Antigen Cocktail Research Articles

An evaluation of different vaccine models to protect mice from visceral leishmaniasis

A soluble cocktail vaccine was prepared from sonicated promastigotes of five Iraqi Leishmania isolates with some adjuvants (BCG and Alum), was used experimentally to protect Balb/c mice against visceral leishmaniasis. Groups of mice were immunized with a soluble cocktail of antigens given in 75 μg /0.1 ml of phosphate buffer saline with booster at different intervals. Full protection was observed when mice were challenged one month post vaccination ; the challenged mice were rechallenged after six weeks. At the eighth week post challenge, the results exhibited complete resistance to the rechallenge (no parasites in culture media or impression smears of liver and spleen were noticed). A long run follow-up was performed. Five of the twelve vaccinated mice developed infection, which resolved by 16 weeks. Significant cellular and humoral response (Delayed hypersensitivity, Immunoglobulin levels, Lymphocyte subsets, Cytokine levels, Eosinophil cationic protein and Macrophages migration inhibition) to Leishmania donovani were demonstrated in all of the groups that were subjected to 75 μg of antigens. Protein bands of crude antigens of Leishmania isolates by SDS - PAGE and Western blotting and the specific protein bands for leishmanial cocktail was determined to be 18 k Da. Results of vaccination with 75 μg antigens and booster doses revealed that immunization against L.donovani could be a practical method of protection from visceral leishmaniasis.

UV-inactivated vaccinia virus (VV) in a multi-envelope DNA-VV-protein (DVP) HIV-1 vaccine protects macaques from lethal challenge with heterologous SHIV

The pandemic of HIV-1 has continued for decades, yet there remains no licensed vaccine. Previous research has demonstrated the effectiveness of a multi-envelope, multi-vectored HIV-1 vaccine in a macaque-SHIV model, illustrating a potential means of combating HIV-1. Specifically, recombinant DNA, vaccinia virus (VV) and purified protein (DVP) delivery systems were used to vaccinate animals with dozens of antigenically distinct HIV-1 envelopes for induction of immune breadth. The vaccinated animals controlled disease following challenge with a heterologous SHIV. This demonstration suggested that the antigenic cocktail vaccine strategy, which has succeeded in several other vaccine fields (e.g. pneumococcus), might also succeed against HIV-1. The strategy remains untested in an advanced clinical study, in part due to safety concerns associated with the use of replication-competent VV. To address this concern, we designed a macaque study in which psoralen/ultraviolet light-inactivated VV (UV VV) was substituted for replication-competent VV in the multi-envelope DVP protocol. Control animals received a vaccine encompassing no VV, or no vaccine. All VV vaccinated animals generated an immune response toward VV, and all vaccinated animals generated an immune response toward HIV-1 envelope. After challenge with heterologous SHIV 89.6P, animals that received replication-competent VV or UV VV experienced similar outcomes. They exhibited reduced peak viral loads, maintenance of CD4+ T cell counts and improved survival compared to control animals that received no VV or no vaccine; there were 0/15 deaths among all animals that received VV and 5/9 deaths among controls. Results define a practical means of improving VV safety, and encourage advancement of a promising multi-envelope DVP HIV-1 vaccine candidate.

Development of an ultrasensitive polymerase chain reaction–amplified immunoassay based on mycobacterial RD antigens: implications for the serodiagnosis of tuberculosis

Immuno-polymerase chain reaction (I-PCR) combines the versatility of enzyme-linked immunosorbent assay (ELISA) with the exponential amplification power of PCR. The present study was designed to detect antibodies to Mycobacterium tuberculosis complex–specific region of difference (RD) antigens, i.e., early secretory antigenic target-6, culture filtrate protein-10, culture filtrate protein-21, and mycobacterial protein from species tuberculosis-64, as well as antigens in pulmonary tuberculosis patients by I-PCR assay. We could detect ESAT-6 and other RD antigens up to 0.1 fg by I-PCR assay, thus resulting in 10 7 times higher sensitivity than that observed with ELISA. With paired sample analysis based on the detection of antibodies in serum and antigens in sputum of the same individual, the sensitivity of RD multi-antigen cocktail-based I-PCR assay was 72% in smear-negative cases and 91% in smear-positive cases of pulmonary tuberculosis with high specificity values. In extrapulmonary tuberculosis patients, higher sensitivity was observed by detecting cocktail of antigens by I-PCR assay as compared to sensitivity earlier observed in the same samples by ELISA.

