A functionalized Sup35NM nanofibril-assisted oriented antibody capture in lateral flow immunoassay for sensitive detection of dengue type II NS1.

Rapid and sensitive dengue non-structural protein 1 (NS1) detection assay is essential for the treatment of disease and currently releases high medical cost burdens. To address the limitations of conventional LFIA strips, we have developed an improved Sup35NM-Z-based LFIA that immobilizes antibodies on cellulose membranes in an orientated manner to increase the sensitivity of LFIA strips. A dual-functional Sup35NM nanofibril was fabricated by fusion with the antibody binding domain; resultant nanofibril from the amyloid Sup35NM was sprayed on the T-line to orientate the capture antibody and produces fluorescence signals. Antibody binding analysis showed that self-assembly of the Sup35NM monomer does not affect the binding activity of the Z-domain with the antibody. The NS1 for DENV-2 infection was chosen as a model target antigen to assess the feasibility of the Sup35NM-Z-domain-based LFIA platform. Under optimal conditions, the Sup35NM-Z-domain-based LFIA detected NS1 within 15 min with a detection limit of 1.29 ng/ml, while the detection limit of traditional LFIA with the same concentration of anti-NS1-Ab1 on the T-line by conventional physical adsorption was 2.20 ng/ml, 1.7 times higher than that of Sup35NM-Z-domain-based LFIA. As compared to traditional LFIAs, the Sup35NM-Z-based LFIA had a wide detection range of 1.29-625 ng/mL. The LFIA's clinical performance in identifying NS1 was also assessed using 15 clinical samples. The LFIA accurately recognized positive and negative samples, equal to 86.7% accuracy. The developed Sup35NM-Z-domain-based LFIA in this study offers great potential for the identification of target markers because of its greatly improved sensitivity and wider detection range.

Advantages of Lateral Flow Assays Based on Fluorescent Submicrospheres and Quantum Dots for Clostridium difficile Toxin B Detection.

Clostridium difficile colitis is caused by a cytotoxin produced by the anaerobic bacteria C. difficile in the epithelial cells of the large intestine, particularly C. difficile toxin B (TcdB). However, the sensitivity of currently utilized C. difficile endotoxin determination methods has been called into question, and, therefore, more accurate and convenient detection methods are needed. Our study is the first to systematically compare fluorescent submicrosphere-based and quantum-dot nanobead-based lateral fluidity measurement methods (FMs-LFA and QDNBs-LFA) with toxin B quantification in fecal samples via sandwich analysis. The limits of detection (LOD) of FMs-LFA and QDNBs-LFA in the fecal samples were 0.483 and 0.297 ng/mL, respectively. TcdB analyses of the fecal samples indicated that the results of QDNBs-LFA and FMs-LFA were consistent with those of a commercial enzyme-linked immunosorbent assay (ELISA) test kit. The sensitivity of QDNBs-LFA was highly correlated with clinical diagnoses. Therefore, quantum dot nanobeads (QDNBs) are deemed highly suitable for lateral fluidity analyses, which would facilitate the implementation of portable and rapid on-the-spot applications, such as food hygiene and safety tests and onsite medical testing.

Rapid and fully-automated detection of Clostridium difficile Toxin B via magnetic-particle-based chemiluminescent immunoassay.

Clostridium difficile colitis is caused by a cytotoxin produced by the anaerobic bacteria C. difficile on the epithelial cells of the large intestine, particularly C. difficile toxin B (Tcd B). Current C. difficile toxin assays have proven to be insensitive and have thus been ruled out from diagnostic purposes. Therefore, Tcd B detection via sandwich-type chemiluminescent immunoassay was proposed as a straightforward approach with potential diagnostic applicability. Here, two high-affinity anti-Tcd B monoclonal antibodies were successfully identified and implemented in a fully-automated magnetic-particle-based chemiluminescent immunoassay (CLEIA). In this test, toxin B was sandwiched between the anti-toxin B antibody-coated magnetic particles and alkaline phosphate-labeled anti-toxin B antibodies. Compared with traditional techniques, the proposed immunoassay demonstrated high sensitivity for toxin B identification and was further optimized to achieve a linear response ranging from 0.12 to 150 ng/mL with a limit of detection (LOD) of 0.47 ng/mL. Importantly, the entire process could be completed in less than 30 minutes. The proposed assay was used to detect toxin B in 104 randomly-selected human stool samples and delivered similar results to those of a commercial ELISA kit, highlighting its great potential for rapid and efficient toxin B determination in human stool specimens.

A sensitive and rapid chemiluminescence immunoassay for point-of-care testing (POCT) of copeptin in serum based on high-affinity monoclonal antibodies via cytokine-assisted immunization.

Purpose: Antibodies are key reagents in the development of immunoassay. We attempted to develop high-performance CPP immunoassays using high-affinity monoclonal antibodies prepared via cytokine-assisted immunization. Methods: We used fetal liver tyrosine kinase 3 ligand (Flt3L), CC subtype chemokine ligand 20 (CCL20), and granulocyte-macrophage colony-stimulating factor (GM-CSF) to assist traditional subcutaneous immunization of preparing high-affinity monoclonal antibodies, and further to develop high-performance immunoassay methods for CPP. Results: This novel immune strategy significantly enhanced immune response against CPP. Six anti-CPP monoclonal antibodies (mAbs) with high affinity were successfully screened and selected for application in a fully automated magnetic chemiluminescence immunoassay (CLIA). This robust and rapid assay can efficiently detect CPP in the range of 1.2-1250 pmol L-1 with a detection limit of 6.25 pmol L-1. Significantly, the whole incubation process can be completed in 30 min as compared to about 4.5 hr for the control ELISA kit. Furthermore, this assay exhibited high sensitivity and specificity, low intra-assay and inter-assay coefficients of variation (CVs < 15%). The developed assay was applied in the detection of CPP in 115 random serum samples and results showed a high correlation with data obtained using a commercially available ELISA kit (correlation coefficient, 0.9737). Conclusion: Our assay could be applied in the point-of-care testing of CPP in the serum samples, and also the method developed in this study could be adopted to explore the detection and diagnosis of other biomarkers for various diseases.

