Effectivity of crude versus purified mycobacterial secretory proteins as immunogen for optimum antibody production

Monospecific antibodies have been successfully utilized in antigen detection, which is better indicator of active infection. Mycobacterium tuberculosis excretory secretory (M tb ES) antigens such as ES 31, ES 41 and ES 43 (31 kDa, 41 kDa and 43 kDa protein, respectively) have been shown to be present in Mycobacterium tuberculosis H37Ra culture filtrate and are of diagnostic interest. To study the immunogenic potential of crude versus purified antigen, goat was immunized with M tb detergent soluble sonicate (DSS) antigen as well as purified antigen fraction (ESAS 7) containing ES 31 antigen. Both anti-DSS IgG antibody and anti ESAS 7 IgG antibody were found to be reactive with ES 31 antigen upto 1 ng concentration of antibody by ELISA. Crude DSS antigen was found to be quite effective in producing high titre antibodies and showed further high reactivity with other ES antigens (ES 41 and ES 43) of diagnostic interest.     

Evaluation of a recombinant excretory secretory Haemonchus contortus protein for use in a diagnostic enzyme-linked immunosorbent assay

The nematode Haemonchus contortus (H. contortus) is one of the most pathogenic and economically important parasites of sheep. A 24 kDa protein is one of the important components in H. contortus excretory/secretory (ES), which was shown to have important biological function. In our research, the cDNA of its open reading frame (ES24) was obtained and analyzed. Then the ES24 was sub-cloned into pET-30a expression vector. The recombinant vector that codes hexahistidyl peptide fusion protein (His-ES24) was transformed into Escherichia coli BL21 (DE3) strain. After induction, a high expression level of His-ES24 was found at 6h taking about 26% of the total bacterial protein analyzed by gel thin-layer scanning. The expressed His-ES24 was purified and then used in an enzyme-linked immunosorbent assay (ELISA) to detect specific antibodies in serum samples. The ELISA was able to differentiate between H. contortus-infected sheep serum and Fasciola hepatica-infected sheep serum or non-infected sheep serum. No cross-reaction was observed in sheep sera that have been experimentally infected with F. hepatica. A total of 153 field sheep serum samples conserved in our laboratory were examined using the His-ES24 ELISA, and 82 (53.6%) of them were found seropositive to H. contortus. Our results demonstrate that the prokaryotic-expressed His-ES24 might be a useful diagnostic reagent for epidemiological studies of H. contortus in sheep.     

Purification and partial immunochemical characterization of a low molecular mass, diagnostic Echinococcus granulosus immunogen for sheep hydatidosis

Abstract A hydatid specific antigen of 8 kDa molecular mass was affinity-purified from crude hydatid cyst fluid. Some of the epitopes recognised by antibodies in the sera from sheep with hydatidosis were periodate-sensitive. The purified 8 kDa antigen was observed to be a thermo-stable glycoprotein in its immunochemical characteristics. By immunofluorescence on acetone-fixed protoscolices anti-8 kDa monospecific IgG antibodies indicated the existence of the 8 kDa molecule on the hooklets of protoscolices. The purified antigen was used in an enzyme-linked immunosorbent assay for the detection of specific antibodies in sera from sheep hydatidosis. Eighteen (90%) of 20 sera from sheep hydatidosis had antibodies to purified 8 kDa antigen while none of the sera from other parasitic infections or uninfected animals had any detectable levels of antibodies to 8 kDa antigen. Thus, the data on localization and recognition of hydatid specific 8 kDa molecule suggested that this may be one of the major molecules for specific immunodiagnosis and for modulating the hydatid disease process in infected hosts.     

