Protein-based Enzyme-linked Immunosorbent Assay Research Articles

Abstract 6292: Novel seroprofiling assay reveals previously undescribed autoantibody responses in immunotherapy treated cancer patients

Abstract In contrast to cell-mediated immunity, the potential role of antibody-mediated “humoral” immunity in the context of immunotherapy is greatly under-appreciated. This is in spite of clues that patient autoantibodies can dramatically affect cancer progression. For instance, patients who fortuitously produce autoantibodies that target their tumor (e.g., anti-HER2 and anti-MUC1) have enhanced survival. Autoantibodies also underlie numerous cancer-associated paraneoplastic syndromes and immune-related adverse events. Thus, the humoral immune system represents a rich source of natural products (antibodies) that can exert either anti-neoplastic activity or harmful toxicities. A major barrier to uncovering functional humoral responses in immunotherapy is the paucity of methods to comprehensively determine autoantibody targets, particularly for autoantibodies targeting extracellular proteins that could plausibly mediate disease-modifying effects. To address this, we developed a yeast-surface display-based method that enables rapid discovery of seroreactivities. In short, we created a library of ~3,500 extracellular proteins, with each protein displayed on genetically-barcoded yeast clones. We incubate a small sample (~50µL) of patient serum or plasma with the library, use magnetic selection to isolate antibody-coated yeast clones, and use next-generation sequencing of genetic barcodes to identify bound proteins. Hundreds of samples can be screened against thousands of proteins simultaneously. Compared to platforms such as peptide arrays and phage-display libraries, yeast-surface display enables sampling of conformational epitopes that benefit from enhanced folding in a eukaryotic secretory environment. Using this assay, we screened a longitudinal cohort of 63 NSCLC patients treated primarily with anti-PD-L1 and anti-PD-1 checkpoint inhibition along with a variety of other antibody immunotherapies (anti-OX40, anti-VEGFA, anti-TNF, etc.). We successfully detected the therapeutic antibodies administered to these patients, which served as internal positive controls. We also identified novel autoantibody reactivities against proteins that have not yet been described in the context of cancer and that could potentially have disease-modifying effects. These include autoantibodies targeting chemokines (e.g., CXCL1/2/3), type 1 interferons, growth factors (e.g., VEGFB), and adhesion receptors (e.g., MADCAM1). We validated many of these reactivities using recombinant protein-based enzyme-linked immunosorbent assays and luciferase immunoprecipitation systems. Ultimately, these data indicate that humoral immunity could have wide-ranging functional effects in the context of immunotherapy and may provide not only insights into potential therapeutic targets but also novel patient-derived immunotherapeutic antibody agents. Citation Format: Eric Y. Wang, Connor E. Rosen, Yile Dai, Miguel F. Sanmamed, Aaron M. Ring. Novel seroprofiling assay reveals previously undescribed autoantibody responses in immunotherapy treated cancer patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6292.

Detection of autoantibodies against alpha-2-macroglobulin-like 1 in paraneoplastic pemphigus sera utilizing novel green fluorescent protein-based immunoassays

BackgroundParaneoplastic pemphigus (PNP) is a devastating autoimmune multiorgan syndrome associated with autoantibodies against several autoantigens, including the alpha-2-macroglobulin-like-1 (A2ML1). A2ML1 is recognized by up to 70 % of PNP sera. The currently recommended techniques for serological diagnosis of PNP are inadequate to detect anti-A2ML1 antibodies. ObjectivesTo develop novel assays which allow to easily and reliably detect anti-A2ML1 autoantibodies in PNP sera. MethodsWe produced full-length A2ML1 in fusion with enhanced green fluorescent protein (EGFP-A2ML1) in transfected human embryonic kidney 293 T cells. The recombinant protein was used as fluorescent ligand for immunoprecipitation studies. We further developed an enzyme-linked immunosorbent assay (ELISA) by immobilizing EGFP-A2ML1 on 96-well plates. ResultsA2ML1-positive PNP sera were able to immunoprecipitate EGFP-A2ML1. Direct measurement of fluorescence in immunoprecipitates correlates with the relative levels of anti-A2ML1 antibodies in the PNP sera. By the novel ELISA, based on the determined best cut-off value, 61 % of the tested 36 PNP sera were A2ML1 positive with a specificity of 88.9 % and a sensitivity of 95 %. The 20 tested normal sera (NHS) were negative, while 2 (10 %) of 20 pemphigus vulgaris and 3 (15 %) of 20 bullous pemphigoid sera showed borderline values. ConclusionsOur novel immunoassays enable rapid stratification of PNP patients. The novel green fluorescent protein-based ELISA utilizing an active eukaryotic A2ML1 is highly sensitive and reliable and, hence, is useful for a better understanding of the immunological background of PNP. This approach may be easily applied for the rapid detection of antibodies to various other antigens.

