A Recombinant Secondary Antibody Mimic as a Target-specific Signal Amplifier and an Antibody Immobilizer in Immunoassays.
ABSTRACT: We construct a novel recombinant secondary antibody mimic, GST-ABD, which can bind to the Fc regions of target-bound primary antibodies and acquire multiple HRPs simultaneously. We produce it in tenth of mg quantities with a bacterial overexpression system and simple purification procedures, significantly reducing the manufacturing cost and time without the use of animals. GST-ABD is effectively conjugated with 3 HRPs per molecule on an average and selectively bind to the Fc region of primary antibodies derived from three different species (mouse, rabbit, and rat). HRP-conjugated GST-ABD (HRP-GST-ABD) is successfully used as an alternative to secondary antibodies to amplify target-specific signals in both ELISA and immunohistochemistry regardless of the target molecules and origin of primary antibodies used. GST-ABD also successfully serves as an anchoring adaptor on the surface of GSH-coated plates for immobilizing antigen-capturing antibodies in an orientation-controlled manner for sandwich-type indirect ELISA through simple molecular recognition without any complicated chemical modification.
Project description:BACKGROUND:Feline panleukopenia virus (FPV) is an etiologic pathogen of feline panleukopenia that infects all members of Felidae including tigers (Panthera tigris). Vaccinations against FPV among wild felid species have long been practiced in zoos worldwide. However, few studies have assessed the tiger immune response post-vaccination due to the absence of a serological diagnostic tool. To address these limitations, this study aimed to develop an in-house indirect enzyme-linked immunosorbent assay (ELISA) for the monitoring of tiger antibody levels against the feline panleukopenia vaccine by employing the synthesized subunit capsid protein VP2. An in-house horseradish peroxidase (HRP) conjugated rabbit anti-tiger immunoglobulin G (IgG) polyclonal antibody (HRP-anti-tiger IgG) was produced in this study and employed in the assay. It was then compared to a commercial HRP-conjugated goat anti-cat IgG (HRP-anti-cat IgG). Sensitivity and specificity were evaluated using the Bayesian model with preferential conditional dependence between HRP-conjugated antibody-based ELISAs and hemagglutination-inhibition (HI) tests. RESULTS:The posterior estimates for sensitivity and specificity of two indirect ELISA HRP-conjugated antibodies were higher than those of the HI test. The sensitivity and specificity of the indirect ELISA for HRP-anti-tiger IgG and HRP-anti-cat IgG were 86.5, 57.2 and 86.7%, 64.6%, respectively, while the results of the HI test were 79.1 and 54.1%. In applications, 89.6% (198/221) and 89.1% (197/221) of the tiger serum samples were determined to be seropositive by indirect ELISA testing against HRP-anti-tiger and HRP-anti-cat, respectively. CONCLUSION:To the best of our knowledge, the specific serology assays for the detection of the tiger IgG antibody have not yet been established. The HRP-anti-tiger IgG has been produced for the purpose of developing the specific immunoassays for tigers. Remarkably, an in-house indirect ELISA based on VP2 subunit antigen has been successfully developed in this study, providing a potentially valuable serological tool for the effective detection of tiger antibodies.
Project description:The association of low endothelial shear stress (ESS) with high-risk plaque (HRP) has not been thoroughly investigated in humans. We investigated the local ESS and lumen remodelling patterns in HRPs using optical coherence tomography (OCT), developed the shear stress score, and explored its association with the prevalence of HRPs and clinical outcomes.A total of 35 coronary arteries from 30 patients with stable angina or acute coronary syndrome (ACS) were reconstructed with three dimensional (3D) OCT. ESS was calculated using computational fluid dynamics and classified into low, moderate, and high in 3-mm-long subsegments. In each subsegment, (i) fibroatheromas (FAs) were classified into HRPs and non-HRPs based on fibrous cap (FC) thickness and lipid pool size, and (ii) lumen remodelling was classified into constrictive, compensatory, and expansive. In each artery the shear stress score was calculated as metric of the extent and severity of low ESS. FAs in low ESS subsegments had thinner FC compared with high ESS (89 ± 84 vs.138 ± 83 µm, P < 0.05). Low ESS subsegments predominantly co-localized with HRPs vs. non-HRPs (29 vs. 9%, P < 0.05) and high ESS subsegments predominantly with non-HRPs (9 vs. 24%, P < 0.05). Compensatory and expansive lumen remodelling were the predominant responses within subsegments with low ESS and HRPs. In non-stenotic FAs, low ESS was associated with HRPs vs. non-HRPs (29 vs. 3%, P < 0.05). Arteries with increased shear stress score had increased frequency of HRPs and were associated with ACS vs. stable angina.Local low ESS and expansive lumen remodelling are associated with HRP. Arteries with increased shear stress score have increased frequency of HRPs and propensity to present with ACS.
