CN101290318B - ELISA reagent kit for diagnosing liver cancer - Google Patents
ELISA reagent kit for diagnosing liver cancer Download PDFInfo
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- CN101290318B CN101290318B CN2007100395627A CN200710039562A CN101290318B CN 101290318 B CN101290318 B CN 101290318B CN 2007100395627 A CN2007100395627 A CN 2007100395627A CN 200710039562 A CN200710039562 A CN 200710039562A CN 101290318 B CN101290318 B CN 101290318B
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- antibody
- gpc3
- heparin sulfate
- sulfate proteoglycan
- epitope
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Abstract
The invention discloses a detection kit for liver cancer. The detection kit contains a polyclonal antibody combined with the epitope of the heparan sulfate proteoglycans 3 between the position of 379 and 393, and a monoclonal antibody or a polyclonal antibody combined with the epitope except the heparan sulfate proteoglycans 3 between the position of 379 and 393. The kit has high detection sensitivity and short detection time, helps a doctor diagnose a liver cancer patient as early as possible to ensure that the patient can receive the immediate treatment and improve the survival rate. Moreover, compared with the only detection of the AFP marker, the kit detects GPC3 marker at the same time when the AFP marker is detected, thereby evidently improving the diagnosis sensitivity of the liver cancer.
Description
Technical Field
The invention relates to the fields of biotechnology and immunology, in particular to an antibody and a kit for detecting a liver cancer marker heparin sulfate proteoglycan 3(GPC3) and application thereof.
Background
Primary liver cancer is the most common cancer at 6 th worldwide and 3 rd china. According to the estimation of the international research center for cancer (IARC), the number of liver cancer attacks in the world in 2000 is 56.4 thousands of people, about 55% of the liver cancer attacks in China, namely 30.6 thousands of people with liver cancer attacks in China, 30.0 thousands of liver cancer deaths and the second place of tumor deaths of residents in China. When liver cancer is manifested as symptoms, it is usually in middle and late stages, and has high recurrence and metastasis rate after resection. Therefore, early diagnosis of liver cancer is of great significance in prolonging the survival time of patients and reducing the death rate of liver cancer.
At present, imaging diagnosis, cell and histology diagnosis and chemical diagnosis are three main methods for tumor diagnosis. The imaging diagnosis plays an important role in liver cancer diagnosis, but has certain limitations in the diagnosis of small liver cancer and the differentiation of benign and malignant nodules. The positive result of ultrasonic examination or CT is combined with the detection result that the level of serum alpha-fetoprotein (AFP) is higher than 400ng/ml, so that the liver cancer can be diagnosed. However, often when these conditions are met, the best opportunity for treatment has been missed. Whether it be ultrasonography, CT scanning or nuclear magnetic resonance, there are limitations to the identification of small lesions, especially the hepatocirrhotic nodules have many similarities with small liver cancer nodules in imaging. Therefore, although imaging techniques have been greatly advanced in this year, there is still a need to combine molecular markers of liver cancer to distinguish benign and malignant liver diseases in more complicated cases. In addition, the clinical stage and size of the tumor are the determining factors for the treatment effect of liver cancer, so the tumor marker can also be used for the general investigation of liver cancer risk people (such as HBV chronic carriers and liver cirrhosis patients).
The most common marker for liver cancer diagnosis at present is alpha-fetoprotein (AFP), which is abnormal several months before symptoms and imaging changes occur. The AFP content in normal human serum is generally less than 10 ng/ml. When the diagnostic value of AFP is set as 20ng/ml, the sensitivity is 50-60%, but in the case of smaller tumors, the sensitivity is remarkably reduced, and is only 40% reported. Another problem with the use of AFP alone as a diagnostic marker for liver cancer is the lack of specificity, and the AFP content also reaches 20-200ng/ml in a considerable number of patients with chronic hepatitis, especially in patients with liver cirrhosis. At present, the markers which can be complementarily diagnosed with AFP are considered to be AFP heteroplasmon, gamma-glutamyltransferase isozyme II, abnormal prothrombin and the like, but because of the defects of sensitivity and specificity and the complex detection method, the markers always stay at the laboratory research level and cannot be converted into clinical routine examination items. Therefore, the search for new tumor molecular markers and the establishment of corresponding detection methods which are easy to popularize are still one of the important subjects in the field of liver cancer research at present.
Heparin sulfate proteoglycan 3(Glypican-3, GPC3) is a molecule found in recent years to be associated with liver cancer. By applying a difference display technology, Hsu and the like find that MXR7 gene with cDNA 100% homologous to GPC3 has abnormal expression in liver cancer tissues for the first time. Of 191 hepatocellular carcinoma (HCC) tissues, 143 (74.8%) were able to detect MXR7 mRNA, while of 156 paracancerous "non-neoplastic" liver tissues, only 5 (3.2%) were positive, these 5 cases all having portal or distant metastases. Subsequently, several laboratories have confirmed this result in succession. In HCC tissue, the positive detection rate of GPC3mRNA and protein is more than 73%, the average level of mRNA can be 21.7 times higher than that of normal liver tissue, and the mRNA is negative in normal liver, liver benign tumor, chronic hepatitis, liver cirrhosis and the like, which shows that the protein expression has higher tumor cell specificity, and is a novel liver cancer marker.
However, because the content of GPC3 in normal human peripheral blood is weak, the content of GPC3 in the serum of a tumor patient is still at a low level although the content is obviously increased, and the establishment of a detection system with high sensitivity is very difficult, so that the tumor marker cannot be clinically used so far.
Therefore, there is an urgent need in the art to develop a detection system and a detection method capable of sensitively detecting the GPC3 level, which provides a good approach for clinical tumor diagnosis.
Disclosure of Invention
The invention aims to provide a detection reagent which can sensitively and accurately detect the content of heparin sulfate proteoglycan 3 in a sample.
The invention also aims to provide a detection kit for detecting the content of the heparin sulfate proteoglycan 3 in the sample.
In a first aspect of the present invention, there is provided a test strip for detecting heparin sulfate proteoglycan 3, said test strip comprising:
a solid support; and
a polyclonal antibody against heparin sulfate proteoglycan 3 coated on the solid phase carrier, wherein the polyclonal antibody specifically binds to the epitope in 379-393 of the heparin sulfate proteoglycan 3 but not to the epitope in 1-378 or 394-580 of the heparin sulfate proteoglycan 3.
In another preferred embodiment, the solid phase carrier is an enzyme-labeled reaction plate.
In a second aspect of the present invention, there is provided a detection kit comprising said test strip, or
The kit comprises: (a) a solid support;
(b) a container a and a polyclonal antibody against heparin sulfate proteoglycan 3 as a first antibody in the container a, wherein the polyclonal antibody specifically binds to the epitope in 379-393 of the heparin sulfate proteoglycan 3 but not to the epitope in 1-378 or 394-580 of the heparin sulfate proteoglycan 3; and
(c) a reagent for coating said first antibody on said solid support.
In another preferred embodiment, the kit is further loaded with:
a container b, wherein the container b contains a second antibody, and the second antibody is a polyclonal antibody, and the polyclonal antibody specifically binds to the epitope except for the 379-position 393 in the heparin sulfate proteoglycan 3.
In another preferred embodiment, the kit is further loaded with:
and a container b ', wherein the container b' is filled with a second antibody, and the second antibody is a monoclonal antibody, and the monoclonal antibody specifically binds to the epitope in the heparin sulfate proteoglycan 3 except the 379-393 position.