Development and application of real-time RT-PCR and S1 protein-based indirect ELISA for porcine deltacoronavirus

Porcine deltacoronavirus (PDCoV) has been recently recognized as an emerging viral pathogen that causes diarrhea in newborn piglets. A total of 254 small intestinal or fecal samples collected from 10 provinces including Henan, Hunan, Zhejiang, Jiangxi, Anhui, Hebei, Heilongjiang, Jiangsu, Shandong and Shanghai between 2014 and 2015, were screened by quantitative RT-PCR targeting the viral M gene. Eleven PDCoV positive samples were identified with a total positive rate of 4.33%. An indirect enzyme-linked immunosorbent assay (ELISA) was developed based on the recombinant S1 protein of PDCoV. This assay was used to test 609 serum samples of pigs with diarrhea symptoms collected from 10 provinces between 2015 and 2016. The positive rate of PDCoV antibody was 44.17% (269/609). The two methods can be used to monitor the PDCoV epidemiology in the levels of PDCoV specific RNA or antibody, helping better prevent and control PDCoV.

Antigenic regions of African swine fever virus phosphoprotein P30.

African swine fever (ASF) is one of the most complex and lethally haemorrhagic viral diseases of swine, affecting all breeds and ages of pigs. In the absence of ASF vaccines, reliable laboratory diagnosis and restricted biosecurity are critical for disease prevention and control. A detection of ASF-specific antibodies in an unvaccinated pig is a good marker for the diagnosis of ASF. The immunoperoxidase test (IPT) is a sensitive test for detecting ASF virus (ASFV) antibodies. However, due to the complexity of the procedure, the IPT is only suitable to be used as a confirmatory test. The ASFV p30 protein-based enzyme-linked immunosorbent assay (ELISA) is widely used for ASFV antibody screening, but the sensitivity is not comparable to the IPT. It is essential to have a better understanding of the antigenic properties of ASFV p30 to improve p30-based serologic tests. In this study, we developed a panel of 21 monoclonal antibodies (mAbs) against ASFV p30. With 14 out of the 21 mAbs, we defined 4 antigenic regions that contain at least 4 linear epitopes. Nine of the 14 mAbs mapped to antigenic regions 3 and 4 reacted with p30 in all serologic methods tested in this study, such as indirect immunofluorescence assay (IFA), ELISA and Western blot. The antigenic regions 3 and 4 are highly conserved and immunodominant in host antibody response. These mAbs and the defined p30 antigenic regions 3 and 4 provide valuable tools for the development and improvement of ASF serologic assays.

Development and Validation of a S1 Protein-Based ELISA for the Specific Detection of Antibodies against Equine Coronavirus.

Equine coronavirus (ECoV) is considered to be involved in enteric diseases in foals. Recently, several outbreaks of ECoV infection have also been reported in adult horses from the USA, France and Japan. Epidemiological studies of ECoV infection are still limited, and the seroprevalence of ECoV infection in Europe is unknown. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) method utilizing ECoV spike S1 protein was developed in two formats, and further validated by analyzing 27 paired serum samples (acute and convalescent sera) from horses involved in an ECoV outbreak and 1084 sera of horses with unknown ECoV exposure. Both formats showed high diagnostic accuracy compared to virus neutralization (VN) assay. Receiver-operating characteristic (ROC) analyses were performed to determine the best cut-off values for both ELISA formats, assuming a test specificity of 99%. Employing the developed ELISA method, we detected seroconversion in 70.4% of horses from an ECoV outbreak. Among the 1084 horse sera, seropositivity varied from 25.9% (young horses) to 82.8% (adult horses) in Dutch horse populations. Further, sera of Icelandic horses were included in this study and a significant number of sera (62%) were found to be positive. Overall, the results demonstrated that the ECoV S1-based ELISA has reliable diagnostic performance compared to the VN assay and is a useful assay to support seroconversion in horses involved with ECoV outbreaks and to estimate ECoV seroprevalence in populations of horses.