Antibody profiles by EITB and ELISA of cattle and sheep infected with Fasciola hepatica

In evaluating potential mechanisms of immunity in fascioliasis we compared the time-course analysis of the antibody responses between a resistant (cattle) and a susceptible model (sheep). Sera from sheep and cows experimentally infected with F. hepatica were reacted with both somatic (FhWWE) and excretory-secretory (ES) antigens in order to evaluate the antibody repertoires in the 2 different hosts. Analysis of these sera by ELISA showed a significant increase in antibody levels by 2 wk in most infected cattle using both somatic and ES antigens, whereas with most infected sheep antibodies are not clearly detected until week 4. By EITB, both infected sheep and cows recognize major somatic polypeptides in a molecular weight range of 30-38 kDa by 8 wk. Cattle recognized 3 additional major antigens of 56, 64, and 69 kDa as early as 6 wk. Various polypeptides of 20-25 kDa are prominently detected by most sheep and very faintly, if at all, by the cow sera. The sera from both sheep and cows also identify ES polypeptides of 20-28 kDa. The patterns of polypeptides recognized by sheep infected with S. mansoni and challenged with F. hepatica in EITB are almost identical to those with a simple F. hepatica primary infection. No significant differences were detected in the antibody kinetics in ELISA between these 2 groups. Differences and similarities between these models could eventually help determine which antibodies may be predictive of resistance or susceptibility in fascioliasis.     

Purification and characterization of E. histolytica antigen for diagnosis of amoebiasis

Entamoeba histolytica soluble crude antigen was fractionated by gel filtration on Sephacryl S-300 into four fractions, viz. F1(669 kDa); F2(51.2 kDa); F3(25.1 kDa) and F4(10.5 kDa). F1 fraction was observed to be more sensitive and specific for the detection of antibody in amoebiasis than the crude and other fractions of purified antigens employing IHAT and ELISA. ELISA was found to be better than IHAT since it could detect antibody in the sera (3/6) of asymptomatic cyst passers. The cross reaction of crude antigen with toxocariasis (1/4) and toxoplasmosis (2/5) sera were associated with F4 fraction. F3 and F4 were having low molecular weight and were not sensitive in detection of antibody in amoebiasis. Biochemical characterization revealed glycoprotein nature of the specific (F1) antigen fraction.     

Purification of antigenic protein of sparganum by immunoaffinity chromatography using a monoclonal antibody

The quality improvement of antigen (crude saline extract) of Spirometra mansoni pleroceroid (sparganum) was investigated by protein purification. The crude extract was fractionated by gel filtration through Sephacryl S-300 Superfine. Its third fraction was purified by affinity chromatography using a monoclonal antibody as ligand. When observed by SDS-PAGE, the purified protein was composed of 2 bands of 36 kDa and 29 kDa which were found already as the most sensitive components in the crude extract by immunoblots with patients sera. The quality of the purified antigen was evaluated in comparison with the crude extract by enzyme-linked immunosorbent assay (ELISA) for the specific (IgG) antibody in sera of human sparganosis, other parasitic and neurologic diseases, and normal control. When the purified antigen was used, the sensitivity was not altered but remained high (96.4%) while the specificity was increased from 86.8% to 96.9%.     

Kinetics of antibody response by Dot-ELISA in rabbits immunized with adult Haemonchus contortus antigen

Whole adult soluble extract of Haemonchus contortus as an antigen along with Freund's complete adjuvant, was used to immunize rabbits. Antisera from immunized rabbits were collected at intervals of 30, 60, 90, 120, 150 and 180 days. For the detection and titration of anti-H. contortus antibodies in these sera, Dot-ELISA was developed. Sera collected 30 days post-immunization exhibited a titre of 1:5,000 in all the rabbits except one, where a titre of 20,000 was recorded. Later, all the rabbits attained the highest titre of 40,000 at different periods of post-immunizations, which were maintained 150-180 days. These high titre sera can be of immense use in the identification and characterization of immunodominant antigens of adult H. contortus.     

