Synthesis of polypeptide antibody from anti-human myocardial troponin I, its production and use

A technology for cardiac troponin and synthetic peptides, applied in the direction of anti-animal/human immunoglobulin, material inspection products, biological testing, etc., can solve difficulties, high extraction and purification costs, difficult to meet early diagnosis of acute myocardial infarction, etc. question

Inactive Publication Date: 2007-06-20
INST OF RADIATION MEDICINE CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] However, since human myocardial tissue is difficult to obtain, it is extremely difficult to extract and p

Method used

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  • Synthesis of polypeptide antibody from anti-human myocardial troponin I, its production and use
  • Synthesis of polypeptide antibody from anti-human myocardial troponin I, its production and use
  • Synthesis of polypeptide antibody from anti-human myocardial troponin I, its production and use

Examples

Experimental program
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Embodiment 1

[0045] Synthesis of Anti-Human Cardiac Troponin I Polypeptide

[0046] Use the Hopp&Woods model in Protscale software to predict the hydrophilicity, antigenic type and homology analysis of cTnI molecules, select the 98-110th amino acid at the N-terminal of cTnI as a polypeptide with 13 amino acids, and synthesize the peptide mainly through solid-phase peptide synthesis Method preparation. Using Fmoc-protected amino acids as raw materials, the amino acid sequence of the required peptide RQLHARVDKVDEE was synthesized in a stepwise condensation manner on a solid-phase support (resin). Finally, the peptide is "cleaved" from the carrier by an appropriate method, and all protective groups are removed. The polypeptide can be purified up to 95% by high performance liquid chromatography (see Figure 1 for details), and a pure synthetic peptide antigen can be obtained.

Embodiment 2

[0048] Ligation of synthetic peptides to carrier protein (KLH) (MBS ligation method)

[0049] (1) Dissolve 5 mg of KLH in 0.5 ml of PBS, place in a dialysis bag, dialyze with PBS overnight at 4°C, and then transfer to a glass test tube. (2) Add 70ul of MBS / DMF and stir at room temperature for 30 minutes. (3) Take the PD-10 chromatographic column pre-equilibrated with PB, add the sample slowly, and elute with PB, collect about 12-20 tubes, each tube is 0.5ml, and measure the 280nm OD value of the eluent by a UV-visible spectrophotometer. The first peak represents the MBS / KLH conjugate, which is flocculent; the second peak contains free MBS. The MBS / KLH conjugate fractions were pooled. (4) Dissolve 5mg of synthetic peptide in PBS, if the synthetic peptide is insoluble in PBS, it can be dissolved in 6mol / 0.1mol / lPB. (5) Mix the synthetic peptide solution with the MBS / KLH conjugate, adjust the pH to 7.3 with 0.1mol / l hydrochloric acid or 0.1mol / l sodium hydroxide, stir magnetic...

Embodiment 3

[0051] Preparation of Polyclonal Antibody to cTnI Synthetic Peptide Antigen

[0052] The specific process of polyclonal antibody preparation technology includes: animal immunization, antiserum titer detection, harvesting of antiserum, antibody purification and antibody quality identification, which are described as follows:

[0053] 1. Animal immunization: New Zealand big-eared white rabbits weighing 1.5-1.8Kg were selected, and human cTnI polypeptide synthetic antibodies were prepared after initial and multiple booster immunizations;

[0054] (1). Detection of immune effect: 10-14 days after the second booster immunization, a small amount of blood was collected from the rabbit's ear vein, the serum was separated, and the antibody titer in the rabbit serum was detected by indirect ELISA method.

[0055] (2). Collection of antiserum: a large number of bloodletting at one time. When the titer of antiserum reaches the maximum and no longer increases (or begins to decline), the s...

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Abstract

CTnI synthetic polypeptide antibody, its production and use are disclosed. The process is carried out by synthesizing polypeptide by 98-110 amino-acid at N end of amino-acid sequence RQLHARVDKVDEE, taking it as partial antigen to connect with protein carrier, preparing artificial immunogen, immunizing for animal, inducing organism effectively, generating immune response to obtain antibody, specific combined reacting cTnI synthetic polypeptide antibody with natural myoepicardial protein I molecule and coating cellulose nitrate film by the antibody, and reducing by tri-sodium citrate to obtain the final product. It's fast and has better sensitivity and specificity, and it can be used for acute myocardial infarction early diagnostic technology.

Description

technical field [0001] The invention relates to a biological product for in vitro experiments characterized by antibodies, more specifically, an anti-human cardiac troponin I (cTnI) synthetic polypeptide antibody, a preparation method thereof and a colloidal gold immunoassay card for measuring cTnI. Background technique [0002] At present, in industrialized countries, cardiovascular disease is one of the main diseases that threaten human life, with high morbidity and mortality. Early diagnosis and treatment of such diseases are of great significance. The diagnosis of heart disease includes clinical manifestations, changes in electrocardiogram and laboratory detection of myocardial injury biochemical markers, among which the detection of myocardial injury biochemical markers plays an important role in the diagnosis of myocardial injury and acute myocardial infarction. Finding and screening early, sensitive and specific myocardial injury markers and simple, fast and sensitive...

Claims

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Application Information

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IPC IPC(8): C07K16/18G01N33/53
Inventor 张春明王德芝陈冀莹赵启仁穆传杰秦丽莉李玉彬胡晓林
Owner INST OF RADIATION MEDICINE CHINESE ACADEMY OF MEDICAL SCI
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