Development and application of a fiber2 protein-based indirect ELISA for detection of duck adenovirus 3

Duck adenovirus 3 (DAdV-3) is a newly identified duck adenovirus that has recently emerged in China. The incidence of duck infection caused by this virus is very high, with very large economic losses to the poultry industry. Thus, there is an urgent need for a serological assay for the specific detection of DAdV-3. To this end, prokaryotic expression of the fiber2 protein of DAdV-3 was used as a coating antigen to establish an indirect enzyme linked immunosorbent assay (ELISA) method for the specific detection of antibodies against DAdV-3. The method was found to be specific, repeatable and more sensitive than the agarose gel precipitation test (AGP). This indirect ELISA method based on the recombinant fiber2 protein may be used for the clinical detection of DAdV-3 infection and for monitoring antibody levels after vaccine immunization and is of great significance for the effective prevention and control of the disease.

Comparison of enzyme-linked immunosorbent assay systems using rift valley fever virus nucleocapsid protein and inactivated virus as antigens

BackgroundRift Valley Fever (RVF) is a mosquito-borne viral zoonosis. To detect RVF virus (RVFV) infection, indirect immunoglobulin G (IgG) and immunoglobulin M (IgM) enzyme linked immunosorbent assays (ELISAs) which utilize recombinant RVFV nucleocapsid (RVFV-N) protein as assay antigen, have reportedly been used, however, there is still a need to develop more sensitive and specific methods of detection.MethodsRVFV-N protein was expressed in Escherichia coli (E. coli) and purified by histidine-tag based affinity chromatography. This recombinant RVFV-N (rRVFV-N) protein was then used as antigen to develop an IgG sandwich ELISA and IgM capture ELISAs for human sera. Ninety six serum samples collected from healthy volunteers during the RVF surveillance programme in Kenya in 2013, and 93 serum samples collected from RVF-suspected patients during the 2006–2007 RVF outbreak in Kenya were used respectively, to evaluate the newly established rRVFV-N protein-based IgG sandwich ELISA and IgM capture ELISA systems in comparison with the inactivated virus-based ELISA systems.ResultsrRVFV-N protein-based-IgG sandwich ELISA and IgM capture ELISA for human sera were established. Both the new ELISA systems were in 100% concordance with the inactivated virus-based ELISA systems, with a sensitivity and specificity of 100%.ConclusionsRecombinant RVFV-N is a safe and affordable antigen for RVF diagnosis. Our rRVFV-N-based ELISA systems are safe and reliable tools for diagnosis of RVFV infection in humans and especially useful in large-scale epidemiological investigation and for application in developing countries.

Development and application of a recombinant protein-based indirect ELISA for the detection of serum antibodies against Cystoisospora suis in swine

The apicomplexan parasite Cystoisospora suis which causes neonatal porcine coccidiosis is one of the predominant parasite in suckling piglets. Currently, the immunofluorescence antibody test (IFAT) is the only available serological tool for detecting serum antibodies against C. suis which has several limitations, including bias from interpretation and low throughput. In the present study, an indirect enzyme-linked immunosorbent assay (ELISA) was developed using a previously characterized recombinant merozoite protein for the detection of specific immunoglobulin G (IgG) and IgA against C. suis. The recombinant protein was expressed in Escherichia coli as a N-terminal histidine fusion protein, and its specificity was confirmed in an immunoblot probed with highly positive anti-C. suis sera from experimentally infected piglets. Optimal dilutions of recombinant protein, sera and conjugate were determined by checkerboard titrations, and the serum dilution that gave the greatest ratio between the positive and the negative sera was selected for subsequent analyses. Agreement between the IFAT and the newly developed ELISA was assessed with kappa statistics. The receiver operating characteristic (ROC) curve analysis based on 185 serum samples with known C. suis exposure previously tested in the reference IFAT was used to determine the cut-off value, sensitivity and specificity of the ELISA. For IgG, the ELISA had an estimated cut-off value of 0.82 and sensitivity and specificity values of 94.7% and 98%, respectively, whereas for IgA the estimated cut-off value was 0.41 and sensitivity and specificity values were both100%. According to kappa coefficient, an excellent correlation (κ > 0.8) was found between IFAT and ELISA for both isotypes. The diagnostic accuracy of the test measured as the area under the ROC curve index scaled between 0.98 and 1.0, indicating high discriminatory capacity and its possible application as a serological tool for detecting antibody response in the host following C. suis exposure/immunization and large-scale surveillance studies.

