Preparation method and application of anti-alpha-enolase antibody chromatography test strip

The technology of enolase and immunochromatographic test paper is applied in the field of detection of anti-alpha-enolase antibody immunochromatographic test paper, which can solve the problems such as not being published, and achieve the effects of short detection time, high sensitivity and high detection performance.

Inactive Publication Date: 2015-07-08
苏州和锐生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In the diagnosis of autoimmune diseases, many immunochromatographic test strips have appeared, but the immunochromatographic test strips for anti-NNE antibodies have not yet come out in the domestic and foreign markets

Method used

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  • Preparation method and application of anti-alpha-enolase antibody chromatography test strip
  • Preparation method and application of anti-alpha-enolase antibody chromatography test strip
  • Preparation method and application of anti-alpha-enolase antibody chromatography test strip

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1. NNE antigenic protein preparation

[0046] Recombinant protein method: The NNE antigen protein applied to this test paper is to construct a recombinant gene through gene cloning technology, and then use a prokaryotic expression vector or a eukaryotic expression vector. The prokaryotic expression vector used in the present invention is pET28a (+), and the expression bacteria is BL21 large intestine Bacillus; the eukaryotic expression vector is pcDNA3.1(+), and the expression bacteria is yeast. Both expression techniques can successfully express the NNE antigen protein that is all human. For the operation method, please refer to the "Molecular Cloning Experiment Guide". Among them, the NNE antigen protein is derived from the genetic engineering antigen protein obtained by affinity purification and desalting process.

[0047] NNE antigen protein preparation method 2: Screen 2-6 NNE important antigenic epitope polypeptides, use a peptide synthesizer to synthe...

Embodiment 2

[0048] Example 2 Antibody Preparation

[0049] Anti-human IgG monoclonal and polyclonal antibodies coated with antibodies for the conjugate pad and detection zone, as well as monoclonal and polyclonal antibodies from other animal sources for the quality control zone and conjugate pad, can be obtained by immunizing animals.

[0050] 1. The specific operation method of monoclonal antibody preparation is as follows:

[0051] 1) The injection solution is obtained by mixing 0.05mg~5mg of immune preparations (respectively for goats, mice, guinea pigs, rabbits, horses, and camels) with 1 volume of Freund's complete adjuvant, and the injection solution is added to the animal skin. site injection;

[0052] 2) One month later, the Freund's complete adjuvant mixture for animals was subcutaneously injected into multiple sites to boost the immunization. After 7 to 14 days, the animals were bled, and the antiserum titers were measured.

[0053] 3) The animals were boosted until the titer...

Embodiment 3

[0059] Embodiment 3 preparation of colloidal gold solution

[0060] 1) Add 0.01% (w / v) HAuCl 4 Heat the solution to boiling, quickly add every 100mL HAuCl 4 Add an appropriate amount of reducing agent solution to the solution, and the color changes from blue, then light blue, to blue, then red after heating, and transparent orange-red after boiling for 7-10 minutes. Then filter with ultrafiltration or microporous membrane (0.45μM) to remove polymers and other impurities that may be mixed therein. The prepared colloidal gold should be pure, translucent, free of sediment and floating matter, and discard when oily matter and a large amount of black granular precipitate impurities appear on the liquid surface.

[0061] 2) The reducing agent used therein can be trisodium citrate, tannic acid-trisodium citrate, white phosphorus, preferably trisodium citrate, more preferably 1% (w / v) trisodium citrate.

[0062] 3) The glass containers used should be absolutely clean and pickled be...

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Abstract

The invention discloses an anti-alpha-enolase antibody chromatography test strip, and a preparation method and application thereof. The test strip includes a sample pad (1), a combination pad (2), an analysis membrane (3), a water absorption pad (6), a base plate (7), a test zone T line (4) and a quality control C line (5). The upper part of the base plate (7) is adhered to the analysis membrane (3), and the the upper two ends of the analysis membrane (3) are respectively adhered to the combination pad (2) and the water absorption pad (6); one upper end of the combination pad (2) is adhered to the sample pad (1); the test zone T line (4) and the quality control C line (5) are arranged on the analysis membrane (3). The invention employs indirect immunodetection and alpha-enolase antigen protein to realize high sensitivity, high specificity and rapid detection of alpha-enolase antibody protein and provide the basis for auxiliary diagnosis of clinical autoimmune diseases such as inflammatory bowel disease.

Description

technical field [0001] The invention relates to the field of immunoassay detection. Specifically, the present invention relates to an immunochromatographic detection test paper for detecting anti-alpha-enolase antibody, a preparation method and an application thereof. Background technique [0002] Enolase (enolase) was discovered in 1934 by Lohman and Mayerhof in the study of the conversion of phosphoglycerate to pyruvate in muscle extracts. It can not only catalyze the conversion of 2-phospho-D-glycerate (PGA) to phospho-enolpyruvate (PEP) in the process of glycolysis, but also catalyze the reverse reaction in the process of glycogen synthesis, that is, as phosphoric acid Pyruvate hydratase converts PEP to PGA, so enolase plays an important role in cellular energy metabolism [J Biol Chem, 2000, 275(8): 5958-5965]. In vertebrates, there are three isozymes of enolase: α, β, γ. α-enolase exists in many tissues, β-enolase is almost only found in muscle tissue, and γ-enolase ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/531G01N33/532
CPCG01N33/68G01N33/531G01N33/532
Inventor 陈菲李振军霍如松周阳
Owner 苏州和锐生物科技有限公司
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