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Human lactoferrins double-antibody sandwich ELISA kit

An enzyme-linked immunosorbent assay, human lactoferrin technology, applied in the biological field, can solve the problems of low sensitivity and specificity of antibodies and cannot meet the needs of human lactoferrin, etc., and achieve the effect of high sensitivity and specificity

Active Publication Date: 2015-12-16
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection methods of genetically modified organisms and their processed foods mainly include the detection of gene level and the detection of protein level. In terms of protein detection, the current detection method of human lactoferrin is mainly ELISA detection method, but the existing anti-human lactoferrin Due to the low sensitivity and specificity of the protein antibody, it cannot meet the requirements of double-antibody sandwich enzyme-linked immunosorbent assay for human lactoferrin

Method used

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  • Human lactoferrins double-antibody sandwich ELISA kit
  • Human lactoferrins double-antibody sandwich ELISA kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] This example is used to illustrate the screening of human lactoferrin monoclonal antibody pairs.

[0023] The protein sequence of human lactoferrin (hLF) is shown in NCBIGenbank sequence number: U95626. According to the method in the literature (Penghua Yang, et al., Cattle Mammary Bioreactor Generated ya Novel Procedure of Transgenic Cloning for Large-Scale Production of Functional HumanLactoferrin., PLoSONE, 2008, 3(10), e3453), human lactoferrin (hLF) transgenic cows were obtained.

[0024] 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 7th day of injection, the udders of the transgenic cows were massaged with w...

Embodiment 2

[0033] Carry out the HRP (horseradish peroxidase) labeling operation of the second monoclonal antibody (human lactoferrin monoclonal antibody 3B4, preservation number is CGMCCNO.10595) according to the product manual of PierceHRP labeling kit (CatNo31488), to obtain horseradish over- Oxidase-labeled secondary monoclonal antibody.

[0034] Enzyme-linked immunosorbent assay (ELISA) detection steps are as follows: (1) coating: with the phosphate buffer that contains 0.08 volume % Tween-20 as coating buffer, the first monoclonal antibody (human lactoferrin monoclonal antibody) The cloned antibody 5C5, whose preservation number is CGMCCNO.10594) was diluted to 5 μg / mL with coating buffer, added 100 μL / well to the enzyme-linked plate, placed at 37°C for 2 hours, and washed 3 times with coating buffer; (2) blocking: add 150 μL Blocking solution per well (phosphate buffered saline containing 0.08% by volume Tween-20 and 6% by weight of casein), placed at 37°C for 1 hour, washed 3 time...

Embodiment 3

[0039] 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 genetic cows), a total of 162 samples.

[0040] With reference to the same method of Example 2, the sample to be tested is detected, wherein the sample to be tested is listed in Table 2, and the OD value obtained by corresponding detection is also shown in Table 2

[0041] Table 2

[0042]

[0043] According to the data in Table 2, it can be seen that the detection of the milk of transgenic cows with human lactoferrin in the present invention has a sensitivity of 1000 times of dilution,...

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Abstract

The invention discloses a human lactoferrins double-antibody sandwich ELISA kit. The kit comprises an enzyme-linked plate, a coating buffer, a first monoclonal antibody, a blocking solution, anenzyme-labelled second monoclonal antibody, a chromogenic substrate and a stopping solution; the first monoclonal antibody is secreted by a hybridoma cell strain with the preservation number of CGMCC NO. 10594; and the second monoclonal antibody is secreted by a hybridoma cell strain with the preservation number of CGMCC NO. 10595. According to the above technical scheme, detection on human lactoferrins can reach the sensitivity of 1 ng / mL by employing the double-antibody sandwich ELISA kit, cross reactions do not happen aiming at cattle lactoferrin and sheep lactoferrin, also cross reactions do not happen aiming at human lysozyme transgenic cattle and human alpha-whey alhumin transgenic cattle, and the double-antibody sandwich ELISA kit possesses relatively high sensitivity and specificity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a human lactoferrin double-antibody sandwich enzyme-linked immunoassay kit. Background technique [0002] The advent of "Shuoshu" (Palmite et al., 1982) has accelerated the research on genetically modified organisms. The birth of the cloned sheep "Dolly" (Wilmute et al., 1997) has promoted the widespread application of transgenic technology in mammals. At present, a variety of transgenic pigs, cattle, and sheep (Schnieke et al., 1997; Toledo et al., 2006), which are used to produce medicinal or edible protein, increase lean meat percentage, improve nutrition, and enhance disease resistance, have been successfully developed. Their potential economic benefits It has huge social benefits and has good commercialization prospects. 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] In ord...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/535
CPCG01N33/535G01N33/6803
Inventor 林祥梅王勤刘晓飞付伟冯春燕李晓琳仇松寅刘丹丹
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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