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Immune colloidal gold test stripe for detecting leucomalachite green in aquatic product and preparation method

The technology of a recessive malachite green and colloidal gold test paper is applied in the fields of immune colloidal gold and veterinary drug residue detection, and food safety. The effect of simple process, low cost and convenient on-site operation

Active Publication Date: 2010-02-24
江西中德生物工程股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

High-performance liquid chromatography is costly, takes a long time to operate, and has many and complicated steps, so it cannot achieve real on-site detection

Method used

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  • Immune colloidal gold test stripe for detecting leucomalachite green in aquatic product and preparation method
  • Immune colloidal gold test stripe for detecting leucomalachite green in aquatic product and preparation method
  • Immune colloidal gold test stripe for detecting leucomalachite green in aquatic product and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Hapten and Complete Antigen (Immunogen) Synthesis

[0025] 1.1 Preparation of recessive malachite green ethyl ketone

[0026] Add 20 mL of dichloromethane and 18 g (0.135 mol) of anhydrous aluminum trichloride into a 100 mL three-necked flask. Cool in an ice-salt bath to -10°C, add dropwise a mixture of 7.1g (0.07mol) acetic anhydride and 21.61g (0.065mol) recessive malachite green, and control the internal temperature to not exceed -5°C. After the dropwise addition, the stirring was continued for 15h, and the solution turned from yellow to dark red. The reaction solution was poured into a mixture of 50 mL of concentrated hydrochloric acid and 50 g of crushed ice, then the water layer was separated with a separatory funnel, and extracted twice with dichloromethane. The organic phases were combined, washed with water until neutral, and dried over anhydrous sodium sulfate. The solvent was distilled off to obtain 9.8 g of a yellow oil. The crude product can b...

Embodiment 2

[0032] Example 2 Preparation and Titer Detection of Whole Antigen Monoclonal Antibody

[0033] 2.1 Preparation of monoclonal antibodies

[0034] BSA-hapten was used as the immunogen to immunize 4 BALB / C mice, and 100 μg of the immunogen was taken from each mouse, mixed with an equal volume of Freund's complete adjuvant, emulsified evenly, and injected into the peritoneal membrane along the groin. Four weeks later, booster immunization was performed with the same dose and Freund's incomplete adjuvant as the adjuvant. After boosting the immunization three times, blood was collected to measure the titer. After the serum titer stopped rising, mice were immunized with twice the dose of antigen without adjuvant. Three days later, the spleen cells and mouse myeloma cells were collected under aseptic conditions and pressed for 5- Mix in a 50mL centrifuge tube at a ratio of 10:1, add 30mL of serum-free IPMI1640 medium, centrifuge at 1200r / min for 10min, discard the supernatant, shake ...

Embodiment 3

[0040] Embodiment 3 Preparation of goat (rabbit) anti-mouse IgG in the present invention

[0041] Select healthy male New Zealand white rabbits or goats, use OVA-hapten as the immunogen and the same amount of Freund's complete adjuvant to mix into a water-in-oil emulsion by syringe pumping method, and carry out the first immunization at the amount of 1mg / kg body weight , Take multi-point injections under the skin on the back. Booster immunization every two weeks, with Freund's incomplete adjuvant instead of Freund's complete adjuvant, the dose and method are the same as the first immunization. From the third immunization, 10 days after each immunization, 1 mL of blood was collected from the ear vein for antibody titer detection. When the antibody titer no longer increased, the last (7th) immunization was performed without adjuvant. Thigh intramuscular injection, carotid artery bloodletting after 7 days, coagulated at room temperature for 2 hours and overnight at 4°C, centrifu...

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Abstract

The invention discloses an immune colloidal gold test stripe for detecting leucomalachite green in aquatic products and a preparation method; the colloidal gold test stripe provided by the invention detects leucomalachite green with a direct competition method, and comprises a sample pad, a colloidal gold combined pad, a nitrocellulose membrane (NC), a water adsorption pad and a PVC back lining, one end of the PVC back lining is sequentially adhered with the sample pad and the colloidal gold combined pad, and the middle thereof is adhered with the NC layer, and the other end thereof is sequentially adhered with water adsorption pad; the immune colloidal gold test stripe has the characteristics that colloidal gold combined pad is enveloped with colloidal gold marker which is monoclonal antibody of the specificity of the leucomalachite green, and the NC is adsorbed with leucomalachite green-OVA and sheep anti-mouse IgG. The invention has high detection sensitivity and low price and is simple and rapid, the whole reaction only needs 30 minutes; the immune colloidal gold test stripe can be used for large-batch screening of samples and can rapidly detect the residual leucomalachite green in the aquatic products at site.

Description

technical field [0001] The invention belongs to the field of detection of immune colloidal gold and veterinary drug residues in the field of food safety. Specifically, the invention relates to an immune colloidal gold test strip suitable for detecting residual malachite green in aquatic products. Background technique [0002] Malachite green (Malachite green) molecular formula is: C 23 h 25 ClN 2 , is a triphenylmethane triphenylmehtane dye for industrial use, because it has the effect of antibacterial and anti-gram-positive bacteria, and the price is cheap, so it is widely used in fish farming to prevent saprolegniasis, gill mold, and small melon bug disease, etc., and in order to prolong the life of fish with damaged scales, malachite green is often used during transportation and in storage ponds. When the aquaculture water contains malachite green, fish, shrimp, etc. will quickly absorb malachite green and then metabolize into recessive malachite green (Leμcomalachite...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/558G01N33/532G01N33/566C07K14/765C07K14/77C07K1/113C07K16/44
Inventor 赖卫华黄启明杨晓慧陈媛魏华熊勇华
Owner 江西中德生物工程股份有限公司
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