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Preparation method and application of human-human chimeric antiviral IgG antibody positive control

A positive quality control, chimeric technology, applied in antiviral immunoglobulins, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of complexity, prolonging the preparation period of quality control substances, and being too late.

Pending Publication Date: 2021-06-04
天津佰恒生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although the method of DNA recombination can solve the disadvantages of traditional quality control materials such as the lack of sources and potential biological infectivity, the experimental operation involves hybridoma technology, recombinant DNA technology, etc., which is relatively complicated and prolongs the preparation cycle of quality control materials. , and the most important thing is that chimeric monoclonal antibodies are formed by chimerizing different methods of variable regions derived from other species and constant regions of human origin, so the monoclonal antibody chimera and real human serum samples Compared with specific antibodies, it is inferior in many ways

Method used

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  • Preparation method and application of human-human chimeric antiviral IgG antibody positive control
  • Preparation method and application of human-human chimeric antiviral IgG antibody positive control
  • Preparation method and application of human-human chimeric antiviral IgG antibody positive control

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Extraction of Total RNA from Peripheral Blood Mononuclear Cells of Hepatitis C Positive Patients and Reverse Transcription to Obtain cDNA

[0045] (1) Collect 20-30 tubes (2-3mL / tube) of peripheral blood from hepatitis C positive patients, and separate peripheral blood mononuclear cells (PBMC) with polyvinylpyrrolidone silica gel (Percoll) separation method;

[0046] (2) The isolated mononuclear cell pellet is used for total RNA extraction, and stored at -80°C for later use;

[0047] (3) Perform reverse transcription on the extracted total RNA to obtain cDNA.

Embodiment 2

[0048] Example 2: Construction of phage antibody library

[0049] (1) Use the ImMunoGene Tics database to design primers, see Attached Table 1;

[0050] (2), PCR amplification obtains the light chain variable region fragment V of the antibody L (comprising Vκ and Vλ) and heavy chain variable region V H ;

[0051] (3), by overlapping PCR, V H and V L Assembled into scFv, the linker is GGGGSGGGGSGGGGS (as shown in SEQ ID NO.37);

[0052] (4) The product of PCR is recovered and purified after being separated by 1% agarose electrophoresis.

[0053] (5) Clone the scFv fragment into the pCANTAB 5E vector, electrotransform into TG1 competent cells under the condition of 15kv / cm, 5ms, and coat the SOB-AG plate;

[0054] (6) Single colonies were counted on the SOB-AG plate, single clones were picked for enzyme digestion identification, and the library capacity of the phage antibody library was calculated. A total of about 6×10 7 clone library.

[0055] Table 1

[0056]

[0...

Embodiment 3

[0059] Example 3: Screening of Phage Antibody Library and Obtaining Specific Anti-Hepatitis C Monoclonal Antibody

[0060] (1) Coat immunotubes with hepatitis C core, NS3, NS4, and NS5 antigens overnight at 4°C (coating volume: 2ml / tube), set up positive and negative controls, and seal with 5% skimmed milk powder for 2 hours;

[0061] (2) Discard the blocking solution, wash 3 times with 1×PBST (5min / time), add the recombinant phage culture supernatant to the immunotube, incubate at 220rpm / min at 37°C for 30min, and let stand at room temperature for 1.5h;

[0062] (3) Discard the supernatant, wash 3 times with 1×PBST (5min / time), add 0.2M Glycine-HCl (pH2.5) 2ml / tube, rotate gently at 37°C for 6min, then immediately add an equal volume of Tris-His (pH 7.4) buffer to neutralize the eluent, add host bacteria TG1 (OD600=0.5) and shake at 150 rpm / min at 37°C for 1 hour to complete a round of panning;

[0063] (4) After five rounds of panning, four phage antibody libraries targetin...

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Abstract

The invention discloses a preparation method and application of a human-human chimeric antiviral IgG antibody positive control. The preparation method mainly comprises the following steps: collecting peripheral blood samples from viral infection-positive patients, separating peripheral blood mononuclear cells, carrying out total RNA extraction, and performing reverse transcription to obtain cDNA; carrying out PCR amplification to obtain a target fragment scFv and constructing a phage antibody library; screening the phage antibody library and obtaining a specific monoclonal antibody; splicing VH and VL of the scFv with a human IgG1 constant region and a human Ckappa constant region, respectively; and finally obtaining the human-human chimeric antiviral IgG antibody positive control with good specificity and high affinity and stability. The invention can be applicable to various detection kits, and has important use value for clinical diagnosis of virus-infected persons.

Description

technical field [0001] The invention belongs to the technical field of biological immunity, and in particular relates to a preparation method and application of a human-human chimeric antiviral IgG antibody positive quality control product. Background technique [0002] Hepatitis C virus (HCV) infection mainly improves the early detection rate of hepatitis C patients through two serological detection methods for HCV antibodies, enzyme-linked immunoassay and chemiluminescence immunoassay. In order to accurately monitor the influence of factors such as reagents, samples, and experimenter operations on the experimental results during the experiment, a positive control is essential. The traditional positive control mainly comes from the positive serum of hepatitis C patients. Due to the obvious defects in the application of this positive quality control product, such as difficult to obtain and high cost, it involves theoretical problems, poor stability, and potential application...

Claims

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Application Information

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IPC IPC(8): C07K16/10C12N15/13C12N15/10C12N15/70C40B50/06G01N33/576G01N33/577
CPCC07K16/109C07K16/005C12N15/1096C12N15/1037C12N15/70C40B50/06G01N33/5767G01N33/577C07K2317/24C07K2317/622C07K2317/92C07K2317/94G01N2333/186G01N2469/10
Inventor 王世兴李天宁
Owner 天津佰恒生物科技有限公司
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