SEVA TB ELISA – Multi antigen and antibody assays for serodiagnosis of suspected cases of pulmonary and extra pulmonary tuberculosis in tertiary care hospital –A retrospective study

ObjectiveTo assess the usefulness of in-house developed multiantigen and antibody assays, in diagnosis of both pulmonary and extra-pulmonary tuberculosis. MethodClinically suspected cases of 31 pulmonary and 171 extra-pulmonary tuberculosis (TB) were screened by ELISA using cocktail (ES-31 + EST-6) antigen and their specific antibodies (anti ES-31 + anti EST-6 IgG) for detection of antibody and and antigen (circulating antigen and immune complexed antigen) respectively and correlated with antituberculosis therapy in retrospective study. ResultsOut of 31 cases of pulmonary TB screened, 15 patients showed ELISA positivity out of which five cases were given antituberculosis therapy. Out of 171 cases of EPTB screened, 76 cases showed ELISA positivity out of which 18 were given antituberculosis therapy. Further 4 EPTB cases which showed AFB negativity were given ATT. The data was further analyzed based on PTB & EPTB, adults and children, OPD and IPD patients to understand false positivity in clinically suspected PTB and EPTB cases. There was significant correlation (108/202 cases) with ELISA negativity and no ATT advised in clinically suspected PTB and EPTB patients. ConclusionsIn house developed multi antigen and antibody assays have been observed to be quite useful as adjunct test in serodiagnosis of suspected cases of tuberculosis in particular extrapulmonary tuberculosis.

Search forMycobacterium aviumSubspeciesparatuberculosisAntigens for the Diagnosis of Paratuberculosis

The aim of this study was to evaluate a wide panel of antigens of Mycobacterium avium subsp. paratuberculosis (MAP) to select candidates for the diagnosis of paratuberculosis (PTB). A total of 54 recombinant proteins were spotted onto nitrocellulose membranes and exposed to sera from animals with PTB (n = 25), healthy animals (n = 10), and animals experimentally infected with M. bovis (n = 8). This initial screening allowed us to select seven antigens: MAP 2513, MAP 1693, MAP 2020, MAP 0038, MAP 1272, MAP 0209c, and MAP 0210c, which reacted with sera from animals with PTB and showed little cross-reactivity with sera from healthy animals and animals experimentally infected with M. bovis. The second step was to evaluate the antigen cocktail of these seven antigens by ELISA. For this evaluation, we used sera from animals with PTB (n = 25), healthy animals (n = 26), and animals experimentally infected with M. bovis (n = 17). Using ELISA, the cocktail of the seven selected MAP antigens reacted with sera from 18 of the 25 animals with PTB and did not exhibit cross-reactivity with healthy animals and only low reactivity with animals with bovine tuberculosis. The combined application of these antigens could form part of a test which may help in the diagnosis of PTB.

Comparative Evaluation of MPT83 (Rv2873) for T Helper-1 Cell Reactivity and Identification of HLA-Promiscuous Peptides in Mycobacterium bovis BCG-Vaccinated Healthy Subjects

