Human VEGF detection combination reagent, reagent box and use method thereof

A reagent and antibody detection technology, which is applied in the field of immunology, can solve problems such as lack of sensitivity, failure to meet the needs of clinical testing or diagnosis, and low specificity of antibodies

Active Publication Date: 2015-04-22
AMPSOURCE BIOPHARMA (SHANGHAI) INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] There are many sources of VEGF detection reagents, but none of them have been standardized, and there are no VEGF diagnostic reagents registered and approved by the CFDA. The data obtained by various laboratories are very different, such as the normal value range is nearly 10 times different, and the specificity of antibodies is different. High, low sensitivity, poor stability and repeatability, etc. Although some of these ELISA kits can detect elevated VEGF concentrations in cancer patients, they lack the sensitivity required to measure VEGF blood levels in normal individuals, so they cannot reach clinical testing or diagnostic needs

Method used

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  • Human VEGF detection combination reagent, reagent box and use method thereof
  • Human VEGF detection combination reagent, reagent box and use method thereof
  • Human VEGF detection combination reagent, reagent box and use method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Embodiment 1, the preparation of anti-human VEGF mouse monoclonal antibody

[0093] Four-week-old BALB / c mice were subcutaneously injected with 20 μg of recombinant human VEGF (hVEGF, PEPROTECH) in complete adjuvant. Five injections are given every three to four weeks. Finally, a single injection of 50 μg hVEGF was given intraperitoneally. After serum testing, mice with high levels of anti-human VEGF antibody serum were identified. The mouse spleen was taken out and fused with the mouse myeloma Sp2 / 0 cell line. Mix 5×10 8 Sp2 / 0 cells with 5 × 10 8 Splenocytes were fused in a solution of 50% polyethylene glycol (PEG, molecular weight 1450) and 5% dimethyl sulfoxide (DMSO). Adjust the number of spleen cells to 7.5×10 with Iscove medium (containing 10% fetal bovine serum, 100 units / mL penicillin, 100 μg / mL streptomycin, 0.1 mM hypoxanthine, 0.4 μM aminopterin, and 16 μg thymidine). 5 / mL, add 0.2mL into the wells of the 96-well culture plate. Place at 37°C, 5% CO ...

Embodiment 2

[0094] Example 2. Competitive binding assay of anti-human VEGF mouse antibody and Avastin antibody

[0095] Avastin (Roche), a fully humanized antibody against human VEGF labeled with horseradish peroxidase (HRP), was used as a reagent. RhVEGF (50 μL, 0.1 μg / mL) was coated on a microtiter plate (Corning) and left overnight at room temperature. The coating solution was discarded, the wells were blocked with skimmed milk dissolved in phosphate buffered saline for 0.5 h, and the wells were washed with PBS containing 0.05% Tween 20. Then, a mixture of 50 μL of growth medium (DMEM+5% FBS, Invitrogen) and 50 μL of HRP-labeled Avastin antibody (250 ng / mL) was added to each well. Unlabeled Avastin and an irrelevant antibody against another antigen (Irrelevant mAb) were used as positive and negative controls. Among them, murine antibodies TM12-8-21 and TM12-13-31 competed for binding to EC 50 The values ​​are 0.4μg / mL, that is, when the concentration is as high as 0.4μg / mL, only ab...

Embodiment 3

[0096] Example 3, Cloning the heavy chain and light chain of TM12-15-1 murine antibody

[0097] In order to express the C12-15-1 antibody, the DNA fragments encoding the variable regions of the heavy and light chains of the anti-VEGF murine antibody TM12-15-1 must first be obtained. cDNA was prepared by isolating mRNA from TM12-15-1 mouse hybridoma cells using the mRNA Purification Kit (NEB Corp.) (SMARTer RACE Kit, Clontech Corp.). By polymerase chain reaction (PCR) using a 5'-primer mix (Long (0.4 μM):

[0098] 5'-ctaatacgactcactatagggc AAGCAGTGGTATCAACGCAGAGT-3' (SEQ ID NO: 11, primer 1) and

[0099] Short (2 μM): 5'-ctaatacgactcactatagggc-3' (SEQ ID NO: 12, primer 2) with 3'-primer 5'-catcccagggtcaccatggagtta-3' (SEQ ID NO: 13, primer 3) for isolation of heavy chain from cDNA Variable region DNA fragments. The 3'-primer is homologous and antisense to the mouse IgG1 heavy chain constant region. These resulting DNA fragments were cloned into TOPO TA vector (Invitrogen) (...

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Abstract

The invention discloses a murine monoclonal antibody resistant to human vascular endothelial growth factors (VEGFs), a combination reagent containing the murine monoclonal antibody and used for detecting the human VEGF level in a biological sample, a reagent box containing the combination reagent and a use method of the reagent box. The combination KD value of the murine monoclonal antibody and the human VEGFs reaches 0.01 nM, and the combination reagent containing the murine monoclonal antibody and the reagent box containing the combination reagent have the advantages of being high in detection sensitivity and specificity.

Description

technical field [0001] The invention relates to the field of immunology, in particular to an anti-human VEGF mouse monoclonal antibody and a combined reagent, a kit and a use method thereof for detecting the level of human VEGF in a biological sample. Background technique [0002] Among the more than 20 polypeptide vascular growth factors discovered so far, vascular endothelial growth factor (VEGF, also written as VEGF-A) is the key regulator with the highest specificity for endothelial cells and the strongest effect on promoting angiogenesis Factors are highly expressed in a variety of primary malignant tumor tissues. Furthermore, increased VEGF in the aqueous and vitreous humor of the eye has been shown to be strongly associated with various retinopathy (Aiello LP et al., N Engl J Med , 1994, 331:1480-1487). [0003] VEGF is a highly conserved homodimeric glycoprotein, which consists of two single chains with a molecular weight of 24 kDa and a disulfide bond to form a di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/22G01N33/68G01N33/577
Inventor 李强孙见宇李媛丽武翠高永娟孙乃超
Owner AMPSOURCE BIOPHARMA (SHANGHAI) INC
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