Blood serum detection method of annexin A2 as well as detection kit and application thereof

A detection kit and combined detection technology, applied in the field of biology, can solve the problems of poor prognosis, AnnexinA2 verification, and survival rate of less than 10%

Active Publication Date: 2016-03-16
CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI +1
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  • Abstract
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Problems solved by technology

Due to its high degree of malignancy and poor prognosis, the five-year survival rate is less than 10%.
However, these two studies only screened the expression differences between HCC and healthy liver from the perspective of genome and proteome, and did not perform any validation on AnnexinA2 in clinical samples, and currently there is no AnnexinA2 that can be expressed in human serum detected and reported in association with human disease

Method used

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  • Blood serum detection method of annexin A2 as well as detection kit and application thereof
  • Blood serum detection method of annexin A2 as well as detection kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Serum indirect double-sandwich ELISA detection method using human ANXA2

[0035] In order to detect biological samples, the following ELISA detection method is carried out, and the specific steps of the method are:

[0036] 1. Coating: Use 50mM carbonate buffer (15mMNa 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 4°C refrigerator overnight (>16h). The next day, use 350 μl of 150 mM PBST buffer (137 mM NaCl, 2 mM KH 2 PO 4 ,10mMNa 2 HPO 4 , adding 0.05% Tween-20, adjusted to pH 7.4) immersion washing plate, 3 min × 3 times.

[0037] 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.

[...

Embodiment 2

[0046] Example 2: Clinical application of serum indirect double-sandwich ELISA detection method for human ANXA2

[0047] 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 match , 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%.

[0048] Table 1: OD value of setting and detection of ANXA2 standard curve

...

Embodiment 3

[0051] Embodiment 3: the parallel detection that adopts control method to carry out

[0052] 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 value of these two proteins was 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 these t...

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Abstract

The invention relates to a blood serum detection method of annexin A2 as well as a detection kit and application thereof, and particularly relates to a method for detecting HCC (Hepatocellular Carcinoma) by independently using a new marker annexin A2 or detecting the HCC by the combination of the annexin A2 and AFP (Alpha Fetal Protein) as well as the detection kit and the application thereof.

Description

[0001] This application is a divisional application of the invention application with the application number 200810089657.4 and the invention title of "serum detection method, detection kit and application of annexin A2". technical field [0002] The present invention relates to the field of biology, in particular to a method for detecting HCC using a new marker alone or in combination with AFP, a detection kit and an application thereof. Background technique [0003] Liver cancer (Liver cancer) is a malignant tumor that seriously endangers human health. In 2000, there were about 564,000 new cases worldwide, ranking fifth among all malignant tumors. Due to its high degree of malignancy and poor prognosis, the five-year survival rate is less than 10%. China is a high-incidence area of ​​liver cancer, where about 54% of the new cases in the world are concentrated, among which hepatocellular carcinoma (HCC) accounts for more than 90% of primary liver cancer [1,2] . According ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/68
CPCG01N33/57438G01N33/6893
Inventor 赵晓航孙玉琳乔媛媛
Owner CANCER INST & HOSPITAL CHINESE ACADEMY OF MEDICAL SCI
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