Project description:The purpose of this study is to optimize ELISA conditions to quantify the colorectal cancer antigen GA733 linked to the Fc antibody fragment fused to KDEL, an ER retention motif (GA733-FcK) expressed in transgenic plant. Variable conditions of capture antibody, blocking buffer, and detection antibody for ELISA were optimized with application of leaf extracts from transgenic plant expressing GA733-FcK. In detection antibody, anti-EpCAM/CD362 IgG recognizing the GA733 did not detect any GA733-FcK whereas anti-human Fc IgG recognizing the human Fc existed in plant leaf extracts. For blocking buffer conditions, 3% BSA buffer clearly blocked the plate, compared to the 5% skim-milk buffer. For capture antibody, monoclonal antibody (MAb) CO17-1A was applied to coat the plate with different amounts (1, 0.5, and 0.25 ?g/well). Among the amounts of the capture antibody, 1 and 0.5 ?g/well (capture antibody) showed similar absorbance, whereas 0.25 ?g/well of the capture antibody showed significantly less absorbance. Taken together, the optimized conditions to quantify plant-derived GA733-FcK were 0.5 ?g/well of MAb CO17-1A per well for the capture antibody, 3% BSA for blocking buffer, and anti-human Fc conjugated HRP. To confirm the optimized ELISA conditions, correlation analysis was conducted between the quantified amount of GA733-FcK in ELISA and its protein density values of different leaf samples in Western blot. The co-efficient value R(2) between the ELISA quantified value and protein density was 0.85 (p<0.01), which indicates that the optimized ELISA conditions feasibly provides quantitative information of GA733-FcK expression in transgenic plant.
Project description:Detection of specific antibodies has numerous research, therapeutic and diagnostic applications. Short peptide ligands that bind specifically to antibodies with continuous epitopes can be derived from epitope mapping experiments. Short peptide ligands (mimotopes) specific to antibodies with discontinuous epitopes can be identified by screening complex peptide libraries. In an effort to enhance practical utility of such peptide ligands, we describe here a simple approach to turn such target antibody-specific peptide ligands into specific ELISA detection reagents. We show that a simple addition of biotinylated peptide ligands to commonly available horseradish peroxidase (HRP)-labeled streptavidin (or HRP-anti-biotin antibody), or digoxigenin-labeled peptides to HRP-anti-digoxigenin antibody detection reagents transformed these generic detection reagents into sensitive target antibody-specific reagents. ELISA assays performed using these reagents exhibited excellent analytical properties indicating their practical utility for antibody detection. One generic detection reagent can be readily transformed into many different specific ELISA reagents by a simple mix and match design using an appropriate target-specific peptide ligand. Simplicity of preparation of these ELISA reagents for detecting antibodies should facilitate their practical applications.
Project description:A fusion protein SBP-Cap?41, consisting of Cap?41 (without 41 amino acids at the N-terminus) protein of porcine circovirus 2 (PCV2) and a streptavidin binding peptide (SBP), was constructed. This fusion protein binds to HRP-labeled streptavidin (HRP-SA) through high affinity between SBP and SA, forming an HRP-streptavidin bound antigen (Hsb-Ag) with both immunoreactivity and enzymatic activity, which can be used in a double-antigen sandwich ELISA for detection of PCV2 antibodies. Comparison of the characteristics of the HSb-Cap?41 and chemical conjugates of the recombinant Cap?41 protein showed that the HSb-Cap?41 based double-antigen sandwich ELISA (HBDS-ELISA) had higher specificity and sensitivity. Use of the HBDS-ELISA detected PCV2-IgG in 9 injected pigs as early as 10 days p.i., 3 days earlier than both a double-antigen sandwich ELISA (DS-ELISA) based on a chemically conjugated antigen, and a commercial indirect ELISA kit.
