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A kind of detection method and detection kit for transpeptidase activity

A detection kit and detection method technology, applied in biological testing, measuring devices, material testing products, etc., can solve the problems of slow hydrolysis reaction rate of transpeptidase A, and achieve low quantitative lower limit, simple operation and high throughput. Effect

Active Publication Date: 2022-07-19
GENEQUANTUM HEALTHCARE SUZHOU
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Existing detection methods are only used to detect the hydrolysis function of transpeptidase A, while transpeptidase A used in coupling technology includes two functions of hydrolysis and coupling, and the main functional sites of hydrolysis and coupling are also separated ; The hydrolysis reaction rate of transpeptidase A is much slower than its coupling rate

Method used

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  • A kind of detection method and detection kit for transpeptidase activity
  • A kind of detection method and detection kit for transpeptidase activity
  • A kind of detection method and detection kit for transpeptidase activity

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Embodiment 1

[0052] 1. Main materials and equipment

[0053] A full-wavelength microplate reader from Molecular Device, a 96-well V-bottom dilution plate, and a 96-well microplate.

[0054] 2. Sources and preparation methods of biological and chemical reagents

[0055] The first peptide substrate: biotin-PEG4-LPETGG (20 mg / mL) and the second peptide substrate: GGGGG-PEG4-His-His-His-His-His-His (20 mg / mL) The preparation method is mainly for:

[0056] Both the first peptide substrate and the second peptide substrate use dichloro resin as the starting material, and are synthesized by conventional steps such as deprotection and coupling; Molecular PEG and 1 molecule of biotin, and finally use trifluoroacetic acid cutting solution to cut the peptide from the resin, prepare, purify and freeze-dry it; the second peptide substrate is first solid-phase synthesized GGGGG, and then solid-phase added 4 molecules of PEG and 6 Molecular His is finally obtained by cleaving the polypeptide from the r...

Embodiment 2

[0075] (1) Dilute 6×His-tag monoclonal antibody (Thermo Fisher) solution with carbonate buffer (pH9.6) to 2 µg / mL, add 100 µL per well to 96-well microtiter plate, 2-8 Incubate overnight at ℃;

[0076] (2) Shake off the liquid in the plate, wash three times with PBST (300 µL / well), shake at 350 rpm for 30 sec; pat dry, add PBST containing 5% BSA, 250 µL / well, incubate at 25°C for 2 h;

[0077] (3) After blocking, wash 3 times with PBST (300µL / well each time), and pat dry for later use;

[0078] (4) Dilute transpeptidase A with Tris-HCl buffer, and prepare standard and quality control products respectively. The concentration gradient of transpeptidase A standard S1-S8 is 5000 ng / mL, 2500 ng / mL, and 1250 ng / mL. mL, 625 ng / mL, 313 ng / mL, 156 ng / mL, 78 ng / mL, 39 ng / mL, and the concentration gradients of the quality controls were 2000 ng / mL, 500 ng / mL, 200 ng / mL, 78 ng / mL; the upper and lower limits of quantification are 2500 ng / mL and 78 ng / mL, respectively, where S1 and S8 are ...

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Abstract

The present invention provides a detection method and detection kit for transpeptidase activity, which comprise the following steps: (1) respectively mixing a first peptide substrate, a second peptide substrate and a transpeptidase A standard or a sample to be tested Incubate to form peptide complexes; the mass ratio of the first peptide substrate and the second peptide substrate is 10-150:1; (2) the peptide complexes are attached to the support; (3) the addition of compounds capable of binding to biotin and The complex of the enzyme capable of catalyzing the color development of the substrate and the chromogenic substrate detect the signal value emitted by the reaction system, and evaluate the enzyme activity of the sample to be tested according to the detection result of the transpeptidase A standard. The method of the invention can simultaneously reflect the hydrolysis and coupling functions in the actual technological application of the transpeptidase, and has high detection sensitivity, lower quantitative lower limit, low detection cost, high throughput and simple operation.

Description

technical field [0001] The invention belongs to the fields of biological analysis, immune detection and enzyme activity detection, and in particular relates to a detection method and detection kit for transpeptidase activity. Background technique [0002] In the increasingly hot field of biological targeted drugs, Antibody Drug Conjugates (ADC) has attracted much attention due to its unique structural characteristics, high efficiency and low toxicity. However, the success of ADC drugs is inseparable from the development of conjugation technology. progress. The traditional chemical coupling technology has problems such as complex preparation process, damage to protein activity, and non-uniform connection products in the ADC coupling process. The ADC coupling technology mediated by transpeptidase (Sortase) can be well overcome. A series of problems of traditional chemical ligation methods, and obvious advantages such as mild reaction conditions, high reaction efficiency, and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/573G01N33/577G01N33/58G01N21/78
CPCG01N33/573G01N33/577G01N33/581G01N21/78G01N2333/96466
Inventor 任朋亮王俊皓孙亚军秦刚
Owner GENEQUANTUM HEALTHCARE SUZHOU
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