In another preferred embodiment, the monoclonal antibody binds to the following epitope of heparin sulfate proteoglycan 3:
an epitope in positions 25-358 of heparin sulfate proteoglycan 3;
an epitope in position 350-364 of heparin sulfate proteoglycan 3; or
An epitope in position 444-516 of heparin sulfate proteoglycan 3.
More preferably, the monoclonal antibody binds to an epitope in positions 25-358 of heparin sulfate proteoglycan 3.
In another preferred embodiment, the second antibody is detectably labeled.
In another preferred embodiment, the detectable label is selected from the group consisting of: horseradish peroxidase (HRP), alkaline phosphatase.
More preferably, the label is horseradish peroxidase.
In another preferred embodiment, the kit is further loaded with:
a container c containing a substrate corresponding to the label;
the container d is filled with a color developing agent;
a container e, wherein the container e is filled with washing liquid; and/or
And a container f, wherein the container f is filled with stop solution.
In a third aspect of the present invention, there is provided a method for in vitro detection of heparin sulfate proteoglycan 3, comprising the steps of:
(a) adding a sample to be detected to a solid phase carrier coated with a first antibody, so that the heparin sulfate proteoglycan 3 in the sample to be detected is combined with the first antibody on the solid phase carrier to form the solid phase carrier with a binary compound of the heparin sulfate proteoglycan 3-the first antibody; the first antibody is a polyclonal antibody which specifically binds to the epitope in 379-393 position of the heparin sulfate proteoglycan 3, but does not bind to the epitope in 1-378 position or 394-580 position of the heparin sulfate proteoglycan 3;
(b) applying a second antibody to the solid phase carrier obtained in (a), thereby forming a solid phase carrier with a ternary complex of "second antibody-heparin sulfate proteoglycan 3-first antibody"; the second antibody is a monoclonal antibody or a polyclonal antibody which is specifically bound to an epitope other than the 379-position 393 of heparin sulfate proteoglycan 3, and the second antibody carries a detectable marker; and
(c) detecting the detectable label in the ternary complex, thereby determining the presence or absence and amount of heparin sulfate proteoglycan 3 in the sample to be detected.
In the fourth aspect of the present invention, there is provided a polyclonal antibody, which specifically binds to the epitope in positions 379-393 of heparin sulfate proteoglycan 3 but does not bind to the epitope in positions 1-378 or 394-580 of heparin sulfate proteoglycan 3, and which is obtained by immunizing an animal with the protein fragment in positions 379-393 of heparin sulfate proteoglycan 3.
In another preferred embodiment, the animal is: rabbits.
In another aspect, the present invention also provides the use of the polyclonal antibody as described above, for the preparation of a kit for the detection of heparin sulfate proteoglycan 3.
Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.
Drawings
FIG. 1 shows the coding gene of 25-358aa amino acid fragment of GPC3 protein.
FIG. 2 shows the amino acid sequence of GPC3 protein 25-358.
FIG. 3 shows the small-scale expression identification of the pProEX Hta-GPC3(73-1074) plasmid.
FIG. 4 shows the SDS-PAGE gel electrophoretic identification of GPC3(25-358aa) protein after Ni column purification.
FIG. 5 shows Western Blot detection of purified GPC3(25-358aa) protein. Wherein M represents a molecular weight marker; w-18 represents the result of detection using a polyclonal antibody W-18 (directed against protein N-terminal protein epitopes); anti-His indicates the results of detection using anti-His antibody.
FIG. 6 shows the Western Blot results of cell lysates of placenta and tumor cells detected using murine anti-GPC 3(25-358aa) monoclonal antibody gp2 #. Wherein, 1, placenta tissue, 2, L02 cell, 3, MCF-7 cell and 4, Hela cell.
FIG. 7 shows the results of WesternBlot in cell lysates for detection of tumor cells using rabbit anti-GPC 3-Ag3 polyclonal antibody. Wherein, 1.Huh-7 cell; HepG2 cells; MCF-7 cells; hela cells.
Figure 8 shows the GPC3 quantitative standard curve.
FIG. 9 shows the serum GPC3 levels of normal persons, hepatitis liver cirrhosis patients, and liver cancer patients.
Figure 10 shows the GPC3 subject operating characteristic curve (ROC curve).
Detailed Description
The present inventors have made extensive studies and experiments, and have unexpectedly found that a polyclonal antibody corresponding to the epitope at positions 379-393 of the amino acid sequence of heparin sulfate proteoglycan 3(GPC3) can excellently recognize the GPC3 antigen; when GPC3 is captured using this polyclonal antibody as the primary antibody, a secondary antibody (including a monoclonal antibody or a polyclonal antibody) which corresponds to an epitope in a region other than the 379-393 amino acids of GPC3 can bind extremely efficiently to GPC3, thereby allowing GPC3 antigen to be detected with high sensitivity by a double antibody sandwich method. In contrast, when the polyclonal antibody bound to other sections (e.g., 444-516) of GPC3 was used as the first antibody, the detection sensitivity was low.
In addition, the inventors have also surprisingly found that when a polyclonal antibody specifically binding to epitope 379-393 of GPC3 is used as the first antibody, the binding detection of the GPC3 liver cancer marker with the AFP liver cancer marker has higher complementarity diagnostic value. Compared with the method for detecting the AFP marker alone, the kit for detecting the GPC3 marker can obviously improve the diagnosis sensitivity of liver cancer while detecting the AFP marker. The present invention has been completed based on this finding.
It is understood by those skilled in the art that an antigen may contain multiple epitopes (epitopes) and thus, more than one antibody (including monoclonal or polyclonal) may be obtained for the same antigen, and the binding properties (e.g., specificity, etc.) of these antibodies to the antigen may vary. Thus, the skilled person needs to perform repeated comparisons, screens and identifications for the same antigen in order to find antibodies suitable for specific and sensitive binding. Since the GPC3 protein is a long polypeptide, most of its epitopes are contained within the spatial structure, and it is therefore more difficult to find an antibody suitable for binding to it.
In view of the above technical problems, the present inventors prepared various monoclonal antibodies and polyclonal antibodies corresponding to GPC3, and examined the binding specificity and sensitivity of various monoclonal antibodies or polyclonal antibodies to GPC3 through a number of experiments. As a result, it was found that the polyclonal antibody corresponding to the epitope at 379-393 of the amino acid sequence of GPC3 recognized the GPC3 antigen particularly well.
Furthermore, the inventors prepared two-by-two combinations of different monoclonal antibodies and/or different polyclonal antibodies (based on the principle of double antibody sandwich method) to test the binding specificity and sensitivity of the combination of the monoclonal antibodies and the polyclonal antibodies to GPC 3. Finally, the following findings are found: the GPC3 antigen can be detected particularly sensitively by using, as a detection reagent based on the double antibody sandwich method, a polyclonal antibody (as a first antibody) that binds to an epitope in the amino acid sequence 379-393 of GPC3, and a monoclonal antibody or a polyclonal antibody (as a second antibody) that binds to an epitope other than the amino acid sequence 379-393 of GPC 3. When the polyclonal antibody which is combined with the epitope other than 379-393 is compatible with other monoclonal antibodies or polyclonal antibodies, the sensitivity and the specificity are not high; when the monoclonal antibody is used in combination with a monoclonal antibody, the sensitivity is high, but the specificity is poor.
