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Taq DNA polymerase monoclonal antibody and application thereof

A technology of monoclonal antibody and polymerase, applied in application, transferase, anti-enzyme immunoglobulin, etc., can solve the problems of time-consuming, laborious and complicated operation, and achieve good amplification efficiency and sensitivity, good stability and good sealing effect of effect

Active Publication Date: 2022-07-12
GUANGDONG HECIN SCI INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Experimenters often improve the sensitivity and specificity of PCR detection by optimizing the primer probe, annealing temperature, magnesium ion concentration, etc. in the reaction system, but the operation is complicated and time-consuming

Method used

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  • Taq DNA polymerase monoclonal antibody and application thereof
  • Taq DNA polymerase monoclonal antibody and application thereof
  • Taq DNA polymerase monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1 Preparation of Taq DNA polymerase monoclonal antibody

[0087] Taq DNA polymerase monoclonal antibodies were produced using hybridoma technology as follows:

[0088] 1) Preparation of immunogen: According to the protein sequence of NCBI wild-type Taq DNA polymerase (SEQ ID NO: WP_156303260), after mutating three amino acids G59W / V155I / E507K, Taq DNA polymerase (anti-reverse enzyme) was obtained. The gene of Taq DNA polymerase (anti-reverse enzyme) was ligated into the prokaryotic expression vector pet28a, and the expression was induced by IPTG. After the bacteria were broken by ultrasonic, the supernatant was heat inactivated at 75°C and then purified by nickel column. The impurity protein was washed with 20 mM imidazole, and the protein eluted with 200 mM imidazole was collected. The eluted product was precipitated with ammonium sulfate and dialyzed through DEAE column. The elution peaks were identified by SDS-PAGE, and the results were as follows figure 1 ...

Embodiment 2

[0092] Example 2 Titer and sensitivity of ascites obtained by ELISA detection

[0093] 1) Ascites titer: The immunogen obtained in Example 1--Taq DNA polymerase was diluted with carbonate buffer as a coating solution, coated on a 96-well plate, 100ng / well, incubated at 37°C for 1.5h, and incubated After the completion, the plate was washed once with PBS-T; the ascites (diluted by sample diluent at different times, blank control) induced by different monoclonal cell lines (F10-6A5, F10-3F6, F10-5G1) obtained in Example 1 was added. The sample diluent was added to the group, and the sample diluent was a PBS solution containing 1v / v% BSA), 100uL / well, incubated at 37°C for 30 min, and the plate was washed 5 times with PBS-T after the incubation; Goat anti-mouse IgG-HRP) was diluted 1000 times, 100 μL / well; incubated at 37°C for 30 min; after incubation, the plate was washed 5 times with PBS-T; TMB color development, A solution: B solution = 1:1, ready to use; 100 μL / well, incuba...

Embodiment 3

[0099] Example 3 Taq DNA polymerase monoclonal antibody specificity detection

[0100] 1) ELISA method to detect monoclonal antibody specificity: ELISA method is used to detect whether Taq DNA polymerase monoclonal antibodies (6A5, 3F6, 5G1) non-specifically bind to other proteins (such as reverse transcriptase) in the PCR system, so as to detect antibodies specificity. Antigen (Taq DNA polymerase (anti-reverse enzyme), reverse transcriptase, reverse transcriptase purchased from Promega, Cat. No. M170A) was diluted with carbonate coating solution and then coated on a 96-well plate, 100ng / well, heated at 37°C Incubate for 2 h, wash the plate once with PBS-T after incubation; add the above Taq DNA polymerase monoclonal antibody (6A5, 3F6, 5G1) monoclonal antibody (diluted to a concentration of 1 mg / mL, and use the sample diluent at 1:100 , 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800), incubate at 37°C for 30 min; after incubation, wash the plate 5 times with PBS-T; Di...

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Abstract

The invention belongs to the technical field of antibodies, and discloses a Taq DNA polymerase monoclonal antibody and application thereof. The Taq DNA polymerase monoclonal antibody has a good sealing effect on Taq DNA polymerase, can well seal the activity of the Taq DNA polymerase under the conditions of 37 DEG C and 55 DEG C, deactivates and releases the activity of the Taq DNA polymerase under the condition of PCR denaturation at 94 DEG C, and reduces non-specific amplification at low temperature; meanwhile, the hot start enzyme containing the Taq DNA monoclonal antibody has better amplification efficiency and sensitivity, and the detection sensitivity is about 1.5 copies and is higher than the sensitivity in the prior art; and the detection effect is not influenced after the hot start enzyme containing the TaqDNA monoclonal antibody is repeatedly frozen and thawed or stored at 37 DEG C for 7 days, and the hot start enzyme has good stability and has important significance for deviating from normal storage conditions for a short time in the processes of use, transportation and the like.

Description

technical field [0001] The invention belongs to the technical field of antibodies, in particular to a Taq DNA polymerase monoclonal antibody and its application. Background technique [0002] PCR detection is an important means of molecular diagnosis. There are many types of clinical samples in the PCR detection process. Some pathogen samples are not only low in target gene concentration but also rich in inhibitors. Among them, saliva, sputum, nasopharyngeal swab, blood, etc. have strong inhibitory effects on the reaction, which often leads to difficulties in amplification . In daily testing, detection reagents with higher sensitivity and specificity are beneficial to the detection of low-concentration samples and the reduction of non-specific amplification of PCR reactions, thereby reducing false positives and false negatives in the testing process and improving the reliability of experimental results. Experimenters often improve the sensitivity and specificity of PCR det...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C12N15/13C12N5/20C12N9/12C12Q1/686
CPCC07K16/40C12N9/1252C12Q1/686C12Y207/07007C07K2317/565C07K2317/56C12Q2521/101C12Q2563/107Y02A50/30
Inventor 李小锋李晨阳李园枚
Owner GUANGDONG HECIN SCI INC
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