MPT83 (Rv2873), a surface lipoprotein excreted in the culture of Mycobacterium tuberculosis, is immunoreactive in antibody assays in humans and animals and provides protection as a combined DNA vaccine in mice and cattle. This study was undertaken to determine the reactivity of MPT83 in T helper 1 (Th1)-cell assays, i.e., antigen-induced proliferation and gamma interferon (IFN-γ) secretion, using peripheral blood mononuclear cells (PBMCs) obtained from Mycobacterium bovis bacillus Calmette-Guérin (BCG)-vaccinated and/or M. tuberculosis-infected healthy subjects. PBMCs were tested with complex mycobacterial antigens and pools of synthetic peptides corresponding to MPT63, MPT83, MPB70, LppX, PPE68, CFP10, and ESAT-6. The results showed that MPT83 is among the strongest Th1 cell antigens of M. tuberculosis, and it was recognized equally strongly by BCG-vaccinated and by BCG-vaccinated and M. tuberculosis-infected healthy subjects. Furthermore, HLA heterogeneity of the responding donors suggested that MPT83 was presented to Th1 cells by several HLA-DR molecules. The analysis of the mature MPT83 sequence (amino acids [aa] 1 to 220) and its 14 overlapping synthetic peptides for binding prediction to HLA class II molecules and actual recognition of the peptides by PBMCs from HLA-DR-typed subjects in antigen-induced proliferation and IFN-γ assays suggested that Th1 cell epitopes were scattered throughout the sequence of MPT83. In addition, the HLA-promiscuous nature of at least three peptides, i.e., P11 (aa 151 to 175), P12 (aa 166 to 190), and P14 (aa 196 to 220), was suggested by HLA-DR binding predictions and recognition by HLA-DR heterogeneous donors in Th1 cell assays. These results support the inclusion of MPT83 in an antigen cocktail to develop a new antituberculosis vaccine.

Assessment of in vivo and in vitro tuberculosis diagnostic tests in Mycobacterium caprae naturally infected caprine flocks

Caprine tuberculosis in Spain is mainly caused by Mycobacterium caprae although the progression of the disease and lesion severity is similar to that caused by Mycobacterium bovis. In this study, the sensitivity of the gamma-interferon (IFN-γ) assay using an antigen cocktail containing early secretory antigenic target-6 kDa (ESAT-6) and culture filtrate protein 10 (CFP-10) peptides for stimulation was determined and compared with those obtained in single intradermal tuberculin (SIT) and single intradermal cervical comparative tuberculin (SICCT) tests and IFN-γ assay using purified protein derivative (PPD) in three different flocks infected with M. caprae under different epidemiological conditions. Correlation between specific IFN-γ production and severity of lesions was also evaluated. Sensitivities of the diagnostic tests varied greatly in the three flocks studied, with higher values in those where higher lesion scores were observed. The results show that IFN-γ assay applied in goats using PPD or the ESAT-6/CFP-10 peptides cocktail for stimulation yielded similar sensitivity values. A significant yet weak positive correlation between specific IFN-γ production and lesion scores was detected after the stimulation with PPDs ( p = 0.004) whereas when the blood samples were stimulated with ESAT-6/CFP-10 peptides, the correlation was not significant ( p > 0.05). Therefore, specific-IFN-γ production after the stimulation with PPDs or ESAT-6/CFP-10 was not an accurate indicator of lesion severity in naturally tuberculosis infected goats with M. caprae.

Evaluation of the Abbott ARCHITECT Chagas prototype assay

Serologic tests are established tools for the diagnosis of Chagas disease applied to support a safe blood supply in endemic countries. However, sensitivity and specificity of most commercially available enzyme-linked immunosorbent assays (ELISAs) are not regarded as adequate enough to rely on a single assay to determine the Trypanosoma cruzi infection status of a blood donor or a patient. The overall assay performance is driven by the general choice of antigens and the actual antigen cocktail provided in the test. In this report we describe key performance data of the Abbott ARCHITECT Chagas prototype assay in comparison to the well-recognized bioMérieux ELISA cruzi assay. The ARCHITECT assay demonstrated superior specificity (99.99% versus 99.93%) and sensitivity (99.85% versus 98.38%), along with excellent precision, thus showing the potential to serve as single assay to determine the T. cruzi status of a given blood unit or diagnostic specimen on a fully automated instrument platform.

