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SARS early diagnostic method and reagent box

A cross-linking agent and antibody technology, applied in chemical instruments and methods, biological testing, instruments, etc., can solve the problem of lack of pathogens

Inactive Publication Date: 2009-03-11
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently a lack of specific antibodies against pathogens

Method used

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  • SARS early diagnostic method and reagent box
  • SARS early diagnostic method and reagent box
  • SARS early diagnostic method and reagent box

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Artificial Synthesis of Peptides

[0053] According to the amino acid sequence shown in Table 1, the peptide was synthesized on a peptide synthesizer. After the synthesis, it was verified by mass spectrometry, which proved that the synthesized peptide was consistent with the design.

[0054] Table 1: Amino acid sequence of the polypeptide of the present invention

[0055] Numbering: sequence SEQ ID NO: S protein S1-(34-54) TSSMRGVYYPDEIFRSDTLYL 1 S2-(94-113) KSNVVRGWVFGSTMNNKSQS 2 S3-(197-216) YKGYQPIDVVRDLPSGFNTL 3 S4-(273-293) TDAVDCSQNPLAELKCSVKSF 4 S5-(297-316) KGIYQTSNFRVVPSGDVVRF 5 S6-(332-351) TKFPSVYAWERKKISNCVAD 6 S7-(436-455) YNYKYRYLRHGKLRPFERDI 7 S8-(540-559) PSSKRFQPFQQFGRDVSDFT 8 S9-(731-753) CANLLLQYGSFCTQLNRALSGIA 9 S10-(758-780) RNTREVFAQVKQMYKTPTLKYFG 10

Embodiment 2

[0057] Linkage of the antigen polypeptide to the carrier (preparation of the conjugate)

[0058] For each polypeptide synthesized in Example 1, first dissolve it with PBS (for some insoluble polypeptides, you can first dissolve it with half an amount of PBS; 0.5mg / ml, 1mg / ml) mixed with 15mg BSA (1.5ml), then add a cross-linking agent (for different cross-linking directions, choose a different cross-linking agent, add glutaraldehyde for N-terminal cross-linking, C Add EDAC at the - end, mix and react at room temperature for 5 hours, avoid light and shake constantly; after cross-linking, put it into a dialysis bag, and dialyze against PBS at 4°C overnight; measure the volume, calculate the concentration with BSA, and store at -20°C spare.

Embodiment 3

[0060] Antigen peptide immunization animals to prepare antibodies:

[0061] Take about 0.5ml of each conjugate prepared in Example 2 (the content of the conjugate is 0.1mg, 0.5mg respectively), add 1.5ml of PBS, mix with CFA (complete Freund's adjuvant), push repeatedly in the three-way tube Beat until it feels hard, and after 3-5 minutes, there is no separation of the two phases.

[0062] Rabbits and mice were immunized with antigen emulsified by adjuvant; each rabbit was injected with about 3ml of antigen, and mice were injected with 0.8ml / monkey. Subcutaneously inject the bipedal pads, neck, and back of the animal into multiple points (0.2ml per point).

[0063] Three weeks after the initial immunization, take the same dose of polypeptide antigen and PBS, mix with 2ml of IFA (incomplete Freund's adjuvant), and emulsify the animals for booster immunization, and the injection dose remains unchanged.

[0064] Animals were bled three weeks later and antiserum was collected. ...

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Abstract

A method and reagent kit for the early diagnosis of SARS features that the polypeptide as antigen synthesized artificially from the S protein of SARS virus and the antibody generated by the animal immunized by said antigen is used in enzyme-linked immunoassay to detect the early SARS antibody IgM or IgG or the SARS pathogen from human serum specimen.

Description

technical field [0001] The invention relates to an early diagnosis method and kit for SARS. Specifically, the present invention uses artificially synthesized polypeptides derived from the S protein of SARS virus, and antibodies (mAbs, polyclonal antibodies) produced after the polypeptides are immunized to animals, combined with ELISA and other methods to detect early anti-antibodies in human serum samples. SARS antibody IgM or IgG, or direct detection of SARS pathogens in serum samples. Background technique [0002] The causative agent of atypical pneumonia, Severe Acute Respiratory Syndrome (SARS), has recently been identified as a coronavirus. The virus is a positive-strand RNA virus with a genome of single-stranded RNA containing Poly(A), with a total length of nearly 30kb. The surface of the virus has a rod-shaped structure of about 20nm, which is crown-shaped. The pathogen is highly contagious and contagious. After the virus undergoes an early replication process in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/165C07K16/08G01N33/68G01N33/569G01N33/577G01N33/533
Inventor 孙兵吴家睿裴钢李亦学石铁流杨瑞馥何有裕施木德吕伟季永镛
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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