CN101290318B - ELISA reagent kit for diagnosing liver cancer - Google Patents

Abstract

The invention discloses a detection kit for liver cancer. The detection kit contains a polyclonal antibody that binds to the antigenic epitopes in positions 379-393 of heparan sulfate proteoglycan 3, and a polyclonal antibody that binds to positions 379-393 of heparan sulfate proteoglycan 3. Monoclonal or polyclonal antibodies to epitopes other than 393. The kit of the invention has high detection sensitivity and short detection time, which is helpful for early diagnosis of liver cancer patients, timely treatment and improvement of survival rate. Moreover, compared with only detecting AFP markers, using the kit of the present invention to detect GPC3 markers while detecting AFP markers can significantly improve the diagnostic sensitivity of liver cancer.

Description

A kind of ELISA kit for diagnosing liver cancer

technical field

The invention relates to the fields of biotechnology and immunology, in particular to antibodies, kits and applications for detecting liver cancer marker heparan sulfate proteoglycan 3 (GPC3).

Background technique

Primary liver cancer is the sixth most common cancer in the world and the third most common cancer in China. According to the estimates of the International Cancer Research Center (IARC), in 2000, the global incidence of liver cancer was 564,000, of which about 55% occurred in China, that is, the incidence of liver cancer in China was 306,000, and the death of liver cancer was 300,000, accounting for the second largest number of cancer deaths among Chinese residents. bit. Liver cancer is mostly in the middle and late stage when symptoms appear, and the recurrence and metastasis rate after resection is high. Therefore, early diagnosis of liver cancer is of great significance to prolong the survival time of patients and reduce the mortality of liver cancer.

At present, imaging diagnosis, cell and histological diagnosis and chemical diagnosis are the three main methods of tumor diagnosis. Imaging diagnosis plays an important role in the diagnosis of liver cancer, but it has certain limitations in the diagnosis of small liver cancer and the distinction between benign and malignant nodules. The positive results of ultrasound examination or CT, combined with the detection results of serum α-fetoprotein (AFP) level higher than 400ng/ml, can make a diagnosis of liver cancer. However, usually when these conditions are met, the best time for treatment has been missed. Whether it is ultrasonography, CT scan or MRI, the identification of small lesions has certain limitations, especially in liver cirrhosis nodules, which have many similarities with small hepatocellular carcinoma nodules in imaging. Therefore, although imaging technology has made great progress this year, it is still necessary to combine molecular markers of liver cancer clinically to distinguish benign from malignant liver diseases in more complicated cases. In addition, the clinical stage and tumor size of the tumor are the determinants of the treatment effect of liver cancer. Therefore, tumor markers can also be used for general screening of risk groups of liver cancer (such as chronic carriers of HBV and patients with liver cirrhosis).

At present, the most commonly used marker for the diagnosis of liver cancer is alpha-fetoprotein (AFP), which can appear abnormal several months before symptoms and imaging changes occur. The AFP content in normal human serum is generally less than 10ng/ml. When the diagnostic value of AFP is set at 20ng/ml, its sensitivity is 50-60%, but in cases with small tumors, the sensitivity is significantly reduced, and it has been reported that it is only 40%. Another problem with using AFP alone as a diagnostic marker for liver cancer is the lack of specificity. In a considerable number of patients with chronic hepatitis, especially those with liver cirrhosis, the AFP content can reach 20-200ng/ml. At present, it is believed that the markers that can be complementary to AFP diagnosis include AFP heterogeneity, γ-glutamyl transferase isoenzyme II and abnormal prothrombin, etc., but due to their insufficient sensitivity and specificity, and detection methods The cumbersome and complicated procedures make it remain at the level of laboratory research and fail to be transformed into clinical routine inspection items. Therefore, exploring new tumor molecular markers and establishing corresponding detection methods that are easy to promote are still one of the important topics in the field of liver cancer research today.

Heparan sulfate proteoglycan 3 (Glypican-3, GPC3) is a molecule related to liver cancer discovered in recent years. Using differential display technology, Hsu et al first discovered that the MXR7 gene whose cDNA was 100% homologous to GPC3 was abnormally expressed in liver cancer tissues. In 191 cases of hepatocellular carcinoma (HCC) tissues, 143 cases (74.8%) could detect MXR7 mRNA, while only 5 cases (3.2%) were positive in 156 cases of adjacent "non-tumor" liver tissues. All 5 cases had portal vein or distant metastasis. Subsequently, many laboratories have confirmed this result. In HCC tissue, the positive detection rate of GPC3 mRNA and protein is above 73%, and the average mRNA level can be 21.7 times higher than that in normal liver tissue, while in normal liver, benign liver tumor, chronic hepatitis and liver cirrhosis, etc. Negative, indicating that the protein expression has high tumor cell specificity, and is a new type of liver cancer marker.

However, due to the weak GPC3 content in the peripheral blood of normal people, the GPC3 content in the serum of tumor patients is still at a low level despite a significant increase.

Therefore, there is an urgent need in this field to develop a detection system and a detection method that can sensitively detect the level of GPC3, so as to provide a good way for the diagnosis of clinical tumors.

Contents of the invention

The purpose of the present invention is to provide a detection reagent capable of sensitively and accurately detecting the content of heparan sulfate proteoglycan 3 in a sample.

The object of the present invention is also to provide a detection kit for detecting the content of heparan sulfate proteoglycan 3 in a sample.

In a first aspect of the present invention, there is provided a test piece for detecting heparan sulfate proteoglycan 3, said test piece comprising:

a solid support; and

The anti-heparan sulfate proteoglycan 3 polyclonal antibody coated on the solid phase carrier, the polyclonal antibody specifically binds to the epitope in the 379-393 position of the heparan sulfate proteoglycan 3, and does not bind to sulfuric acid Epitopes in positions 1-378 or 394-580 of heparin proteoglycan 3.

