CN111175520A - Glyphican-3 double-antibody sandwich method detection kit and detection method - Google Patents

Glyphican-3 double-antibody sandwich method detection kit and detection method Download PDF

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CN111175520A
CN111175520A CN202010039984.XA CN202010039984A CN111175520A CN 111175520 A CN111175520 A CN 111175520A CN 202010039984 A CN202010039984 A CN 202010039984A CN 111175520 A CN111175520 A CN 111175520A
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gpc3
antibody
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bsa
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程华
武向丽
孙劲冲
吴萌
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Institute of Biology of Hebei Academy of Sciences
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Abstract

The invention relates to a Glypican-3 double-antibody sandwich method detection kit, which comprises a coated microporous plate coated with a capture antibody and a serum antibody coupled with a labeled enzyme; the capture antibody is 1H5, 2D12, or 3a 6; the serum antibodies include: the anti-GPC antibody GPC3-P1 serum antibody GPC3-S1, anti-GPC 3-P2 serum antibody GPC3-S2, anti-GPC 3-P3 serum antibody GPC3-S3 or anti-GPC 3-P4 serum antibody GPC3-S4, wherein the amino acid sequences of GPC 3-P1-GPC 3-P4 are shown in SEQ ID No. 1-SEQ ID No. 4; the invention also relates to a method for detecting by using the detection kit. The invention has the advantages of convenient detection, low cost and wide detection range.