As used herein, the term "sample" refers to a substance isolated from the human body, including but not limited to: serum, plasma, urine, or other tissue extracts. Preferably, the sample is serum or plasma.
As used herein, the terms "capture antibody", "coating antibody", "first antibody" and "primary antibody" are used interchangeably and refer to the polyclonal antibody that binds to the epitope in amino acid sequence 379-393 of GPC 3.
The first antibody may be coated on a solid support. The solid phase carrier to be used in the present invention is not particularly limited as long as it can be coupled (linked) with the first monoclonal antibody. For example, the solid phase carrier is an enzyme-labeled reaction plate.
As used herein, the terms "detection antibody", "second antibody" and "secondary antibody" are used interchangeably and refer to an antibody that specifically binds to an epitope other than positions 379-393 of GPC3, and can be either a monoclonal antibody or a polyclonal antibody.
Heparin sulfate proteoglycan 3(Glypican-3, GPC3) is located on the surface of cell membrane, is composed of core protein and glycosaminoglycan (heparin and heparan sulfate) side chains, is a multifunctional co-receptor on the cell surface, transmits signals by the interaction with extracellular matrix, growth factors, protease, and the like, and has important functions in the growth, development, differentiation, migration, and the like of cells. The presence of GPC3 in vivo has significant tissue and phase specificity: there is abundant expression of GPC3 in human embryonic, gastrointestinal and mesoderm derived tissues; however, in adults, only a few tissues other than placenta tissue, such as lung, kidney, heart, and ovary, have low expression of GPC3, and the liver is negative. The inventor finds that the average concentration of GPC3 in serum of hepatocellular carcinoma (HCC) patients is obviously higher than that of normal control and liver cirrhosis patients in research. In HCC cases with better differentiation, GPC3 is more sensitive than AFP detection, and the combined detection of the GPC3 and the AFP can greatly improve the positive rate of liver cancer diagnosis.
The amino acid sequence and nucleotide sequence of the GPC3 protein are known in the art, and the amino acid sequence is set forth in SEQ ID NO: 7, and the nucleotide sequence is shown as SEQ ID NO: and 6. Alternatively, one skilled in the art can also see amino acid sequences and nucleotide sequences provided in GenBank accession nos. NP _004475 and NM _ 004484.2.
Antibodies
The present inventors provide a polyclonal antibody that can excellently recognize the GPC3 antigen, and the antibody specifically binds to an epitope in 379-393 position of heparin sulfate proteoglycan 3.
The polyclonal antibody is obtained by immunizing an animal with the 379-position 393 protein fragment of GPC 3. The preparation of polyclonal antibodies is well known to those skilled in the art. Adjuvants can be used to enhance the immune response, such as Freund's adjuvant. For example, the polyclonal antibody can be prepared as follows: the protein fragment at 379-393 of GPC3 is mixed with Freund's adjuvant at a proper ratio (such as 1:1), and the mixture is emulsified sufficiently to immunize animals. The immunization method may use subcutaneous injection. After 1-4 months of animal immunization, antiserum can be harvested from animal venous blood and purified to obtain polyclonal antibodies against the 379-position 393 protein fragment of GPC 3.
In a preferred embodiment of the present invention, the animal is: rabbits.
In the examples of the present invention, the present inventors prepared a variety of polyclonal antibodies (including rabbit anti-human GPC3-Ag3 polyclonal antibody (binding to epitope in 379-393 of GPC3), rabbit anti-human GPC3-Ag1 (binding to epitope in 73-86 of GPC3), and rabbit anti-human GPC3-Ag4 polyclonal antibody (binding to epitope in 444-516 of GPC 3)), and found that in the subsequent ELISA detection, the rabbit anti-human GPC3-Ag3 polyclonal antibody was compatible with other various monoclonal or polyclonal antibodies that do not bind to epitope in 379-393 of GPC3, and could detect the content of GPC3 antigen with particular sensitivity.
Techniques for preparing monoclonal antibodies specific against the GPC3 protein are well known in the art. Herein, "specificity" means that the antibody is capable of binding to the GPC3 protein or a fragment thereof; more particularly, antibodies that bind to GPC3 protein or fragments thereof, but do not recognize and bind to other unrelated antigenic molecules.
The monoclonal antibody is an antibody which specifically binds to an epitope other than 379-393 of GPC 3. It is to be understood that when used as a second antibody for the detection of the GPC3 antigen in combination with the polyclonal antibody that binds to an epitope in positions 379-393 of GPC3 (as a first antibody) (based on the principle of the double antibody sandwich method), the monoclonal antibody may be varied so long as it is capable of specifically binding to an epitope other than positions 379-393 of GPC 3.
Preferably, the epitope other than positions 379-393 of GPC3 is selected from the group consisting of:
epitope corresponding to 25 th to 358 th positions of GPC 3;
GPC3 epitope corresponding to position 350-364; or
Epitope corresponding to position 444-516 of GPC 3.
The monoclonal antibodies of the invention can be prepared using hybridoma technology. The use of hybridoma technology for the preparation of monoclonal antibodies is well known to those skilled in the art and can be found, for example, in Kohler et al, Nature 256; 495, 1975; kohler et al, Eur.J.Immunol.6: 511, 1976; kohler et al, Eur.J.Immunol.6: 292, 1976; hammerling et al, In Monoclonal Antibodies and T Cell hybrids, Elsevier, N.Y., 1981.
The epitope position of GPC3 antigen corresponding to the monoclonal antibody is known, and the monoclonal antibody can be obtained by known methods. For example, when it is desired to prepare a monoclonal antibody against an epitope corresponding to positions 25 to 358 of GPC3, a hybridoma is prepared by immunizing a mouse with a fragment of the 25 th to 358 th protein of GPC3, thereby obtaining the desired antibody.
Detection kit
The inventor prepares a kit which can be used for detecting the GPC3 protein level in a sample according to the principle of a double antibody sandwich method. The double antibody sandwich method is conventionally performed by immobilizing a primary antibody on a carrier, reacting the primary antibody with an antigen, washing, reacting with a secondary antibody (the secondary antibody carries a detectable label or can be combined with a substance carrying a detectable label), and finally performing chemiluminescence or enzyme-linked color reaction to detect a signal. The double antibody sandwich method is particularly suitable for the detection of antigens having two or more epitopes.
The inventor finds that the target antigen GPC3 is adsorbed and positioned by using a polyclonal antibody which is combined with the epitope in 379-393 positions of GPC3 and an antibody which is specifically combined with the epitope other than the 379-393 positions of GPC3, and the positioning and amplification effects are particularly good, so that the target antigen GPC3 has high specificity and precision. In addition, compared with a single-antibody competitive method, the double-antibody sandwich method has better measuring effect, so that a small amount of sample is required for measurement. Therefore, the double antibody sandwich method has more advantages in sensitivity, accuracy, specificity and stability.
The detection kit of the invention contains a test strip, and the test strip comprises: a solid support; and a polyclonal antibody against heparin sulfate proteoglycan 3 coated on the solid phase carrier, wherein the polyclonal antibody specifically binds to the epitope in 379-393 of the heparin sulfate proteoglycan 3 but not to the epitope in 1-378 or 394-580 of the heparin sulfate proteoglycan 3.