In another preferred example, the solid phase carrier is an enzyme-labeled reaction plate.

In the second aspect of the present invention, a detection kit is provided, the kit contains the test piece, or

The kit contains: (a) a solid phase carrier;

(b) container a and the polyclonal antibody against heparan sulfate proteoglycan 3 as the first antibody located in the container a, the polyclonal antibody specifically binds to positions 379-393 of heparan sulfate proteoglycan 3 The epitope that does not bind to the epitope in the 1-378 or 394-580 of heparan sulfate proteoglycan 3; and

(c) a reagent for coating the first antibody on the solid phase carrier.

In another preferred example, the kit is also loaded with:

Container b, said container b is equipped with a second antibody, said second antibody is a polyclonal antibody, and said polyclonal antibody specifically binds to epitopes other than 379-393 in heparan sulfate proteoglycan 3 .

In another preferred example, the kit is also loaded with:

Container b', the container b' is equipped with a second antibody, the second antibody is a monoclonal antibody, and the monoclonal antibody specifically binds to the heparan sulfate proteoglycan 3 except for positions 379-393 gauge.

In another preferred example, the monoclonal antibody binds to the following epitope of heparan sulfate proteoglycan 3:

Epitopes in positions 25-358 of heparan sulfate proteoglycan 3;

an epitope in position 350-364 of heparan sulfate proteoglycan 3; or

Epitope in positions 444-516 of heparan sulfate proteoglycan 3.

More preferably, the monoclonal antibody binds to the epitope at positions 25-358 of heparan sulfate proteoglycan 3.

In another preferred example, the second antibody has a detectable label.

In another preferred example, the detectable marker is selected from the group consisting of horseradish peroxidase (HRP) and alkaline phosphatase.

More preferably, the marker is horseradish peroxidase.

In another preferred example, the kit is also loaded with:

container c, the substrate corresponding to the marker is housed in the container c;

container d, the color developer is housed in the container d;

a container e containing washing liquid; and/or

Container f, said container f is filled with stop solution.

In a third aspect of the present invention, a method for in vitro detection of heparan sulfate proteoglycan 3 is provided, comprising the following steps:

(a) Load the sample to be tested on the solid phase carrier coated with the first antibody, so that the heparan sulfate proteoglycan 3 in the sample to be tested is combined with the first antibody on the solid phase carrier to form a The solid phase carrier of the binary complex of proteoglycan 3-first antibody; the first antibody specifically binds to the epitope in positions 379-393 of heparan sulfate proteoglycan 3, but does not bind to heparan sulfate protein Polyclonal antibodies to epitopes in positions 1-378 or positions 394-580 of polysaccharide 3;

(b) adding the second antibody to the solid phase carrier obtained in (a), thereby forming a solid phase carrier with a ternary complex of "second antibody-heparan sulfate proteoglycan 3-first antibody"; The second antibody is a monoclonal antibody or a polyclonal antibody that specifically binds to an epitope other than the 379-393 position of heparan sulfate proteoglycan 3, and the second antibody carries a detectable label; and

(c) detecting the detectable marker in the ternary complex, thereby determining the presence or absence and the amount of heparan sulfate proteoglycan 3 in the sample to be detected.

In the fourth aspect of the present invention, a polyclonal antibody is provided. The polyclonal antibody specifically binds to the epitope in positions 379-393 of heparan sulfate proteoglycan 3, but does not bind to the epitope of heparan sulfate proteoglycan 3. 1-378 or 394-580, and the polyclonal antibody is obtained by immunizing animals with the 379-393 protein fragment of heparan sulfate proteoglycan 3.

In another preferred example, the animal is: rabbit.

In another aspect, the present invention also provides the use of the above-mentioned polyclonal antibody, which is used to prepare a kit for detecting heparan sulfate proteoglycan 3.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1 shows the gene encoding the 25-358aa amino acid fragment of GPC3 protein.

Figure 2 shows the 25-358 amino acid sequence of the GPC3 protein.

Figure 3 shows the small-scale expression identification of the pProEX Hta-GPC3(73-1074) plasmid.

Fig. 4 shows the SDS-PAGE gel electrophoresis identification of GPC3 (25-358aa) protein purified by Ni column.

Figure 5 shows the Western Blot detection of purified GPC3 (25-358aa) protein. Among them, M represents the molecular weight marker; W-18 represents the result of detection using polyclonal antibody W-18 (for protein N-terminal protein epitope); anti-His represents the result of detection using anti-His antibody.

Figure 6 shows the Western Blot results of cell lysates of placenta and tumor cells detected by mouse anti-GPC3 (25-358aa) monoclonal antibody gp2#. Among them, 1. Placental tissue, 2. L02 cells, 3. MCF-7 cells, 4. Hela cells.

Figure 7 shows the results of Western Blot of the cell lysate of tumor cells detected by rabbit anti-GPC3-Ag3 polyantibody. Among them, 1. Huh-7 cells; 2. HepG2 cells; 3. MCF-7 cells; 4. Hela cells.

Figure 8 shows the GPC3 quantification standard curve.

Figure 9 shows the serum GPC3 content of normal people, hepatitis cirrhosis and liver cancer patients.

Figure 10 shows the receiver operating characteristic curve (ROC curve) of GPC3.