Description

Glyphican-3 double-antibody sandwich method detection kit and detection method
Technical Field
The invention relates to a Glypican-3 double-antibody sandwich method detection kit and a detection method.
Background
In 1996, the Glyphalin-3 (GPC 3) protein was discovered by researchers, and GPC3 is a member of the Glyphalin family, the gene of which is located on human chromosome Xq26, the whole length of the genome structure is greater than 900kb, and the protein is one of the largest genes in the human genome. The 5 'end of the gene faces to a telomere area, the 3' end faces to a central particle area and consists of 8 exons and 7 introns. The promoter region has a number of transcription factor binding sites. The whole length of the cDNA sequence is 2263bp, 1740bp, and the open reading of deceive codes 580 amino acids. The structure of the Glypican-3 has the common characteristics of Glypican families, such as a central spherical structure space, a C-terminal glycosaminoglycan side chain connection site and the like.
At present, the detection method of the Glypican-3 mainly comprises an immune biochemical method and an enzyme-linked immune method, wherein the immune biochemical method is complex in operation, high in requirement on personnel and poor in judgment result. The enzyme-linked immunosorbent assay is relatively simple to operate, but the existing kit has a small detection range and cannot meet clinical requirements. There is a need to develop better detection methods.
Disclosure of Invention
The invention aims to provide a Glypican-3 double-antibody sandwich method detection kit which is convenient to detect, low in cost and wide in detection range and a detection method by using the detection kit.
The invention adopts the following technical scheme:
a detection kit of a Glypican-3 double-antibody sandwich method comprises a coated microporous plate coated with a capture antibody and a serum antibody coupled with a labeled enzyme; the capture antibody is 1H5, 2D12, or 3a 6; the serum antibodies include: a serum antibody GPC3-S1 against GPC3-P1, a serum antibody GPC3-S2 against GPC3-P2, a serum antibody GPC3-S3 against GPC3-P3, or a serum antibody GPC3-S4 against GPC 3-P4; the amino acid sequence of GPC3-P1 is shown as SEQ ID No.1, the amino acid sequence of GPC3-P2 is shown as SEQ ID No.2, the amino acid sequence of GPC3-P3 is shown as SEQ ID No.3, and the amino acid sequence of GPC3-P4 is shown as SEQ ID No. 4.
When the capture antibody is 1H5, the serum antibody is GPC3-S1, GPC3-S2 or GPC 3-S4.
When the capture antibody is 2D12, the serum antibody is GPC3-S1, GPC3-S3 or GPC 3-S4.
When the capture antibody is 3A6, the serum antibody is GPC3-S2 or GPC 3-S4.
Preferably, the capture antibody is 1H 5; the serum antibody is GPC 3-S4.
The detection kit also comprises a washing solution, a developing solution, a Glypican-3 standard substance and a stop solution.
In the detection kit, the washing solution is 0.9% sodium chloride solution.
In the detection kit, the color development liquid is 0.1% of 3,3',5,5' -tetramethylbenzidine solution.
In the detection kit, the stop solution is a 1-mol sulfuric acid solution.
In the detection kit, the labeled enzyme is horseradish peroxidase.
In the detection kit, the serum antibody is prepared by the following method:
(1) respectively coupling GPC3-P1, GPC3-P2, GPC3-P3 and GPC3-P4 with bovine serum albumin to obtain polypeptide conjugates PC3-P1-BSA, GPC3-P2-BSA, GPC3-P3-BSA and GPC 3-P4-BSA;
(2) selecting 4 healthy New Zealand white rabbits (2 kg), and immunizing 1 rabbit with each polypeptide conjugate; before immunization, taking blood serum from ear vein of each rabbit as negative control; GPC3-P1-BSA, GPC3-P2-BSA, GPC3-P3-BSA, and GPC3-P4-BSA were dissolved in physiological saline and diluted to a concentration of 1mg/ml, respectively; freund's complete adjuvant is used for the first immunization, the dosage of the antigen is 300 ug/mouse, and the injection mode is intradermal multiple injection at the back;
(3) carrying out second immunization after 2 weeks, wherein the adjuvant is Freund incomplete adjuvant;
(4) a total of 5 immunizations were performed, and 2 weeks after the last immunization, antibody titers were measured by blood sampling, and antiserum GPC3-S1, GPC3-S2, GPC3-S3, and GPC3-S4 were collected.
A method for detecting the Glypican-3 by using the detection kit of the Glypican-3 double-antibody sandwich method comprises the following steps:
(A) coating the capture antibody on a coating microporous plate at a concentration of 5ug/mL, sealing, adding 200uL 10% (mass fraction) of calf serum (5 mg/mL of bovine serum albumin component 5) as sealing liquid into each well, and keeping the temperature at 37 ℃ for 1 h; washing, returning to room temperature, pouring off the confining liquid, washing for three times, shaking lmin each time, and patting dry;
(B) adding the solution to be detected, and keeping the temperature at 37 ℃ for 1 h; washing, returning to room temperature, pouring off the confining liquid, washing for three times, shaking lmin each time, and patting dry;
(C) 100uL of 1: 1000 serum antibody is added into each hole, and the temperature is 37 ℃ for 1 h; washing, returning to room temperature, pouring off the confining liquid, washing for three times, shaking lmin each time, and patting dry; the titer of GPC3-S1 and GPC3-S2 is 1: 7.68X 105(ii) a The titer of GPC3-S3 and GPC3-S4 is 1: 1.53X 106；
(D) Adding 100uL of goat anti-rabbit secondary antibody-HRP (horse radish peroxidase) with the ratio of 1: 10000 into each hole, and standing at 37 ℃ for 30 min; washing for 3 times, shaking for 1min each time, and patting to dry; the titer of the goat anti-rabbit secondary antibody-HRP is one ten thousandth;
(D) adding 100uL of substrate developing solution into each hole, and carrying out heat preservation and light-proof reaction at 37 ℃ for 15 min;
(E) adding 50uL of stop solution into each hole to stop the reaction;
(F) and reading the optical density value of each hole by using a microplate reader with the detection wavelength of 450 nm.
The invention has the beneficial effects that: the invention utilizes the prepared antibody, adopts a double-antibody sandwich method, specifically recognizes the Glypican-3, has simple operation, low detection cost, lower operation requirement on personnel, easy judgment of results and wide detection range, and meets the clinical requirement.
Drawings
FIG. 1 is a schematic diagram of the detection principle of the double antibody sandwich ELISA detection kit.
FIG. 2 shows the results of the antibody pairing experiment when the capture antibody is 1H 5.
FIG. 3 shows the results of the antibody pairing experiment when the capture antibody was 3A 6.
FIG. 4 shows the results of antibody pairing experiments when the capture antibody was 2D 12.
FIG. 5 shows the result of an antibody pairing experiment when the capture antibody 1H5 and the detection antibody are S4.
FIG. 6 is a test standard curve.
Detailed Description
The embodiments of the present invention are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. The terminology used in the exemplary embodiments illustrated in the accompanying drawings is not intended to be limiting of the invention.
EXAMPLE 1 polyclonal antibody preparation
The synthesized four polypeptides are named as GPC3-P1, GPC3-P2, GPC3-P3 and GPC3-P4 respectively, and the specific sequences are shown in Table 1.
