Kit for simultaneously detecting gynecological tumor markers HER-2 and HE4
A technology for HER-2 and gynecological tumors, applied in measuring devices, scientific instruments, instruments, etc., can solve the problems of low sensitivity and specificity of kits, time-consuming human gynecological tumors, and low reliability of test results. Achieve the effects of screening and diagnosis, good antigenicity, and error reduction
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Embodiment 1
[0039] Example 1 Preparation of HER-2-T antibody and HE4-T antibody
[0040] The amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 were used as the HER-2-T-1 antigen and the HER-2-T-2 antigen to immunize animals, thereby preparing specific HER-2- T-1 and HER-2-T-2 antibodies. In the same way, the amino acid sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 are used as the HE4-T-1 antigen and HE4-T-2 antigen to immunize animals, thereby preparing specific HE4-T-1 using the above antigens. And HE4-T-2 antibody. HER-2-T-1 and HER-2-T-2 are abbreviated as HER-2-T-1 / 2, and HE4-T-1 and HE4-T-2 are abbreviated as HE4-T-1 / 2.
[0041] 1. Immunize animals to prepare monoclonal antibodies:
[0042] 1.1. Take the above-mentioned HER-2-T-1 antigen, HER-2-T-2 antigen, HE4-T-1 antigen and HE4-T-2 antigen (immunogen) respectively with an equal volume of Freund's complete adjuvant (Purchased from Sigma) After mixing thoroughly, immunize Balb / c mice with 50 μg antigen / mouse, and inject th...
Embodiment 2
[0052] Example 2: Specific identification of human HER-2-T-1 / 2 antibody and HE4-T-1 / 2 antibody
[0053] Detected by ELISA. Human HER-2, human HE4, carcinoembryonic antigen (CEA), neuron-specific enolase (NSE) (all purchased from Sigma) were used as the detection antigen to coat the ELISA plate, and then the prepared HER- The 2-T-1 / 2 monoclonal antibody and HE4-T-1 / 2 monoclonal antibody react specifically with the antigen plate. Normal BALB / c mouse serum is used as a negative control, and PBS solution is used as a blank control.
[0054] Results: The HER-2-T-1 / 2 monoclonal antibody only reacts positively with HER-2 (P / N> 2.1), HE4-T-1 / 2 monoclonal antibody only reacts positively with HE4 (P / N> 2.1), and the reaction with CEA and NSE is negative, indicating that the HER-2-T-1 / 2 monoclonal antibody and HE4-T-1 / 2 monoclonal antibody of the present invention have specificity.
[0055] The above method was used to identify the specificity of HER-2-T-1 / 2 polyclonal antibody and HE4-T-1 / 2 p...
Embodiment 3
[0056] Example 3 Preparation of a kit for simultaneous detection of gynecological tumor markers HER-2 and HE4
[0057] This embodiment provides a kit for simultaneously detecting the gynecological tumor markers HER-2 and HE4, which includes individually packaged: a lanthanide-labeled HER-2-T-1 antibody and a lanthanide-labeled HE4-T- 1 Antibody reagents, antibody-coated plates coated with HER-2-T-2 antibody and HE4-T-2 antibody, calibrator solution prepared with a series of concentrations of HER-2 and HE4 antigen, reaction buffer, concentrated Washing liquid and / or enhancing liquid.
[0058] Preparation of lanthanide-labeled HER-2-T-1 antibody solution (Eu-HER-2-T): Add 0.2 mg of europium labeling reagent (Eu-HER-2-T) to 1 mg of HER-2-T-1 monoclonal antibody 3+ -N 2 -[P-Isocyanic acid-benzyl]-diethylenetriaminetetraacetate sodium), mixed and magnetically stirred at 30°C for 20 hours. The next day, the mixture was subjected to cross-linked agarose gel 6B column chromatography, the ...
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