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Double-antibody sandwich colloidal gold detection test strip for human lactoferrin

A human lactoferrin, double antibody sandwich technology, applied in the biological field, can solve the problems of low sensitivity and specificity, unable to meet the requirements of detecting human lactoferrin, poor pairing, etc., and achieve high sensitivity and specificity. Effect

Active Publication Date: 2015-11-04
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The use of double-antibody sandwich test paper for the detection of human lactoferrin can realize the rapid and easy detection of human lactoferrin; however, the existing anti-human lactoferrin antibodies have low sensitivity and specificity and poor pairing Poor, unable to meet the requirements of double-antibody sandwich immunoassay for detecting human lactoferrin

Method used

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  • Double-antibody sandwich colloidal gold detection test strip for human lactoferrin
  • Double-antibody sandwich colloidal gold detection test strip for human lactoferrin

Examples

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Effect test

Embodiment 1

[0033] This example is used to illustrate the screening of antibody pairs used in the double-antibody sandwich colloidal gold detection test strip for human lactoferrin.

[0034] The protein sequence of human lactoferrin (hLF) is shown in NCBI Genbank sequence number: U95626. Human milk was obtained according to the method in the literature (Penghua Yang, et al., Cattle Mammary Bioreactor Generated by a Novel Procedure of Transgenic Cloning for Large-Scale Production of Functional Human Lactoferrin., PLoS ONE, 2008, 3(10), e3453) Ferritin (hLF) transgenic dairy cows.

[0035] Intramuscular injection of prolactin needles (purchased from Xi'an Caotan Pharmaceutical Factory, the infertile cow lactation needles, the product is boxed, 10 No. I needles, 3 No. II needles) to transgenic dairy cows for lactation, of which No. I needles were injected intramuscularly continuously 10 days, once a day. One No. II needle was injected on the 13th, 15th, and 17th days respectively. From the ...

Embodiment 2

[0044] This example is used to illustrate the sensitivity and specificity of the human lactoferrin double-antibody sandwich colloidal gold detection test strip of the present invention (ie, the immune colloidal gold test strip screened in Example 1) when testing actual samples.

[0045] (1) Sample preparation: collected milk, dissolved milk powder and liquid dairy products. Take about 200 μl of milk sample into the sample tube.

[0046] (2) Detection: Take out the immunocolloidal gold test strips screened in Example 1, equilibrate at room temperature for 20 minutes, open the package and take out the test strips, insert the test strips into the sample tube in the direction of the arrow, and let stand for 10-15 minutes ,judgement result.

[0047] (3) Judgment of results: At that time, there was a purple-red quality control line visible to the naked eye on the paper strip, but no visible purple-red detection line, it was judged as negative, and recorded as "-"; Control line, if...

Embodiment 3

[0055] 140 milks known to be negative for human lactoferrin were collected (including 100 non-transgenic cows collected from a dairy farm in Dachang, Hebei, 20 transgenic cows with human lysozyme in a dairy farm at China Agricultural University and 20 human α-lactalbumin transgenic cows located in the dairy farm of China Agricultural University), and 22 known positive human lactoferrin milk (collected from the dairy farm located in China Agricultural University, transgenic human lactoferrin Gene cows), 162 samples in total, carried out blind test with the immune colloidal gold test paper obtained by screening in Example 1, and the measurement results showed that 140 known human lactoferrin-negative milks were all shown as negative for detection, and 22 known human lactoferrin Positive milk was all shown as positive for the test, and the diagnostic specificity of the test strip was 100%. Some of the results of the measurements can be seen in Figure 4 with Figure 5 shown.

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Abstract

The invention provides a double-antibody sandwich colloidal gold detection test strip for human lactoferrin. The immune colloidal gold test strip comprises a water absorption pad, a base film, a gold-labeled pad and a sample pad which are connected in sequence, wherein a line C and a line T are arranged on the base film; the line C is coated with an antibody for goat anti-mouse IgG; the gold-labeled pad contains a colloidal gold labelled first monoclonal antibody; the line T is coated with a second monoclonal antibody; the first monoclonal antibody is secreted by a hybridoma cell strain with the collection number of CGMCC NO.10595; the second monoclonal antibody is secreted by a hybridoma cell strain with the collection number of CGMCC NO.10594. The double-antibody sandwich colloidal gold detection test strip can be used for detecting human lactoferrin by a double-antibody sandwich colloidal gold detection method, so that the sensitivity of 10ng / mL can be achieved, and no cross reaction for bovine lactoferrin and goat lactoferrin is caused; furthermore, no cross reaction for lysozyme transgenic cattle and human alpha-serum albumin transgenic cattle is caused.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a double-antibody sandwich colloidal gold detection test strip for human lactoferrin. Background technique [0002] The advent of "Shuoshu" (Palmiter et al., 1982) has accelerated the research on genetically modified organisms. The birth of the cloned sheep "Dolly" (Wilmut et al., 1997) has promoted the widespread application of transgenic technology in mammals. At present, a variety of transgenic pigs, cattle, and sheep have been successfully developed for the production of medicinal or edible protein, increased lean meat percentage, improved nutrition, and enhanced disease resistance (Schnieke et al., 1997; Toledo et al., 2006). The potential economic and social benefits are huge, and it has a good prospect of commercialization. However, due to the risks of GMOs and their products, the management of GMOs and their products has always been highly valued by governments. [0003] I...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/541G01N33/543G01N33/577G01N33/585G01N33/68G01N2333/79
Inventor 王勤林祥梅刘晓飞冯春燕付伟李晓琳仇松寅刘丹丹
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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