Detailed ways

After extensive research and experiments, the inventors unexpectedly found that the polyclonal antibody corresponding to the 379-393 epitope of the amino acid sequence of heparan sulfate proteoglycan 3 (GPC3) can recognize the GPC3 antigen excellently; After the cloned antibody is used as the first antibody to capture GPC3, the second antibody (including monoclonal antibody or polyclonal antibody) corresponding to the epitope of the segment other than amino acid 379-393 of GPC3 can be used to bind to GPC3 extremely effectively , so as to detect GPC3 antigen with high sensitivity by double antibody sandwich method. In contrast, when using polyclonal antibodies that bind to other segments of GPC3 (such as positions 444-516) as the primary antibody, the detection sensitivity is low.

In addition, the present inventors unexpectedly found that when a polyclonal antibody specifically binding to the epitope 379-393 of GPC3 was used as the primary antibody, the binding detection of the GPC3 liver cancer marker and the AFP liver cancer marker had a higher Complementary diagnostic value. Compared with single detection of AFP markers, using the kit of the present invention to detect GPC3 markers while detecting AFP markers can significantly improve the diagnostic sensitivity of liver cancer. The present invention has been accomplished on this basis.

Those skilled in the art understand that an antigen may contain multiple antigenic epitopes (antigenic determinants), therefore, more than one antibody (including monoclonal antibody or polyclonal antibody) can be obtained against the same antigen. Binding properties (eg, specificity, etc.) may vary. Therefore, for the same antigen, those skilled in the art need to make repeated comparisons, screenings and identifications in order to find antibodies suitable for specific and sensitive binding. Since the GPC3 protein is a long polypeptide, most of its antigenic determinants are contained in the interior of the spatial structure, so it is difficult to find antibodies suitable for binding to it.

In view of the above technical problems, the inventors prepared a variety of monoclonal antibodies and polyclonal antibodies corresponding to GPC3, and tested the binding specificity and sensitivity of different monoclonal antibodies or polyclonal antibodies to GPC3 through a large number of experiments. As a result, it was found that the polyclonal antibody corresponding to the 379th-393rd epitope of the GPC3 amino acid sequence can recognize the GPC3 antigen particularly well.

Moreover, the inventors combined different monoclonal antibodies and/or different polyclonal antibodies pairwise (based on the principle of double-antibody sandwich method) to detect the binding specificity of the combination of these monoclonal antibodies and polyclonal antibodies to GPC3 and sensitivity. Final discovery: use polyclonal antibodies (as primary antibodies) that bind to epitopes in amino acid positions 379-393 of GPC3, and monoclonal antibodies or polyclonal antibodies that bind to epitopes other than amino acid positions 379-393 in GPC3 The antibody (as a secondary antibody) as a detection reagent based on the double-antibody sandwich method can detect the GPC3 antigen particularly sensitively. However, when a polyclonal antibody that binds to an epitope other than 379-393 is used in combination with other monoclonal antibodies or polyclonal antibodies, the sensitivity and specificity are not high; When combined, the sensitivity is high, but the specificity is poor.

As used herein, the "sample" refers to a substance isolated from a 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 "capture antibody", "coating antibody", "first antibody" and "primary antibody" are used interchangeably, and they all refer to the GPC3 amino acid sequence binding at position 379-393 polyclonal antibodies to antigenic epitopes.

The first antibody can be coated on a solid carrier. The present invention has no particular limitation on the solid phase carrier used, 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 "detection antibody", "secondary antibody" and "secondary antibody" are used interchangeably, and all refer to antibodies that specifically bind to antigenic epitopes other than positions 379-393 of GPC3, which can are monoclonal or polyclonal antibodies.

Heparan Sulfate Proteoglycan 3

Heparan sulfate proteoglycan 3 (Glypican-3, GPC3) is located on the surface of the cell membrane and is composed of core protein and glycosaminoglycan (heparin and heparan sulfate) side chains. , growth factors and proteases, etc. to transmit signals, and have important functions in cell growth, development, differentiation and migration. The existence of GPC3 in vivo has obvious tissue specificity and phase specificity: in the human embryonic period, there is a large amount of GPC3 expression in tissues derived from the gastrointestinal tract and mesoderm; but in adults, except for placental tissues, only lung, kidney GPC3 was lowly expressed in a few tissues such as breast, heart and ovary, and it was negative in the liver. The inventors found in the study that the average concentration of GPC3 in the serum of patients with hepatocellular carcinoma (HCC) was significantly higher than that of normal controls and patients with liver cirrhosis. In well-differentiated HCC cases, GPC3 is more sensitive than AFP detection, and the combined detection of the two can greatly increase the positive rate of diagnosis of liver cancer.

The amino acid sequence and nucleotide sequence of the GPC3 protein are known in the art, and its amino acid sequence is shown in SEQ ID NO: 7, and its nucleotide sequence is shown in SEQ ID NO: 6. Alternatively, those skilled in the art can also refer to the amino acid sequence and nucleotide sequence provided by GenBank accession numbers NP_004475 and NM_004484.2.

Antibody

The inventors of the present invention provide a polyclonal antibody that can recognize the GPC3 antigen very well, and the antibody specifically binds to the epitope in positions 379-393 of heparan sulfate proteoglycan 3.

The polyclonal antibody is obtained by immunizing animals with protein fragments at positions 379-393 of GPC3. 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 preparation method of the polyclonal antibody is as follows: mix the 379-393 protein fragment of GPC3 with Freund's adjuvant in an appropriate ratio (such as 1:1), fully emulsify and immunize animals. The method of immunization may use subcutaneous injection. After 1-4 months of animal immunization, the antiserum can be harvested and purified from the venous blood of the animal to obtain the polyclonal antibody against the 379-393 protein fragment of GPC3.

As a preferred mode of the present invention, the animal is: rabbit.