Cross-reactivity of anti-Salmonella egg-yolk antibodies to Salmonella serovars

The cross-reactivity of egg yolk antibodies specific to antigens of Salmonella Enteritidis and Salmonella Typhimurium to killed bacterial cells of common Salmonella serovars were tested using an indirect Enzyme Linked Immunosorbent Assay (ELISA). Egg yolk antibodies were produced against purified fimbriae, flagella and lipopolysaccharide (LPS) of S. Enteritidis strain ATCC13076 and flagella, LPS and outer membrane proteins (OMP) of S. Typhimurium strain ATCC13311. For immunological specificity of egg yolk antibodies against killed bacterial cells, we found that the titers of the anti-S. Enteritidis egg yolk antibodies were higher than those of the anti-S. Typhimurium antibodies. In the evaluation of cross-reactivity of these egg yolk antibodies to various Salmonella serovars, we observed that the anti-S. Enteritidis antibodies exhibited more specific affinity than those of the anti-S. Typhimurium antibodies. All S. Enteritidis strains reacted specifically with the anti-S. Enteritidis fimbrial and flagellar egg yolk antibody whereas anti-S. Enteritidis LPS and anti-S. Typhimurium LPS, OMP and flagellar antibodies displayed non–specific reactivity to all Salmonella serovars used in this study. This finding suggests that it may be possible to design a anti-fimbrial egg yolk antibody of S. Enteritidis as a diagnostic tool and a cocktail of OMP and LPS antigens of S. Enteritidis and S. Typhimurium could be used for administering broad spectrum passive immunity to protect against the colonization of pathogenic Salmonella strains in food animals.

Evaluation of the cocktail recombinant antigens, LipL32 and LipL41 for serodiagnosis of canine leptospirosis

Leptospirosis is one of the diseases with economic impact, so its diagnosis and serosurveillance are very important for any control program. Many tests have been used in the field as screening tests of leptospirosis. The aim of this study was to evaluate the efficacy of combined recombinant antigens (rLipL41 + rLipL32) for serodiagnosis of canine leptospirosis and to compare the efficacy of IgG Enzyme linked immunosorbent assay and Latex agglutination test with standard MAT. A total of 533 canine serum samples were subjected to IgG-ELISA and LAT using recombinant LipL41 and LipL32 antigens in a single and in a combinations which were compared with standard MAT. The potential diagnostic cocktails of combined recombinant antigens (rLipL31 + rLipL41) developed in this study showed higher sensitivity and specificity in IgG ELISA and LAT (94.84, 88.50%; 98.21, 97.7%) when compared to the use of single recombinant antigen in this study. Results indicated that the both assays can be easily performed, and avoids the risk of infection in laboratory workers, and it seems to be a practical and suitable tool for serodiagnosis of leptospirosis.

Outbreak investigations and genetic characterization of foot-and-mouth disease virus in Ethiopia in 2008/2009

The study was conducted in three regional states of Ethiopia: Amhara, Oromia, and Addis Ababa from August 2008 to April 2009 with the objectives of identifying the genetic diversity of serotypes and topotypes in Ethiopia, and determining the attack rate and associations of potential risk factors with foot-and-mouth disease (FMD) seropositivity. A total of 496 cattle were clinically and serologically examined for presence of specific lesions and nonstructural protein for FMD, respectively. Of which, 140 (28.2%) manifested clinical signs and lesions suggestive of FMD, and 219 (44.2%) were seropositive. From a total of 7,781 animals observed and recorded on a designed format in six districts, 1,409 (19.6%) were infected, and 15 (0.12%) died during outbreaks of FMD. Epidemiological investigations revealed that the morbidity rate of the disease was 21.1% in Akaki-kality sub-city, but the mortality rate was <2% in all districts. Furthermore, the mortality and case fatality rates were relatively higher, 1.6% and 8.9% in calves than the other age groups, respectively. From a total of 33 bovine epithelial tissue-cultured samples, 19 (57.6%) showed CPE for FMD virus, in which 16 samples had serotype O and EA-3 topotype, while three samples had found serotype A, Africa topotype, and G-VII strain. Various strains of FMD viruses were isolated in Ethiopia in this study, and therefore, further detailed studies on the evaluation of available vaccines and the development of a vaccine which contains cocktails of antigens of FMD virus strains in the country should be encouraged.