Alternatively, the detection kit of the present invention comprises (a) a solid phase carrier; (b) a container a and a polyclonal antibody against heparin sulfate proteoglycan 3 as a first antibody in the container a, wherein the polyclonal antibody specifically binds to the epitope in 379-393 of the heparin sulfate proteoglycan 3 but not to the epitope in 1-378 or 394-580 of the heparin sulfate proteoglycan 3; and (c) a reagent for coating said first antibody on said solid support.
In a preferred embodiment of the present invention, the kit further comprises:
a container b, wherein the container b contains a second antibody, the second antibody is a polyclonal antibody, and the polyclonal antibody specifically binds to the epitope except for the 379-position 393 in the heparin sulfate proteoglycan 3; or,
and a container b ', wherein the container b' is filled with a second antibody, and the second antibody is a monoclonal antibody, and the monoclonal antibody specifically binds to the epitope in the heparin sulfate proteoglycan 3 except the 379-393 position.
In a preferred embodiment of the present invention, the second antibody is labeled with a detectable label.
As used herein, the term "detectable label" refers to a marker used to determine the presence or absence and amount of GPC3 in a sample to be tested. After the coating antibody and/or the detection antibody used in the kit of the present invention is determined, various labels conventionally used in the art for binding to the detection antibody for detection may be used. The label used in the present invention is not particularly limited, and any label that can bind to the detection antibody and accurately indicate the presence or absence and the amount of GPC3 protein in the sample to be detected after appropriate treatment is usable. The label may be provided directly on the second antibody; alternatively, the label may be provided on an anti-antibody specific to the second antibody, and a person skilled in the art may select an appropriate label depending on the kind and characteristics of the antibody used. For example, the marker may be selected from: horseradish peroxidase (HRP), Alkaline Phosphatase (AP), glucose oxidase, beta-D-galactosidase, urease, catalase, or glucoamylase.
When some enzyme labels as shown above are used, it is also necessary to use some substrate which binds to the corresponding enzyme, so that the presence or amount of the label can be reported by color development or the like. As used herein, the term "substrate corresponding to a label" means a substrate that can be developed by the label to show the recognition signal of the second antibody bound to GPC 3. The substrate is, for example: o-phenylenediamine (OPD), Tetramethylbenzidine (TMB), ABTS for horseradish peroxidase; p-nitrophenylphosphate (p-NPP) for alkaline phosphatase; and so on. The skilled person will be able to select a suitable substrate depending on the kind and nature of the label employed.
In a preferred embodiment of the present invention, the second antibody is directly linked to a label. More preferably, the label is HRP. Compared with the method that the detection antibody is marked by biotin and reacts with streptavidin-HRP, the method that the HRP is directly marked on the detection antibody and the substrate is directly added for color development after the reaction is finished is simpler and more convenient.
In order to obtain quantitative results, a standard containing a plurality of GPC3 proteins at known concentrations may also be set up during the detection. The setting method for the standard may be a conventional method.
In order to eliminate false positive and false negative, quality control (control) can also be set in the detection process.
In addition, in order to facilitate the detection of the kit of the present invention, the kit preferably further comprises other auxiliary reagents, which are conventionally used in ELISA kits, and the characteristics of these reagents and their preparation methods are well known to those skilled in the art. Such agents are for example (but not limited to): color developing agent, washing liquid, stopping liquid, sensitizing liquid and diluting liquid.
In addition, instructions for use may be included in the kit to instruct the method of use of the reagents loaded therein.
The kit of the invention can be applied to:
(A) as an independent detection system, diagnosing liver cancer;
(B) diagnosing liver cancer by combining with imaging examination and/or AFP marker detection;
(C) carrying out differential diagnosis of benign and malignant of AFP (alpha-fetoprotein) heightening liver diseases;
(D) performing prognostic monitoring of a GPC3 positive liver cancer prior to treatment;
(E) screening high risk group of liver cancer; and/or
(F) Basic research related to liver cancer is carried out.
Detection method
The invention provides a method for detecting GPC3 protein in vitro by using the kit, which comprises the following steps:
(a) adding a sample to be detected to a solid phase carrier coated with a first antibody, so that the heparin sulfate proteoglycan 3 in the sample to be detected is combined with the first antibody on the solid phase carrier to form the solid phase carrier with a binary compound of the heparin sulfate proteoglycan 3-the first antibody; the first antibody is a polyclonal antibody which specifically binds to the epitope in 379-393 position of the heparin sulfate proteoglycan 3, but does not bind to the epitope in 1-378 position or 394-580 position of the heparin sulfate proteoglycan 3;
(b) applying a second antibody to the solid phase carrier obtained in (a), thereby forming a solid phase carrier with a ternary complex of "second antibody-heparin sulfate proteoglycan 3-first antibody"; the second antibody is a monoclonal antibody or a polyclonal antibody which is combined with the epitope other than 379-393 of heparin sulfate proteoglycan 3, and the second antibody carries a detectable marker; and
(c) detecting the detectable label in the ternary complex, thereby determining the presence or absence and amount of heparin sulfate proteoglycan 3 in the sample to be detected.
In a preferred embodiment of the present invention, the method for quantitatively detecting the GPC3 protein is specifically as follows:
(i) antigen-antibody reaction: coating the first antibody on a porous plate, and then respectively adding standard substances, quality control substances (optional) or serum samples to be detected with different concentrations into micropores of the porous plate;
(ii) enzyme-linked reaction: adding a solution of a second antibody (provided with a marker) to each well, oscillating, and incubating; washing;
(iii) and (3) color development reaction: adding a substrate and a color developing agent corresponding to the marker into each hole, incubating, adding a reaction stop solution into each hole, and finishing the reaction;
(iv) measuring an OD value by using an enzyme-labeling instrument;
(v) and (4) calculating a result:
A) making a standard curve: taking the concentration of a GPC3 protein standard substance as an abscissa and the measured OD value of the standard substance as an ordinate, and making a standard curve;
B) judging the concentration of the quality control product (optional): reading out a corresponding concentration value from the standard curve according to the OD value of the quality control product; when the concentration value of the quality control substance is in a given range, the measurement is effective;
C) calculating the concentration of the serum sample to be detected: and when the standard curve and the quality control product are judged to be effective, calculating the GPC3 protein concentration of the serum sample to be detected from the standard curve according to the OD value of the sample to be detected.
Because the detection reagent or the kit has high sensitivity and greatly shortens the sample incubation time (about 2 +/-0.5 hours), the quantitative data of GPC3 can be obtained by using the kit only in a short time, and the time is generally 2 to 4 hours. Where conventional reagents or kits are used for detection, the sample is typically incubated overnight to increase sensitivity.
The main advantages of the invention are:
(1) the polyclonal antibody corresponding to the epitope at 379-393 of the amino acid sequence of GPC3 is found to be capable of well recognizing the GPC3 antigen for the first time, and the GPC3 antigen can be particularly sensitively detected by combining the polyclonal antibody with the antibody corresponding to the epitope at 379-393 of the amino acid sequence of GPC3 as a detection reagent based on the double-antibody sandwich method.
(2) Provides a liver cancer detection kit which is convenient to use and high in sensitivity, can sensitively detect when the content of GPC3 in a sample is low (1ng level), and has short time (about 2-4 hours) for completing the detection. The kit is helpful for diagnosing liver cancer patients as early as possible, treating the liver cancer patients in time, improving survival rate and having positive social benefits. Has wide application value in the aspects of liver cancer curative effect follow-up, subclinical metastasis and relapse monitoring, prognosis judgment and the like.
