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Application of ochratoxin A anti-idiotypic nano-antibody used as ochratoxin A standard substitute

An ochratoxin, anti-idiotype technology, applied in the field of molecular biology, can solve the problems of long production cycle, small antibody molecular weight, complex preparation process, etc., and achieve the effect of good accuracy and accurate results

Active Publication Date: 2019-03-29
深圳市金阅检测科技有限责任公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Using polyclonal antibody technology to prepare anti-idiotypic antibody production technology is relatively simple, easy to operate, and low cost, but the later purification of serum is more cumbersome, and the single output is limited
However, the monoclonal antibody method has a long production cycle, complicated preparation process and high cost, and the cell fusion rate and positive rate are far lower than Ab1 class monoclonal antibodies. However, once this method is successful, the resulting hybridoma cell line can be Long-term stable storage enables the preparation of a large amount of AId once and for all, and the method is simple, the antibody is of high quality, and its properties are uniform and stable
The preparation of AId by phage antibody library technology is simple, fast, and economical. The resulting antibody has small molecular weight, low immunogenicity, and consistent genotype and phenotype. It creates a new method for preparing humanized antibodies, but this method requires a large antibody library capacity. High and rich in diversity, the post-screening method should also be suitable, otherwise it is difficult to obtain AId with excellent performance

Method used

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  • Application of ochratoxin A anti-idiotypic nano-antibody used as ochratoxin A standard substitute
  • Application of ochratoxin A anti-idiotypic nano-antibody used as ochratoxin A standard substitute
  • Application of ochratoxin A anti-idiotypic nano-antibody used as ochratoxin A standard substitute

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of Phage Display Nanobody Immune Library

[0035] 1. Alpaca immunity

[0036] Take 200 μg of ochratoxin A monoclonal antibody (dissolved in PBS7.4), emulsify it with an equal volume of Freund’s incomplete adjuvant, and inject it into three-year-old male alpaca (Alpaca) subcutaneously, and then every two weeks One immunization, a total of 8 immunizations, blood testing began after the fourth immunization. Seven to 10 days after the fourth immunization, 10 mL of blood was collected from the jugular vein of an EDTA vacuum blood collection tube. At the same time, the blood collection tube was gently inverted to avoid blood coagulation, and then the blood was processed with the filter in the LeukoLOCK kit, and then sequentially washed with 3 mL of LPBS buffer and 3 mL of RNAlater Wash the filter with the buffer solution, and the required white blood cells will be trapped in the filter, and finally the filter should be sealed and stored at -80°C for RN...

Embodiment 2

[0065] Example 2 Panning and identification of ochratoxin A anti-idiotype nanobody

[0066] (1) Panning of ochratoxin A anti-idiotypic nanobodies

[0067] Taking ochratoxin A monoclonal antibody as the target (the said ochratoxin A monoclonal antibody is secreted by the hybridoma cell line 1H2 with the preservation number CCTCC NO.C201329, and the hybridoma cell line 1H2 was released in March 2013 It was preserved in the China Center for Type Culture Collection (CCTCC) on the 7th, and has been disclosed in the patent "Hybridoma Cell Line 1H2, Anti-Ochratoxin A Monoclonal Antibody Produced by It and Its Application", application number: 201310115921.8), passed Reduce the concentration of the original coating and the concentration of ochratoxin A standard substance for competitive elution round by round, alternately use blocking reagents, and perform affinity enrichment panning to obtain antibodies against ochratoxin A monoclonal antibodies, namely anti-antibodies, and Known as...

Embodiment 3

[0091] Example 3 Expression and purification of ochratoxin A anti-idiotype nanobody

[0092] (1) Preparation of Top10F' competent cells:

[0093] (1) Pick an appropriate amount of Top10F' to streak on the LB-tetracycline plate, and culture overnight at 37°C;

[0094] (2) Pick the Top10F' monoclonal into 5mL LB medium, culture at 37°C, 250rpm until OD 600 0.6-0.8;

[0095] (3) Transfer the bacterial liquid to a pre-cooled centrifuge tube (about 1.3mL / tube), centrifuge at 4°C, 8000rpm, 8min;

[0096] (4) Put it on ice immediately after centrifugation, discard the supernatant, and add 1 mL of pre-cooled 0.1M CaCl 2 Resuspend the bacteria in the solution, pipette and mix well, and then ice-bath for 30 minutes;

[0097] (5) At 4°C, centrifuge at 8000rpm for 8min, put it back on ice quickly, suck up the liquid completely, and use 100μL pre-cooled 0.1MCaCl for precipitation 2 The solution is resuspended, which is Top10F'competent cells.

[0098] (2) Extraction and transformatio...

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Abstract

The invention belongs to the field of molecular biology, and particularly relates to an application of an ochratoxin A anti-idiotypic nano-antibody used as a ochratoxin A standard substitute. The present invention obtains the ochratoxin A anti-idiotypic nano-antibody with an amino acid sequence shown in SEQ ID NO: 1. According to ELISA standard curves of standard ochratoxin A and an ochratoxin A anti-idiotypic antibody, under the same inhibition ratio, corresponding relationship curves of the concentration of the ochratoxin A and the concentration of the ochratoxin A anti-idiotypic antibody are established. Besides, the results are subjected to exponential regression analysis to obtain a corresponding relationship between the the ochratoxin A and the ochratoxin A anti-idiotypic antibody. The corresponding relationship can be applied to the detection and analysis of ochratoxin A in agricultural products. The immunodetection method that the ochratoxin A anti-idiotypic nano-antibody replaces the standard ochratoxin A has accurate and reliable results, and is an effective and feasible method for green immunoassay.

Description

technical field [0001] The invention belongs to the field of molecular biology, and specifically relates to the application of an ochratoxin A anti-idiotype nanobody as a substitute for an ochratoxin A standard product. Background technique [0002] Ochratoxin A (ocbratoxinA, OTA) is a toxic fungal secondary metabolite, mainly produced by Aspergillus and Penicillium, which mainly pollutes food, vegetables, coffee, grapes, cocoa and other economic crops. The globalization of toxin-producing fungi and the continuous development of world trade in crop products have resulted in the widespread spread of ochratoxin A in the world, and has seriously endangered the health of animals and humans. It has strong nephrotoxicity and hepatotoxicity, and has teratogenicity, carcinogenicity, immunotoxicity and embryotoxicity. Many countries have set limit standards, so it is extremely important to establish a low-cost, fast and effective on-site detection method. [0003] At present, the me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/42C12N15/13G01N33/543
CPCG01N33/543C07K16/4208C07K2317/569
Inventor 李培武王防修唐晓倩姜俊张奇张文王督
Owner 深圳市金阅检测科技有限责任公司
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