiasis.

The literature of the past 4-5 yr on serodiagnosis and seroepidemiology of schistosomiasis is reviewed. A variety of assays with different antigens are being used for serodiagnosis. Several purified antigens appear to be sensitive and specific, but have little if any capability of indicating duration of infection, parasite burden, or effect of chemotherapy. The results of long-term posttherapy field studies indicate that serology has a role in monitoring control programs. Standardized serologic assays and the need for International Standard Reference Sera are emphasized. A standardized enzyme-linked immunosorbent assay based on the Falcon Assay Screening Test system (FAST-ELISA), and involving a standard reference serum pool, is suitable for both serodiagnosis and field studies. Measurement of circulating antigens as a parameter of active infection is considered to have increased potential, compared with antibody measurement, in management of clinical disease and in control programs. Recombinant DNA technology may be useful for producing standard antigens for use in assays measuring antibody or circulating antigen. Time-resolved immunofluorescence involving europium-labeled conjugates may provide the increased assay sensitivity needed for measurement of circulating antigen.

A New Approach to the Preparation of a Toxoplasma gondii Membrane Antigen for Use in ELISA

SummaryA selective Toxoplasma gondii antigen preparation consisting of extracted T. gondii tachyzoite cell membrane proteins arranged into submicroscopical particles (iscoms), was used as coating antigen in an ELISA (iscom ELISA). By means of sodium‐dodecyl‐sulphate polyacrylamid gel electrophoresis (SDS‐PAGE), the Toxoplasma iscoms were found to consist of 4 major proteins with estimated molecular weights of 50 kD, 44 kD, 32 kD and 20 kD. By immunoblot analysis using T. gondii positive sheep sera, 3 major antigenic components with estimated molecular weights of 50 kD, 44 kD and 32 kD were detected. 85 sheep sera submitted for routine T. gondii serology were used to compare the results in the iscom ELISA, the Toxoplasma IFAT (indirect fluorescent antibody test) and an ELISA (FT‐S ELISA) employing a soluble (cytoplasmatic) T. gondii antigen. The iscoms ELISA was found to be as sensitive as the other tests in detecting low levels of T. gondii antibodies and in discriminating positive and negative sera. In strongly positive sera, most likely also containing antibodies to intracellular components, the FT‐S ELISA gave considerably higher titres than did the iscom ELISA and the IFAT. Since positive FT‐S ELISA results have been recorded during recent monoinfection with Sarcocystis cruzi in calves, whereas no such cross‐reaction was observed in the iscom ELISA of IFAT (Uggla et al., 1987), it was concluded that the iscom ELISA combines the specificity of the IFAT with the objectivity and simplicity of the ELISA.ZusammenfassungEine selektive Toxoplasma gondii‐Antigen‐Präparation aus extrahierten Proteinen aus der Zellmembran von T. gondii‐Tachyzoiten, angeordnet in submikroskopischen Partikeln (iscoms, immuno stimulating complexes), wurde als Schicht‐Antigen in einem ELISA (iscom ELISA) verwendet.Mit Hilfe der Sodium‐Dodecyl‐Sulfat Polyacrylamid‐Gel‐Elektrophorese (SDS‐PAGE) wurde nachgewiesen, daß die Partikel aus vier Hauptproteinen mit Molekulargewichten von schätzungsweise 50 kD, 44 kD, 32 kD und 20 kD bestehen. Bei Verwendung von T. gondii‐positiven Schafseren in der Immunoblot‐Analyse wurden drei antigene Hauptkomponenten mit Molekulargewichten von schätzungsweise 50 kD, 44 kD und 32 kD ermittelt. 85 Schafseren, die für routinemäßige T. gondii‐Serologie vorgelegt waren, wurden unter Verwendung eines löslichen (cytoplasmatischen) T. gondii‐Antigens zu einem Vergleich der Ergebnisse im iscom ELISA, im Toxoplasma IFAT (indirekter Fluoreszenz‐Antikörper‐Test) und im konventionellen ELISA (FT‐S ELISA) herangezogen.Der iscom‐ELISA erwies sich als ebenso empfindlich wie die anderen Tests bei der Erfassung niedriger Antikörpertiter gegen T. gondii und bei der Unterscheidung positiver und negativer Seren. In stark positiven Seren, die höchstwahrscheinlich auch Antikörper gegen intrazelluläre Komponenten enthielten, ergab der FT‐S ELISA bedeutend höhere Titer als der iscom ELISA und der IFAT. Nachdem im FT‐S ELISA auch positive Ergebnisse bei einer frischen Monoinfektion mit Sarcocystis cruzi in Kälbern registriert wurden, während im iscom ELISA und IFAT keine derartige Kreuzreaktion zu beobachten war, wurde der Schluß gezogen, und der iscom ELISA die Spezifität des IFAT mit der Objektivität und Einfachheit des ELISA vereint.

Thermostable muscle antigens suitable for use in enzyme immunoassays for identification of meat from various species

AbstractA method for the extraction of thermostable muscle antigens for use in enzyme immunoassays is described. The method yields antigens devoid of contaminating proteins which reduce the adsorption of the antigens on to the plate. The effect of such proteins is stimulated by the addition of gelatin. Gelatin (5 mg ml−1) results in 100% inhibition of the antigen adsorption on to the plate.

Enzyme labeling of steroids by the N-succinimidyl ester method. Preparation of glucose oxidase-labeled antigen for use in enzyme immunoassay.

Enzyme labeling of a steroid with alkaline phosphatase by the N-succinimidyl ester method was investigated. The activated ester of 4-hydroxytestosterone 4-hemiglutarate was treated with alkaline phosphatase to give a labeled antigen. Various molar ratios of steroid to enzyme, ranging from 5 to 200, were employed. The loss of enzymic activity was less than 20% under the coupling conditions used. Satisfactory immunoreactivities with an anti-testosterone antiserum in the enzyme immunoassay procedure were obtained with the labeled antigens prepared at molar ratios higher than 20. The effect of steroid/enzyme molar ratio in the labeling on the sensitivity of the testosterone assay was then examined. It was found that the sensitivity of the assay is significantly influenced by the molar ratio, and a higher ratio results in a decrease in assay is significantly influenced by the molar ratio, and a higher ratio results in a decrease in assay sensitivity. A doserespared at a molar ratio of 30. The active ester method proved to be useful for the preparation of alkaline phosphatase-labeled antigens as well as for β-galactosidase and horseradish peroxidase labelings, because of its simplicity and excellent reproducibility.