In the embodiment of the present invention, the inventors prepared a variety of polyclonal antibodies (including rabbit anti-human GPC3-Ag3 polyclonal antibody (binding to the epitope in GPC3 379-393), rabbit anti-human GPC3-Ag3 Ag1 (binding to the epitope in GPC3 73-86), rabbit anti-human GPC3-Ag4 polyclonal antibody (binding to the epitope in GPC3 444-516)), found in the subsequent ELISA detection , Compatibility of rabbit anti-human GPC3-Ag3 polyclonal antibody with various other monoclonal antibodies or polyclonal antibodies that do not bind to the epitope in positions 379-393 of GPC3 can detect the content of GPC3 antigen particularly sensitively.

Techniques for preparing monoclonal antibodies specific for GPC3 proteins are well known in the art. Here, "specificity" refers to antibodies that can bind to GPC3 protein or its fragments; more specifically, refers to those antibodies that can bind to GPC3 protein or its fragments but do not recognize and bind to other irrelevant antigenic molecules.

The monoclonal antibody is an antibody that specifically binds to antigenic epitopes other than positions 379-393 of GPC3. It should be understood that when used as the second antibody in conjunction with the polyclonal antibody (as the first antibody) that binds to the epitope in GPC3 379-393 for the detection of the GPC3 antigen (based on the double-antibody sandwich method principle), the monoclonal antibody can be of various types, as long as it can specifically bind to antigenic epitopes other than positions 379-393 of GPC3.

Preferably, the antigenic epitopes other than positions 379-393 of GPC3 are selected from:

Antigen epitopes corresponding to positions 25-358 of GPC3;

The antigenic epitope corresponding to position 350-364 of GPC3; or

Antigen epitopes corresponding to positions 444-516 of GPC3.

The monoclonal antibodies of the present invention can be produced using hybridoma technology. Production of monoclonal antibodies using hybridoma technology is well known to those skilled in the art, for example, see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur.J.Immunol.6: 511, 1976; Eur. J. Immunol. 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981 described.

After knowing the epitope position of the GPC3 antigen corresponding to the monoclonal antibody, those skilled in the art can conveniently obtain the monoclonal antibody through known methods. For example, when it is necessary to prepare a monoclonal antibody against the epitope corresponding to the 25th-358th position of GPC3, the mouse is immunized with the protein fragment of the 25th-358th position of GPC3 to prepare a hybridoma, so as to obtain the required antibody.

Detection kit

According to the principle of the double-antibody sandwich method, the inventors prepared a kit that can be used to detect the level of GPC3 protein in a sample. The routine method of the double antibody sandwich method is to immobilize the primary antibody on the carrier, then react the primary antibody with the antigen, and then react with the secondary antibody after washing (the secondary antibody carries a detectable label, or can be combined with an antibody carrying a detectable label). Substance binding), and finally perform chemiluminescence or enzyme-linked color reaction to detect the signal. The double-antibody sandwich method is especially suitable for the detection of antigens with two or more epitopes.

The present inventors found that the target antigen GPC3 can be adsorbed and localized by using a polyclonal antibody that binds to an epitope in positions 379-393 of GPC3 and an antibody that specifically binds to an epitope other than positions 379-393 in GPC3 , its positioning and amplification effects are particularly good, so it has high specificity and precision. Moreover, compared with the single-antibody competition method, the double-antibody sandwich method has a better measurement effect, so only a small amount of sample is required for the measurement. Therefore, the double-antibody sandwich method has more advantages in terms of sensitivity, precision, accuracy, specificity and stability.

The detection kit of the present invention contains a test piece, and the test piece includes: a solid-phase carrier; and a polyclonal antibody against heparan sulfate proteoglycan 3 coated on the solid-phase carrier, and the polyclonal antibody has a specific It binds to the epitope in positions 379-393 of heparan sulfate proteoglycan 3, but does not bind to the epitope in positions 1-378 or 394-580 of heparan sulfate proteoglycan 3.

Alternatively, the detection kit of the present invention contains (a) a solid phase carrier; (b) a container a and a polyclonal antibody against heparan sulfate proteoglycan 3 as the first antibody located in the container a, the polyclonal The antibody specifically binds to the epitope in position 379-393 of heparan sulfate proteoglycan 3, but not to the epitope in position 1-378 or position 394-580 of heparan sulfate proteoglycan 3; and (c) for A reagent for coating the first antibody on the solid phase carrier.

As a preferred mode of the present invention, the kit is also loaded with:

Container b, said container b is equipped with a second antibody, said second antibody is a polyclonal antibody, and said polyclonal antibody specifically binds to epitopes other than 379-393 in heparan sulfate proteoglycan 3 ;or,

Container b', the container b' is equipped with a second antibody, the second antibody is a monoclonal antibody, and the monoclonal antibody specifically binds to the heparan sulfate proteoglycan 3 except for positions 379-393 gauge.

As a preferred mode of the present invention, the second antibody has a detectable label.

As used herein, the "detectable marker" refers to a marker used to determine the presence or absence and the amount of GPC3 in the sample to be detected. After the coating antibody and/or detection antibody used in the kit of the present invention is determined, various markers routinely used in the art for detection in combination with the detection antibody can be used. The present invention has no particular limitation on the marker used, as long as it can bind to the detection antibody and can accurately indicate the presence or absence and the amount of GPC3 protein in the sample to be detected after appropriate treatment. It is available. The label can be directly set on the second antibody; or, the label can also be set on the specific anti-second antibody anti-antibody, those skilled in the art can according to the type of antibody used and characteristics, choose the appropriate markers. For example, the marker can be selected from: horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase.

