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PubMed · 12498211

Nine-analyte detection using an array-based biosensor.

Abstract

A fluorescence-based multianalyte immunosensor has been developed for simultaneous analysis of multiple samples. While the standard 6 x 6 format of the array sensor has been used to analyze six samples for six different analytes, this same format has the potential to allow a single sample to be tested for 36 different agents. The method described herein demonstrates proof of principle that the number of analytes detectable using a single array can be increased simply by using complementary mixtures of capture and tracer antibodies. Mixtures were optimized to allow detection of closely related analytes without significant cross-reactivity. Following this facile modification of patterning and assay procedures, the following nine targets could be detected in a single 3 x 3 array: Staphylococcal enterotoxin B, ricin, cholera toxin, Bacillus anthracis Sterne, Bacillus globigii, Francisella tularensis LVS, Yersiniapestis F1 antigen, MS2 coliphage, and Salmonella typhimurium. This work maximizes the efficiency and utility of the described array technology, increasing only reagent usage and cost; production and fabrication costs are not affected.

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BibTeXRIS

Chris Rowe Taitt, George P Anderson, Brian M Lingerfelt, s Mark J Feldstein, Frances S Ligler. 2002-12-01. Nine-analyte detection using an array-based biosensor.. https://doi.org/10.1021/ac0260185

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Antigens↗

The fibrinogen antigenic turbidimetric assay (FIATA): the X2x test--the corrected chi-square comparison against the control-mean.

Vancomycin precipitates fibrinogen. The turbidity induced by this vancomycin-fibrinogen interaction is used to establish a simple standardized antigenic assay for plasmatic fibrinogen, the FIATA. 1 mM vancomycin or 2 mM chloramine-T inactivates 50% of fibrinogen in human plasma. In contrast to chloramine-T, vancomycin does not react in NaJ-based photometric assay for chloramines,vancomycin does not inactivate the singlet oxygen-sensible antithrombin III, and the vancomycin action against fibrinogen is not changed in spite of the presence of the 1O2 quenchers methionine or ascorbic acid. The FIATA is performed as follows: to 25 microL plasma 50 microL PBS are added and the absorbance (A) at 405 nm is read. Then 50 microL FIATA-reagent, consisting of 4.4 mM vancomycin in PBS, are added. After 2 minutes (RT) DeltaA is determined and standardized against a plasma pool of 100% of norm (2.8 g/L) fibrinogen. The FIATA is nearly linear up to a fibrinogen concentration of about 150% of norm (4.2 g/L), resulting in a DeltaA of about 600 mA. The lower detection limit is 4% of norm (0.1 g/L). The intra-assay and interessay CV values are < 4%. The normal range of FIATA is 100% +/-20% (x- +/- 1 SD). In = 321 or 344 unselected patient plasmas the FIATA (x- = 130%; SD = 52% or 43%) correlated with the functional fibrinogen assays a) modified Clauss-Method (x- = 4.1 g/L; SD =1.7 g/L) with r = 0.755 and b) FIFTA (x- = 124%; SD = 40%) with r = 0.813. The vancomycin/fibrinogen interaction (binding of about 16 molecules of vancomycin/molecule of fibrinogen) can be used to purify fibrinogen out of plasma. Vancomycin also clouds dysfunctional fibrinogen (fibrinogen in presence of EDTA or chloramine-T)or soluble fibrin. Vancomycin-reacted fibrinogen stimulates tissue type plasminogen activator (t-PA) up to about 20-fold. The experimental data are analyzed by a new significance test: the two foldYates-corrected chi-square comparison against the mean value ofthe control-collective, called the Chi2x - Test. The P < .05 significance barrier calculated with the Chi2x - Test is equivalent to that calculated with the Fisher's Exact Test. The FIATA might be considered an interesting screening test for inactive fibrinogen forms or soluble fibrin, as eg in disseminated intravascular coagulation. Fibrinogen precipitation by vancomycin within the blood vessel might explain why vancomycin has to be infused slowly (< 10 mg/min) to prevent nephrotoxicity. The FIATA is of such a simplicity that the determination of fibrinogen antigen in plasma can be performed anywhere--even outside a hospital--within seconds. Thus, the presented FIATA might contribute to extra hospital testing of patients for assessing their risk for myocardial or cerebral ischemia/infarction.

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