Development of a recombinant protein-based ELISA for diagnosis of larval cyathostomin infection.

Cyathostomins are ubiquitous nematodes of horses. Once ingested, they can spend a substantial time as encysted larvae in the intestinal wall. The larvae can comprise up to 90% of the total burden, with up to several million worms reported in individuals. These stages can emerge in large numbers to cause life-threatening colitis. Direct methods for detection of encysted larval burdens in live horses do not exist. Previously, two antigen complexes were identified as promising markers for infection. A component of these, cyathostomin gut associated larval antigen-1 (Cy-GALA-1), was identified following immunoscreening of a complementary DNA library. Serum immunoglobulin G(T) (IgG(T)) responses to Cy-GALA-1 were shown to inform on larval infection. Sequence analysis of polymerase chain reaction products amplified from individual worms indicated that Cy-GALA-1 was derived from Cyathostomum pateratum. As cyathostomin infections always comprise multiple species, a diagnostic test must account for this. Here, segments of the Cy-gala gene were isolated from four common species, Cyathostomum catinatum, Cylicocyclus ashworthi, Cylicostephanus goldi and Cylicostephanus longibursatus, and the associated proteins expressed in recombinant form. The specificity and immunogenicity of each protein was confirmed. Each protein was assessed by enzyme linked immuno sorbent assay (ELISA) for its ability for informing on the presence of encysted larval infection and the level of burden.

Development and Evaluation of Recombinant Nucleocapsid Protein Based Diagnostic ELISA for Detection of Nipah Virus Infection in Pigs

The recombinant viral protein-based indirect enzyme-linked immunosorbent assay (ELISA) is a cost-effective, safe, specific, and rapid tool to diagnose the viral infection. Nipah virus nucleocapsid (NiV-N) protein was expressed in Escherichia coli and purified by histidine tag-based affinity chromatography. The N protein was selected based on its immuno dominance and conservation among different NiV strains. An indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for swine sera was optimized using the recombinant NiV-N protein as an antigen along with negative and positive controls. The background reading was blocked using skim milk powder and chicken serum. A total number of 1709 swine serum samples from various states of India were tested with indirect ELISA and Western blot. The test was considered positive only when its total reactivity reading was higher than 0.2 cut-off value and the ratio of the total reactivity to the background reading was more than 2.0. Since specificity is high for Western blotting it was used as standard test for comparison of results of indirect ELISA. Sensitivity and specificity of indirect ELISA was 100% and 98.7%, respectively, in comparison with Western blotting. Recombinant N protein-based ELISA can be used in screening large number of serum samples for epidemiological investigations in developing countries where high containment laboratories are not available to handle this zoonotic virus.

Application of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for the detection of SFTSV-specific human IgG and IgM antibodies by indirect ELISA.