(3) The inventor also finds that the GPC3 liver cancer marker and the AFP liver cancer marker have complementary diagnostic value. GPC3 and AFP are combined for use, which can not only improve the positive rate of early diagnosis of liver cancer, but also can perform differential diagnosis of benign and malignant lesions of a large number of patients with low or moderate increase of AFP clinically. Compared with the method for detecting the AFP marker alone, the kit for detecting the GPC3 marker can obviously improve the diagnosis sensitivity of liver cancer while detecting the AFP marker.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
Example 1 prokaryotic preparation of polypeptide antigens GPC3(25-358), GPC3(444-516)
A polypeptide antigen GPC3(25-358) having the full length amino acid sequence of GPC3 as bit sequence 25-358 was prepared as follows:
(1) extraction of human placental total RNA
Approximately 1g of human placenta tissue was taken, 1ml of TRAZOL reagent was added to a 1.5ml centrifuge tube, the tissue was broken with a glass homogenizer, and RNA was extracted.
(2) Reverse transcription
Mu.g of total RNA was collected and subjected to reverse transcription with the aid of an Amersham RT-PCR System (GIBCO BRL) kit, oligo (dT)20 as a primer and AMV reverse transcriptase, to synthesize cDNA, in accordance with the instructions of the reverse transcription kit.
(3) Construction of expression vectors
The following primers were designed:
GPC3-73(SEQ ID NO：3)：
5 'cgcgaattcCAGCCCCCGCCGCCGCCGCC 3' and
GPC3-1074(SEQ ID NO：4)：
5’gcgctcgagTTATCTATATTGGCGTTGTTGAGAATGGGCACATAAC3’
and (3) amplifying the N-terminal coding sequence (the part of the 24aa signal peptide is removed) nt.73-1074 of the GPC-3 protein by using newly synthesized human placenta cDNA as a template and the designed primer as a primer. The amplification system is 10 mu L; the amplification conditions were: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; further extension was carried out at 72 ℃ for 10 min. The PCR product was identified by 10g/L agarose gel electrophoresis and recovered by a PCR fragment recovery kit.
The PCR product was cloned into the pProEX HTa vector (purchased from Invitrogen, USA) at EcoRI and Xho I sites to obtain plasmid pProEX Hta-GPC3(73-1074) expressing GPC-3 protein 25-358aa, sequencing identified point mutations in the gene sequence that did not affect amino acid changes (FIG. 1, SEQ ID NO: 1), and the deduced amino acid sequence was shown in FIG. 2(SEQ ID NO: 2).
(4) Expression and purification of pProEX Hta-GPC3(73-1074)
The recombinant plasmid pProEX Hta-GPC3(73-1074) was transformed into competent E.coli strain BL21-codonplus (DE3) -RP (from Stratagene), plated on ampicillin-resistant agar plates, grown overnight at 37 ℃ in an inverted state, 3 clones were picked with toothpicks on ampicillin-containing agar plates in 3ml of LB containing ampicillin, and shaken at 37 ℃ overnight. The overnight cultured bacteria were grown as described in 1:100 portions were transferred to 3ml of LB containing ampicillin and cultured with shaking at 37 ℃ for 3 hours to mid-log phase (A)5500.5). Adding isopropyl thio-beta-d-galactoside (IPTG) to a final concentration of 1mmol/L, continuing shaking culture for 3h, and inducing GPC3 protein expression. Taking each induced bacterium and non-induced control bacterium, the amount is equivalent to A550Mu.l of the suspension was suspended in 10. mu.l of 1 XSDS loading buffer after precipitation on a 1-day basis and boiled in a boiling water bath for 5 min. Stored at-20 ℃ or immediately subjected to SDS-PAGE gel electrophoresis. SDS-PAGE electrophoresis identified protein expression. As a result, the induced bacteria had a specific protein expression band (GPC3) at 40KD, as shown in FIG. 3.
According to the small sample induction conditions, 200ml of pProEXHta-GPC3(73-1074) -codonplus expression protein was induced and purified with Ni column, followed by SDS-PAGE gel electrophoresis identification. As a result, the major band of protein of about 40KD is found to account for more than 90% of the total protein, as shown in FIG. 4.
pProEXHta-GPC3(73-1074) expressed protein was localized in inclusion bodies and purified according to the purification procedure for insoluble protein:
(a) the column was washed with GuNTA-0Buffer (20mmol/L Tris-HCl pH7.9, 0.5mol/L NaCl, 8mol/L Urea) in 10 TNA volumes.
The sample is applied to a TNA chromatographic column, the flow rate is controlled at about 15ml/h, and the penetrating fraction is collected and used for SDS-PAGE analysis of protein binding.
The chromatography was performed with a 5-fold TNA volume of GuNTA-0Buffer, and the flow rate was controlled to be about 30 ml/h.
(b) Elution was carried out with 5-fold TNA volume of GuTNA-209(20mmol/L Tris-HCl pH7.9, 0.5mol/L NaCl, 8mol/L Urea, 20mmol/L), GuTNA-40(20mmol/L Tris-HCl pH7.9, 0.5mol/L NaCl, 8mol/L Urea, 40mmol/L Imidazole), GuTNA-60(20mmol/L LTris-HCl pH7.9, 0.5mol/L NaCl, 8mol/L Urea, 60mmol/L Imidazole), GuTNA-100(20mmol/L Tris-HCl pH7.9, 0.5mol/L NaCl, 8mol/L Urea, 100mmol/L idazole), GuTNA-500(20mmol/L Tris-HCl pH7.9, 0.5mol/L NaCl, 8mol/L Urea, 500mmol/L Imidazole), 15ml flow rate control, the eluate was collected, one TNA volume per tube.
The purified product was subjected to a conventional Western Blot assay using anti-His antibody and anti-GPC 3 polyclonal antibody W-18 (against the N-terminal protein epitope of the protein) from Santa Cruz, and the expressed product was identified as GPC3 protein, as shown in FIG. 5.
The present inventors also prepared the polypeptide antigen GPC3(444-516) at position 444-516 of the full-length amino acid sequence of GPC3, named GPC3-Ag4, using a prokaryotic expression method similar to that described previously for the preparation of the polypeptide antigen GPC3 (25-358).
Example 2
Synthesis and coupling of polypeptide antigen GPC3(379-393), GPC3(73-86) and GPC3(350-364)
The antigenicity of the full-length GPC3 protein is analyzed by using software DNAStar, and the polypeptide antigen GPC3(379-393) at the amino acid position ETLSSRRRELIQKLK (SEQ ID NO: 5) of the full-length amino acid sequence of GPC3 is selected mainly according to two parameters of the antigenicity and accessibility of the protein and is named as GPC3-Ag 3. The polypeptide is prepared by adopting an artificial synthesis method, synthesized by GPC3-Ag3 and coupled with KLH (keyhole limpet hemocyanin) by adopting a glutaraldehyde method to obtain GPC3-Ag 3-KLH.
In addition, the inventor synthesizes the 73 th to 86 th polypeptide antigen GPC3(73-86) selected from the full-length amino acid sequence of GPC3, and the name is GPC3-Ag 1; the polypeptide antigen GPC3(350-364) selected from the 350-364 th position of the full-length amino acid sequence of GPC3 is named as GPC3-Ag 2. Also, GPC3(73-86) was coupled with KLH to give GPC3-Ag 1-KLH.