Prospective study of SEVA TB peroxidase assay for cocktail antigen and antibody in the diagnosis of Tuberculosis in suspected patients attending a tertiary care hospital located in rural area

Objective To evaluate inhouse developed SEVA TB peroxidase enzyme immunoassay using cocktail of mycobacterial excretory-secretory antigens (ES-31, ES-43 & EST-6) for antibody detection and their affinity purified antibodies for antigen detection in tuberculosis suspected patients. Methods Inhouse developed SEVA TB peroxidase enzyme immunoassay was evaluated prospectively in 73 suspected pulmonary and 46 extra-pulmonary tuberculosis patients during November 2008∼March 2009 in a tertiary hospital located in rural area. Results Assay on prospective analysis showed 100% correlation of pulmonary tuberculosis (PTB) and extra-pulmonary tuberculosis (EPTB) acid fast bacilli positivity and antitubercular treatment in 11 cases. Thirty nine PTB and 12 EPTB cases showed negative for ELISA test and were also not given antitubercular therapy. However 30 PTB and 27 EPTB cases showing ELISA positivity were neither acid fast bacilli positive nor antitubercular therapy treated. These cases may possibly have dormant infection and need further diagnosis. In EPTB cases ELISA was observed to be more useful than AFB smear test. Conclusions This inhouse developed user-friendly peroxidase ELISA can be used as an adjunct test of smear microscopy or culture techniques for routine screening of patients suspected of PTB or EPTB.

Attempts to immunize sheep against Haemonchus contortus using a cocktail of recombinant proteases derived from the protective antigen, H-gal-GP

The objective of this study was to determine whether an antigen cocktail containing recombinantly expressed versions of most of the protective proteases of H-gal-GP, a known protective antigen from Haemonchus contortus, would confer any protection to lambs in a vaccine-challenge trial. Haemonchus contortus metalloendopeptidases, MEP1, MEP3 and MEP4, were expressed as soluble recombinant proteins in insect cells, but attempts to express the H. contortus aspartyl proteases, PEP1 and PEP2, by the same techniques were not successful. Recombinant H. contortus PEP1 was therefore expressed in Escherichia coli and refolded. Groups of sheep were immunized thrice with either native H-gal-GP, a cocktail of recombinantly expressed proteins (rMEP1, rMEP3, rMEP4 and rPep1), or adjuvant only (QuilA in PBS). All sheep were challenged with 5000 infective larvae 1 week after the final vaccination. High levels of serum antibodies that recognized H-gal-GP were detected in both the native antigen and recombinant cocktail-immunized groups by the time of challenge, but protective immunity was only observed in the group immunized with native H-gal-GP.

Serodiagnostic Efficacy of Mycobacterium tuberculosis 30/32-kDa Mycolyl Transferase Complex, ESAT-6, and CFP-10 in Patients with Active Tuberculosis

Elimination of tuberculosis (TB) largely depends upon definitive rapid diagnosis and treatment. Widely used diagnostic tests do not qualify for use in a developing country due to lack of either desired accuracy or their cost. In the present study an enzyme-linked immunosorbent assay was used to evaluate the diagnostic potential of an immuno-dominant 30/32-kDa mycolyl transferase complex (Ag85 complex) and Mycobacterium tuberculosis-specific proteins (ESAT-6 and CFP-10) of the RD1 region. Higher sensitivity (84.1%) with Ag85 complex was observed compared with ESAT-6 (64.9%) and CFP-10 (66%), with almost similar specificity (Ag85: 85.2%, ESAT-6: 88.9%, CFP-10: 85.2%), whereas the individual components of Ag85 complex, i.e. Ag85A, Ag85B, and Ag85C, showed sensitivities of 44.6, 34, and 80.9% and specificities of 55.6, 74.1, and 40.7% respectively. A cocktail of Ag85 complex, ESAT-6, CFP-10, Ag85A, Ag85B, and Ag85C antigens also could not help in increasing either sensitivity (51.1%) or specificity (85.2%). Furthermore, immunoblot analysis using clinical isolates as well as a standard strain (H37Rv) of M. tuberculosis also showed strong reactivity of sera from TB patients to Ag85 complex and, to a lesser extent, also to ESAT-6. To conclude, use of Ag85 complex along with ESAT-6 and CFP-10 seems to be promising in minimizing the heterogeneous sero-responses of adult TB cases.