Project description:Immunoassay usually deal with the antibody labeling with various reporter molecules, one such useful reporter molecule is horseradish peroxidase (HRPO). Conjugating enzyme with antibody without losing its enzymatic activity is a challenging task. Our aim is to modify existing classical method of conjugating antibodies with HRP to enhance immunoassay techniques with better sensitivity. We used chemicals such as sodium meta periodate to generate aldehyde group by oxidation of carbohydrate moieties on HRPO. The activated form of HRPO is lyophilized and then mixed with 1 mg/ml concentration of antibodies to be conjugate.After confirming chemical modification of conjugates via UV-Spec and SDS-PAGE independent molecules were used for conjugation and HRP-antibody conjugate. Finally, enzymatic activity of HRP-antibody conjugate was confirmed by performing direct ELISA. Functional properties were analyzed using ELISA with dilution of 1:5000, whereas the conjugate prepared by existing method of conjugation worked with as low dilution of 1:25 with a p value highly significant (<?0.001) for classical verses modified method of conjugation preparation. Collectively, this study showed the enhanced ability of antibody to bind more number of HRPO with an additional step of lyophilization in the regular conjugation protocol. Future exploration are necessary on wide range of IgG antibodies.
Project description:Accurate antigen detection is imperative for clinicians to diagnose disease, assess treatment success, and predict patient prognosis. The most common technique used for the detection of disease-associated biomarkers is the enzyme linked immunosorbent assay (ELISA). In an ELISA, primary antibodies are incubated with biological samples containing the biomarker of interest. Then, detectible secondary antibodies conjugated with horseradish peroxidase (HRP) bind the primary antibodies. Upon addition of a color-changing substrate, the samples provide a colorimetric signal that directly correlates to the targeted biomarker concentration. While ELISAs are effective for analyzing samples with high biomarker content, they lack the sensitivity required to analyze samples with low antigen levels. We hypothesized that the sensitivity of ELISAs could be enhanced by replacing freely delivered primary antibodies with antibody-nanoparticle conjugates that provide excess binding sites for detectible secondary antibodies, ultimately leading to increased signal. Here, we investigated the use of nanoshells (NS) decorated with antibodies specific to epidermal growth factor receptor (EGFR) as a model system (EGFR-NS). We incubated one healthy and two breast cancer cell lines, each expressing different levels of EGFR, with EGFR-NS, untargeted NS, or unconjugated EGFR antibodies, as well as detectable secondary antibodies. We found that EGFR-NS consistently increased signal intensity relative to unconjugated EGFR antibodies, with a substantial 13-fold enhancement from cells expressing high levels of EGFR. Additionally, 40x more unconjugated antibodies were required to detect EGFR compared to those conjugated to NS. Our results demonstrate that antibody-nanoparticle conjugates lower the detection limit of traditional ELISAs and support further investigation of this strategy with other antibodies and nanoparticles. Owing to their enhanced sensitivity, we anticipate that nanoparticle-modified ELISAs can be used to detect low levels of biomarkers found in various diseases, such as cancers, tuberculosis, and rheumatoid arthritis, and may ultimately enable earlier diagnosis, better prognostication, and improved treatment monitoring.
Project description:Detection of specific antibodies against hepatitis C virus (HCV) is the most widely available test for viral diagnosis and monitoring of HCV infections. However, narrowing the serologic window of anti-HCV detection by enhancing anti-HCV IgM detection has remained to be a problem. Herein, we used LD5, a novel evolved immunoglobulin-binding molecule (NEIBM) with a high affinity for IgM, to develop a new anti-HCV enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase-labeled LD5 (HRP-LD5) as the conjugated enzyme complex. The HRP-LD5 assay showed detection efficacy that is comparable with two kinds of domestic diagnostic kits and the Abbott 3.0 kit when tested against the national reference panel. Moreover, the HRP-LD5 assay showed a higher detection rate (55.9%, 95% confidence intervals (95% CI) 0.489, 0.629) than that of a domestic diagnostic ELISA kit (Chang Zheng) (53.3%, 95% CI 0.463, 0.603) in 195 hemodialysis patient serum samples. Five serum samples that were positive using the HRP-LD5 assay and negative with the conventional anti-HCV diagnostic ELISA kits were all positive for HCV RNA, and 4 of them had detectable antibodies when tested with the established anti-HCV IgM assay. An IgM confirmation study revealed the IgM reaction nature of these five serum samples. These results demonstrate that HRP-LD5 improved anti-HCV detection by enhancing the detection of anti-HCV IgM, which may have potential value for the early diagnosis and screening of hepatitis C and other infectious diseases.
