Development of an enzymatic chromatography strip with nicotinamide adenine dinucleotide-tetrazolium coupling reactions for quantitative l-lactate analysis.

In this study, a dry assay of l-lactate via the enzymatic chromatographic test (ECT) was developed. An l-lactate dehydrogenase plus a nicotinamide adenine dinucleotide (NADH) regeneration reaction were applied simultaneously. Various tetrazolium salts were screened to reveal visible color intensities capable of determining the lactate concentrations in the sample. The optimal analysis conditions were as follows. The diaphorase (0.5 µl, 2(-6)U/µl) was immobilized in the test line of the ECT strip. Nitrotetrazolium blue chloride (5 µl, 12 mM), l-lactate dehydrogenase (1 µl, 0.25U/µl), and NAD(+) (2µl, 1.5×10(-5)M) were added into the mobile phase (100 µl) composed of 0.1% (w/w) Tween 20 in 10mM phosphate buffer (pH 9.0), and the process was left to run for 10 min. This detection had a linear range of 0.039 to 5mM with a detection limit of 0.047 mM. This quantitative analysis process for l-lactate was easy to operate with good stability and was proper for the point-of-care testing applications.

Numerical analysis of an immunochromatographic test strip reader in abused drugs screening.

A point-of-care immunoassay strip reader, Uniscan™, was applied to detect methamphetamine, opiate, and marijuana in human urine by providing numerical apparent drug concentrations. Calibration curves were determined by a nonlinear regression. The cutoff was verified using spiked controls. Clinical samples were analyzed and compared with enzyme multiplied immunoassay technique (EMIT). The discrepant results were confirmed by gas chromatography-mass spectrometry (GC-MS). The impacts of interference and cross-reactivity were determined for numerous compounds. The coefficients of the calibration curves had a high correlation coefficient. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and total recovery all had high values for spiked controls. For the 19 discrepant results of clinical samples, GC-MS confirmed that Uniscan and EMIT were correct for 11 and eight samples, respectively. For both methamphetamine and opiate, Uniscan had a lower false positive rate, a higher true negative rate, and a higher total recovery rate than EMIT. For marijuana, Uniscan had a higher true positive rate and a lower false negative rate than EMIT. The Uniscan performed excellently when compared to EMIT. It is advantageous for Uniscan to interpret the test result based on digital read-out, rather than subjective visual judgment.

Evaluation on the use of reactive dye-modified polylysine as the biomarker in immunochromatographic test application.

An improved dye immunochromatographic test (DICT) using polylysine (PL) as conjugate spacer loading dye molecules to enhance chromophor color intensity with the potential of a simultaneous multicolored assay has been developed. To construct this new effective chromophor, a dyeing process coupling a reactive dye, Procion Blue MX-7RX (PB7RX), with PL of different molecular weights was performed. The optimal conjugate condition between PB7RX and PL was studied. It showed that under the optimized dyeing conditions, a PL molecular weight of 189.4 kDa and a molar ratio (mol dye/mol amine group in PL) of 1.5 were obtained. The resulting dyed PL chromophor, used as both a spacer and a color intensifier, was further labeled to a model antibody, anti-human serum albumin (anti-HSA), to build a PB7RX-PL-anti-HSA (PPA) conjugate. The PPA obtained in this way generated the highest color intensity of 19,455 assayed by immunochromatographic test strip under densitometer scanning. A competitive DICT for determination of HSA was carried out. A linear range between 0 and 18.77 µg/ml with a detection limit of 0.49 µg/ml was observed. A test for using dyed PL chromophors as biomarkers was also performed to demonstrate the feasibility of a multianalyte immunoassay.

Development of a chromatographic strip assay for detection of porcine antibodies to 3ABC non-structural protein of foot-and-mouth disease virus serotype O.

A chromatographic strip assay was developed for rapid detection of serum antibodies to non-structural protein of foot-and-mouth disease virus. The assay was based on Escherichia coli-expressed 3ABC non-structural protein and an immunochromatographic technique, which shortened the detection time to about one hour. The sensitivity of the assay was determined to be 96.8% for infected pigs; its specificity was 100% for naïve pigs and 98.8% for vaccinated pigs. In the experimentally infected pigs, anti-3ABC antibodies were detectable from eight days post-infection until the end of the study, 34 days post-infection. The performance of this assay was comparable to that of two commercial ELISA kits, Ceditest FMDV-NS and UBI FMDV NS EIA, and was better than that of CHEKIT FMD-3ABC po. Given its advantages of instant testing and quantitative measurement, this assay has potential as a useful tool for rapid on-farm diagnosis of foot-and-mouth disease.

Development of a disperse dye immunochromatographic test for the detection of antibodies against infectious bursal disease virus.

For investigating the feasibility of using disperse dyes as an immunoassay chromogenic marker, a disperse dye, DADISPERSE NAVY BLUE SP, was selected in analyzing antibody against infectious bursal disease virus (anti-IBDV). With the color intensity revealed in the disperse dye immunochromatographic test (DICT) strip as the objective function, the optimal dyeing conditions were found as follows: dye concentration absorbance (at lambda(max)=587nm)=3, pH 7, 50 degrees C, for 10min. Under these conditions, the resultant dyed-antibody (rabbit anti-chicken) can produce an optimal color intensity reading of 55,054 on the strip. For performing qualitative immunoassay, chicken sera samples taken from different farms were used for the anti-IBDV titre assessment. The results of DICT strips showed very high sensitivity and specificity as compared to that analyzed by FlockChek enzyme linked immunosorbent assay (F-ELISA) kits. For quantitative immunoassay, it was found that the color intensity measured with DICT was linearly correlated to that of F-ELISA titre (r(2)=0.9687). Therefore, DICT was further applied to the detection of chicken anti-IBDV sera under vaccination in the farms. The average titres of the sampling groups exhibited a strong agreement to that of F-ELISA. Accordingly, the DICT method developed in this study, shown to be reliable, cheap and simple in both qualitative and quantitative immunoassays, is particularly suitable for point-of-need testing (PONT) in agricultural applications.

Optimized dyeing conditions of immunoprotein with reactive dye Procion Blue MX-7RX.

For investigating the feasibility of using reactive dyes as an immunoassay marker, a dichlorine triazine dye, Procion Blue MX-7RX, was employed to stain the antibody against human serum albumin (anti-HSA). With the color intensity revealed in the immunochromatographic test strip as the objective variables, the optimal dyeing conditions were found as follows: pH 11.4, temperature 35.7 degrees C, molar ratio 188 (mol dye/mol antibody), and reaction time 45.6 min. The dyed-anti-HSA revealed a maximal color intensity of 8738 without apparent loss of antigen binding affinity.

Evaluation and application of conducting polymer entrapment on quartz crystal microbalance in flow injection immunoassay.

An immunosensor employing conducting polymer entrapment (CPE) method to immobilize immuno-protein on the quartz crystal microbalance (QCM) for clinical flow-injection-analysis (FIA) purpose was exploited. By comparing the CPE approach and the conventional physical immobilization (PI) method, a frequency shift of the former was 47% higher than that of the latter when measuring at 0.5 mg/ml human serum albumin antibody concentration. This implied that CPE was a feasible approach for developing QCM-FIA process. An immunoassay of anti-pseudorabies virus antibody in mouse sera further exemplified its practical potential in diagnostic implication.