Western blot antibody determination in sera from patients diagnosed with Anisakis sensitization with different antigenic fractions of Anisakis simplex purified by affinity chromatography

Using Western blot techniques, the specificities of crude and purified (PAK and PAS) Anisakis simplex antigens were compared against 24 sera from patients diagnosed with Anisakis sensitization. All patients recognized a 60 kDa protein against the A. simplex crude extract, while 37.5% and 12.5% reacted with proteins of 40 and 25 kDa, respectively, when IgG was tested. In the case of IgE determination, 41.6% of sera were negative, while 12.5% and 20.8% appeared to cross-react against Toxocara canis and Ascaris suum, respectively. When the PAK antigen (A. simplex antigen purified by means of a column of IgG anti-A. simplex) was tested, immune recognition towards the 60, 40 and 25 kDa proteins increased in 83.3%, 16.7% and 4.2%, respectively, when the Ig antibodies were tested. In the case of the PAS antigen (PAK antigen purified by means of a column of IgG anti-A. suum), the reaction against the 40 and 25 kDa proteins increased to 45.8% and 25%, respectively, when Ig antibodies were used. Finally, when the EAS antigen (eluted from the anti-A. suum column after PAK purification) was tested, 83.3% of the assayed sera reacted against the 14 kDa protein, when the Ig antibodies, IgG and IgM immunoglobulins were measured. With the IgE determination, the reactions were observed in 41.7% of patients with proteins between 60 and 35 kDa against the PAS antigen. With the EAS antigen, reactive bands of 184, 84 and 14 kDa appeared. In conclusion, in the purification process of the A. simplex larval crude extract, the proteins implicated in cross-reactions with Ascaris and Toxocara were eliminated, with an important concentration of proteins responsible for the induction of specific responses.     

Characterization of a Toxocara canis species-specific excretory-secretory antigen (TcES-57) and development of a double sandwich ELISA for diagnosis of visceral larva migrans

This study describes the isolation of a Toxocara canis species-specific excretory-secretory (ES) antigen and the development of an enzyme-linked immunosorbent assay (ELISA) based on this antigen. Analysis of the ES antigens of T. canis, Toxocara vitulorum, Ascaris lumbricoides and Necator americanus larval antigen was performed by SDS-PAGE followed by western blotting. A 57 kDa T. canis-specific antibody fraction (TcES-57) was identified by western blotting and labelling with anti-Toxocara antibodies (from experimental rabbits and human patients) and tracing with anti-human or anti-rabbit peroxidase conjugate. No protein fraction of 57 kDa was detected in ES or larval antigens collected from T. canis, T. vitulorum, A. lumbricoides and N. americanus. Using TcES-57, a specific antiserum was produced in rabbits and a double sandwich ELISA was developed. This test was validated using known seropositive sera from toxocariasis patients, sera from A. lumbricoides or N. americanus patients, and 50 serum samples from cats. These tests revealed that TcES-57 antigen is specific to T. canis infection and does not cross react with sera of other related infections. Thus, ELISA based on TcES-57 antigen was proven to be an effective tool in the diagnosis of toxocariasis and studies on the role of T. canis in the epidemiology of human toxocariasis.     

Analysis of excretory-secretory and somatic antigens of Gastrothylax crumenifer

The excretory/secretory (ES) metabolic products released by Gastrothylax crumenifer (Trematoda: Digenea) during in vitro incubations and the somatic extract of the adult parasite were analysed using polyacrylamide gel electrophoresis (PAGE). Immunogenicity of ES and somatic extracts were evaluated by immunoblotting and ELISA using sera raised against ES and somatic antigens in rabbits. The electropherograms of ES and somatic extracts have been resolved into 38 and 41 polypeptides, respectively. The apparent molecular weights of these polypeptides range from < 29 to > 205 kDa. A total of 14 polypeptides were found to be common to both of the samples. The analysis of immunoblot results revealed 22 and 27 antigenic polypeptides in ES and somatic extracts respectively. Only 11 and 13 antigenic polypeptides were found specific to ES and tissue extract respectively. The molecular weights of these specific polypeptides were calculated to be < 14.4, 16, 20, 25, 33, 42, 119, 125 and > 205 kDa for metabolic products and < 14.4, 25, 30, 35, 78, 84 and > 205 kDa for the tissue extracts, respectively. Analysis of ELISA results revealed that a dilution of up to 1:3200 of the test sera could react with the ES product. Further, when the ES antigens were allowed to react with antisomatic extracts in hyperimmune sera the titre of IgG increased up to a dilution of 1:12800. The potential importance of these antigens in the immunodiagnosis of amphistomiasis is discussed.     

