Anti-idiotypic nano antibody based zearalenone green immunoassay method

A zearalenone and nanobody technology, applied in the field of molecular biology, can solve the problems of long production cycle, small antibody molecular weight, complicated preparation process, etc., and achieve the effects of good accuracy, high library capacity and good diversity

Inactive Publication Date: 2018-01-19
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
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  • 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, with the monoclonal antibody method, the production cycle is long, the preparation process is relatively complicated, the cost is high, and the cell fusion rate and positive rate are much lower than that of Ab 1 However, once this method is successful, the obtained hybridoma cell line can be stored stably for a long time, and a large amount of AId can be prepared once and for all. 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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  • Anti-idiotypic nano antibody based zearalenone green immunoassay method

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Construction of Phage Display Nanobody Immune Library

[0043] (1) Alpaca immunization: Take 150 μg of anti-zearalenone monoclonal antibody 2D3 (dissolved in conventional phosphate buffer solution), emulsify with an equal volume of Freund’s incomplete adjuvant, and treat three-year-old male alpacas ( Alpaca) were injected subcutaneously at multiple points, and then immunized once every two weeks for a total of six times. After the fourth immunization, blood samples were collected for testing. 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, and 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 conventional phosphate buffer saline Wash the filter with 3mL RNAlater buffer, the required white blood cells will be trapped i...

Embodiment 2

[0056] Example 2 Panning and Identification of Zearalenone Anti-idiotypic Nanobodies

[0057] (1) Panning of zearalenone anti-idiotypic nanobodies

[0058] Using the 2D3 monoclonal antibody as the target, the antibody against the 2D3 antibody was obtained by reducing the concentration of the original coating and the concentration of the standard zearalenone that was eluted by competition, and alternately using blocking reagents for affinity enrichment panning. That is, anti-antibody, also known as anti-idiotypic antibody, the panning steps are as follows:

[0059] (1) Coating: Coating with 2D3 monoclonal antibody, 20 μg / mL, 100 μL / well, covering 6 wells, can be diluted with coating buffer; desorbed wells, coated with 3% BSA-regular phosphate buffer, 100 μL / well Wells, pack 6 wells; overnight coating at 4 hours is better than incubation at 37°C for 2 hours;

[0060] (2) Blocking: 3% conventional phosphate buffer M, 300uL / well, after incubation at 37°C for 2h, hand wash the pl...

Embodiment 3

[0083] Example 3 Expression and purification of zearalenone anti-idiotype nanobody

[0084] (1) Preparation of Top10F' competent cells:

[0085] (1) Pick an appropriate amount of Top10F' to streak on the LB-tetracycline plate, and culture overnight at 37°C;

[0086] (2) Pick the Top10F' monoclonal into 5mL LB medium, culture at 37°C, 250rpm until OD 600 0.6-0.8;

[0087] (3) Transfer the bacterial liquid to a pre-cooled centrifuge tube (about 1.3mL / tube), centrifuge at 4°C, 8000rpm, 8min;

[0088] (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;

[0089] (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 0.1M CaCl for precipitation 2 The solution is resuspended, which is Top10F'competent cells.

[0090] (2) Extraction and transformatio...

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Abstract

Belonging to the field of molecular biology, the invention in particular relates to an anti-idiotypic nano antibody based zearalenone green immunoassay method. According to the invention, the zearalenone monoclonal antibody 2D3 is employed to immunize alpaca, and the leukocyte total RNA is extracted to construct a phage displayed nano antibody library; the 2D3 antibody is adopted as the target, four rounds of affinity enrichment panning are conducted to lower the coating concentration and competitive elution concentration round by round, and a positive phage zearalenone anti-idiotypic nano antibody with good specificity can be obtained. The invention employs the zearalenone anti-idiotypic nano antibody substitute zearalenone standard ELISA method to determine naturally polluted corn and feed samples, and performs comparison with the HPLC detection result, the two methods have good correlation, and R2 is 0.997, thus proving that the zearalenone anti-idiotypic nano antibody substitute standard method has accurate and reliable result and is an effective and feasible green immunoassay method.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to an anti-idiotype nanobody-based zearalenone green immunoassay method. Background technique [0002] Zearalenone (ZEN) is a mycotoxin produced by Fusarium fungus, which mainly contaminates corn, wheat, sorghum and other cereal crops. It has reproductive toxicity, immunotoxicity and potential carcinogenicity. The toxin can enter the human and animal body by contaminating grain and its products or meat and milk with ZEN residue, posing a serious threat to the development of animal husbandry and human health. In view of the widespread contamination of zearalenone in agricultural products and foods and the exposure level of residents' diets, most countries have established the maximum allowable amount of ZEN in grains and products. [0003] Currently, methods for ZEN detection include thin layer chromatography, high performance liquid chromatography, immunoassay and elect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/42G01N33/64
Inventor 李培武姜俊王督唐晓倩张兆威李慧喻理张文张奇
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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