Unlock instant, AI-driven research and patent intelligence for your innovation.

Glypican-3 double-antibody sandwich method detection kit and detection method

A detection kit and double-antibody sandwich technology, which is applied in the field of Glypican-3 double-antibody sandwich method detection kit, can solve the problems of complex operation of immunobiochemical method, inability to meet clinical needs, and small detection range of the kit, and achieve the cost of detection Low, easy results, wide detection range effect

Pending Publication Date: 2020-05-19
BIOLOGY INST OF HEBEI ACAD OF SCI
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the detection methods of Glypican-3 mainly include immunobiochemical method and enzyme-linked immunosorbent method. Among them, the immunobiochemical method is complicated to operate, has high requirements for personnel, and the results are not easy to judge.
ELISA is relatively simple to operate, but the detection range of existing kits is small and cannot meet clinical needs

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Glypican-3 double-antibody sandwich method detection kit and detection method
  • Glypican-3 double-antibody sandwich method detection kit and detection method
  • Glypican-3 double-antibody sandwich method detection kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of polyclonal antibody

[0038] The synthesized four polypeptides were named GPC3-P1, GPC3-P2, GPC3-P3, and GPC3-P4 respectively, and the specific sequences are shown in Table 1.

[0039] Table 1 Selected epitope sequences

[0040] .

[0041] Select 4 healthy New Zealand white rabbits weighing 2 kg, and immunize one rabbit with each polypeptide conjugate. Serum was collected from the ear vein of each rabbit before immunization as a negative control. The above four polypeptides were respectively coupled with bovine serum albumin (BSA) to obtain polypeptide conjugates GPC3-P1-BSA, GPC3-P2-BSA, GPC3-P3-BSA and GPC3-P4-BSA were dissolved and diluted with normal saline To a concentration of 1 mg / ml, the specific coupling method is a known technique.

[0042] Freund's complete adjuvant was used for the first immunization, the dose of antigen was 300ug / monkey, and the injection method was multi-point intradermal injection on the back. The second im...

Embodiment 2

[0043] Preparation, purification and titer determination of embodiment 2 polyclonal antibody

[0044] Purify the polyclonal antibody using the existing mature method of affinity column chromatography.

[0045] The antisera GPC3-S1, GPC3-S2, GPC3-S3, and GPC3-S4 obtained in Example 1 were tested for potency, and the results are shown in Table 2.

[0046] Table 2 Rabbit polyclonal antibody titer

[0047] .

Embodiment 3

[0048] Example 3 Development of double-antibody sandwich method ELISA detection kit

[0049] 1. The first round of antibody pairing experiments

[0050] The kit of the present invention adopts the double-antibody sandwich method (such as figure 1 ), two antibodies that can be paired are required, so for the 4 polyclonal antibodies (GPC3-S1, GPC3-S2, GPC3-S3, GPC3-S4) and 3 monoclonal antibodies (1H5, 2D12, 3A6) obtained in Example 2 Perform pairwise pair experiments to determine the most suitable pairing combination.

[0051]The specific steps are: coat 3 monoclonal antibodies at a concentration of 5ug / mL, block, add 200uL of 10% calf serum (commercially available 5mg / ml bovine serum albumin component 5) to each well as a blocking solution, and keep at 37°C for 1h; Wash (0.9% sodium chloride solution), return to room temperature, pour off the blocking solution, wash three times, shake for 1 min each time, pat dry; add 50ng / mL Glypican-3 solution, 37°C for 1 hour; wash, retur...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a Glypican-3 double-antibody sandwich method detection kit. The detection kit comprises a coated microporous plate coated with a captured antibody and a serum antibody coupledwith labeling enzyme, wherein the capture antibody is 1H5, 2D12 or 3A6; the serum antibody comprises a serum antibody GPC3-S1 of anti-GPC3-P1, a serum antibody GPC3-S2 of anti-GPC3-P2, a serum antibody GPC3-S3 of anti-GPC3-P3 or a serum antibody GPC3-S4 of anti-GPC3-P4, the amino acid sequences of the GPC3-P1 to GPC3-P4 are as shown in SEQ ID No. 1 to SEQ ID No. 4. The invention also relates to adetection method using the detection kit. The method has the advantages of convenience in detection, low cost and wide detection range.

Description

technical field [0001] The invention relates to a Glypican-3 double antibody sandwich method detection kit and detection method. Background technique [0002] The Glypican-3 (GPC3) protein was discovered by researchers in 1996. GPC3 is a member of the Glypican family. Its gene is located on human chromosome Xq26. The genome structure is more than 900kb in length and is one of the largest genes in the human genome. Its 5' end faces the telomeric region, and its 3' end faces the centriole region, consisting of 8 exons and 7 introns. There are many transcription factor binding sites in the promoter region. The full length of its cDNA sequence is 2263bp, and the open reading of 1740bp encodes 580 amino acids. The structure of Glypican-3 has the common characteristics of the Glypican family, such as the central globular structure space, the C-terminal glycosaminoglycan side chain connection site, etc. [0003] At present, the detection methods of Glypican-3 mainly include immu...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/535
CPCG01N33/68G01N33/535G01N2333/82
Inventor 程华武向丽孙劲冲吴萌
Owner BIOLOGY INST OF HEBEI ACAD OF SCI