Epitope sequences selected from Table 1
Figure 406491DEST_PATH_IMAGE002
。
4 healthy New Zealand white rabbits (2 kg) were selected and 1 rabbit was immunized with each polypeptide conjugate. Before immunization, ear veins of each rabbit were bled and used as a negative control. The four polypeptides are respectively coupled with Bovine Serum Albumin (BSA) to obtain polypeptide conjugates of GPC3-P1-BSA, GPC3-P2-BSA, GPC3-P3-BSA and GPC3-P4-BSA which are respectively dissolved and diluted by normal saline to the concentration of 1mg/ml, and the specific coupling method is a known technology.
The first immunization was performed with Freund's complete adjuvant at a dose of 300 ug/mouse, and the injection was performed as a back intradermal multiple injection. The second immunization was performed after 2 weeks intervals, and the adjuvant was Freund's incomplete adjuvant. The immunization is carried out for 5 times in total, blood is taken 2 weeks after the last immunization to measure the antibody titer, and antiserum is collected. The antiserum was designated as GPC3-S1, GPC3-S2, GPC3-S3, and GPC3-S4, respectively. The Freund's complete adjuvant and Freund's incomplete adjuvant are both commercially available products.
EXAMPLE 2 preparation, purification and potency assay of polyclonal antibodies
The existing mature method of affinity column chromatography is utilized to purify the polyclonal antibody.
The antiserum GPC3-S1, GPC3-S2, GPC3-S3, and GPC3-S4 obtained in example 1 were subjected to titer measurement, and the results are shown in Table 2.
TABLE 2 Rabbit Multi-antibody Titers
Figure DEST_PATH_IMAGE003
。
Example 3 double antibody Sandwich ELISA detection kit development
First round of antibody pairing experiment
The kit of the invention adopts a double-antibody sandwich method (as shown in figure 1) and needs two antibodies capable of matching, so that two-by-two matching experiments are carried out on 4 polyclonal antibodies (GPC 3-S1, GPC3-S2, GPC3-S3 and GPC 3-S4) obtained in example 2 and 3 monoclonal antibodies (1H 5, 2D12 and 3A 6) to determine the most suitable matching combination.
The method comprises the following specific steps: coating 3 monoclonal antibodies with the concentration of 5ug/mL, sealing, adding 200uL 10% calf serum (commercially available 5mg/mL bovine serum albumin component 5) as sealing solution into each well, and keeping the temperature at 37 ℃ for 1 h; washing (0.9% sodium chloride solution), returning to room temperature, pouring off the confining liquid, washing for three times, shaking lmin each time, and patting dry; adding 50ng/mL of a Glypican-3 solution, and keeping the temperature at 37 ℃ for 1 h; washing, returning to room temperature, washing for three times, shaking lmin each time, and patting dry; add 100uL of polyclonal antibody diluted at l: 1000 to each well (while adding diluent to other wells as control), 1h at 37 ℃; washing, returning to room temperature, washing for three times, shaking lmin each time, and patting dry; adding 100uL of goat anti-rabbit secondary antibody-HRP diluted by l: 10000 into each hole, and standing at 37 ℃ for 30 min; washing for 3 times, shaking for 1min each time, and patting to dry; developing, adding 100uL (0.1% of 3,3',5,5' -tetramethyl benzidine solution) of substrate developing solution into each well, and carrying out heat preservation and light-proof reaction at 37 ℃ for 15 min; stopping the reaction, namely adding 50uL of stop solution (1 mol of sulfuric acid solution) into each hole to stop the reaction; determination of OD450nmThe optical density of each well was read using a microplate reader with a detection wavelength of 450 nm.
After the four existing antibodies are paired pairwise, the results are shown in fig. 2-fig. 4 and table 3, and as can be seen from table 3, GPC3-S4 and the three monoclonal antibodies can be paired, and when the antibodies are paired with 1H5, the OD value is highest, so that the pair is selected for the second test.
TABLE 3 table of antibody pairings
Figure 713844DEST_PATH_IMAGE004
。
Second and third round of antibody pairing experiments
The optimal pairing of antibodies was optimized a second time. The specific steps are that monoclonal antibody 1H5 is coated with 5ug/mL concentration, sealed, 200uL 10% calf serum is added into each hole as sealing liquid, and the temperature is 37 ℃ for 1H; washing, returning to room temperature, pouring off the confining liquid, washing for three times, shaking for l min each time, and patting dry; adding 20, 40, 60, 80 and 100ng/mL of Glypican-3 solution, and keeping the temperature at 37 ℃ for 1 h; the subsequent steps are matched with the first round of antibody.
The results of the second pairing experiment are shown in fig. 5 and table 2, and it can be seen that when 1H5 is used as a capture antibody and S4 is used as a detection antibody, the detection sensitivity can be lower than 10ng/mL, and the detection level of the existing like products in the market is reached, so that the pairing combination is determined to be used for assembling the Glypican-3 detection kit.
TABLE 4 results of antibody pairing assays
Figure DEST_PATH_IMAGE005
。
Drawing a standard curve
The main characteristic of quantitative analysis is to establish a metering-reaction curve, and to establish a metering-reaction relation by the known concentration of standard substance and the corresponding reaction amount. Then, the concentration of the sample to be measured is calculated from the reaction amount of the sample to be measured.
The specific process is that 5ug/mL antibody is coated and sealed, 200uL 10% calf serum is added into each hole as sealing liquid, and the temperature is 37 ℃ for 1 h; washing, returning to room temperature, pouring off the confining liquid, washing for three times, shaking lmin each time, and patting dry; antigen solutions of serial concentrations, i.e., 0ng/mL, 10ng/mL, 25ng/mL, 50ng/mL, 100ng/mL, 150ng/mL, 200ng/mL, 300ng/mL, were added in the standard solutions of Glypican-3. 1h at 37 ℃; the subsequent steps are matched with the first round of antibody.
The double-antibody sandwich ELISA method established in the method is adopted for repeated detection for 4 times, and the result shows that the detection interval with good linear relation is 10-300 ng/mL. The standard curve is shown in FIG. 6.
Example 4
Random samples were tested according to the test procedure provided in example 3, see table 5.
TABLE 5 comparison of the results of the clinical tests with the kit of the invention
Figure DEST_PATH_IMAGE006
。
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
SEQUENCE LISTING
<110> institute of biological research of academy of sciences of Hebei province
<120> detection kit and detection method for Glypican-3 double-antibody sandwich method
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Cys Ser Arg Lys Met Glu Glu Lys Tyr Gln Leu Thr Ala
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Ser Ile Gln Tyr Val Gln Lys Asn Ala Gly Lys Leu Thr
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Gln Gln Arg Gln Tyr Arg Phe Ala Tyr Tyr Pro Glu Asp Leu Phe
1 5 10 15
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Val Glu Arg Tyr Ser Gln Lys Ala Ala Arg Asn Gly Met
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Claims (6)