EXAMPLE 3 preparation of polyclonal antibodies
a. Preparation of rabbit anti-human GPC3-Ag3 polyclonal antibody
Antigen: GPC3-Ag 3-KLH.
Animals: new Zealand white rabbits, one male, one female, 4-6 weeks, 2.5-3.0kg, were purchased from Shanghai laboratory animal center of Chinese academy of sciences.
(1) One male and female healthy new zealand white rabbit were immunized simultaneously. Collecting 1-2ml blood via rabbit marginal vein before immunization, separating serum, packaging, and storing at-80 deg.C. At the time of primary immunization, 1mg of antigen was injected per rabbit. Freund's complete adjuvant was mixed with 1ml of antigen (1mg) in equal volume, emulsified well and injected via multiple injection into the dorsal intradermal tissue.
(2) The subsequent booster immunization is performed once every 2 weeks, two times in total, the antigen amount is unchanged, Freund incomplete adjuvant is used, and the injection method is unchanged. On day 7 after the second boost, the titer of the antibody in the rabbit serum was determined by ELISA. And (3) after the serum titer meets the requirement, anesthetizing the rabbit with ether, performing carotid intubation, taking out blood of the New Zealand white rabbit, standing at room temperature for 30min, standing in a refrigerator at 4 ℃ overnight, sucking the separated rabbit serum, centrifuging the residual blood at 4 ℃ at 4000rpm for 10min, and sucking the upper serum to obtain the rabbit anti-human GPC3-Ag3 polyclonal antibody.
The serum titers of rabbits 5 weeks after immunization with GPC3-Ag3-KLH are shown in Table 1.
TABLE 1
Western Blot detection was performed on cell lysates using purified rabbit serum. As a result, the cell lysates of liver cancer cells Huh-7(ATCC) and HepG2(ATCC) are used as antigens, and the rabbit serum is used as an antibody, so that a specific band is formed at the molecular weight of 60KD (full-length GPC 3); and the lysates of MCF-7 (human breast cancer cell, ATCC) and Hela (human cervical cancer cell, ATCC) were used as antigens, and the rabbit serum was used as an antibody, and the result was negative, as shown in FIG. 7. Therefore, the rabbit anti-human GPC3-Ag3 polyclonal antibody has higher specificity. Further experiments also showed that the polyclonal antibody does not bind to the epitope located in positions 1-378 or 394-580 of GPC 3.
b. Preparation of rabbit anti-human GPC3-Ag1 and rabbit anti-human GPC3-Ag4 polyclonal antibodies
The antigens used were GPC3-Ag1-KLH and GPC3-Ag4, and the animals used were the same as those used in a above.
According to the same method as that of a, rabbit anti-human GPC3-Ag1 polyclonal antibody and rabbit anti-human GPC3-Ag4 polyclonal antibody were prepared. The rabbit serum titers after 7 weeks of GPC3-Ag1-KLH immunization are shown in Table 2; the serum titers of rabbits 5 weeks after GPC3-Ag4 immunization are shown in Table 3.
TABLE 2
TABLE 3
EXAMPLE 4 preparation and purification of anti-GPC 3 monoclonal antibody
a. Preparation and purification of mouse anti-GPC 3(25-358) monoclonal antibody
Taking 6-8 weeks old BALB/c mice, mixing purified GPC3(25-358) protein for primary immunization with equal volume of Freund's complete adjuvant, performing subcutaneous immunization on the back and inguinal multipoint, performing subcutaneous immunization every 2 weeks later, mixing the same dose of antigen and Freund's incomplete adjuvant uniformly, and performing subcutaneous immunization on the back multipoint. After 7 days from the 3 rd immunization, blood was collected from the rat tail vein and the production of specific antibodies was determined by indirect ELISA. 3 days before fusion, the same dose of antigen without adjuvant was used to boost the peritoneal cavity 1 time.
Three days after the boost, the mice were sacrificed by cervical dislocation, the spleen was removed under aseptic conditions, about 0.2-0.5ml of serum-free culture medium was injected with a syringe to swell it, the spleen membrane was punctured at multiple points with a curved injection needle, lymphocytes were escaped therefrom by squeezing, immune spleen cell suspensions were prepared and counted, and washed 2 times with incomplete culture medium. Taking myeloma cells with logarithmic growth, centrifuging at SP2/0,1000rpm/min for 5min, discarding the supernatant, suspending the cells with incomplete culture solution, counting, taking the required number of cells, and washing with incomplete culture solution for 2 times. Myeloma cells and spleen cells were mixed together at a ratio of 1:10, washed 1 time with incomplete culture medium in a 50ml plastic centrifuge tube, and centrifuged at 1200rpm/min for 8 min. Discard the supernatant, and use the dropper to suck up the residual liquid, so as to avoid affecting the concentration of PEG. Lightly flick the bottom of the tube to loosen the cell pellet. Fusion at room temperature: adding preheated 1ml 45% PEG1000 containing 5% DMSO into 30s while stirring; action for 90 s; ③ adding the preheated incomplete culture solution to terminate the PEG action, and adding 1ml, 2ml, 3ml, 4ml, 5ml and 10ml respectively every 2 min. Centrifugation at 800rpm/min for 6 min. The supernatant was discarded and first suspended gently with about 6ml of 20% calf serum RPMI1640 without blowing hard to disperse the fused cells. Complete medium was added to 10ml of a 96-well plate depending on the number of 96-well plates used. Adding the fused cell suspension to a medium containing feeder cells96 well plates, 100. mu.l/well, 37 ℃ C, 5% CO2And (5) incubator culture. Typically, a 96-well plate contains 1X 107Spleen cells. HAT selection medium was added 24h after fusion. After two weeks of HAT selection medium maintenance culture, HT medium was used, and then two weeks of maintenance culture were carried out, using general medium. Detection of specific antibodies can begin when hybridoma cells have spread across the area of the bottom 1/10 of the well. Diluting GPC3 protein obtained by genetic engineering to 5 μ g/ml with coating solution, coating the ELISA plate with 100 μ l per well, standing at 4 deg.C overnight, discarding the supernatant, washing the coated ELISA plate with washing solution 3 times for 5min each time, adding 200 μ l blocking solution (3% BSA PBST) per well, standing at 37 deg.C for 2h, decanting the blocking solution, washing as above, adding 100 μ l supernatant of culture solution per well, incubating at 37 deg.C for 1.5h, discarding the supernatant, adding 1: incubating 100 μ l of goat anti-mouse IgG-HRP diluted at 1000 deg.C for 1H at 37 deg.C, discarding supernatant in wells, washing the same, and adding 100 μ l of developing solution (OPD-H) to each well2O2) After the reaction is finished by adding 50 mul of stop solution into each hole after the reaction is performed for 10-20min in a dark color at room temperature, and reading A by a microplate reader490And (5) nm value. Cloning the positive clone by a limiting dilution method, and screening out the required hybridoma cell line after three times of cloning. As a result, 11 positive clones were obtained and named gp1# -gp11 #.
Intraperitoneal injection of liquid paraffin F1Mice were replaced by intraperitoneal injection of 1X 10 after 2 weeks5Generating ascites 7-10 days after inoculating the hybridoma cells, closely observing the health condition and ascites signs of the animals, killing the mice before the mice are endangered as much as possible and sucking the ascites into the test tube by using a dropper.
