Competition law-based nanometer immunometric chromatographic detection card for lactoferrin in milk

A technology of lactoferrin and flow measurement chromatography, which is applied in the direction of measuring devices, analytical materials, biological testing, etc., can solve the problems of rapid detection of lactoferrin and other problems, and achieve the effect of convenient and fast detection method

Active Publication Date: 2019-07-30
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the operation of these methods requires the operation of laboratory technicians and the pretreatment of samples, and some of them also use expensive experimental instruments, so the rapid detection of lactoferrin in samples has brought certain troubles

Method used

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  • Competition law-based nanometer immunometric chromatographic detection card for lactoferrin in milk
  • Competition law-based nanometer immunometric chromatographic detection card for lactoferrin in milk
  • Competition law-based nanometer immunometric chromatographic detection card for lactoferrin in milk

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Purification and identification of monoclonal antibody 3E2 of the present invention

[0020] 1) Preparation of monoclonal antibody ascites

[0021] Balb / c mice aged 9-10 weeks were selected, and each mouse was sensitized by intraperitoneal injection of 500 μL of liquid paraffin. 7-10 days after the sensitization, the hybridoma cells in good growth state in the 96-well cell culture plate were selected for cell counting (about 10 5 ~10 6 ) into the peritoneal cavity of sensitized mice. If the injection operation is improper, it will lead to the death of the mice. After injecting the cells, the state of the mice should be observed every day. About 10-12 days, the mouse's abdomen was obviously swollen and moved slowly, indicating that the hybridoma cells in the abdominal cavity had proliferated in large numbers and produced ascites. When the abdomen of the mouse continues to swell and grow until the mouse is dying, a sterile needle can be used to puncture the ...

Embodiment 2

[0030] Example 2 Preparation and Identification of Nanoflower Immunochromatography Detection Card of the Present Invention

[0031] 1) Preparation of nanoflower gold solution

[0032] The antibody solution (0.01 M PBS) after purification and dialysis will contain a large amount of salt ions, and the concentration of these salt ions will affect the stability of the colloidal gold solution and even denature the colloidal gold. So the monoclonal antibody needs to be in ddH 2 O was dialyzed for 24 h, and the dialysate was changed every 6 h. After dialyzing, it was concentrated with PEG-20000 for later use. with 100 mL ddH 2 O dissolves 1 g HAuCl 4 The prepared chloroauric acid solution was divided into 1.5 mL sterilized centrifuge tubes with 1 mL per tube, sealed with tinfoil and stored at -20 °C for later use. Take 750 μL of chloroauric acid solution and add 100 mL ddH 2 O, add 500 μL colloidal gold solution and mix well, then add 300 μL trisodium citrate (1wt%) and shake we...

Embodiment 3

[0041] Example 3 The detection application of the nanoflower immunochromatography detection card of the present invention

[0042] 1) Specificity determination of nanoflower immunochromatographic detection card

[0043] According to the optimal lactoferrin concentration and goat anti-mouse IgG concentration, the antigen and secondary antibody were streaked on the NC membrane on the streak gold system. Then place the streaked NC membrane in a 37°C constant temperature incubator to dry for 10 minutes, and add 5.2.13 optimal amount of nanoflower gold-labeled antibody to each gold-labeled pad and dry it in a 37°C constant-temperature incubator 10 min. Dilute 10 different complete antigens (BSA, OVA, KLH, Casein, HSA, IFN-γ, TLH, PBSM, GFP, LF) with 0.01 M PBS to a final concentration of 20 μg / mL. Then, 100 μL of diluted different antigens were sequentially added to the sample pad of the assembled nanoflower gold label detection card, and the reaction time was 10 min, and the dis...

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Abstract

The invention belongs to the field of antibody engineering and immunoassay, and particularly relates to a competition law-based nanometer immunometric chromatographic detection card for lactoferrin inmilk. The nanometer immunometric chromatographicdetection card comprises a sample pad, a nanometer gold label pad, a nitrocellulose membrane (NC membrane), a water absorbent pad and a plastic card holder. The NC membrane is provided with a C line and a T line; the C line is sprayed with a goat anti-mouse IgG antibody; and the T line is coated with a bovine lactoferrin antigen. The gold label padcontains a nanometer gold-labeled murine monoclonal IgG antibody. Through the competition law-based nanometer immunometric chromatographic detection card, the lactoferrin in the milk can be rapidly detected, the detection sensitivity is 2.4 ng / mL, no cross reaction occurs among the main casein, serum albumin and skim milk in the milk; and the established detection method is convenient and quick and can be directly used for the rapid detection of lactoferrin contents in dairy products.

Description

technical field [0001] The invention belongs to the field of antibody engineering and immune detection, and in particular relates to a lactoferrin competition method nanoflower immunoassay flow chromatography detection card in milk. Background technique [0002] Lactoferrin (Lactoferrin, LF), also called lactoferrin, is a multifunctional protein of the transferrin family, with a relative molecular mass of about 80 kDa, present in the secretions of all mammals, especially in bovine milk most widely present. Lactoferrin has nutritional, antibacterial, antiviral, anti-inflammatory, anti-oxidative, anti-tumor, and immune-regulating effects, and has been widely used in food, medicine, and cosmetics. In addition, it can be used as an important index to evaluate the quality of milk products in food safety testing, so it is particularly important for the rapid detection of lactoferrin content in dairy products. [0003] Currently, methods for detecting lactoferrin content in sampl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/543G01N33/531
CPCG01N33/531G01N33/54346G01N33/6803
Inventor 王荣智王俊程汪世华刘海梅李秀兰凌素美贾坤志
Owner FUJIAN AGRI & FORESTRY UNIV
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