Recombinant Antigen-based ELISA Research Articles

Testing for Lyme borreliosis: could serology tell more?

Purpose: Remnant antibodies might be prevalent in the general population, thus recognizing relevant seropositivity might be challenging. The sequential nature of the anti-Borrelia antibody response might be diagnostically utilized. We aimed to specify details which might potentially be useful in orientating the diagnostic schedule. Methods: We processed the sera of 1304 patients using a recombinant antigen-based ELISA between Aprils of 2017 and 2019. Seroreactivity (when coherent with the anamnestic data) was confirmed with a line immunoassay (LIA). ELISA testing (IgG and/or IgM) was reactive in 539 cases. 107 patients with persistent symptoms tested positive or borderline with IgG ELISA. Results: A significant difference was observed (Mann-Whitney U-test p=0.003) between the LIA scores of patients with characteristic (arthritis, acrodermatitis, neuropathy, other objective neurologic disorder; n=83; median LIA score: 16) and non-specific symptoms (entirely subjective complaints, other known disease, lone subfebrility, uveitis; n=24; median LIA score: 6). 101 of the 107 patients tested positive for IgG against any specific protein by LIA. Those with a LIA score reaching the group median of 15 (n=51) displayed strong anti-VlsE IgG positivity or a typical late IgG antibody (against p100, p18 or p39) more often than those below (88,2% vs. 30% and 100% vs. 38% respectively, Χ2 p<0.0001). Conclusions: Weak LIA positivity (especially anti-VlsE IgG) in combination with the lack of late antibodies in patients with persistent symptoms might suggest the presence of remnant antibodies and prompt scrupulous differential diagnosis.

Evaluation of the immunodiagnostic potential of a recombinant surface protein domain from Acanthamoeba castellanii.

Acanthamoeba spp. are free-living protists widely distributed in environment, able to cause keratitis, encephalitis and skin lesions in humans and animals. Acanthamoeba spp. exist in two forms: an infective trophozoite and a dormant cyst. Several factors contribute to the pathogenesis of Acanthamoeba spp. The parasite adhesion to the host cell is the primary step for infection and is mediated by a mannose binding-protein, expressed in the surface and considered the main pathogenicity factor in Acanthamoeba spp. So far, there was no evidence of another surface protein of Acanthamoeba spp. relevant for host invasion or infection by these organisms. The aims of this study were to identify and characterize an Acanthamoeba castellanii surface protein and to evaluate its diagnostic potential. In silico predictions of surface proteins allowed to identify the A. castellanii calreticulin as a possible surface antigen. The coding sequence of a predicted extracellular domain of A. castellanii calreticulin was cloned by in vivo homologous recombination and the recombinant polypeptide (AcCRT29-130) was produced. Its immunodiagnostic potential was assessed in a recombinant antigen-based ELISA with sera from experimentally infected rats that developed keratitis and encephalitis, and sera from patients with encephalitis. The AcCRT29-130 was significantly more recognized by sera from encephalitis infected rats in comparison with the non-infected controls. Human sera from encephalitis patients, however presented no significant response. These results showed the AcCRT29-130 potential for A. castellanii infection immunodiagnosis in animals, with further studies being required for assessment of its use for human infections.

Tandem repeat protein as potential diagnostic antigen for Trypanosoma evansi infection

Trypanosoma evansi infection (surra) causes significant losses in livestock production in tropical and sub-tropical areas. The current ELISA recommended by OIE for diagnosis of the disease is based on trypanosome lysate antigen. However, antigenic variation and unstable nature of cell lysate antigen make it difficult to standardize the assay. Thus, there are needs to develop recombinant antigen-based ELISA that improve stability, sensitivity, and specificity of the test. Since tandem repeat (TR) proteins of trypanosomatid parasites generally possess high antigenicity, they have been considered to be the promising antigens for trypanosomosis and leishmaniosis. In this study, IgG responses against 14 recombinant TR proteins of trypanosomes were examined by ELISA. Serum samples were obtained from three water buffaloes experimentally infected with T. evansi. Since Trypanosoma congolense GM6 (TcoGM6) elicited highest IgG responses to all water buffaloes, we further bioinformatically and molecular biologically identified Trypanosoma brucei brucei GM6 (TbbGM6) and T. evansi GM6 (TeGM6) TR genes, respectively. As expected, predicted amino acid sequences of TbbGM6 and TeGM6 were identical while the nucleic acid sequence homology between TbbGM6 and TcoGM6 was 63.8%. All buffaloes became clearly positive in recombinant TbbGM6 (rTbbGM6)-based ELISA at 48 days post-infection, suggesting that rTbbGM6 is usable as a serodiagnostic antigen for chronic T. evansi infection.

Seroreactivity against raw insect-derived recombinant KMPII, TRYP, and LACK Leishmania infantum proteins in infected dogs

The recombinant proteins KMPII, TRYP, and LACK of Leishmania infantum were produced in baculovirus-infected Trichoplusia ni larvae and used to analyze the seroreactivity of 165 dog serum samples by the multiple-well ELISA technique (57 infected dogs with clinical signs, 46 naturally infected and 11 experimentally infected; and 108 non-infected dogs, 76 from non-endemic areas and 32 from endemic areas). Recombinant (r) KMPII was the most recognized antigen, as the majority of infected dogs seroreacted against it (0.75). This is the first report of seroreactivity against rTRYP (0.51) and rLACK (0.42) in L. infantum-infected dogs, since previous studies using recombinant TRYP and LACK proteins produced in prokaryotic systems failed to detect specific seroreactivity. All non-infected dogs were negative for rTRYP and rLACK, and only one of the 32 from endemic areas seroreacted against rKMPII. The results demonstrate that L. infantum-infected dogs develop humoral immunity against rKMPII, rTRYP, and rLACK antigens. There was substantial agreement between crude total L. infantum antigen (CTLA)-based ELISA and rKMPII ELISA ( κ = 0.664), although this was higher than that found between the CTLA-based ELISA and rTRYP ( κ = 0.427) or rLACK ( κ = 0.343) ELISA, which can be interpreted as fair and moderate agreement, respectively. Ninety-three percent of the infected dogs analyzed developed specific antibodies against at least one of these three recombinant antigens. When the three recombinant antigen-based ELISA techniques were evaluated in parallel, almost perfect agreement ( κ = 0.880) with CTLA-based ELISA was observed, with a specificity of 0.97 and a sensitivity of 0.93 in relation to CTLA-based ELISA. Further studies using purified recombinant antigens in a single-well test or individually, depending on the objective of the study, are warranted.