When using some of the above-mentioned enzyme markers, it is also necessary to use some substrates that bind to the corresponding enzymes, so that the presence or amount of the markers can be reported by means of color development and the like. As used herein, the "substrate corresponding to the label" refers to a color that can be catalyzed by the label to display the recognition signal of the binding of the second antibody to GPC3. Described substrate is for example: o-phenylenediamine (OPD), tetramethylbenzidine (TMB), ABTS for horseradish peroxidase; p-nitrophenyl phosphate, p-NPP); and so on. Those skilled in the art can select an appropriate substrate according to the type and characteristics of the marker used.

As a preferred mode of the present invention, the second antibody is directly linked to the label. More preferably, the marker is HRP. Compared with labeling the detection antibody with biotin and then reacting with streptavidin-HRP, it is simpler and more convenient to directly label HRP on the detection antibody and directly add the substrate to develop the color after the reaction is completed.

In order to obtain quantitative results, standards containing multiple GPC3 proteins at known concentrations can also be set during the detection process. A conventional method can be used for the setting method of the standard.

In order to eliminate false positives and false negatives, a quality control (control) can also be set during the detection process.

In addition, in order to make the kit of the present invention more convenient during detection, the kit preferably also contains other auxiliary reagents, and the auxiliary reagents are some reagents routinely used in ELISA kits, and the characteristics of these reagents and their preparation methods are well known to those skilled in the art. The reagents are, for example (but not limited to): chromogenic reagent, washing solution, stop solution, sensitization solution, diluent.

In addition, the kit may also contain an instruction manual for explaining the method of using the reagents contained therein.

Test kit of the present invention can be applied to:

(A) as an independent detection system for the diagnosis of liver cancer;

(B) Combined with imaging examination and/or AFP marker detection, etc., to diagnose liver cancer;

(C) differential diagnosis of benign and malignant liver diseases with elevated AFP;

(D) Prognostic monitoring of GPC3-positive liver cancer before treatment;

(E) Screening of high-risk populations for liver cancer; and/or

(F) Carry out basic research related to liver cancer.

Detection method

The invention provides a method for using the kit of the invention to detect GPC3 protein in vitro, comprising the following steps:

(a) Load the sample to be tested on the solid phase carrier coated with the first antibody, so that the heparan sulfate proteoglycan 3 in the sample to be tested is combined with the first antibody on the solid phase carrier to form a The solid phase carrier of the binary complex of proteoglycan 3-first antibody; the first antibody specifically binds to the epitope in positions 379-393 of heparan sulfate proteoglycan 3, but does not bind to heparan sulfate protein Polyclonal antibodies to epitopes in positions 1-378 or positions 394-580 of polysaccharide 3;

(b) adding the second antibody to the solid phase carrier obtained in (a), thereby forming a solid phase carrier with a ternary complex of "second antibody-heparan sulfate proteoglycan 3-first antibody"; The second antibody is a monoclonal antibody or a polyclonal antibody that binds to an epitope other than the 379-393 position of heparan sulfate proteoglycan 3, and the second antibody carries a detectable label; and

(c) detecting the detectable marker in the ternary complex, thereby determining the presence or absence and the amount of heparan sulfate proteoglycan 3 in the sample to be detected.

As a preferred mode of the present invention, the method for quantitatively detecting the GPC3 protein is as follows:

(i) Antigen-antibody reaction: Coat the first antibody of the present invention on a multiwell plate, and then add different concentrations of standard, quality control (optional), or serum samples to be tested in the microwells of the multiwell plate ;

(ii) Enzyme-linked reaction: adding the solution of the second antibody (with a marker on it) to each well, shaking and incubating; washing;

(iii) Color reaction: Add substrate and chromogenic agent corresponding to the marker to each well, incubate, add reaction termination solution to each well, and end the reaction;

(iv) Microplate reader measures OD value;

(v) Result calculation:

A) Make a standard curve: take the concentration of the GPC3 protein standard substance as the abscissa, and measure the OD value of the standard substance as the ordinate, and make a standard curve;

B) Judging the concentration of the quality control substance (optional): read the corresponding concentration value from the standard curve according to the OD value of the quality control substance; when the measured concentration value of the quality control substance is within a given range, the determination is valid;

C) Calculating the concentration of the serum sample to be tested: when both the standard curve and the quality control substance are determined to be valid, the GPC3 protein concentration of the serum sample to be tested is calculated from the standard curve according to the OD value of the sample to be tested.

Due to the high sensitivity of the detection reagent or kit of the present invention, the incubation time of the sample is greatly shortened (about 2 ± 0.5 hours), so the quantitative data of GPC3 can be obtained in a very short time using the kit, which is generally in 2-4 hours. When using conventional reagents or kits for detection, samples usually need to be incubated overnight to increase sensitivity.

The main advantages of the present invention are:

(1) For the first time, it was found that the polyclonal antibody corresponding to the 379-393 amino acid epitope of GPC3 can well recognize the GPC3 antigen, and it was used in combination with the antibody corresponding to the antigen epitope other than the 379-393 amino acid of GPC3 As a detection reagent based on the double-antibody sandwich method, it can detect GPC3 antigen particularly sensitively.

(2) An easy-to-use and highly sensitive liver cancer detection kit is provided, which can detect sensitively when the GPC3 content in the sample is low (1ng level), and the time required to complete the detection is very short (about 2-4 Hour). The kit is helpful for early diagnosis and timely treatment of liver cancer patients, improving survival rate and having positive social benefits. It also has wide application value in follow-up of liver cancer efficacy, monitoring of subclinical metastasis and recurrence, and prognosis judgment.