Project description:The enzyme-linked immunosorbent assay (ELISA) is the diagnostic test most commonly used in efforts to control paratuberculosis in domestic ruminants. However, commercial ELISAs have not been validated for detecting antibodies against Mycobacterium avium subsp. paratuberculosis in wild animals. In this study, we compared the sensitivities and specificities of five ELISAs using individual serum samples collected from 41 fallow deer with or without histopathological lesions consistent with paratuberculosis. Two target antigenic preparations were selected, an ethanol-treated protoplasmic preparation obtained from a fallow deer M. avium subsp. paratuberculosis isolate (ELISAs A and B) and a paratuberculosis protoplasmic antigen (PPA3) (ELISAs C and D). Fallow deer antibodies bound to the immobilized antigens were detected by using a horseradish peroxidase (HRP)-conjugated anti-fallow deer IgG antibody (ELISAs A and C) or HRP-conjugated protein G (ELISAs B and D). A commercially available assay, ELISA-E, which was designed to detect M. avium subsp. paratuberculosis antibodies in cattle, sheep, and goats, was also tested. Although ELISAs A, C, and E had the same sensitivity (72%), ELISAs A and C were more specific (100%) for detecting fallow deer with lesions consistent with paratuberculosis at necropsy than was the ELISA-E (87.5%). In addition, the ELISA-A was particularly sensitive for detecting fallow deer in the latent stages of infection (62.5%). The antibody responses detected with the ELISA-A correlated with both the severity of enteric lesions and the presence of acid-fast bacteria in gut tissue samples. In summary, our study shows that the ELISA-A can be a cost-effective diagnostic tool for preventing the spread of paratuberculosis among fallow deer populations.
Project description:The Pseudomonas syringae hrp and hrmA genes controlling pathogenicity and elicitation of the hypersensitive response and the avr genes controlling host range have been shown previously to be regulated by carbon, nitrogen, pH, osmolarity, and hypothetical plant factors. In P. syringae pv. syringae Pss61, inactivation of hrp complementation groups II and XIII reduced expression of a plasmid-borne hrmA'-lacZ fusion. The hrp regions II and XIII were cloned on separate plasmids and shown to enhance the activity of the hrmA promoter in Escherichia coli MC4100 transformants at least 100-fold. The nucleotide sequence of region XIII revealed two open reading frames (hrpR and hrpS) whose deduced products share homology with P. syringae pv. phaseolicola NPS3121 HrpS and are both related to the NtrC family of two-component signal transduction systems. HrpR and HrpS differ from most members of the protein family by lacking an amino-terminal domain which modulates the regulatory activity. A single open reading frame, hrpL, whose product shares homology with AlgU, a putative alternate sigma factor of P. aeruginosa, as well as with the related alternate sigma factors was identified within region II. Key domains are partially conserved. Inactivation of hrpS in Pss61 repressed expression of a plasmid-borne hrpL'-lacZ fusion carried by pYXPL1R, and transformation of MC4100(pYXPL1R) with a plasmid carrying hrpRS increased hrpL promoter activity at least 200-fold. Neither hrpS nor hrpR, when cloned on separate plasmids, activated the hrpL promoter activity individually. The expression of hrpL when directed by a lac promoter was sufficient to express a set of plasmid-borne hrmA'-, hrpJ'-, and hrpZ'-lacZ fusions independently of other hrp genes. The results indicate that hrpRS and hrpL are part of a regulatory cascade in which HrpR and HrpS activate expression of hrpL and HrpL, a putative sigma factor, induces expression of HrpL-responsive genes.