BackgroundSevere fever with thrombocytopenia syndrome (SFTS) is an emerging disease that was first reported in China in 2011. It is caused by SFTS virus (SFTSV) which is a member of the Phlebovirus genus in the Bunyaviridae family. SFTSV has been classified as a BSL3 pathogen. There is a need to develop safe and affordable serodiagnostic methods for proper clinical management of infected patients.MethodsThe full length nucleocapsid (N) gene of SFTSV Yamaguchi strain was amplified by RT-PCR and cloned to an expression vector pQE30. The recombinant (r) SFTSV-N protein was expressed by using Escherichia coli (E. coli) expression system and purified under native conditions. rSFTSV-N protein based indirect IgG and IgM enzyme linked immunosorbent assay (ELISA) systems were established to detect specific human IgG and IgM antibodies, respectively. One hundred fifteen serum samples from clinically suspected-SFTS patients were used to evaluate the newly established systems and the results were compared with the total antibody detecting sandwich ELISA system.ResultsThe native form of recombinant (r) SFTSV-N protein was expressed and purified. Application of the rSFTSV-N protein based indirect IgG ELISA to the 115 serum samples showed results that perfectly matched those of the total antibody sandwich ELISA with a sensitivity and specificity of 100 %. The rSFTSV-N protein based indirect IgM ELISA missed 8 positive samples that were detected by the total antibody sandwich ELISA. The sensitivity and specificity of rSFTSV-N-IgM capture ELISA were 90.59 and 100 %, respectively.ConclusionsThe rSFTSV-N protein is highly immunoreactive and a good target for use as an assay antigen in laboratory diagnosis. Its preparation is simpler in comparison with that used for the total antibody sandwich system. Our rSFTSV-N protein-based IgG and IgM ELISA systems have the advantage of distinguishing two types of antibodies and require small volume of serum sample only. They are safe to use for diagnosis of SFTS virus infection and especially fit in large-scale epidemiological investigations.

Development of a multi-epitope antigen of S protein-based ELISA for antibodies detection against infectious bronchitis virus.

An indirect enzyme-linked immunosorbent assay (ELISA) method based on a novel multi-epitope antigen of S protein (SE) was developed for antibodies detection against infectious bronchitis virus (IBV). The multi-epitope antigen SE protein was designed by arranging three S gene fragments (166-247 aa, S1 gene; 501-515 aa, S1 gene; 8-30 aa, S2 gene) in tandem. It was identified to be approximately 32 kDa as a His-tagged fusion protein and can bind IBV positive serum by western blot analysis. The conditions of the SE-ELISA method were optimized. The optimal concentration of the coating antigen SE was 3.689 μg/mL and the dilution of the primary antibodies was identified as 1:1000 using a checkerboard titration. The cut-off OD450 value was established at 0.332. The relative sensitivity and specificity between the SE-ELISA and IDEXX ELISA kit were 92.38 and 89.83%, respectively, with an accuracy of 91.46%. This assay is sensitive and specific for detection of antibodies against IBV.

Recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibody to turkey coronavirus

Nucleocapsid (N) protein gene of turkey coronavirus (TCoV) was expressed in a prokaryotic system and used to develop an enzyme-linked immunosorbent assay (ELISA) for detection of antibody to TCoV. Anti-TCoV hyperimmune turkey serum and normal turkey serum were used as positive or negative controls for optimization of the ELISA. Goat anti-turkey IgG (H+L) conjugated with horseradish peroxidase was used as detector antibody. Three hundred and twenty two turkey sera from the field were used to evaluate the performance of ELISA and determine the cut-off point of ELISA. The established ELISA was also examined with serum samples obtained from turkeys experimentally infected with TCoV. Those serum samples were collected at various time intervals from 1 to 63 days post-infection. The optimum conditions for differentiation between anti-TCoV hyperimmune serum and normal turkey serum were recombinant TCoV N protein concentration at 20μg/ml, serum dilution at 1:800, and conjugate dilution at 1:10,000. Of the 322 sera from the field, 101 were positive for TCoV by immunofluorescent antibody assay (IFA). The sensitivity and specificity of the ELISA relative to IFA test were 86.0% and 96.8%, respectively, using the optimum cut-off point of 0.2 as determined by logistic regression method. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the recombinant N protein coated on the ELISA plates was not detected. These results indicated that the established antibody-capture ELISA in conjunction with recombinant TCoV N protein as the coating protein can be utilized for detection of antibodies to TCoV in turkey flocks.

