A kit and method for quantitatively detecting analyte in whole blood samples
A kit and material technology, applied in the field of immunological detection, can solve the problems of affecting the stability of reagents, increasing the cost of reagents, and affecting the detection speed, so as to achieve the effect of increasing reagent cost, reducing reagent cost and improving detection speed
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preparation example 1
[0154] Preparation Example 1: Preparation of whole blood C-reactive protein detection kit
[0155] In this preparation example, use goat anti-human CRP antibody (Guilin Inmate Biotechnology Co., Ltd.) to sensitize latex reagents (JSR Life Sciences) with an average diameter of 68nm and 198nm respectively, and then mix the above reagents in a certain proportion. as the reaction reagent. Specific steps are as follows:
[0156] Add 3mL of MES buffer pH5.8 to 1mL of carboxylated latex particles (taking the average diameter of latex particles as 68nm and solid content of 5% as an example), and then add carbodiamine hydrochloride (Aladdin Reagent Co., Ltd.) 10mg / mL 300μL , then add N-hydroxysuccinimide (Sinopharm Chemical Reagent Co., Ltd.) 10mg / mL 600uL, stir at 25°C for 20min, then add 0.2mol / L sodium carbonate solution 400μL, then add 5mL containing 1.6mg / mL goat antibody The 0.02MPBS solution of human C-reactive protein antibody was stirred at 25°C for 2h, and 2mL of 2% N6-PEG ...
Embodiment 1
[0159] Embodiment 1: Determination method of C-reactive protein content based on immunoturbidimetry
[0160] Add 250 μL of the reaction reagent obtained in Preparation Example 1 to the cuvette, incubate at 37°C, then add 8 μL of CRP calibrator with different concentrations, mix the calibrator and the reaction reagent thoroughly, and use the analyzer at 6 seconds and 60 seconds ( Guilin Youlit Medical Electronics Co., Ltd., BH-5360CRP) respectively measured the absorbance (A1, A2) of the reaction system at a wavelength of 850nm, and calculated the difference between the two (A2-A1). The absorbance difference is taken as the ordinate, and the corresponding CRP concentration is taken as the abscissa to make a standard curve.
[0161] Whole blood samples from patients were determined in the same way, and the content of C-reactive protein in the samples was calculated by the standard curve.
[0162] The preparation concentration is the CRP calibrator of 175.0mg / L, 120.0mg / L, 60.0m...
Embodiment 2
[0163] Embodiment 2: the impact of different blocking agents on detection results
[0164] Using N6-PEG obtained in Preparation Example 1 as the reaction reagent of the blocking agent, and bovine serum albumin (BSA) as the contrast reaction reagent of the blocking agent, and using the method of Example 1 to measure the relative concentration of C-reactive protein in series respectively Standard product, make CRP standard curve. The results are shown in Table 1 and figure 2 As shown, when N6-PEG was used as the blocking agent, the sensitivity and upper limit of detection of the reagent were significantly improved, and the linear range was wider. However, when BSA was used as the blocking agent, the reaction intensity at the lowest CRP value of 5 mg / mL was only 0.6 of that of N6-PEG. times, the HOOK effect appears earlier at a high value, and the upper limit of detection is low. It can be seen that N6-PEG is significantly better than BSA as a blocking agent.
[0165] Table 1...
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