(3) The inventors also found that the combined detection of GPC3 liver cancer marker and AFP liver cancer marker has complementary diagnostic value. The combined application of GPC3 and AFP can not only increase the positive rate of early diagnosis of liver cancer, but also make differential diagnosis of benign and malignant lesions in a large number of patients with low or moderately increased AFP. Compared with single detection of AFP markers, using the kit of the present invention to detect GPC3 markers while detecting AFP markers can significantly improve the diagnostic sensitivity of liver cancer.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory guide (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions. Percentages and parts are by weight unless otherwise indicated.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can also be applied in the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

Example 1. Prokaryotic preparation of polypeptide antigens GPC3 (25-358), GPC3 (444-516)

The polypeptide antigen GPC3 (25-358) having the 25th-358th sequence of the full-length amino acid sequence of GPC3 is prepared by the following method:

(1) Extraction of human placenta total RNA

Take about 1g of human placental tissue, put it into a 1.5ml centrifuge tube, add 1ml TRAZOL reagent, smash the tissue with a glass homogenizer, and extract RNA.

(2) reverse transcription

Take 5 μg of total RNA, use the ThermoScript RT-PCR System (GIBCO BRL company) kit, refer to the instructions of the reverse transcription kit, use oligo(dT)20 as a primer, and AMV reverse transcriptase to synthesize cDNA by reverse transcription.

(3) Construction of expression vector

Design the following primers:

GPC3-73 (SEQ ID NO: 3):

5'cgcgaattcCAGCCCCCGCCGCCGCCGCC3' and

GPC3-1074 (SEQ ID NO: 4):

5' gcgctcgagTTATCTATATTGGCGTTGTTGAGAATGGGCACATAAC3'

Using the newly synthesized human placenta cDNA as a template and the previously designed primers as primers, the N-terminal coding sequence of GPC-3 protein (with the 24aa signal peptide removed) nt.73-1074 was amplified. The amplification system was 10 μL; the amplification conditions were: pre-denaturation at 94°C for 3 minutes; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, a total of 30 cycles; extension at 72°C for 10 min. The PCR product was identified by 10g/L agarose gel electrophoresis, and the PCR product was recovered with a PCR fragment recovery kit.

The PCR product was cloned into the pProEX HTa vector (purchased from Invitrogen, USA) with EcoRI and Xho I sites to obtain the plasmid pProEX Hta-GPC3 (73-1074) expressing GPC-3 protein 25-358aa, and the sequencing identified no influence on the gene sequence The point mutation of amino acid change (Fig. 1, SEQ ID NO: 1), the deduced amino acid sequence is 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 Escherichia coli strain BL21-codonplus (DE3)-RP (purchased from Stratagene), coated with ampicillin-resistant agar plate, and grown upside down at 37°C overnight, in the ampicillin-containing On the agar plate, pick 3 clones with a toothpick and put them in 3ml LB containing ampicillin, and shake them overnight at 37°C. The bacteria cultured overnight were inoculated into 3 ml of LB containing ampicillin at a ratio of 1:100, and cultured with shaking at 37°C for 3 hours until mid-logarithmic phase (A ₅₅₀ =0.5). Isopropylthio-β-d-galactoside (IPTG) was added to a final concentration of 1 mmol/L, and shaking culture was continued for 3 hours to induce GPC3 protein expression. Take each induced bacterium and uninduced control bacterium, the amount is equivalent to 100 μl bacterial liquid when A ₅₅₀ =1, suspend in 10 μl 1×SDS loading buffer after sedimentation, boil in boiling water bath for 5 minutes. Store at -20°C or use immediately for SDS-PAGE gel electrophoresis. The expression of protein was identified by SDS-PAGE electrophoresis. Results The induced bacteria had a specific protein expression band (GPC3) at 40KD, as shown in Figure 3.

According to the small sample induction conditions, 200ml pProEXHta-GPC3(73-1074)-codonplus expression protein was induced, purified with Ni column, and then identified by SDS-PAGE gel electrophoresis. It was found that the main band of about 40KD protein accounted for more than 90% of the total protein, as shown in Figure 4.

The protein expressed by pProEXHta-GPC3(73-1074) is located in the inclusion body and purified according to the purification steps of insoluble protein:

(a) The chromatographic column was washed with GuNTA-0Buffer (20mmol/L Tris-HCl pH7.9, 0.5mol/L NaCl, 8mol/L Urea) with 10 times the volume of TNA.

The sample was added to the TNA chromatography column, the flow rate was controlled at about 15ml/h, and the breakthrough fraction was collected for SDS-PAGE analysis of protein binding.

The chromatography was washed with GuNTA-0Buffer 5 times the volume of TNA, and the flow rate was controlled at about 30ml/h.

(b) 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/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 Imidazole), GuTNA-500 (20mmol/L Tris-HCl pH7.9, 0.5mol/LNaCl , 8mol/L Urea, 500mmol/L midazole) for elution, the flow rate was controlled at 15ml/h, and the eluate was collected, and one volume of TNA was collected in each tube.

Routine Western Blot detection was performed on the purified product with anti-His antibody and anti-GPC3 polyantibody W-18 from Santa Cruz Company (for the protein N-terminal protein epitope), and the results confirmed that the expressed product was GPC3 protein, as shown in Figure 5.

Using the prokaryotic expression method similar to the aforementioned preparation of polypeptide antigen GPC3 (25-358), the inventors also prepared a polypeptide antigen GPC3 (444-516) selected from the 444-516th position of the full-length amino acid sequence of GPC3, named GPC3 -Ag4.