Validation of a Recombinant Based Antibody ELISA for Diagnosis of Human and Canine Leishmaniasis

In this study, a recombinant chimeric antigen (CA) ELISA was validated as a single test for both human and dog leishmaniasis. Serum panels included 327 human and 339 canine IFAT-positive and 1113 human and 1078 canine IFAT-negative samples. CA-ELISA was carried out using the same serum dilution, and labelled protein A as secondary reagent. Test performances were calculated using ROC analysis. For the human panel, the test showed diagnostic accuracy (DA) 0.974, specificity (Sp) 97.12%, sensitivity (Se) 91.44%, and concordance (K) 0.88. The dog panel showed DA 0.998, Sp 99.54%, Se 98.54%, and K 0.98. The proposed method is the best recombinant antigen-based ELISA, and can be used as IFAT substitute for mass screening.

A Recombinant Antigen-Based Elisa For The Simultaneous Differential Serodiagnosis Of Mycoplasma Gallisepticum, Mycoplasma Synoviae, And Mycoplasma Meleagridis Infections

We have previously identified species-specific DNA fragments, referred to as MS2/28 and Mm14, of Mycoplasma synoviae and Mycoplasma meleagridis, respectively. In the present study, we extended our analysis of the MS2/28 fragment that was found to encode a species-specific antigenic site, and we demonstrated the specificity of the Mycoplasma gallisepticum hemagglutinin protein encoded by pMGA1.2 (a member of the vlhA gene family). Then, we combined the Escherichia coli-expressed products of MS2/28, Mm14, and pMGA1.2, to develop a recombinant antigen-based enzyme-linked immunosorbent assay (recELISA), for the simultaneous and specific detection of antibodies to the three aforementioned major avian mycoplasma species. For comparative purposes, a novel in-house crude antigen capture ELISA (capELISA) was developed in parallel. In the latter protocol, the microtiter wells were enriched in species-specific antigens by capturing sonicated crude antigens on coated rabbit polyclonal antibodies that had been extensively adsorbed with the whole antigen of the heterologous species. With regard to rapid serum agglutination, both ELISA tests were highly specific, and they showed a significant correlation when field sera from naturally infected birds were tested. recELISA proved to be highly specific because absorbance values, with the heterologous species, were significantly lower (P < 0.001) than those obtained with capELISA. Given its cost-effectiveness and simplicity, the recombinant antigen-based ELISA seems to represent a valid tool for the specific screening of the three major avian mycoplasma species. recELISA will be particularly useful with regard to trade control because a large number of samples from various fields could be rapidly processed.

Antibodies against hepatitis E virus in Old World monkeys.

To examine whether Indian monkeys are infected with hepatitis E virus (HEV) in nature, serum samples from wild rhesus (Macaca mullata), bonnet (M. radiata) and langur (Presbytes entellus) monkeys were screened for anti-HEV IgG antibodies in recombinant antigen-based ELISA assays. The positivity rates were 36.7%, 19.1% and 2% respectively. The protection of such antibodies against human HEV was studied in four rhesus monkeys. Of the two rhesus monkeys with anti-HEV titres of 100 and 1000 respectively which were inoculated with the KOL-91 strain of HEV, the former demonstrated a 10-fold rise in anti-HEV titres. Anti-HEV titre in the second rhesus monkey remained unchanged. Neither of the monkeys showed any rise in serum alanine transaminase (ALT) or presence of virus in the faeces, as tested by polymerase chain reaction (PCR). Two other rhesus monkeys with anti-HEV titres of 10,000 and 100 respectively were inoculated with the AKL-90 strain of HEV. Serum ALT levels and anti-HEV titres remained unchanged in the first monkey. Excretion of virus in faeces was not noted (PCR). The second monkey developed a typical HEV infection. HEV infection could be produced in anti-HEV negative control monkeys inoculated with both strains of HEV. These results show that either human or simian HEV, or a closely related agent, is circulating among Indian macaques. Titre-dependent protection of naturally occurring anti-HEV antibodies supports this view.

Immunity to onchocerciasis: identification of a putatively immune population in a hyperendemic area of Ecuador.

The existence of immunity to Onchocerca volvulus (Ov) infection is suggested by the presence of uninfected persons in hyperendemic areas. A major barrier to the study of immunity has been the correct identification of putatively immune (PI) subjects. To identify a PI group in a hyperendemic area in Ecuador, clinical and epidemiologic information was combined with a polymerase chain reaction (PCR)-based assay identifying Ov DNA in skin snips and a recombinant antigen-based ELISA. Comparison of immune responses revealed that PI subjects had significantly lower levels of Ov-specific IgG, IgG subclasses, and IgE than infected (INF) subjects. Female subjects were significantly more likely to be PI than male subjects, and INF female subjects had significantly lower levels of Ov-specific IgG, IgG1, and IgG3 than INF male subjects. Thus, the use of molecular-based techniques has helped to define more precisely the PI state in onchocerciasis.