1. A Glyphain-3 double antibody sandwich method detection kit is characterized by comprising a coated microporous plate coated with a capture antibody and a serum antibody coupled with a labeled enzyme; the capture antibody is 1H5, 2D12, or 3a 6; the serum antibodies include: a serum antibody GPC3-S1 against GPC3-P1, a serum antibody GPC3-S2 against GPC3-P2, a serum antibody GPC3-S3 against GPC3-P3, or a serum antibody GPC3-S4 against GPC 3-P4; the amino acid sequence of GPC3-P1 is shown in SEQ ID No.1, the amino acid sequence of GPC3-P2 is shown in SEQ ID No.2, the amino acid sequence of GPC3-P3 is shown in SEQ ID No.3, and the amino acid sequence of GPC3-P4 is shown in SEQ ID No. 4.
2. The Glypican-3 double antibody sandwich assay kit as claimed in claim 1, wherein the capturing antibody is 1H 5; the serum antibody is GPC 3-S4.
3. The Glypican-3 double antibody sandwich assay kit according to claim 1 or 2, which further comprises a washing solution, a developing solution, a Glypican-3 standard, and a stop solution.
4. The Glypican-3 double antibody sandwich assay kit according to claim 1 or 2, characterized in that the labeled enzyme is horseradish peroxidase.
5. The Glypican-3 double antibody sandwich assay kit according to claim 1, characterized in that the serum antibody is prepared by the following method:
(1) respectively coupling GPC3-P1, GPC3-P2, GPC3-P3 and GPC3-P4 with bovine serum albumin to obtain polypeptide conjugates PC3-P1-BSA, GPC3-P2-BSA, GPC3-P3-BSA and GPC 3-P4-BSA;
(2) selecting 4 healthy New Zealand white rabbits (2 kg), and immunizing 1 rabbit with each polypeptide conjugate; before immunization, taking blood serum from ear vein of each rabbit as negative control; GPC3-P1-BSA, GPC3-P2-BSA, GPC3-P3-BSA, and GPC3-P4-BSA were dissolved in physiological saline and diluted to a concentration of 1mg/ml, respectively; freund's complete adjuvant is used for the first immunization, the dosage of the antigen is 300 ug/mouse, and the injection mode is intradermal multiple injection at the back;
(3) carrying out second immunization after 2 weeks, wherein the adjuvant is Freund incomplete adjuvant;
(4) a total of 5 immunizations were performed, and 2 weeks after the last immunization, antibody titers were measured by blood sampling, and antiserum GPC3-S1, GPC3-S2, GPC3-S3, and GPC3-S4 were collected.
6. A method for detecting the Glypican-3 by using the Glypican-3 double-antibody sandwich method detection kit as defined in any one of claims 1 to 5, which is characterized by comprising the following steps:
(A) coating the capture antibody on a coating microporous plate at the concentration of 5ug/mL, sealing, adding 200uL 10% calf serum into each well as sealing solution, and keeping the temperature at 37 ℃ for 1 h; washing, returning to room temperature, pouring off the confining liquid, washing for three times, shaking lmin each time, and patting dry;
(B) adding the solution to be detected, and keeping the temperature at 37 ℃ for 1 h; washing, returning to room temperature, washing for three times, shaking lmin each time, and patting dry;
(C) adding 100uL of serum antibody into each hole, and keeping the temperature at 37 ℃ for 1 h; washing, returning to room temperature, washing for three times, shaking lmin each time, and patting dry;
(D) adding 100uL of goat anti-rabbit secondary antibody-HRP into each hole, and standing at 37 ℃ for 30 min; washing for 3 times, shaking for 1min each time, and patting to dry;
(D) adding 100uL of substrate developing solution into each hole, and carrying out heat preservation and light-proof reaction at 37 ℃ for 15 min;
(E) adding 50uL of stop solution into each hole to stop the reaction;
(F) and reading the optical density value of each hole by using a microplate reader with the detection wavelength of 450 nm.
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Publication number Priority date Publication date Assignee Title
CN113391070A (en) * 2021-06-03 2021-09-14 山东大学 Improved quantitative detection method for phosphatidylinositol proteoglycan 3

