Membrane associated protein A2 blood serum detection method, detection reagent kit and its uses
A detection kit and combined detection technology, applied in the field of biology, can solve the problems of AnnexinA2 verification, poor prognosis and less than 10% survival rate
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Embodiment 1
[0033] Example 1: Serum indirect double-sandwich ELISA detection method using human ANXA2
[0034] In order to detect biological samples, the following ELISA detection method is carried out, and the specific steps of the method are:
[0035] 1. Coating: Use 50mM carbonate buffer (15mM Na 2 CO 3 ; 35mM NaHCO 3 ) as a diluent, a goat anti-human ANXA2 antibody (Santa Cruz Biotechnology, Cat. No. sc-1924) was diluted to 2 μg / ml. Add 50 μl of coating solution to the microwells of a polystyrene 96-well ELISA plate, seal it with plastic wrap, and place it in a refrigerator at 4°C overnight (>16h). The next day, use 350μl 150mM PBST buffer (137mM NaCl, 2mM KH 2 PO 4 , 10mM Na 2 HPO 4 , adding 0.05% Tween-20, adjusted to pH 7.4) immersion wash plate, 3 min × 3 times.
[0036] 2. Blocking: add 300 μl of 2% BSA (Sigma-Aldrich Company, Cat. No. A7906) to each well, and block for 4 hours at room temperature. Wash the plate with 350 μl PBST buffer immersion, 1 min×3 times.
[0037...
Embodiment 2
[0045] Example 2: Clinical application of serum indirect double-sandwich ELISA detection method for human ANXA2
[0046] Utilize the above-mentioned method of embodiment 1, 30 routine healthy adults (average age 51 years old, maximum age 70 years old, minimum age 33 years old) and 98 routine HCC patients (average age 54 years old, maximum age 83 years old) of age, gender matching , minimum age 15 years) serum ANXA2 concentrations were measured. The test results and the concentration gradient of the standard curve and the OD value subtracted from the background are shown in Table 1. The linear correlation coefficient between the two is 0.971, indicating that the concentration gradient basically falls within the linear range of the detection reaction. It also shows that the established system is relatively stable and effective for all cases, and the CV values of most samples are controlled within 10%.
[0047] Table 1: OD value of setting and detection of ANXA2 standard curve...
Embodiment 3
[0050] Embodiment 3: the parallel detection that adopts control method to carry out
[0051] The serum AFP concentration of all ELISA samples of the foregoing embodiment 2 was measured by electrochemiluminescence method (method routinely used clinically), and the results showed that the average serum AFP concentration of healthy adults and HCC patients was 3.5875ng / ml and 31.415ng respectively / ml. Draw the receiver curve (ROC curve) of these two proteins for HCC detection respectively ( figure 1 ), it was found that the area under the curve of AFP was 0.81, and that of ANXA2 was 0.78, and the diagnostic values of these two proteins were basically the same (chi-square test, p=0.6096). At this time, the serum AFP concentration to obtain the best diagnostic effect was 11 ng / ml, the diagnostic sensitivity was 93.3%, and the specificity was 63.5%. While the threshold of ANXA2 is 27.93μg / ml, the diagnostic sensitivity and specificity are 96.7% and 52.1%, respectively. When the...
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