Example 2

Synthesis and coupling of polypeptide antigens GPC3(379-393), GPC3(73-86), GPC3(350-364)

Use the software DNAStar to analyze the antigenicity of the full-length GPC3 protein, and select the polypeptide antigen GPC3 (379-393) amino acids located at the 379-393th position of the full-length amino acid sequence of GPC3 mainly according to the two parameters of protein antigenicity and accessibility ETLSSRRRELIQKLK (SEQ ID NO: 5), named GPC3-Ag3. The polypeptide is prepared by artificial synthesis. After GPC3-Ag3 is synthesized, it is coupled with KLH (keyhole limpet hemocyanin) by glutaraldehyde method to obtain GPC3-Ag3-KLH.

In addition, the inventors also synthesized the polypeptide antigen GPC3 (73-86) selected from the 73-86th position of the full-length amino acid sequence of GPC3, named GPC3-Ag1; selected from the 350-364th position of the full-length amino acid sequence of GPC3 The polypeptide antigen GPC3 (350-364), named GPC3-Ag2. Also, GPC3(73-86) was coupled to KLH to obtain GPC3-Ag1-KLH.

The preparation of embodiment 3 polyclonal antibody

a. Preparation of rabbit anti-human GPC3-Ag3 polyclonal antibody

Antigen: GPC3-Ag3-KLH.

Animals: New Zealand white rabbits, one male and one female, 4-6 weeks old, 2.5-3.0 kg, were purchased from Shanghai Experimental Animal Center, Chinese Academy of Sciences.

(1) A male and a female healthy New Zealand white rabbit were immunized at the same time. Before immunization, 1-2ml of blood was collected from the rabbit's ear vein, the serum was separated, aliquoted, and stored at -80°C until use. For the first immunization, each rabbit was injected with 1 mg of antigen. Mix Freund's complete adjuvant with 1ml of antigen (1mg) in equal volume, fully emulsify, and inject at multiple points through the intradermal tissue of the back.

(2) Afterwards, a booster immunization was given every 2 weeks, for a total of two times. The amount of antigen remained unchanged, and Freund's incomplete adjuvant was used instead, and the injection method remained unchanged. On the 7th day after the second boost, the antibody titer in the rabbit serum was measured by ELISA method. After the serum titer reached the requirement, the rabbit was anesthetized with ether, and the carotid artery was intubated. The blood of the New Zealand white rabbit was collected and kept at room temperature for 30 minutes, and then placed in a refrigerator at 4°C overnight. The separated rabbit serum was absorbed, and the remaining blood was centrifuged at 4°C and 4000rpm. After 10 minutes, the supernatant serum was drawn, and the rabbit anti-human GPC3-Ag3 polyclonal antibody was obtained.

See Table 1 for the serum titers of rabbits 5 weeks after GPC3-Ag3-KLH immunization.

Table 1

The purified rabbit serum was used for Western Blot detection of the cell lysate. As a result, it was found that the cell lysates of liver cancer cells Huh-7 (ATCC) and HepG2 (ATCC) were used as antigens, and the rabbit serum was used as antibodies, and there were specific bands at molecular weight 60KD (full-length GPC3); The lysates of -7 (human breast cancer cells, ATCC) and Hela (human cervical cancer cells, ATCC) were used as antigens, and the rabbit serum was used as antibodies, and the results were negative, as shown in FIG. 7 . It can be seen that the rabbit anti-human GPC3-Ag3 polyclonal antibody has high specificity. Moreover, further tests also showed that the polyclonal antibody does not bind to the epitopes located in positions 1-378 or positions 394-580 of GPC3.

b. Preparation of rabbit anti-human GPC3-Ag1 and rabbit anti-human GPC3-Ag4 polyclonal antibodies

The antigens used are GPC3-Ag1-KLH and GPC3-Ag4, and the animals used are the same as the above-mentioned a.

Prepare rabbit anti-human GPC3-Ag1 polyclonal antibody and rabbit anti-human GPC3-Ag4 polyclonal antibody according to the same method as above a. See Table 2 for the titer of rabbit serum 7 weeks after GPC3-Ag1-KLH immunization; see Table 3 for the titer of rabbit serum 5 weeks after GPC3-Ag4 immunization.

Table 2

table 3

Example 4 Preparation and Purification of Anti-GPC3 Monoclonal Antibody

a. Preparation and purification of mouse anti-GPC3(25-358) monoclonal antibody

Take 6-8 week-old BALB/c mice, mix purified GPC3(25-358) protein with an equal volume of Freund's complete adjuvant for the first immunization, and subcutaneously immunize multiple points on the back and groin, and then every 2 weeks For one immunization, the same dose of antigen was mixed with Freund's incomplete adjuvant and subcutaneously immunized at multiple points on the back. Seven days after the third immunization, blood was collected from the tail vein of the rats, and the production of specific antibodies was measured by indirect ELISA. Three days before the fusion, the same dose of antigen without adjuvant was used to boost the intraperitoneal immunization once.