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CN102634487A (en) * 2012-03-28 2012-08-15 南方医科大学 GPC3 (glypican-3) monoclonal antibody hybridoma strain 8G6, and preparation method and application thereof
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CN104610441A (en) * 2015-03-04 2015-05-13 首都医科大学 Artificial hapten used for preparing glypican-3 (GPC3) monoclonal antibody, preparation method and obtained monoclonal antibody
CN104829704A (en) * 2014-12-15 2015-08-12 河北省科学院生物研究所 Phosphatidylinositol proteoglycan GPC3 protein fragment, application thereof and hybridoma cell strain prepared therewith

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CN102634487A (en) * 2012-03-28 2012-08-15 南方医科大学 GPC3 (glypican-3) monoclonal antibody hybridoma strain 8G6, and preparation method and application thereof
CN102633864A (en) * 2012-03-28 2012-08-15 南方医科大学 GPC3 antigen polypeptide, anti-GPC3 polyclonal antibody and application thereof
CN104829704A (en) * 2014-12-15 2015-08-12 河北省科学院生物研究所 Phosphatidylinositol proteoglycan GPC3 protein fragment, application thereof and hybridoma cell strain prepared therewith
CN104610441A (en) * 2015-03-04 2015-05-13 首都医科大学 Artificial hapten used for preparing glypican-3 (GPC3) monoclonal antibody, preparation method and obtained monoclonal antibody

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Title
吴萌 等: "肝细胞癌标志物GPC3 表位肤抗体制备及其特性测定" *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113391070A (en) * 2021-06-03 2021-09-14 山东大学 Improved quantitative detection method for phosphatidylinositol proteoglycan 3

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Application publication date: 20200519