Cytotoxin antibody-based colourimetric sensor for field-level differential detection of elapid among big four snake venom

Development of a rapid, on-site detection tool for snakebite is highly sought after, owing to its clinically and forensically relevant medicolegal significance. Polyvalent antivenom therapy in the management of such envenomation cases is finite due to its poor venom neutralization capabilities as well as diagnostic ramifications manifested as untoward immunological reactions. For precise molecular diagnosis of elapid venoms of the big four snakes, we have developed a lateral flow kit using a monoclonal antibody (AB1; IgG1 – κ chain; Kd: 31 nM) generated against recombinant cytotoxin-7 (rCTX-7; 7.7 kDa) protein of the elapid venom. The monoclonal antibody specifically detected the venoms of Naja naja (p < 0.0001) and Bungarus caeruleus (p<0.0001), without showing any immunoreactivity against the viperidae snakes in big four venomous snakes. The kit developed attained the limit of quantitation of 170 pg/μL and 2.1 ng/μL in spiked buffer samples and 28.7 ng/μL and 110 ng/μL in spiked serum samples for detection of N. naja and B. caeruleus venoms, respectively. This kit holds enormous potential in identification of elapid venom of the big four snakes for effective prognosis of an envenomation; as per the existing medical guidelines.

Imprinted Zeolite Modified Carbon Paste Electrode as a Selective Sensor for Blood Glucose Analysis by Potentiometry

Imprinted zeolite modified carbon paste (carbon paste-IZ) electrode had been developed as a sensor to analyze blood glucose content by potentiometry. The used zeolite was Lynde Type A (LTA) that synthesized with a mole ratio of Na2O, Al2O3, SiO2 and H2O of 4:1:1.8:270, respectively while non-imprinted zeolite was prepared with a mole ratio of glucose/Si of 0.0306. Glucose was then extracted from the zeolite framework using hot water (80 °C) to produce imprinted zeolite (IZ). The carbon paste-IZ electrode prepared from activated carbon, paraffin pastilles, and IZ with a mass ratio of 5:4:1 showed the best performance. The modified electrode demonstrated the measurement range of 10–4-10–2 M, the Nernst factor of 29.55 mV/decade, the response time less than 120 s, and the detection limit of 5.62 × 10–5 M. Ascorbic acid, uric acid, urea and creatinine did not interfere on the glucose analysis by potentiometry. Comparison test with spectrophotometry showed an accuracy of (90.7 ± 1.4)% (n = 5), while the application of the electrode to analyze five spiked serum samples showed recovery of (92.2 ± 1.3)% (n = 5). The electrode was stable for up to 9 weeks (168 times usage). Based on its performance, the developed electrode can be applied to analyze glucose in human serum sample and recommended for used in the medical field.

Synthesis of Atenolol-Imprinted Polymers with Methyl Methacrylate as Functional Monomer in Propanol Using Bulk and Precipitation Polymerization Method

Atenolol is one of the beta-1 blocker drugs that is misused by athletes to increase their performance during competition. Therefore, it is important to analyze atenolol levels in blood selectively. The preparation method that can be used in separating atenolol in sample is molecular imprinting solid-phase extraction (MI-SPE) because it has good selectivity and sensitivity. This study aims to examine the characteristics and analytical performance of imprinted polymers synthesized from functional monomer methyl methacrylate. The stages of this study include the determination of association constants, synthesis of sorbent MI-SPE atenolol using the bulk polymerization method, and precipitation with atenolol as the template, methyl methacrylate as the functional monomer, and propanol as the porogen. The template was extracted from a polymer, and then, the adsorption ability, capacity, and selectivity of MI-SPE and finally the application of the best MI-SPE to spiked serum samples were determined. MI-SPE was also characterized by using Fourier-transform instrument infrared (FTIR) and scanning electron microscope (SEM). The result of characterization with FTIR and SEM showed that MIP made by the precipitation polymerization method was completely polymerized, more porous, and produced smaller particle size with an average value of 0.274 μm. It had better analytic performances than MIP made by bulk polymerization, with affinity value 0.3607 mg/g and homogeneity value 1.3246, and good selectivity toward atenolol with imprinting factor value 22.519. Application of MI-SPE to spiked serum samples has an excellent recovery percentage of 95.46% over 0% for the nonimprinting one. Based on the result of study, MIP made by precipitation polymerization could be used to extract atenolol on serum samples toward drug analysis.