Potential Target Antigens for a Universal Vaccine in Epithelial Ovarian Cancer

The prognosis of epithelial ovarian cancer (EOC), the primary cause of death from gynaecological malignancies, has only modestly improved over the last decades. Immunotherapeutic treatment using a cocktail of antigens has been proposed as a “universal” vaccine strategy. We determined the expression of tumor antigens in the context of MHC class I expression in 270 primary tumor samples using tissue microarray. Expression of tumor antigens p53, SP17, survivin, WT1, and NY-ESO-1 was observed in 120 (48.0%), 173 (68.9%), 208 (90.0%), 129 (56.3%), and 27 (11.0%) of 270 tumor specimens, respectively. In 93.2% of EOC, at least one of the investigated tumor antigens was (over)expressed. Expression of MHC class I was observed in 78.1% of EOC. In 3 out 4 primary tumors, (over)expression of a tumor antigen combined with MHC class I was observed. These results indicate that a multiepitope vaccine, comprising these antigens, could serve as a universal therapeutic vaccine for the vast majority of ovarian cancer patients.

Genetic variation in Staphylococcus aureus surface and immune evasion genes is lineage associated: implications for vaccine design and host-pathogen interactions

BackgroundS. aureus is a coloniser and pathogen of humans and mammals. Whole genome sequences of 58 strains of S. aureus in the public domain and data from multi-strain microarrays were compared to assess variation in the sequence of proteins known or putatively interacting with host.ResultsThese included 24 surface proteins implicated in adhesion (ClfA, ClfB, Cna, Eap, Ebh, EbpS, FnBPA, FnBPB, IsaB, IsdA, IsdB, IsdH, SasB, SasC, SasD, SasF, SasG, SasH, SasK, SdrC, SdrD, SdrE, Spa and SraP) and 13 secreted proteins implicated in immune response evasion (Coa, Ecb, Efb, Emp, EsaC, EsxA, EssC, FLIPr, FLIPr like, Sbi, SCIN-B, SCIN-C, VWbp) located on the stable core genome. Many surface protein genes were missing or truncated, unlike immune evasion genes, and several distinct variants were identified. Domain variants were lineage specific. Unrelated lineages often possess the same sequence variant domains proving that horizontal transfer and recombination has contributed to their evolution. Surprisingly, sequenced strains from four animal S. aureus strains had surface and immune evasion proteins remarkably similar to those found in human strains, yet putative targets of these proteins vary substantially between different hosts. This suggests these proteins are not essential for virulence. However, the most variant protein domains were the putative functional regions and there is biological evidence that variants can be functional, arguing they do play a role.ConclusionSurface and immune evasion genes are candidates for S. aureus vaccines, and their distribution and functionality is key. Vaccines should contain cocktails of antigens representing all variants or they will not protect against naturally occurring S. aureus populations.

Assessment of four recombinant mycobacterial antigens as serodiagnostic markers for pulmonary tuberculosis

An accurate laboratory diagnosis is essential for the effective management of pulmonary tuberculosis patients. This study is precisely aimed at developing an immunodiagnostic assay, using recombinant mycobacterial antigens for the accurate laboratory diagnosis of pulmonary tuberculosis. An indirect ELISA was standardized to measure serum IgG antibody levels against four recombinant mycobacterial antigens (ESAT-6, PlcA, HspX and Tb8.4) in sputum culture-negative, smear negative patients with pulmonary tuberculosis. The sensitivity of the ELISA with PlcA, HspX, Tb8.4 and ESAT-6 were 41.7, 63.3, 45 and 53.3% respectively. A higher sensitivity (76.7%) in ELISA could be achieved when a multiantigen cocktail, consisting of all the above four antigens, was used. These antigens did not give false positive results in the sera of healthy control subjects. The specificity of PlcA, HspX, Tb8.4, ESAT-6 and multiantigen cocktail in ‘disease controls’ were 95.7, 93.5, 94.6, 93.5 and 94.6%. High specificity and sensitivity of ELISA with these recombinant mycobacterial antigens in cocktail indicates that they can be used for the diagnosis of pulmonary tuberculosis particularly in patients. Key words: Mycobacterium tuberculosis, recombinant mycobacterial antigens, serology, tuberculosis