Development of a Recombinant Protein-Based Enzyme-Linked Immunosorbent Assay for Diagnosis of Mycoplasma bovis Infection in Cattle

Mycoplasma bovis causes a range of diseases in cattle, including mastitis, arthritis, and pneumonia. However, accurate serological diagnosis of infection remains problematic. The studies described here aimed to identify an antigen that might be used to develop a more specific and sensitive diagnostic assay. A 226-kDa immunogenic protein was consistently detected in Western blots by antibodies in sera from calves experimentally infected with M. bovis. This protein was shown to be a membrane protein with lipase activity and was named mycoplasma immunogenic lipase A (MilA). Different regions of MilA were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins and recombinant products from the amino-terminal end shown to have strong immunoreactivity with M. bovis-specific bovine sera. The most immunoreactive fusion protein, GST-MilA-ab, was used to develop indirect IgM and IgG enzyme-linked immunosorbent assays (ELISAs). The IgM ELISA detected M. bovis-specific IgM antibody 2 weeks after infection with 97.1% sensitivity and had a specificity of 63.3%, while the IgG ELISA detected M. bovis-specific IgG 3 weeks after infection with 92.86% sensitivity and had a specificity of 98.7%, demonstrating that the IgG ELISA has potential for use as a sensitive and specific assay for detecting infection in cattle.

Detection of antibodies specific to the non-structural proteins of fowl adenoviruses in infected chickens but not in vaccinated chickens

Antibodies specific to the non-structural proteins of viruses are detected in virus-infected animals and show promise as a reliable diagnostic marker for virus infections. We examined the potential use of two non-structural proteins of fowl adenovirus (FAdV)-based, 100K and 33K, enzyme-linked immunosorbent assays (ELISAs) in the diagnosis of FAdVs. We cloned and expressed the 100K and 33K non-structural protein genes of the FAdVs in the pGEX-4T-1 plasmid vector. Purified 100K and 33K proteins alone or in combination were used as antigens in ELISAs. Antibodies specific to the 100K and 33K non-structural proteins were detected in chickens experimentally infected with FAdVs, but not in chickens vaccinated with inactivated FAdVs. In contrast, the agar gel precipitation (AGP) test detected FAdV-specific antibodies in 70.3% of the vaccinated chickens, suggesting that the non-structural protein-based ELISA could be used in the differential diagnosis of infected and vaccinated chickens. To further validate the 100K and 33K-based ELISA (100K-33K-ELISA) method, we compared its sensitivity and specificity with that of a whole virus-based ELISA and an AGP test in detecting FAdV-specific antibodies in 350 field samples. The results showed that the 100K-33K-ELISA exhibited a higher sensitivity than the AGP test and a comparable sensitivity and specificity to the whole virus ELISA. Overall, the 100K-33K-ELISA method is sensitive, specific and can be used to distinguish an acute FAdV infection from an inactivated virus-based vaccination response.

Detection of Specific Antibodies against Tembusu Virus in Ducks by Use of an E Protein-Based Enzyme-Linked Immunosorbent Assay

We developed an enzyme-linked immunosorbent assay (ELISA) using eukaryotically expressed E protein as the antigen (termed E-ELISA) to detect antibodies to tembusu virus (TMUV) in ducks. The E-ELISA did not react with antisera to other known pathogens, indicating the E protein is specific for recognizing anti-TMUV antibodies. Compared to the serum neutralization test, the specificity and sensitivity of the E-ELISA was 93.2 and 97.8%, respectively. Therefore, this E-ELISA is a sensitive and rapid method for detecting antibodies against TMUV in ducks.

English

  By removing the N-terminal hydrophobic sequence, truncated VP2 (tVP2) genes were cloned into the pET-32a (+) plasmid and subsequently expressed as His fusion proteins. The purified recombinant tVP2 proteins were specific to canine parvovirus (CPV), and one of them was used in an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of CPV antibodies. The minimum detection limit of this method was 1:1280. There was good agreement between tVP2-based indirect ELISA and the commercially available diagnostic kit. The results suggest that the recombinant tVP2 protein-based ELISA could be used to detect CPV antibodies. Key words: Canine parvovirus, recombinant truncated VP2 (tVP2), enzyme-linked immunosorbent assay (ELISA), antibody detection

Evaluation of an Enzyme-Linked Immunosorbent Assay Based on Crude Leishmania Histone Proteins for Serodiagnosis of Human Infantile Visceral Leishmaniasis

