A kind of paraquat hapten, complete antigen, antibody, detection test paper and kit

A complete antigen, detection test strip technology, applied in the field of immunology, can solve the problems of strong and weak affinity specific detection sensitivity, the influence of operating conditions, low paraquat sensitivity, etc., to achieve strong paraquat technical affinity and specificity, The effect of rapid detection and high detection sensitivity

Active Publication Date: 2020-12-29
北京纳百生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Immunological analysis methods include enzyme-linked immunoassay, colloidal gold immunoassay, radioimmunoassay, etc. Chinese patent document CN102633876B discloses a method for synthesizing paraquat antigen and its application. The monoclonal antibody prepared from this antigen can be used to detect paraquat in the sample by ELISA, but ELISA has the defects of unstable immune enzyme activity and being affected by operating conditions in practical applications
In addition, the affinity and specificity of the antibodies used in the detection directly affect the detection sensitivity of the immunological analysis method, but the antigens and antibodies in this patent document are used for the detection of paraquat by ELISA. The sensitivity is still low

Method used

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  • A kind of paraquat hapten, complete antigen, antibody, detection test paper and kit
  • A kind of paraquat hapten, complete antigen, antibody, detection test paper and kit
  • A kind of paraquat hapten, complete antigen, antibody, detection test paper and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Preparation of Paraquat Hapten

[0048] The preparation method of the paraquat hapten described in this example is specifically as follows, and the reaction formula is as follows: Formula 1.

[0049] S1. Take 808mg of 2-chloro-4,4'-bipyridine, dissolve it in acetone, add 400uL methyl iodide under stirring at 4°C, react overnight at 4°C, then centrifuge, wash with acetone, and dry to obtain the compound of formula II;

[0050] S2. Take 0.4 g of the compound of formula II, dissolve it in DMF, add 238 mg of 6-aminocaproic acid, reflux at 120°C for 4 hours, then cool, centrifuge, wash and dry to obtain the paraquat hapten of formula I.

[0051] The NMR spectrum of the hapten 2-((5-carboxypentyl)amino)-1,1'-dimethyl-[4,4'-bipyridine]-1,1'-dinium is as follows:

[0052] H-NMR (DMSO) 1.29 (2H, q), 1.52-1.63 (4H, m), 2.30 (2H, t), 3.35 (2H, t), 4.0 (1H, s), 4.39 (6H, s), 7.69 (1H, s), 8.06 (1H, d), 8.47 (1H, d), 8.84 (2H, d), 8.95 (2H, d), 11.0 (1H, s)

[0053] It ...

Embodiment 2

[0056] Example 2 Preparation of Paraquat Complete Antigen

[0057] The preparation method of the paraquat complete antigen described in this example is specifically as follows, and the reaction formula is as follows: Formula 2.

[0058] S1. Weigh 16 mg of the paraquat hapten of formula I, dissolve it in DMF, add 6 mg of NHS and 20 mg of EDC, and activate overnight to obtain an activation solution;

[0059] S2. Weigh 30 mg of BSA and OVA, dissolve them in 3 mL of 0.01M PBS, respectively, drop half of the activation solution obtained in step S1 to each of them, and react for 2 hours to obtain the complete paraquat antigen of formula III. Carrier proteins BSA, OVA, paraquat hapten and paraquat complete antigen were measured by ultraviolet scanning, and it was found that the absorption curve of paraquat complete antigen was significantly changed compared with BSA, OVA and paraquat hapten, indicating that paraquat hapten and Carrier protein coupling was successful.

[0060]

...

Embodiment 3

[0062] Example 3 Paraquat polyclonal antibody and antibody titer determination

[0063] Use the indirect ELISA method to detect the serum titer, dilute the antiserum from 1:200, and take the highest dilution factor of the serum titer corresponding to the OD450 value greater than or equal to 2 times that of the negative control (P / N≥2) to be the ELISA of the serum potency. The results of the serum titer test are shown in Table 1. The experimental results showed that the highest serum titer of paraquat-BSA in the 1# mouse was 51200 times, and the inhibition rate was 60.32%. The No. 1 mouse was selected for the fusion experiment.

[0064] Table 1

[0065]

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Abstract

The invention provides a paraquat hapten, a complete antigen, an antibody, a test paper and a kit, and the paraquat hapten has a structural formula shown in the following formula I in the specification. The molecular structure of the paraquat hapten has higher similarity with a paraquat raw drug. According to the present invention, the hapten and the protein are coupled to obtain the complete antigen, such that the antibody generated by the complete antigen obtained by coupling the hapten and the protein has strong paraquat original drug affinity and strong paraquat original drug specificity so as to provide the high paraquat detection sensitivity of the kit so as to achieve the rapid detection of paraquat in raw and fresh milk, milk powder, chicken and eggs.

Description

technical field [0001] The invention belongs to the technical field of immunology, and in particular relates to a paraquat hapten, a complete antigen, an antibody, a detection test paper and a kit. Background technique [0002] Paraquat (Paraquat, also known as Paraquat, Gramoxone, CAS No. 1910-42-5), pure product is white crystal, is a bipyridyl cationic salt organic heterocyclic fast-acting herbicide, easily soluble in Water, which is broad-spectrum and contact-killing, plants will die after a few hours after exposure to paraquat. Paraquat can be rapidly and strongly adsorbed by soil colloids to cause passivation without affecting plant roots. Paraquat is extremely toxic to humans. Every year, people die from paraquat poisoning. The main target organ of paraquat is the lung, mainly manifested in intrapulmonary hemorrhage and edema. Currently, there is no specific antidote. The use of paraquat has been banned or strictly restricted by more than 20 countries. Since July 1,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D213/74C12N5/20C07K14/765C07K16/44G01N33/558G01N33/577G01N33/58
CPCC07D213/74C07K14/765C07K16/44G01N33/558G01N33/577G01N33/582G01N33/587G01N2430/20
Inventor 魏单平赵荣茂冯小宇乔天奎
Owner 北京纳百生物科技有限公司
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