Background[1-3]
Alkaline phosphatase (ALP) assay kit (microplate method) is used to detect endogenous alkaline phosphatase activity in cell or tissue lysate or homogenate, plasma, serum, urine and other samples . The PNP colorimetric method is used. The detection principle is that Para-nitrophenyl phosphate (pNPP) is a commonly used phosphatase color substrate. Under acidic conditions, AMP is used as the acceptor substance for acyl phosphate, which can be used in alkaline phosphatase. p-nitrophenol is generated under the action of . Under alkaline conditions, p-nitrophenol transforms into a quinone structure, which is darker yellow. The darker the yellow color of the product, the higher the activity of alkaline phosphatase. Otherwise, the lower the activity of the enzyme, measured by a microplate reader ~ 415 Carbo Absorbance at nm of special white carbon black.
The method of measuring the content of a substance by using the change in color of a solution is called colorimetric analysis. With the development of testing instruments, from early visual colorimetry to spectrophotometry. Spectrophotometry is not only applicable to the visible light region, but also extends to the ultraviolet and infrared light regions. The absorption of light by substances is the basis of colorimetry and spectrophotometry. The law of light absorption: Lambert-Beer’s law is the basis of quantitative measurement. Spectrophotometry uses a spectrophotometer to measure the absorbance of a series of standard solutions, draw a standard curve, and then calculate the concentration or content of the test substance from the standard curve based on the absorbance of the test solution. Spectrophotometry and visual colorimetry are not exactly the same in principle. Spectrophotometry is to compare the absorption of light of a certain wavelength by colored solutions, and visual colorimetry is to compare the intensity of transmitted light.
Alkaline phosphatase (ALP or AKP) is an enzyme that is widely distributed in human liver, bones, intestines, kidneys, placenta and other tissues and is excreted out of the bile through the liver. This enzyme can catalyze the removal of the 5′ phosphate group from nucleic acid molecules, thereby converting the 5′-P end of the DNA or RNA fragment into a 5′-OH end. But it is not a single enzyme, but a group of isoenzymes. Six isozymes, AKP1, AKP2, AKP3, AKP4, AKP5 and AKP6, have been discovered so far. Among them, types 1, 2, and 6 all come from the liver, the third type comes from bone cells, the fourth type is produced from placenta and cancer cells, and the fifth type comes from small intestinal villous epithelium and fibroblasts.
Apply[4][5]
For rapid quantitative detection of prealbumin and bone-specific alkaline phosphatase using fluorescence immunochromatography
Prealbumin (PA) and bone-specific alkaline phosphatase (BAP) are specific biochemical indicators for detecting protein-energy nutrition and bone calcium nutrition status respectively, and can be stably and accurately monitored Nutritional status of the human body.
By optimizing the experimental parameters of the immunochromatographic method, we aim to establish two fluorescence immunochromatographic methods for the rapid and quantitative detection of PA and BAP in human serum, and promote the established methods for clinical application, providing a basis for the determination of human nutritional status. Monitoring provides reliable data.
Using the double-antibody sandwich principle, a fluorescence immunochromatographic method for quantitative detection of PA in serum was established.
Quantitative detection can be achieved based on the ratio of the fluorescence intensity (FIT/FIC) of the test strip test line (T line) and the quality control line (C line), which is proportional to the PA concentration in the sample. The experiment conducted paired screening of anti-PA monoclonal antibodies and polyclonal antibodies.
The fluorescent microsphere-anti-PA monoclonal antibody label was prepared through the EDC one-step mediated method, and the experimental parameters during the labeling process and test strip preparation were optimized, and the optimal experimental conditions were selected to detect PA in the sample. content. The detection limit of the immunochromatography test strip is 1.0 ng/m L, and the detection result can be obtained within 20 minutes. It has a good linear relationship (R2=0.998) in the concentration range of 8.0 ng/m L~110.0 ng/m L. ); there is no obvious cross-reaction with hemoglobin, albumin, etc., and the specificity is good; the coefficient of variation of the spiked recovery experiment is within 10%, and the repeatability is good; the methodology of this method was compared with the immunoturbidimetric method, and the results It shows that the correlation coefficient between the two is 0.94. The experimental results show that the established PA fluorescence immunochromatography test strip is sensitive, specific, and time-saving.