Toxoplasma gondii: Recombinant GRA5 antigen for detection of immunoglobulin G antibodies using enzyme-linked immunosorbent assay

In this study, for the first time, the evaluation of Toxoplasma gondii full-length recombinant GRA5 antigen for the serodiagnosis of human toxoplasmosis is shown. The recombinant GRA5 antigen as a fusion protein containing His-tag at both terminals was obtained using an Escherichia coli expression system. The usefulness of rGRA5 for the diagnosis of toxoplasmosis in an ELISA was tested on a total of 189 sera from patients with different stages of the infection and 31 sera from sero-negative individuals, obtained during routine diagnostic tests. Anti-GRA5 IgG antibodies were detected in 70.9% of all seropositive serum samples. This result was comparable to ELISA using a Toxoplasma lysate antigen (TLA) and six combinations of recombinant antigens. The sensitivity of IgG ELISA calculated from all positive serum samples was similar for TLA (94.2%), rMAG1 + rSAG1 + rGRA5 (92.6%), rGRA2 + rSAG1 + rGRA5 (93.1%) and rROP1 + rSAG1 + rGRA5 (94.2%) cocktails, whereas the sensitivity of cocktails without rGRA5 antigens was lower giving 82.0%, 86.2% and 87.8%, respectively. Thus, the present study showed that the full-length rGRA5 is suitable for use as a component of an antigen cocktail for the detection of anti- T. gondii IgG antibodies.

Utility of a combination of RD1 and RD2 antigens as a diagnostic marker for tuberculosis

We evaluated the diagnostic potential of a cocktail of 4 antigens encoded by regions of difference (RD) 1 and 2 of Mycobacterium tuberculosis, that is, early secretory antigenic target-6, culture filtrate protein-10 (CFP-10), CFP-21, and mycobacterial protein from species tuberculosis-64 (MPT-64) on the basis of antigen and antibody detection by enzyme-linked immunosorbent assay. Parallel detection of antigens and antibodies in the serum samples of pulmonary tuberculosis (PTB) patients resulted in higher sensitivity as compared to either of the single tests in both smear-positive (90%) and smear-negative (60%) PTB patients. In addition, combined detection of antigens and antibodies in the fluids of extrapulmonary tuberculosis (EPTB) patients could detect >90% of the patients with high specificity. These results demonstrate the ability of the combination of antigen and antibody detection assays based on the cocktail of RD antigens to diagnose a substantial number of PTB and EPTB cases with high specificity.

Multiplex Screening of Surface Proteins from Mycoplasma mycoides subsp. mycoides Small Colony for an Antigen Cocktail Enzyme-Linked Immunosorbent Assay

A recombinant antigen cocktail enzyme-linked immunosorbent assay (ELISA) for diagnosis of contagious bovine pleuropneumonia (CBPP) was developed after careful selection of antigens among one-third of the surface proteome proteins of the infectious agent Mycoplasma mycoides subsp. mycoides small colony (M. mycoides SC). First, a miniaturized and parallelized assay system employing antigen suspension bead array technology was used to screen 97 bovine sera for humoral immune responses toward 61 recombinant surface proteins from M. mycoides SC. Statistical analysis of the data resulted in selection of eight proteins that showed strong serologic responses in CBPP-affected sera and minimal reactivity in negative control sera, with P values of <10(-6). Only minor cross-reactivity to hyperimmune sera against other mycoplasmas was observed. When applied in an ELISA, the cocktail of eight recombinant antigens allowed a fivefold signal separation between 24 CBPP-affected and 23 CBPP-free sera from different geographical origins. No false-positive results and only two false-negative results were obtained. In conclusion, the selected recombinant mycoplasma antigens qualified as highly specific markers for CBPP and could be employed in both a suspension bead array platform and a cocktail ELISA setting. This set of proteins and technologies therefore offers a powerful combination to drive and further improve serological assays toward reliable, simple, and cost-effective diagnosis of CBPP.