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Monocloned antibody of O6-methylguanine-DNA methyltransferase preparation process and test kit

A technology of monoclonal antibody and methyltransferase, applied in the preparation of the monoclonal antibody of the present invention, the hybridoma cell line CGMCC 0827 of the monoclonal antibody, detects O6-methylguanine-DNA methyl in the sample to be tested In the field of transferase level, it can solve the problems of isotope pollution, complicated operation and high price

Inactive Publication Date: 2004-06-09
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Regrettably, the isotope tracer method used to detect the MGMT activity of tumor cells at home and abroad has obvious defects: fresh tissue is required, and the MGMT activity of paracancerous tissue and tumor tissue cannot be distinguished; and the price is high and the operation is complicated. Time-consuming, raw materials need to be imported, will cause isotope pollution, and it is difficult to promote clinical application
There is also no detection kit for clinical use prepared by the monoclonal antibody

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] o 6 - Preparation of monoclonal antibody against methylguanine-DNA methyltransferase

[0064] 1. Hybridoma cell line preparation procedure

[0065] 1.1 Synthesis of hapten polypeptides

[0066] Adopt the Novasyn KR resin that Novabiochem Company produces, use solid-phase synthesis method (referring to, Pan Heping et al., " the synthesis of salmon calcitonin and its analogue ", Chinese Journal of Biochemistry and Molecular Biology, volume 14 the 4th period 1998 August 463-466) synthesized 14 peptides (C) KRTTLDSPLGKLE of the key antigenic epitope of human methyltransferase, in which cysteine ​​was added to the amino terminal to provide a sulfhydryl group for linking to the carrier protein KLH. The purity of the synthetic peptide is above 90%.

[0067] 1.2 Crosslinking of Hapten Peptides and Carrier Proteins

[0068] Keyhole limpet hemocyanin was selected as the hapten carrier. The MBS / KLH junction is formed by first linking the carrier protein to MBS as described in...

Embodiment 2

[0104] Embodiment 2 has the detection kit of monoclonal antibody of the present invention

[0105] I) the kit that adopts APAAP method to detect

[0106] ① Anti-human MGMT monoclonal antibody solution (BSA, 10g; 1% sodium azide, 10ml; anti-human

MGMT monoclonal antibody, 10ml; 0.1M PBS (Ph7.2-7.4) to make up to 100ml

3.0ml / bottle 1 bottle

② Special blocking solution (composition: 0.01MPBS (7.2~7.4) 80ml;

Periodic acid, 0.1g; calf serum, 10ml;

1%NaN 3 , 10ml)

3.0ml / bottle 1 bottle

③Secondary antibody (goat anti-mouse IgG antiserum: BSA, 10g; 1% sodium azide, 10ml;

Goat anti-mouse IgG antiserum (purchased from the Institute of Basic Medical Sciences, Academy of Military Medical Sciences), 10ml;

0.1M PBS (pH7.2-7.4) to 100ml

3.0ml / bottle 1 bottle

④ Enzyme APAAP complex (purchased from Institute of Basic Medical Sciences, Academy of Military Medical Sciences)

3.0ml / bottle 1 bottle

⑤ substrate (solid...

Embodiment 3

[0141] Embodiment 3 Stability of detection kit of the present invention

[0142] 1 Kit 37°C Accelerated Destruction Test

[0143] The three batches of kits produced continuously were placed in a 37°C incubator, and they were taken out on the 1st, 3rd, 5th, 7th, and 9th day respectively. The kits stored at 37°C for 0 days were used as controls, and 5 cases of positive reference products and Negative reference test. The test results showed that after the three batches of products were placed at 37°C for 1 to 9 days, the test results were consistent with those of the control group, the coincidence rate of the positive reference product and the negative reference product were both 100%, and the sensitivity and specificity met the requirements of product quality standards . According to the Arrhenius equation, the kit is stored at 37°C, and every stable day is equivalent to 1.6 months at 4°C, and 9 days at 37°C is equivalent to 14 months at 4°C.

[0144] 2 kit 4 ℃ stability test...

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Abstract

The present invention relates to a monoclonal antibody of specific conjugated O6-methylguanine-DNA methyltransferase KRTTLDSPLGKLE(C) sequence and hybridoma cell line CGMCC 0827 capable of secreting said monoclonal antibody. Said invention also relates to the method for preparing said monoclonal antibody and the application of the described monoclonal antibody in preparation of horizontal detection kit for identifying O6-methylguarine-DNA methyltransferase in the sample. Said invention also relates to the method for utilizing said kit.

Description

technical field [0001] The present invention relates to a hybridoma secreted by the preservation number of CGMCC 0827, which specifically binds O 6 - Monoclonal antibody to the KRTTLDSPLGKLE (C) sequence on methylguanine-DNA methyltransferase (MGMT). The present invention also relates to the secretion of specific binding O 6 - Hybridoma cell line CGMCC 0827 with monoclonal antibody to the epitope KRTTLDSPLGKLE (C) on methylguanine-DNA methyltransferase. The present invention also relates to a method for preparing the monoclonal antibody of the present invention, and the monoclonal antibody is used to prepare and identify O in samples 6 - the use of the kit for detecting the level of methylguanine-DNA methyltransferase. The present invention further relates to a method of using the kit to detect O in the sample to be tested. 6 - Methylguanine-DNA methyltransferase level method. Background of the invention [0002] Malignant tumors have long been one of the major diseases...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44C12N5/12G01N33/577
Inventor 章扬培任会明由英季守平
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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