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P16 immune cell chemical reagent kit and polypeptide sequence in preparing same

An immunocytochemistry and kit technology, applied in the fields of peptides, peptide sources, biological tests, etc., can solve the problem of insufficient sensitivity of single cell detection

Active Publication Date: 2015-04-15
何以丰 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The mouse anti-human P16 monoclonal antibody purchased in the market is mainly suitable for immunochemical labeling of tissue sections rather than cell smears, which is not sensitive enough for single cell detection

Method used

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  • P16 immune cell chemical reagent kit and polypeptide sequence in preparing same
  • P16 immune cell chemical reagent kit and polypeptide sequence in preparing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, kit assembly

[0048] Three conjugates of keyhole limpet hemocyanin and polypeptides A, B and C (with glutaraldehyde as the coupling agent) were used to immunize mice respectively, and prepared according to the common monoclonal antibody preparation method against the above three polypeptide antigens Mouse anti-human P16 protein IgG monoclonal antibody production series (each production series contains 3 optimized and selected antibodies). Dilute the purified monoclonal antibody stock solution in phosphate buffer, prepare antibody mother solution at 1 mg / ml, and then mix any three antibody mother solutions at 1:1:1 (volume ratio) according to different combinations to ensure Each mixture combination contains and only contains three antibody members, and the three are from three different antibody preparation series. Then, using the obtained tri-antibody mixture (total antibody concentration: 1 mg / ml) and the three individual members of the mixture (ie: 1 ...

Embodiment 2

[0056] Embodiment two, the detection process of the sample to be tested

[0057] Take the 70% ethanol suspension containing women's cervical exfoliated cells, spread it on a glass slide (the coating area must be less than 5 square centimeters), and dry it at room temperature to become a cervical cell smear to be tested.

[0058] Take 2 microliters of component No. (1), add 98 microliters of component No. (6), dilute to become the primary antibody working solution, add dropwise to the cervical cell smear to be tested, covering all the cell coating area. Incubate for 1 hour at room temperature.

[0059] Use 10 ml of phosphate buffer solution (prepared by the experimenter, not provided in this kit) to wash away the above primary antibody working solution, and repeat three times.

[0060] Take 2 microliters (2) component, add 98 microliters (6) component, dilute to become the second antibody working solution, add dropwise to the cervical cell smear to be tested, covering the enti...

Embodiment 3

[0064] Embodiment three, the detection process of positive and negative reference substance

[0065] Cut open the plastic bags for positive and negative control substances, pour off all the 70% ethanol fixative solution, and gently rinse each petri dish three times with 10 ml of phosphate buffer solution (prepared by the experimenter, not provided in this kit).

[0066] Take 2 microliters of component (1), add 98 microliters of component (6), dilute to become the primary antibody working solution, and add dropwise to the petri dish of component (4) (ie: positive control substance) ; Also take 2 microliters of component (1), add 98 microliters of component (6), dilute to become the primary antibody working solution, and add dropwise to the culture of component (5) (ie: negative control substance) in the dish. Incubate for 1 hour at room temperature.

[0067] For each petri dish, wash away the above primary antibody working solution with 5 ml of phosphate buffer solution (prep...

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Abstract

The invention relates to a P16 immune cell chemical reagent kit and a polypeptide sequence in preparing the same. The kit comprises the following components (1), a first antibody, (2) a second antibody, (3) an enzyme substrate, (4) positive controlled substances, (5) negative controlled substances, and (6) a buffer solution. The kit can be used for marking abnormally expressed P16 protein in human cancer cells and pre-neoplastic cells to allow the cells to develop inordinately so as to distinguish from normal cells or same-type cancer cells where covariation of P16 protein coding gene is not further generated.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a method for preparing an immunocytochemical kit for marking P16 protein in human cells and several polypeptide components used for preparing related monoclonal antibodies in the kit. Background technique [0002] Human P16 protein is a protective protein in cells, which can prevent human cells from becoming cancerous and prevent excessive cell proliferation. The gene encoding human P16 protein is CDKN2A, which is located on the short arm of chromosome 9. P16 protein can bind to cyclin-dependent kinase 4 and inhibit its activity, so that cells stay in the G1 phase and cannot continue to proliferate. Therefore, P16 is a tumor suppressor protein. In cells, the expression of P16 protein is usually regulated by two other tumor suppressor proteins: P53 and RB, and is at a low level. [0003] In cervical cancer and its precancerous cells, the expression of P16 protein often increases abnorm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68C07K7/08C07K14/47
Inventor 狄文何以丰张梅莹
Owner 何以丰
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