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High-stability sulfanilamide drug antibody hAb 4D11 and application thereof

A sulfonamide drug, high stability technology, applied in the fields of biotechnology and immunodetection, can solve the problems of organic solvent tolerance and poor thermal stability, and achieve the effects of shortening detection time, good application prospects and improving detection efficiency

Active Publication Date: 2021-04-30
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Monoclonal antibodies (mAbs) prepared by immunization of mice, fusion of spleen cells and tumor cells, and monoclonal screening usually have poor tolerance to organic solvents and thermal stability

Method used

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  • High-stability sulfanilamide drug antibody hAb 4D11 and application thereof
  • High-stability sulfanilamide drug antibody hAb 4D11 and application thereof
  • High-stability sulfanilamide drug antibody hAb 4D11 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1 Preparation and Identification of Recombinant Antibody hAb 4D11

[0087] 1. The amino acid sequence of the heavy chain of the sulfonamide recombinant antibody hAb 4D11 is shown in SEQ ID NO.3, on the basis of SEQ ID NO.1, Q1E, Q5V, Q6E, A9G, E10G, A12V, R13Q , A16G, K23A, T28N, T30K, K38R, R40A, Q43K, Q67R, A68F, T71S, R74T, S76K, S77N, L83M, S84N, A87R, S88A, S91T, S114L replacement; the sulfonamide drug recombinant antibody hAb 4D11 The amino acid sequence of the light chain is shown in SEQ ID NO.4, V3Q, A9S, A12S, V13A, L15V, Q17D, A19V, S22T, Q46K, I62V, A64S, R72G, P73T, S78T, N80S, P81S, V82L, E83Q, A84P, D85E, V87F, G104Q, L108V, A113T.

[0088] 2. Choose total synthesis and synthesize the recombinant genes of heavy chain and light chain respectively.

[0089] 3. According to the multiple cloning site of the expression vector pcDNA5 and the restriction site on the heavy chain plasmid, use Hind III and Pac I to carry out double digestion, and clone the...

Embodiment 2

[0097] Example 2 Determination of Thermal Stability of Recombinant Antibody hAb 4D11

[0098] 1. Add hAb 4D11 and mAb 4D11 (the heavy chain sequence is shown in SEQ ID NO.1, and the light chain sequence is shown in SEQ ID NO.2) into Uni tubes respectively.

[0099] 2. As the temperature increases, use a protein stability analyzer to measure the change of the endogenous fluorescence spectrum.

[0100] 3. Software fitting to obtain the change law curve of fluorescence spectrum with temperature.

[0101] 4. Calculate a mediation number, and its peak value is the Tm value.

[0102] 5. The result is as follows figure 2 As shown, the Tm value of mAb 4D11 was 65.7°C, while the Tm value of hAb 4D11 was 76.3°C, an increase of 10.6°C.

Embodiment 3

[0103] Example 3 Determination of Organic Solvent Tolerance of Recombinant Antibody hAb 4D11

[0104] 1. Add a certain amount of acetonitrile or methanol and other reagents into the reaction buffer of FPIA to prepare buffers containing 0%, 1%, 2%, 5%, 10% and 20% organic solvents.

[0105] 2. Using sulfamethazine (SMZ) as the representative SAs and HS-5AF as the fluorescently labeled hapten, the above buffers were used to prepare standard solutions containing different amounts of organic solvents.

[0106] 3. Establish the calibration curve of SMZ under different organic solvent contents, and the IC 50 value (half inhibitory concentration), δFP value (the difference between the maximum and minimum FP value) and δFP / IC 50 The value is used as the judgment standard to examine the tolerance of hAb and mAb to organic solvents.

[0107] 4. The results are shown in Table 1. Compared with mAb 4D11, the tolerance of hAb 4D11 to methanol increased by 10%, and the tolerance to acetoni...

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Abstract

The invention relates to the field of biotechnology and immunodetection, in particular to a high-stability sulfanilamide drug antibody hAb 4D11 and an application thereof. The antibody hAb 4D11 comprises a heavy chain and a light chain, wherein the heavy chain is obtained by carrying out amino acid residue replacement at a specific site on an amino acid sequence shown in SEQ ID NO.1; wherein the light chain is obtained by performing amino acid residue replacement of a specific site on an amino acid sequence shown in SEQ ID NO. 2. The antibody hAb 4D11 disclosed by the invention has heat resistance and organic solvent resistance at the same time under the condition that the performance of identifying sulfonamides is not changed, and the antibody also has a relatively wide identification range. By utilizing the recombinant antibody hAb 4D11 provided by the invention, nitrogen blowing and redissolution are not needed during sample pretreatment, and the recombinant antibody hAb 4D11 can be used for detection only by dilution after extraction, so that the detection time is favorably shortened, and the detection efficiency is improved.

Description

technical field [0001] The invention relates to the fields of biotechnology and immune detection, in particular to a highly stable sulfonamide drug antibody hAb 4D11 and its application. Background technique [0002] Sulfonamides (SAs) are a class of chemically synthesized antibacterial agents with a wide antibacterial spectrum, low price and many types. They are widely used in veterinary clinics, animal husbandry and aquaculture to prevent and treat bacterial infectious diseases. However, its unreasonable use or even abuse may easily cause SAs residues in animal-derived foods, and then threaten human health and food safety. In addition to controlling the irrational use of SAs from the source, the detection and monitoring of drug residues in animal-derived foods is an important measure to ensure food safety. Therefore, it is necessary to establish a simple, rapid, sensitive and broad-spectrum residue detection method. [0003] At present, the physical and chemical analysis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44G01N33/53
CPCC07K16/44G01N33/9446C07K2317/51C07K2317/515C07K2317/94C07K2317/33
Inventor 王战辉沈建忠温凯于雪芝江海洋李成龙
Owner CHINA AGRI UNIV