Gastric parietal cell antigens of 60-90, 92, and 100-120 kDa associated with autoimmune gastritis and pernicious anemia. Role of N-glycans in the structure and antigenicity of the 60-90-kDa component

Thirty-four human sera containing parietal cell autoantibodies (PCA) specifically immunoprecipitated two antigens, with apparent molecular masses of 60-90 kDa and 100-120 kDa under nonreducing conditions and 60-90 kDa and 120-150 kDa under reducing conditions, from porcine gastric membrane extracts. A third antigen of 92 kDa was only observed in immunoprecipitates analyzed under reducing conditions. By immunoblotting, 24 of the 34 PCA-positive sera reacted with only the 60-90-kDa antigen, three reacted with a broad 60-120-kDa smear, one reacted only with a 92-kDa antigen and six did not react. Reactivity with the 60-90-kDa antigen was observed with gastric membranes from dog, pig, rat, and rabbit. Twenty PCA-negative sera did not react with these components by immunoprecipitation or immunoblotting. PCA reactivity with the 60-90-kDa antigen was abolished when the gastric membranes were (a) digested with Pronase, (b) reduced with 100 mM dithiothreitol, (c) treated with sodium periodate, or (d) digested with N-glycanase. The 60-90-kDa and 100-120-kDa components were insensitive to neuraminidase treatment. N-glycanase digestion of 125I-labeled antigens purified by immunoprecipitation and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis collapsed the 60-90-kDa antigen to a sharp 34-kDa band; the 100-120-kDa component was unaffected. These observations suggest that (i) parietal cell antigens comprise three components of 60-90, 92, and 100-120 kDa; (ii) the epitopes differ in conformational sensitivity; (iii) the 60-90-kDa antigen is a conserved molecule comprising a 34-kDa core protein extensively glycosylated with N-linked oligosaccharides; (iv) sialic acid residues are not present in the 60-90- and 100-120-kDa molecules, and (v) the carbohydrate and protein moieties of the 60-90-kDa molecule are required for antibody binding.     

Analysis of excretory-secretory and somatic antigens of Gastrothylax crumenifer

The excretory/secretory (ES) metabolic products released by Gastrothylax crumenifer (Trematoda: Digenea) during in vitro incubations and the somatic extract of the adult parasite were analysed using polyacrylamide gel electro-phoresis (PAGE). Immunogenicity of ES and somatic extracts were evaluated by immunoblotting and ELISA using sera raised against ES and somatic antigens in rabbits. The electropherograms of ES and somatic extracts have been resolved into 38 and 41 polypeptides, respectively. The apparent molecular weights of these polypeptides range from <29 to > 205 kDa. A total of 14 polypeptides were found to be common to both of the samples. The analysis of immunoblot results revealed 22 and 27 antigenic polypeptides in ES and somatic extracts respectively. Only 11 and 13 antigenic polypeptides were found specific to ES and tissue extract respectively. The molecular weights of these specific polypeptides were calculated to be <14.4, 16, 20, 25, 33, 42, 119, 125 and > 205 kDa for metabolic products and <14.4, 25, 30, 35, 78, 84 and > 205 kDa for the tissue extracts, respectively. Analysis of ELISA results revealed that a dilution of up to 1:3200 of the test sera could react with the ES product. Further, when the ES antigens were allowed to react with antisomatic extracts in hyperimmune sera the titre of IgG increased up to a dilution of 1:12800. The potential importance of these antigens in the immunodiagnosis of amphistomiasis is discussed.     

Single step purification of potent antigenic protein from sparganum by gelatin-affinity chromatography

Out of many component proteins in crude saline extract of Spirometra mansoni plerocercoid (sparganum), 36 kDa and 29 kDa proteins were found to be the most antigenic and were already purified by immunoaffinity chromatography using monoclonal antibody as a ligand. In this study, a single step purification of these potent antigenic proteins of sparganum extract was investigated. When the crude saline extract was charged to gelatin-Sepharose 4B affinity column, 36 kDa and 29 kDa protein fractions were bound. SDS-polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE/immunoblot confirmed that the bound protein to gelatin was serologically pure. When evaluated by ELISA with patients sera, the purified protein of 36 and 29 kDa also showed improved antigenicity.     