Validation of galectin-7 using backscattering interferometry.

15 Background: More than 60% of lung cancer patients are diagnosed with advanced disease due to a lack of early diagnosis tools. These patients are ineligible for surgical resection and have a poor prognosis. This situation could be avoided if we could quantify lung cancer specific biomarkers at sufficiently low concentrations, providing an indication of disease threat. Backscattering Inerferometry (BSI) is a label-free sensor with a simple optical train that is used to quantify biomarkers in complex matrices at picomolar to femtomolar levels. Here we demonstrate that BSI can enable lung cancer biomarker validation in complex, volume constrained samples and at detection limits significantly better than ELISA. Methods: A BSI dose response curve for Galectin-7 was constructed by incubating increasing concentrations (0-20 ng/mL) of recombinant galectin-7 with 100 ng/mL of polyclonal antibody. The samples were mixed at 300 RPM for 2 before being injected into the BSI instrument and measured in triplicate each day. All reagents used were obtained from a commercial ELISA kit. An ELISA dose response curve was similarly constructed using spiked serum and spiked plasma according to the manufacturer’s recommended procedure. Finally, 9 patient serum samples were quantified using BSI and ELISA for direct comparison of the two technologies. Results: BSI was used to quantify galectin-7 in spiked serum and patients samples. The lower limit of detection with standards was determined to be 0.5 ng/mL for ELISA in serum, 10 ng/mL for ELISA in plasma, and 0.04 ng/mL for BSI in serum. In the analysis of 9 patient serum samples, all were quantifiable using BSI, which enjoys low pg/mL detection limits, yet with ELISA only 5 samples contained a galectin-7 concentration high enough to be measured. In this small sample set there was a good correlation between disease state and galectin-7 concentration as measured by BSI. Conclusions: This study demonstrates that BSI is well-suited for biomarker detection and validation, having a greater dynamic operating range and much lower limits of detection than standard commercially available ELISA kits. Future work will focus on detecting other NSCLC biomarkers that are undetectable with currently available technology.

Comparison of Multiplex Immunoassay Platforms

Background: Candidate biomarkers discovered with high-throughput proteomic techniques (along with many biomarkers reported in the literature) must be rigorously validated. The simultaneous quantitative assessment of multiple potential biomarkers across large cohorts presents a major challenge to the field. Multiplex immunoassays represent a promising solution, with the potential to provide quantitative data via parallel analyses. These assays also require substantially less sample and reagents than the traditional ELISA (which is further limited by its ability to measure only a single antigen). We have measured the reproducibility, reliability, robustness, accuracy, and throughput of commercially available multiplex immunoassays to ascertain their suitability for serum biomarker analysis and validation. Methods: Assay platforms MULTI-ARRAY (Meso Scale Discovery), Bio-Plex (Bio-Rad Laboratories), A2 (Beckman Coulter), FAST Quant (Whatman Schleicher & Schuell BioScience), and FlowCytomix (Bender MedSystems) were selected as representative examples of technologies currently used for high-throughput immunoanalysis. All assays were performed according to protocols specified by the manufacturers and with the reagents (diluents, calibrators, blocking reagents, and detecting-antibody mixtures) included with their kits. Results: The quantifiable interval determined for each assay and antigen was based on precision (CV &lt; 25%) and percentage recovery (measured concentration within 20% of the actual concentration). The MULTI-ARRAY and Bio-Plex assays had the best performance with the lowest limits of detection, and the MULTI-ARRAY system had the most linear signal output over the widest concentration range (105 to 106). Cytokine concentrations in unspiked and cytokine-spiked serum samples from healthy individuals were further investigated with the MULTI-ARRAY and Bio-Plex assays. Conclusions: The MULTI-ARRAY and Bio-Plex multiplex immunoassay systems are the most suitable for biomarker analysis or quantification.