Diluting ascites or immunized rabbit serum with PBS (0.01mol/L, pH7.4, containing 0.15mol/L NaCl), adding saturated ammonium sulfate (90g ammonium sulfate is added into 100ml distilled water, dissolving at 80 deg.C, filtering while hot, cooling to room temperature to precipitate crystals, adjusting pH to 7.0 with sulfuric acid), standing at 4 deg.C for 4 hr, centrifuging at 10000rpm/min for 15min, discarding supernatant, dissolving precipitate with 1-5ml PBS, transferring into dialysis bag, dialyzing in PBS (0.01mol/L, pH7.4, containing 0.15mol/L NaCl) at 4 deg.C overnight, transferring Protein solution in dialysis bag into Protein A Sepharose CL-4B column, washing the loaded Protein A column with Tris-HCl (0.05mol/L, pH8.0, containing 0.15mol/L NaCl) for several times, and eluting Protein again on ultraviolet chromatograph, then, the antibody was eluted with a dissociation solution (0.01mol/L, glycine-HCl, pH3.0, containing 0.15mol/L NaCl), the pH of the eluate was immediately adjusted to 7.2 with 1mol/L Tris-HCl (pH8.0), and the eluate was dialyzed overnight at 4 ℃ in PBS (0.01mol/L, pH7.4, containing 0.15mol/L NaCl) to obtain a purified monoclonal antibody against GPC3(25-358) for protein quantification and specificity verification. The titers and the types of the obtained monoclonal antibodies are shown in Table 4.
TABLE 4
The gp2# monoclonal antibody was selected for subsequent experiments, and after ascites purification, it recognized about 40kD soluble GPC3 in the placenta lysate rich in GPC3, but the band was not seen in L02 (embryonic liver cells, ATCC), MCF-7 (human breast cancer cells, ATCC) and Hela (human cervical cancer cells, ATCC) (FIG. 6), and it was found that the antibody had strong specificity.
b. Preparation of mouse anti-GPC 3(350-364) and GPC3(444-516) monoclonal antibodies
10 hybridoma cell lines, namely 2G7, 3D4, 3F5, 3G1, 3G6, 4B9, 5H10, 6H10, 7C8 and 9D9, were prepared by the same method as the above a with polypeptide antigen GPC3(350-364) as the antigen. The results of direct ELISA after dilution of 1:10000 of the monoclonal antibody positive ascites fluid prepared from these hybridomas are shown in Table 5.
TABLE 5
| 2G7 | 3D4 | 3F5 | 3G1 | 3G6 | 4B9 | 5H10 | 6H10 | 7C8 | 9D9 |
| 1.459 | 2.456 | 1.824 | 2.142 | 2.56 | 1.500 | 1.124 | 1.443 | 2.39 | 1.112 |
Using polypeptide antigen GPC3(444-516) as antigen, 7 hybridoma cell lines were prepared in the same manner as in a, and named 1C6, 1F4, 2D2, 2F2, 2G9, 3C6 and 4F3, respectively. The results of direct ELISA after dilution of 1:10000 of the monoclonal antibody positive ascites fluid prepared from these hybridomas are shown in Table 6.
TABLE 6
| Ascites (ascites) | 1C6 | 1F4 | 2D2 | 2F2 | 2G9 | 3C6 | 4F3 | PBS |
| A490nm | 0.491 | 1.211 | 0.888 | 1.249 | 0.633 | 1.099 | 0.588 | 0.103 |
Example 5 Horse Radish Peroxidase (HRP) labeling of antibodies
Weighing HRP0.6mg and dissolving in 120 μ l water; adding NaIO4120 μ l (12.8mg/ml) at 4 ℃ for 30 min; adding ethylene glycol (9 μ l in1ml)120 μ l at room temperature for 30 min; mixing 240 μ l of 1mg/ml antibody, placing into dialysis bag, slowly stirring and dialyzing with carbonate buffer (0.05M pH9.6) overnight to combine; adding NaBH the next day424 μ l (5mg/ml) at 4 ℃ for 2 h; the mixture was packed in a dialysis bag, dialyzed against 0.05M/L PBS (pH7.2) at 4 ℃ overnight, centrifuged at 4 ℃ and the supernatant was collected to examine the titer.
Example 6ELISA detection
1. ELISA using GPC3-Ag3 polyclonal antibody as primary antibody and HRP-labeled mouse anti-GPC 3 monoclonal antibody (gp2#) as secondary antibody
Diluting purified rabbit anti-GPC 3 polyclonal antibody of rabbit anti-GPC 3-Ag3 with coating buffer ((0.01mol/L phosphate buffer pH7.6)) to 5 μ g/ml, coating enzyme-labeled reaction plate, incubating at 4 deg.C overnight and washing 1 time (washing buffer PBST: 0.05ml Tween-20 in 100ml PBS (pH7.2)); adding 200 μ l of blocking solution (blocking solution: 3g BSA in 100ml PBST) to block the enzyme-labeled reaction plate, incubating at 37 deg.C for 2h, and washing for 1 time; the sample to be tested was added to each well in an amount of 100. mu.l (serum: PBS 1: 3) (0.02mol/L PBS buffer pH7.2), incubated at 22 ℃ for 2 hours, and washed 5 times; adding 1:100 mul of 150-diluted horse radish peroxidase-labeled mouse anti-GPC 3 monoclonal antibody (gp2# -HRP) per well, incubating for 30min at 22 ℃, and washing for 6 times; mu.l of substrate solution (50. mu.l each of solution A and solution B) (solution A: 24.3ml of citric acid 0.2mol/L (19.2 g); Na 0.2 mol/L) were added to each well2HPO(28.4g)425.7ml；H2O20.1 percent; add ddH2O to 1200 ml; and B, liquid B: TMB3.9 g; 10.52g of citric acid; EDTA1.86g; 300ml of glycerol; heating to dissolve, and adding distilled water to 10000 ml). The reaction was stopped by adding stop solution to each well after development in the dark at 37 ℃ for 15min, and the OD (wavelength 450nm) of each well was read with an ELX800 microplate reader from Bio-Tek.
As a result, a sandwich ELISA method is established by using the mouse anti-GPC 3 monoclonal antibody (gp2#) and the GPC3-Ag3 polyclonal antibody, and GPC3 recombinant protein diluted in 25% normal human serum is detected, so that the minimum detection concentration is 2ng/ml, and the content of GPC3 and O.D. degree have a good linear relation in the range of 5ng-125ng, and the figure is shown in FIG. 8.
Furthermore, the present inventors performed GPC3 on sera from 106 normal persons, 96 hepatitis liver cirrhosis patients and 364 liver cancer patients by the ELISA method described above, and found that the average value of normal persons was 6.04. + -. 9.21ng/ml and the average value of hepatitis liver cirrhosis was 11.7. + -. 12.23 ng/ml. The serum GPC3 concentration of hepatocarcinoma patients is in biased distribution, and the average mean value is 87.22ng/ml, wherein 3 people with the concentration more than 2000ng/ml exist, and the highest concentration is 5432 ng/ml. The GPC3 difference between normal and hepatitis liver cirrhosis and liver cancer was very significant (P <0.000), and the difference between normal and hepatitis liver cirrhosis was not significant (fig. 9).
According to the working curves of the subjects (figure 10) for the two groups of patients with liver cancer and hepatitis cirrhosis, when the positive value is 36.8ng/ml, the sensitivity and specificity of the diagnosis are 32% and 2%, respectively; when a positive value of 27.1ng/ml was taken, the sensitivity and specificity of the diagnosis were 45.1% and 8.4%, respectively (Table 7).