Human visceral leishmaniasis (VL) is routinely diagnosed by detecting IgG that specifically binds to Leishmania antigens. The enzyme-linked immunosorbent assay (ELISA) remains a widely used method. However, the biggest challenge remains the choice of antigen with the highest specificity and sensitivity. This study is aimed at assessing the diagnostic performances of crude Leishmania histone (CLH) protein-based ELISAs in Mediterranean VL patients. The CLH proteins were biochemically purified from promastigote nuclear extracts. Their reactivities were analyzed by Western blotting (WB) using rabbit polyclonal antibodies against Leishmania recombinant histones and sera from VL patients, respectively. Then, the diagnostic potential of CLH proteins was validated by the CLH-based ELISA using 42 infantile VL patients' sera and 70 control subjects. The CLH-based ELISA performance was compared to that of the soluble Leishmania antigen (SLA)- and the recombinant K39 (rK39)-based ELISAs. Analysis of the WB profile with the use of polyclonal antibodies confirmed the histone origin of low molecular mass proteins (12 to 16 kDa). All VL samples tested presented antibodies reacting against different antigen fractions; however, recognition patterns were different depending on the reactivity of each serum. CLH-based ELISA showed an excellent ability to discriminate between VL cases and healthy controls (97.6% sensitivity and 100% specificity). It had a diagnostic performance similar to that of rK39-based ELISA (97.6% sensitivity and 97.1% specificity, P = 0.5) and a better serodiagnosis accuracy than the SLA-based ELISA (85.7% sensitivity and 90% specificity, P < 0.05). Therefore, crude Leishmania histone extract could be a valuable antigen for clinical use.

Evaluation of Three Commercial Varicella-Zoster Virus IgG Enzyme-Linked Immunosorbent Assays in Comparison to the Fluorescent-Antibody-to-Membrane-Antigen Test

Commercial serologic assays for varicella-zoster virus (VZV), which enable reliable determination of VZV immune status and are amenable to automation, are needed. The present study compares the automated performance of the VZV whole-cell enzyme-linked immunosorbent assay (ELISA) Enzygnost anti-VZV/IgG, the Euroimmun anti-VZV ELISA (IgG) based on highly purified viral proteins, and the VZV glycoprotein (gp)-based Serion ELISA Classic VZV IgG. The fluorescent-antibody-to-membrane-antibody (FAMA) test was used as a reference. A total of 638 serum samples from VZV-negative children, blood donors, varicella vaccinees, and bone marrow transplant recipients were included. The Enzygnost anti-VZV/IgG and the Serion ELISA Classic VZV IgG showed sensitivities of 99.6% and 99.2%, respectively, and the Euroimmun anti-VZV ELISA (IgG) had a significantly lower sensitivity of 90.5%. Specificity was calculated as 100% for both the Euroimmun anti-VZV ELISA (IgG) and for the Enzygnost anti-VZV/IgG, and the Serion ELISA Classic VZV IgG had a significantly lower specificity of 89.4%. Quantitative results of all ELISAs correlated well, but there was a poor quantitative correlation between the ELISAs and FAMA. In conclusion, this study does not show any superiority of a gp- and a protein-based ELISA compared to a whole-cell ELISA for the automated detection of VZV-specific IgG. The automated performance of the Enzygnost anti-VZV/IgG assay correlated best with the FAMA reference assay.

Field application of a recombinant protein-based ELISA during the 2010 outbreak of foot-and-mouth disease type A in South Korea

A recombinant protein-based enzyme-linked immunosorbent assay (RP ELISA) exists for the detection of antibodies to foot-and-mouth disease virus (FMDV) type A. In this study, the efficacy of the RP ELISA was compared to that of other current tests by examining sera collected in the field during an FMD type A outbreak in South Korea in 2010. The RP ELISA detected early antibodies to FMDV with the same sensitivity as the liquid-phase blocking ELISA (LPB ELISA), identifying FMD farm outbreaks correctly on a herd basis. In addition, the two assays exhibited a high correlation coefficient (γ(2)=0.83) when testing thirty seven sera from one outbreak farm exhibiting various antibody titers. The sensitivity and specificity of the RP ELISA relative to the LPB ELISA were 84% and 97%, respectively, and excellent agreement (kappa=0.82) was observed between the two tests. Taken together, the RP ELISA should be a useful alternative to the LPB ELISA for the detection of early antibodies to FMDV type A during an outbreak.