Development of anti-Müllerian hormone immunoassay based on biolayer interferometry technology.

Anti-Müllerian hormone (AMH) is a biomarker for the assessment of female fertility. The accurate measurement of the concentration of AMH is relevant for the success of assisted reproductive therapies and diagnosis of clinical cases. In this study, we show that cytokines such as fetal liver tyrosine kinase 3 ligand (Flt3L), CC subtype chemokine ligand 20 (CCL20), granulocyte-macrophage colony-stimulating factor (GM-CSF), and ß2-microglobulin (ß2M) significantly enhance the immune response against AMH. Two anti-AMH monoclonal antibodies (mAbs) with high affinity were selected by biolayer interferometry (BLI) technology for application in a fully automated magnetic chemiluminescence immunoassay (CLIA). This robust and rapid assay can efficiently detect AMH in the range of 0.125~20 ng mL-1 with a detection limit of 0.099 ng mL-1. This immunoassay showed high specificity with no cross-reaction with structurally related proteins and some of the other members of the TGF-ß super family, such as inhibin A, activin A, follicle-stimulating hormone, and luteinizing hormone. The average recovery rates of three different batches were 100.19%, 102.72%, and 103.59%, respectively, with coefficients of variation of less than 12%. The developed assay was applied in the detection of AMH in 69 serum samples from randomly selected patients. Our data showed a high correlation with those obtained using commercially available ELISA kits (correlation coefficient, 0.9831). Hence, we suggest that this immunoassay could find application in the development of POCT for the diagnosis of AMH in clinical samples. Graphical abstract.

Evaluation of a newly developed quantitative heart-type fatty acid binding protein assay based on fluorescence immunochromatography using specific monoclonal antibodies.

OBJECTIVES: To develop a rapid, sensitive and specific assay for quantification of serum heart-type fatty acid binding protein (H-FABP) based on immunofluorescence of specific monoclonal antibodies. DESIGN AND METHODS: We generated novel H-FABP-directed monoclonal antibodies by cloning of spleen cells of mice immunized with H-FABP. Epitopes were mapped and antigen affinity was assessed by surface plasmon resonance (SPR). The H-FABP specific monoclonal antibodies were coupled to fluorescent beads and sprayed onto a nitrocellulose membrane facilitating quantification of H-FABP by immunofluorescence. Reagent cross-reactivity, interference resistance, accuracy and sensitivity were examined. A total of 103 clinical samples were used to compare the sensitivity and specificity of the new assay to a commercially available Randox kit. RESULTS: This new assay could be finished within 15 min, with sensitivity reaching 1 ng/ml. In a trial of 103 clinical serum samples, the new testing kit results were highly correlated with those from the Randox kit (R(2) = 0.9707). Using the Randox kit as the reference kit, the sensitivity of the new assay was 98.25%, and specificity was 100%. CONCLUSIONS: An immunofluorescence-based H-FABP assay employing novel monoclonal antibodies could rapidly, specifically and sensitively detect H-FABP in serum samples, providing an effective method for rapid clinical assessment of H-FABP index in the clinic.

Plasmodium vivax aldolase-specific monoclonal antibodies and its application in clinical diagnosis of malaria infections in China.

BACKGROUND: Most rapid diagnostic tests (RDTs) currently used for malaria diagnosis cannot distinguish the various Plasmodium infections. The development of a Plasmodium vivax specific RDTs with high sensitivity to sufficiently differentiate the two most common Plasmodium infections would be very crucial for disease treatment and control. METHOD: Plasmodium vivax aldolase gene (PvALDO) was amplified from the extracted genomic DNA and constructed into pET30a vector. Plasmodium vivax aldolase protein was successfully expressed in Escherichia coli in soluble form and the overall purity was over 95% after one-step affinity chromatography purification. The purified products were used for the immunization of mice and rabbits. Rabbit polyclonal antibodies generated were deployed to develop a novel antibody-capture ELISA for hybridoma screening. RESULTS: Three PvALDO specific mAbs (14C7, 15F1 and 5H7) with high affinities were selected and used in immunochromatographic test strips. Clinical blood samples (n=190) collected from Yunnan (China) were used for evaluation and the RDT's sensitivity for P. vivax was 98.33% (95% Confidence Interval (CI): 91.03% to 99.72%) compared with microscopic examination. There was specificity of 99.23% (95% CI: 95.77% to 99.87%) for P. vivax. Only one Plasmodium falciparum sample was detected among the P. falciparum samples (n=20). All Plasmodium malariae samples (n=2) as well as healthy uninfected samples (n=108) were negative. Overall performance of this RDT was excellent with positive predictive value (PPV) and negative predictive value (NPV) of 98.33% and 99.23%, respectively, at 95% CI and a very good correlation with microscopic observations (kappa value, K=0.9757). Test strips show high sensitivity even at 6.25 ng/ml of recombinant P. vivax aldolase (rPvALDO). CONCLUSION: This study further elucidates the possibility of developing aldolase-specific RDTs which can differentiate the different Plasmodium infections and improve accurate diagnosis of malaria. This RDT could adequately differentiate between P. vivax and P. falciparum infections. The novel mAb screening method developed here could find application in the screening of highly specific antibodies against other antigens.