Preparation method of anti-aristololactam FI and aristolochic acid IVa monoclonal antibody, and application of monoclonal antibody

A technology of aristolochid lactam and monoclonal antibody, applied in chemical instruments and methods, anti-plant immunoglobulin, instruments, etc., can solve the problems of high requirements for operators and instrument maintenance, complicated sample pretreatment, and environmental protection question

Inactive Publication Date: 2013-11-13
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The problems of AL's HPLC detection method are that the equipment is expensive, the requirements for operators and equipment maintenance are high, the sample pretreatment is complicated, and toxic organic reagents need to be consumed, which is not environmentally friendly, and it is not conducive to on-site detection and high-throughput detection
However, monoclonal antibodies that simultaneously recognize AL compounds and AA-IVa have not been reported yet.

Method used

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  • Preparation method of anti-aristololactam FI and aristolochic acid IVa monoclonal antibody, and application of monoclonal antibody
  • Preparation method of anti-aristololactam FI and aristolochic acid IVa monoclonal antibody, and application of monoclonal antibody
  • Preparation method of anti-aristololactam FI and aristolochic acid IVa monoclonal antibody, and application of monoclonal antibody

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Experimental program
Comparison scheme
Effect test

Embodiment 1A

[0036] Synthesis of embodiment 1AL artificial hapten, immune antigen and coating antigen

[0037] 1. Synthesis of artificial hapten SAL-FI

[0038] Dissolve 70 mg of AL-FI (>98%) in 7 mL of pyridine, stir until completely dissolved, add 20 times the amount of succinic anhydride and 0.5 times the amount of catalyst 4-dimethylaminopyridine (DMAP), and stir magnetically at room temperature for 111 hours . After the reaction is completed, add 10% concentrated hydrochloric acid aqueous solution dropwise to the reaction liquid to adjust the pH to 3 to neutralize pyridine, then extract the reaction liquid several times with an equal volume of ethyl acetate, and finally wash the ethyl acetate layer with pure water several times to pH was 7, the ethyl acetate layers were combined and the solvent was recovered to obtain a crude product. Purify by preparative liquid chromatography to obtain the artificial hapten SAL-FI.

[0039] 2. Preparation of artificial immune antigen (SAL-FI-KLH)...

Embodiment 2

[0043] The preparation of embodiment 2 monoclonal antibody

[0044] 1. Animal immunity

[0045] The 8M urea solution containing 5mg / mL SAL-FI-KLH was diluted 25 times with PBS, mixed with an equal volume of Freund's complete adjuvant to form an emulsion, and several 7-week-old male BALB / c mice were injected intraperitoneally, each 0.5 mL (50 μg). Two weeks after the first immunization, the same dose was used to boost the immunization, and the adjuvant was replaced by Freund's incomplete adjuvant. After the second immunization, the PBS solution of SAL-FI-KLH (without adjuvant) was injected intraperitoneally, 0.5 mL (100 μg) per mouse, and immunized twice with an interval of 2 weeks between each time. After the second and fourth immunizations, the titer of antiserum and the competitive inhibition rate of AL-FI were detected by ELISA method, and the one with better immune effect was selected, that is, the one with a serum titer of 1187 and 10 μg / mL AL-FI. Spleen cells (6×10 8...

Embodiment 3

[0059] Identification of embodiment 3 monoclonal antibody

[0060] 1. Monoclonal antibody purity test, antibody typing and potency (Ka) determination

[0061] The purity of the above-mentioned monoclonal antibody 1F9-9B-4G determined by the sandwich ELISA method was 33.38%; according to the instructions of the mouse antibody heavy chain isotype determination kit and the mouse antibody κ and λ chain rapid detection test strips, The purified monoclonal antibody solution was taken for operation, and the heavy chain type of the monoclonal antibody 1F9-9B-4G was determined to be IgG1, and the light chain type was κ. Referring to Friguet et al. [21] The antibody titer was detected by the method, and the affinity constant (Ka) of monoclonal antibody 1F9-9B-4G to AL-FI was 1.941×10 9 m -1 .

[0062] 2. Antibody specificity

[0063] Competitive ELISA was used to measure the specificity of the monoclonal antibody of the above invention and the cross-reactivity with similar compounds ...

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Abstract

The invention belongs to the biotechnical field and the traditional Chinese medicinal detection field, and discloses a preparation method of an anti-aristololactam FI (AL-FI) and aristolochic acid IVa (AA-IVa) monoclonal antibody, and an application of the monoclonal antibody The anti-AL-FI monoclonal antibody is prepared by synthesizing the artificial immune antigen and the coating antigen of the AL-FI through the structural reconstruction of the AL-FI, and a competitive enzyme linked immunosorbent assay method is established through the monoclonal antibody. The anti-AL-FI and AA-Iva monoclonal antibody is a first monoclonal antibody aiming at AL compounds and has a high antibody sensitivity, and the affinity constant (Ka) of the monoclonal antibody to the AL-FI is 1.941*10<9>M<-1>. The monoclonal antibody can simultaneously identify the AA-IVa, and the cross reaction rate of the monoclonal antibody is 42.27%. A process for the qualitative/quantitative analysis of the AL-FI and/or the AA-IVa having renal cytotoxicity in relevant medicinal materials and biological samples through utilizing the monoclonal antibody based enzyme linked immunosorbent assay method has the advantages of simple, sensitive and rapid sample pretreatment, environmental protection and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology and the field of traditional Chinese medicine detection, and in particular relates to a preparation method of anti-aristollactam FI (AL-FI) and aristolochic acid IVa (AA-IVa) monoclonal antibody and a method based on the monoclonal antibody The competitive enzyme-linked immunosorbent assay (ELISA) detection method can be used for high-sensitivity and high-throughput detection of nephrotoxic compounds AL-FI and / or AA-IVa in medicinal materials and biological samples. Background technique [0002] Taking aristolochic acid (AA) compounds or Chinese herbal medicines containing such compounds can lead to aristolochic acid nephropathy (AAN), which has aroused great concern at home and abroad. Aristolochic acid has been proved to be nephrotoxic, teratogenic, and carcinogenic [1，2] , and our previous research found that another main component of Aristolochiaceae medicinal plants: aristolochic lactam (AL) als...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D209/80C07K14/795C07K14/765C07K16/16G01N33/577G01N33/543
Inventor 蔡少青正山征洋黎晓维森永纪宇都拓洋王璇
Owner PEKING UNIV
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