TABLE 7
| GPC3 values (ng/ml) | Sensitivity of the composition | 1-specificity |
| 1.5 | 0.864 | 0.663 |
| 5.2 | 0.814 | 0.589 |
| 10.3 | 0.716 | 0.463 |
| 15.5 | 0.655 | 0.379 |
| 20.3 | 0.572 | 0.295 |
| 25 | 0.473 | 0.137 |
| 26.5 | 0.458 | 0.116 |
| 27.1 | 0.451 | 0.084 |
| 30.3 | 0.398 | 0.063 |
| 33.3 | 0.371 | 0.042 |
| 36.8 | 0.322 | 0.021 |
| 37.8 | 0.318 | 0.011 |
| 40.8 | 0.295 | 0.011 |
| 45.1 | 0.25 | 0.011 |
| 48.6 | 0.216 | 0.011 |
| 50.1 | 0.212 | 0 |
| 104.1 | 0.114 | 0 |
2. Compatibility test between monoclonal antibody and monoclonal antibody, between monoclonal antibody and polyclonal antibody, and between polyclonal antibody and polyclonal antibody
The present inventors established a sandwich ELISA method using various monoclonal antibodies and polyclonal antibodies prepared as described above, with different monoclonal antibodies or polyclonal antibodies as the first antibody or second antibody for compatibility, for detecting the antigen GPC 3. The ELISA detection procedure used was similar to that described in 1.
As a result, it was found that the detection sensitivity was far different between the various combinations when used for establishing the sandwich ELISA method. When two kinds of polyclonal antibodies except the rabbit anti-human GPC3-Ag3 polyclonal antibody are used as the first antibody and the second antibody respectively, the sensitivity and the specificity are poor. If a rabbit anti-human GPC3-Ag4 is used for coating, and an anti-human GPC3-Ag1 is used for detection, the OD450 reading of a sample with the concentration of 100ng/ml is 0.234, and the OD450 reading of a negative sample is also 0.204, so that the requirements of a diagnostic kit cannot be met.
When the monoclonal antibody is combined with the monoclonal antibody (such as 1F4+ gp2#), the sensitivity is high, and the lowest detection concentration reaches 0.5 ng/ml. However, the specificity is poor, the serum of normal people is easy to generate false positive results, and the false positive rate is even up to 18%.
When the monoclonal antibody is compatible with polyclonal antibody, the compatibility detection result of rabbit anti-human GPC3-Ag3 polyclonal antibody (first antibody) and mouse anti-GPC 3 monoclonal antibody (gp2#) (second antibody) is accurate, and the OD reading of the same sample is close to that of the former group when the polyclonal antibody anti-human GPC3-Ag3 polyclonal antibody (first antibody) and 3C6 monoclonal antibody (second antibody) are used for detecting the same sample. When the polyclonal anti-rabbit anti-human GPC3-Ag4 (primary antibody) and the monoclonal antibody 7C8 (secondary antibody) are used for detection, the OD reading in the detection of the same sample is 10-15% lower than the compatibility of the polyclonal anti-human GPC3-Ag3 and the monoclonal antibody gp2# of the mouse anti-GPC 3, and the accuracy is poor. And when the polyclonal anti-rabbit anti-human GPC3-Ag1 and a plurality of monoclonal antibodies or polyclonal antibodies prepared by the method are used for detection, the accuracy is poorer.
Example 7 complementation diagnosis of GPC3 with AFP
Establishing an ELISA double-sandwich kit by adopting the optimal compatible rabbit anti-human GPC3-Ag3 polyclonal antibody and mouse anti-GPC 3 monoclonal antibody (gp2#) obtained by the inventor; and an AFP detection radioimmunity kit (purchased from Shanghai biological products institute) for detecting AFP indexes is adopted to simultaneously detect 151 liver cell liver cancers with clear pathological diagnosis of AFP and GPC 3.
As a result, when the positive determination values of AFP and GPC3 were set to 20ng/ml and 30ng/ml, respectively, the former positive diagnosis rate was 49% (74/151) and the latter was 40% (61/151) (Table 8). Significantly, of 77 AFP-negative liver cancers, 34 (44%) of GPC3 were positive, indicating that GPC3 is a liver cancer marker complementary to AFP. The detection of AFP and GPC3 can improve the diagnostic sensitivity of the former from 49% to 72%.
TABLE 8
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
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Claims (9)
1. A test strip for detecting heparin sulfate proteoglycan 3, comprising:
a solid support; and
a polyclonal antibody against heparin sulfate proteoglycan 3 coated on the solid phase carrier, wherein the polyclonal antibody specifically binds to the epitope in 379-393 of the heparin sulfate proteoglycan 3 but not to the epitope in 1-378 or 394-580 of the heparin sulfate proteoglycan 3, and is obtained by immunizing an animal with the protein fragment in 379-393 of the heparin sulfate proteoglycan 3.
2. The test strip of claim 1, wherein the solid support is an enzyme-labeled reaction plate.
3. A test kit comprising the test strip according to claim 1, or
The kit comprises: (a) a solid support;
(b) a container a and a polyclonal antibody against heparin sulfate proteoglycan 3 as a first antibody in the container a, wherein the polyclonal antibody specifically binds to the epitope in 379-position 393 of the heparin sulfate proteoglycan 3 but not to the epitope in 1-378 or 394-position 580 of the heparin sulfate proteoglycan 3, and is obtained by immunizing an animal with the protein fragment in 379-position 393 of the heparin sulfate proteoglycan 3; and
(c) a reagent for coating said first antibody on said solid support.
4. The kit of claim 3, wherein the kit is further loaded with:
a container b, wherein the container b contains a second antibody, and the second antibody is a polyclonal antibody, and the polyclonal antibody specifically binds to the epitope except for the 379-position 393 in the heparin sulfate proteoglycan 3.
5. The kit of claim 3, wherein the kit is further loaded with:
and a container b ', wherein the container b' is filled with a second antibody, and the second antibody is a monoclonal antibody, and the monoclonal antibody specifically binds to the epitope in the heparin sulfate proteoglycan 3 except the 379-393 position.
6. The kit of claim 5, wherein the monoclonal antibodies bind to the following epitopes of heparin sulfate proteoglycan 3:
an epitope in positions 25-358 of heparin sulfate proteoglycan 3;
an epitope in position 350-364 of heparin sulfate proteoglycan 3; or
An epitope in position 444-516 of heparin sulfate proteoglycan 3.
7. The kit of claim 4 or 5, wherein the second antibody is detectably labeled.
8. The kit of claim 7, wherein the detectable label is selected from the group consisting of: horseradish peroxidase, alkaline phosphatase.
9. A polyclonal antibody, characterized in that the polyclonal antibody specifically binds to the epitope in 379-393 position of the heparin sulfate proteoglycan 3 but not to the epitope in 1-378 position or 394-580 position of the heparin sulfate proteoglycan 3, and the polyclonal antibody is obtained by immunizing an animal with the protein fragment in 379-393 position of the heparin sulfate proteoglycan 3.
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| CN102183660A (en) * | 2011-03-09 | 2011-09-14 | 倪温慨 | Time resolved fluoroimmunoassay kit for detecting Glypican-3 |
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| CN107604062A (en) * | 2017-09-01 | 2018-01-19 | 北京启辰生生物科技有限公司 | A kind of more antigen detection methods for liver cancer immunity treatment |
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