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CEA detection kit based on bimolecular fluorescence complementation technology, preparation method and use method thereof
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A detection kit and bimolecular technology, applied in the field of bimolecular fluorescence complementation, can solve the problems of long operation time, radioactive contamination, and reduction of reagent precision in enzyme-linked immunosorbent assay.
Inactive Publication Date: 2018-08-10
NANJING TZONE BIOLOGICAL SCI & TECH
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The enzyme-linked immunosorbent assay takes a long time to operate, the process needs to be cleaned, and the steps are cumbersome
Magnetic particle chemiluminescence is a heterogeneous reaction, and cleaning is required during the operation, which reduces the precision of the reagents
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Embodiment 1
[0028] The anti-CEA antibody is coupled to the N-terminal fragment of fluorescent protein, taking the yellow fluorescent protein (YFP) 1-154 amino acid fragment YFPN as an example, the specific implementation process is as follows:
[0029] 1) Add 0.1mg of YFPN protein into a centrifuge tube, and prepare with 0.05mol / L pH9.5 carbonate buffer (CB) to dilute the YFPN protein to 1mg / mL.
[0031] 3) Reaction in a water bath at 37°C for 2 hours.
[0032] 4) Dialyze with desalting column Sephadex G-25 or 0.05mol / L pH9.5 CB to remove excess glutaraldehyde.
[0033] 5) Take 0.1mg anti-CEA antibody, prepare 1mg / mL antibody with 0.05mol / L pH9.5 CB, and mix the activated YFPN protein and antibody.
[0034] 6) React overnight at 4°C.
[0035] 7) Blocking: add 50 μL of 0.2 mol / L lysine solution, and block for 2 hours at room temperature to block residual aldehyde groups and terminate the reaction.
[0036]...
Embodiment 2
[0039] The anti-CEA antibody is coupled to the fluorescent protein C-terminal fragment, taking the yellow fluorescent protein (YFP) 155-238 amino acid fragment YFPC as an example, the specific implementation process is as follows:
[0040] 1) Add 0.1mg of YFPC protein into a centrifuge tube, and prepare with 0.05mol / L pH9.5 carbonate buffer (CB) to dilute the YFPC protein to 1mg / mL.
[0042] 3) Reaction in a water bath at 37°C for 2 hours.
[0043] 4) Dialyze with desalting column Sephadex G-25 or 0.05mol / L pH9.5CB to remove excess glutaraldehyde.
[0044] 5) Take 0.1mg of anti-CEA antibody, prepare 1mg / mL antibody with 0.05mol / L pH9.5 CB, and mix the activated YFPC protein and antibody.
[0045] 6) React overnight at 4°C.
[0046] 7) Blocking: add 50 μL of 0.2 mol / L lysine solution, and block for 2 hours at room temperature to block residual aldehyde groups and terminate the reaction.
[0047...
Embodiment 3
[0050] The main components of the kit:
[0051] 1) N-terminal fragment of fluorescent protein coupled with anti-CEA antibody;
[0052] 2) C-terminal fragment of fluorescent protein coupled with anti-CEA antibody.
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Abstract
The invention provides a CEA diagnosis kit based on a bimolecular fluorescence complementation technology, wherein the kit comprises an anti-CEA antibody conjugated fluorescent protein N-terminal fragment and an anti-CEA antibody conjugated fluorescent protein C-terminal fragment. The invention further discloses a preparation method of the CEA diagnosis kit, wherein the preparation method comprises: preparation of an anti-CEA antibody conjugated fluorescent protein N-terminal fragment, and preparation of an anti-CEA antibody conjugated fluorescent protein C-terminal fragment. The invention further discloses a use method the kit. According to the present invention, the kit has advantages of simple operation, no cleaning, high precision, high accuracy and the like, is conveniently suitable for clinical detection, can increase the accuracy of the diagnosis of tumors in the monitoring of tumors, and has great market value.
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