Evaluation of immunodiagnostic antigens in the excretory-secretory products of Fasciola hepatica

The metabolic antigens of F. hepatica have been shown to be a source of potential immunodiagnostic antigens. We have fractionated F. hepatica excretory-secretory (ES) antigens by conventional gel filtration and HPLC, analyzed these fractions in PAGE, and evaluated their immunogenicity by ELISA with sera from experimentally infected rabbits to identify potential serodiagnostic antigens for fascioliasis. A fraction enriched in high molecular weight components of ca. 150-160 kDa was found to be very reactive with sera from early fascioliasis. This fraction was successfully adapted to the DOT-ELISA, where titers up to 1:16,000 still appeared visually as positive. Both acute and chronic fascioliasis sera also recognized, in the enzyme-linked immunoelectrotransfer blot technique (EITB), prominent 25-30-kDa polypeptides that have previously been shown to be recognized by infected rabbits, cows, and sheep. We have therefore employed conventional gel filtration and HPLC gel exclusion chromatography as a 1-step procedure to obtain fractions enriched in antigens recognized in early fascioliasis. In addition, these antigens have been successfully applied to a sensitive, visual immunodiagnostic technique that can be easily employed in field studies.     

Comparative diagnostic potentiality of ELISA and dot-ELISA in prepatent diagnosis of experimental Fasciola gigantica infection in cattle

A glycoprotein (27 kDa) was isolated from crude somatic antigen of Fasciola gigantica by two steps affinity chromatography and was used in early detection of experimental fasciolosis in cattle by indirect ELISA and in dot-ELISA formats. Although, anti-27 kDa antibodies could be detected after 3 weeks post infection (WPI) by dot - ELISA which was one week later than indirect ELISA. The test, dot-ELISA, was more convenient in field application. By the test (dot-ELISA) the infection could be equally detected in animals infected with 100, 200 and 300 metacercariae of F. gigantica with high sensitivity. Further, the antigen (27 kDa) was not found to react with goat sera infected with Paramphistomum epiclitum, which are giving strong reaction to homologous immature and mature fluke antigens of P. epiclitum.     

Amblyomma americanum: physiochemical isolation of a protein derived from the tick salivary gland that is capable of inducing immune resistance in guinea pigs

Crude salivary gland derived proteins from Amblyomma americanum ticks were analyzed by physiochemical (gel filtration and ion exchange chromatography) and immunochemical guinea pig IgG1 (anti-tick immunoaffinity column) techniques for the presence of antigens responsible for the induction of host immune resistance responses. Gel filtration (G-75 Sephadex) and ion exchange (diethyl aminoethyl cellulose) chromatography of crude salivary gland antigen yielded multiple fractions, but only one fraction from each procedure induced significant cutaneous anaphylaxis bluing reactions when used for skin tests in tick sensitized animals treated intravenously with 0.5% Evans blue dye. Salivary gland antigen (200 ng) eluted from the immunoaffinity column by 0.2 M Na2CO3, pH 11.3, and emulsified with incomplete Freund's adjuvant conferred a significant level of tick rejection (24%, P less than 0.001) on naive guinea pigs compared with that seen in controls, but less than (P less than 0.01) the level of immunity conferred by crude salivary gland antigen (380 micrograms). The immunizing dose of immunoaffinity purified salivary gland antigen was 1/1900 the dose of the crude antigen preparation representing 99.9% purification. Furthermore, engorged ticks from animals immunized with salivary gland antigen exhibited a significant decrease (P less than 0.001) in weight compared with ticks from naive animals. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I labeled proteins in the Na2CO3 eluate and the skin reactive fraction from gel filtration and ion-exchange chromatography, after immunoprecipitation with a guinea pig IgG1 antibody to the tick that transferred resistance, revealed the presence of a 20 kDa weight protein reported previously to be the antigen responsible for the induction of host resistance. These studies present physiochemical and immunochemical procedures for the purification of an important tick protein that induces skin reactions in tick sensitized guinea pigs, is recognized by antibody to the tick, and most importantly, is capable of immunizing naive guinea pigs against tick challenge.     

