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Screening test paper strip for human lactoferrin in milk and preparation method

A technology of human lactoferrin and test strips is applied in the field of detection of genetically modified ingredients in milk, which can solve the problems of inconvenient grass-roots promotion, high false positive rate, time-consuming and labor-intensive, etc., and achieves low detection cost, good specificity, and less time-consuming. Effect

Active Publication Date: 2013-01-16
HUAZHONG AGRI UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

Many protein detection methods can be roughly divided into four categories: (1) detection based on the physical and chemical properties of proteins, such as two-dimensional electrophoresis, mass spectrometry, chromatography, etc.; (2) detection based on the interaction between nucleic acid and protein, such as Proximity ligation technology is a detection technology that relies on aptamers (aptamers, the general term for DNA or RNA that can specifically recognize and bind proteins) (Mats G., Simon F., Michael T., et al.A Sense of closeness: protein detection by proximity ligation [J]. Current Opinion in Biotechnology, 2003, 14: 82-86; Simon F., Mats G., Jonas J., et al. Protein detection using proximity-dependent DNA ligation assays [J].Nature, 2002, 20:473-477); (3) Detection based on the interaction between proteins, such as commonly used immunoassay methods, including western blot, dot blot (no special instrument, However, the operation is cumbersome and not suitable for batch detection), ELISA (high sensitivity, strong specificity, simple and fast, good stability, convenient for automatic detection, etc., and strong applicability), and immunohistochemical staining (with strong specificity, The characteristics of high sensitivity and accurate positioning are convenient for functional research, but the operation is complicated and the false positive rate is high); (4) the detection of protein is converted into the detection of nucleic acid, such as Bio-bar code assay, which has high sensitivity (Khan S , Klein W, Mirkin CA, et al. Fluorescent and scanometric ultrasensitive detection technologies with the bio-bar code assay for alzheimer's disease[J].Nanoscape, 2005, 2(1): 7-15)
The methods mentioned above are time-consuming and labor-intensive, and require professional technicians and specialized instruments and equipment. Basically, they can only be completed in laboratories, and are not easy to promote at the grassroots level.

Method used

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  • Screening test paper strip for human lactoferrin in milk and preparation method
  • Screening test paper strip for human lactoferrin in milk and preparation method
  • Screening test paper strip for human lactoferrin in milk and preparation method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 (preparation embodiment)

[0042] Preparation method of immunocolloidal gold test strip for detecting hLF

[0043] 1. Synthesis of antigenic polypeptide and acquisition of rabbit anti-human lactoferrin antibody

[0044] Analyze human lactoferrin, select a segment with good hydrophilicity, on the molecular surface, good flexibility and low homology with bovine lactoferrin, and artificially synthesize an antigenic polypeptide, the protein sequence of which is shown in the sequence table SEQ ID NO : 2, it was cross-linked to the carrier protein keyhole limpet hemocyanin (KLH for short, purchased from Sigma Company, article number: H9035), and then the composite protein was used as an antigen to immunize New Zealand white rabbits (June age, body weight 3-4 kg, purchased from Tianjin Keda Breeding Center), obtained positive serum after affinity purification to obtain rabbit anti-hLF polyclonal antibody.

[0045] 2. Preparation of anti-human lactoferrin polyclo...

Embodiment 2

[0062] Embodiment 2 (application embodiment)

[0063] The use method of PBS buffer solution screening immune colloidal gold test strip in milk

[0064] 1. Preparation of milk samples:

[0065] Use PBS buffer (0.01M pH7.4 phosphate buffer, formula: NaCl 8g, KCl 0.2g, NaCl 2 HPO 4 12H 2 O 2.9g, KH 2 PO 4 0.2g, dilute to 1000ml with distilled water) Dilute the milk sample (collected in April 2010, stored at -80°C, with a shelf life of 14 months) 10 times, and at the same time use the formula: NaCl 8g, KCl 0.2g, NaCl 2 HPO 4 12H 2 O 2.9g, KH 2 PO 4 0.2g, dilute to 1000ml with distilled water Recipe: NaCl 8g, KCl 0.2g, NaCl 2 HPO 4 12H 2 O 2.9g, KH 2 PO 4 0.2g, distilled water to 1000ml human lactoferrin solution standard (same source) and PBS solution as positive and negative controls.

[0066] 2. Detection

[0067] Take the positive standard, negative standard and 100 μL of each sample to be tested and add them to the microwell plate for detection, insert the c...

Embodiment 3

[0070] Embodiment 3 (verification test)

[0071] The collected milk samples were detected by Western blot, and this method was used as a control. The steps are as follows:

[0072] 1. Gel Preparation

[0073] Between two clean glass plates, pour 12% separating gel (10mL gel recipe: deionized water 3.3mL, 30% acrylamide solution 4.0mL, 1.5mol / L Tris-HCl (pH8.8) 2.5mL , 10% sodium dodecyl sulfate (SDS) 100 μL, 10% ammonium persulfate 100 μL, N, N, N', N'-tetramethylethylenediamine (TEMED) 8 μL) and 5% stacking gel (4.5mL gel formula: deionized water 3.0mL, 30% acrylamide solution 0.75mL, 1.0mol / L Tris-HCl (pH6.8) 2.5mL, 10% SDS 60μL, 10% ammonium persulfate 45μL, TEMED 6μL ).

[0074] 2. Electrophoresis

[0075] Take 2 μL milk sample, add 4 μL loading buffer (5mL solution formula: 1.0mol / L Tris-HCl (pH6.8) 1.25mL, glycerol 2.5mL, SDS powder 0.5g, bromophenol blue 25mg, β-mercaptoethanol 125μL) and 14 μL of deionized water was denatured at 98°C for 10 minutes on a PCR instru...

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Abstract

Belonging to the technical field of transgenic component detection and relating to immunocolloidal gold technologies, the invention discloses an immunocolloidal gold test paper strip for rapid detection of human lactoferrin (hLF) gene milk and a preparation method thereof. The test paper strip provided in the invention includes a sample pad, a bonding pad, a nitrocellulose membrane, an absorbent pad and a PVC backing. The test paper strip is characterized in that the sample pad, the bonding pad, the nitrocellulose membrane and the absorbent pad are adhered to the PVC backing in order; the bonding pad is coated with the anti-hLF polyclonal antibody-colloidal gold marker, and the polyclonal antibody is prepared by artificial synthesis of hLF polypeptide and then immunization of domestic rabbits. The nitrocellulose membrane is coated with a detection line (5) composed of hLF protein and a quality control line (6) of goat anti-rabbit IgG respectively. The test paper strip provided in the invention has the advantages of strong specificity, simple operation, and fast detection.

Description

technical field [0001] The invention belongs to the technical field of detection of genetically modified components of milk and is related to the detection technology of immune colloidal gold. In particular, it relates to a fast and simple immune colloidal gold detection test strip for transfecting human lactoferrin (human lactoferrin, referred to as hLF) gene components and a preparation method thereof. Background technique [0002] Genetically modified animals and genetically modified animal products have shown strong vitality with their attractive economic prospects, and their marketization will be an irreversible trend of the times. However, in view of biosafety issues, all aspects of transgenic animal production must be strictly controlled. Therefore, a sound, scientific and standardized safety evaluation system is essential. Among them, reliable and accurate genetically modified detection methods are an important link in the safety evaluation system. [0003] Profes...

Claims

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Application Information

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IPC IPC(8): G01N33/558G01N33/544G01N33/532
Inventor 刘榜刘楚新翟珊莉张庆德
Owner HUAZHONG AGRI UNIV
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