Idiotype stimulation of T lymphocytes from Trypanosoma cruzi-infected patients

Anti-Trypanosoma cruzi epimastigote antibodies (anti-epi) from pooled and individual sera from patients with chronic Chagas' disease were purified on immunoaffinity columns of epimastigotes antigens (epi) coupled to activated Sepharose 4B. SDS-PAGE analysis of purified anti-epi preparations showed only the presence of human IgG H and L chains. These antibodies preparations showed similar Western blotting profiles as the sera pools from which they originated. The main polypeptides recognized by anti-epi had apparent molecular masses 31, 46, 51, 75 and 85 kDa. No difference in these patterns were detected between anti-epi from pooled sera of cardiac (anti-epiC) and indeterminate (anti-epiI) clinical forms. Anti-epi preparations (20 to 60 micrograms/ml) of pooled and individual sera stimulated proliferation of homologous and autologous PBMN or T-lymphocyte-enriched population. The stimulatory ability was dependent upon the PBMN-anti-epi combinations. There is no direct correlation between the level of PBMN response to epi and anti-epi stimuli. Comparison of the stimulatory activities of anti-epiC vs anti-epiI on PBMN of either cardiac or indeterminate group of patients indicate that anti-epiC is significantly more active than anti-epiI (p less than 0.025). These data demonstrate the presence of auto-anti-idiotypic-T cells in chagasic patients and lead to the possibility that idiotype/anti-idiotype interactions may play a role in determining the pathogenesis of chagasic cardiopathy.     

Enzyme-linked immunosorbent assay and Western blot antibody determination in sera from patients diagnosed with different helminthic infections with Anisakis simplex antigen purified by affinity chromatography

An evaluation of the sensitivity and the specificity of the Anisakis simplex antigens purified by affinity chromatography was performed using sera from patients diagnosed with Anisakis sensitisation and sera from patients previously diagnosed with different helminthic infections. Only the sera of the patients diagnosed with Schistosoma mansoni or Onchocerca volvulus parasitic infections were negative against the A. simplex antigen and its purified fractions (PAK antigen: A. simplex antigen purified using columns prepared with anti-A. simplex rabbit IgG and PAS antigen: PAK antigen purified using columns prepared with anti-Ascaris suum rabbit IgG). However all the sera were positive against the A. suum antigen. In all the sera from the patients diagnosed with Anisakis sensitisation, the antibody levels detected using the purified antigens (PAK and PAS antigens) were lower than the observed using the A. simplex crude extract with the highest diminution in the case of the IgG. When these same sera were tested against the A. simplex crude extract by Western blot, several bands of high molecular masses were observed as well as, intense bands at 60 and/or 40 kDa. A concentration of these last proteins was observed in the PAK and the PAS antigens. When the sensitivity and the specificity determinations were performed, only seven of the 38 patients diagnosed of Anisakis sensitisation were positive, as well as, the sera from the patients diagnosed with parasitisms by Echinococcus granulosus or Fasciola hepatica.     

Avidity analysis of the human immune response to a chitin binding protein of Toxoplasma gondii.

A 30-33 kDa electroeluted fraction of T. gondii tachyzoites improved discrimination between acute and chronic phase sera when used instead of the whole tachyzoite extract in an avidity-ELISA. In order to identify the components of these fractions, crude tachyzoite antigen was fractionated by anionic exchange chromatography. The 30-33 kDa antigen cluster eluted in the not-bound fraction could account for a large proportion of the antibody response against the 30-33 kDa electroeluted fraction. According to the N-terminal sequence data, this antigen fraction is composed mainly of SAG1 and another protein with high homology to chitin binding proteins from plants.