Three days after the booster immunization, the mice were killed by neck dislocation, the spleen was taken out under aseptic conditions, and about 0.2-0.5ml of serum-free culture solution was injected with a syringe to make it swell, and then the splenic membrane was punctured at multiple points with a curved injection needle. Squeeze the lymphocytes out of it, prepare the immune spleen cell suspension, count it, and wash it twice with incomplete culture medium. Take logarithmic growth myeloma cells SP2/0, centrifuge at 1000rpm/min for 5min, discard the supernatant, suspend the cells with incomplete culture medium and count them, get the required number of cells, wash twice with incomplete culture medium. Mix myeloma cells and splenocytes at a ratio of 1:10, wash once with incomplete culture medium in a 50ml plastic centrifuge tube, and centrifuge at 1200rpm/min for 8min. Discard the supernatant, and suck up the residual liquid with a dropper, so as not to affect the concentration of PEG. Gently flick the bottom of the centrifuge tube to loosen the cell pellet slightly. Fusion at room temperature: ① Add 1ml of preheated 45% PEG1000 containing 5% DMSO within 30s, and stir while adding; ② React for 90s; ③ Add preheated incomplete culture medium to terminate the PEG effect, add 1ml and 2ml every 2min , 3ml, 4ml, 5ml and 10ml. Centrifuge, 800rpm/min, 6min. Discard the supernatant, and gently suspend it with about 6ml of 20% calf serum RPMI1640, and do not blow hard to avoid dispersing the fused cells. According to the number of 96-well culture plates used, add complete culture medium, 10ml per 96-well plate. The fused cell suspension was added to a 96-well plate containing feeder cells, 100 μl/well, and cultured in a 37°C, 5% CO ₂ incubator. Generally, a 96-well plate contains 1×10 ⁷ splenocytes. Add HAT selection medium 24h after fusion. After the HAT selection medium was maintained for two weeks, the HT medium was changed to the HT medium, and after another two weeks of maintenance, the normal medium was used. When the hybridoma cells cover 1/10 of the area of the bottom of the well, the specific antibody can be detected. Dilute the GPC3 protein obtained by genetic engineering to 5 μg/ml with the coating solution, coat the microplate with 100 μl per well, put it at 4°C overnight, discard the supernatant, and wash the coated microplate with the washing solution 3 times, 5 min each time, then add 200 μl blocking solution (3% BSA PBST) to each well, place at 37°C for 2 hours, pour off the blocking solution, wash as above, add 100 μl culture supernatant to each well, incubate at 37°C for 1.5h, discard For the supernatant, wash as above, add 100 μl of goat anti-mouse IgG-HRP diluted 1:1000 to each well, incubate at 37°C for 1 h, discard the supernatant in the well, wash as above, add 100 μl of chromogenic solution (OPD-H ₂ O ₂ ), after 10-20 min of color development at room temperature in the dark, 50 μl of stop solution was added to each well to terminate the reaction, and the A ₄₉₀ nm value was read with a microplate reader. Positive clones were cloned by the limiting dilution method, and the required hybridoma cell lines were screened out after three clones. As a result, 11 positive clones were obtained, named gp1#-gp11# respectively.

Inject liquid paraffin intraperitoneally into the F1 _generation mice, and inject 1×10 ⁵ hybridoma cells intraperitoneally after 2 weeks, produce ascites 7-10 days after inoculating the cells, closely observe the health status of the animals and signs of ascites, wait until ascites is as much as possible, Before the mice were dying, the mice were sacrificed, and the ascites was sucked into the test tube with a dropper.

Take ascites or rabbit serum after immunization and dilute it with an equal volume of PBS (0.01mol/L, pH7.4, containing 0.15mol/L NaCl), then add an equal volume of saturated ammonium sulfate (90g ammonium sulfate to 100ml Dissolve in distilled water at 80°C, filter while hot, and crystals will precipitate after cooling down to room temperature, adjust to pH 7.0 with sulfuric acid), place at 4°C for 4h, then centrifuge at 10,000rpm/min for 15min, discard the supernatant, and use 1- Dissolve 5ml of PBS, transfer it into a dialysis bag, dialyze in PBS (0.01mol/L, pH7.4, containing 0.15mol/L NaCl) overnight at 4°C, transfer the protein solution in the dialysis bag to the Protein A Sepharose CL-4B column for circulation Wash the loaded Protein A column with Tris-HCl (0.05mol/L, pH8.0, containing 0.15mol/L NaCl) several times until no protein is eluted on the UV chromatographic recording paper. Then the antibody was eluted with a dissociation solution (0.01mol/L, glycine-HCl, pH3.0, containing 0.15mol/L NaCl), and the eluent was immediately adjusted to pH with 1mol/L Tris-HCl (pH8.0). 7.2. After dialysis overnight at 4°C in PBS (0.01mol/L, pH7.4, containing 0.15mol/L NaCl), perform protein quantification and specificity verification to obtain purified anti-GPC3(25-358) monoclonal antibody. The obtained monoclonal antibody titers and types are shown in Table 4.

Table 4

The gp2# monoclonal antibody was selected for subsequent experiments. After purification of ascitic fluid, it can recognize soluble GPC3 of about 40KD in GPC3-rich placental lysate, but it is not effective in L02 (embryonic liver cells, ATCC), MCF-7 (human breast cancer Cells, ATCC) and Hela (human cervical cancer cells, ATCC) did not see this band (Figure 6), showing that the antibody has strong specificity.

b. Preparation of mouse anti-GPC3(350-364), GPC3(444-516) monoclonal antibodies

Using the polypeptide antigen GPC3 (350-364) as the antigen, 10 hybridoma cell lines were prepared using the same method as in the above a, and were named 2G7, 3D4, 3F5, 3G1, 3G6, 4B9, 5H10, 6H10, 7C8 and 9D9. Table 5 shows the direct ELISA results of the monoclonal antibody-positive ascites prepared by these hybridomas after dilution of 1:10000.

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 the polypeptide antigen GPC3 (444-516) as the antigen, 7 hybridoma cell lines were prepared using the same method as in the above a, which were named 1C6, 1F4, 2D2, 2F2, 2G9, 3C6 and 4F3. Table 6 shows the direct ELISA results of the monoclonal antibody-positive ascitic fluid prepared by these hybridomas after 1:10000 dilution.

Table 6

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

The horseradish peroxidase (HRP) labeling of embodiment 5 antibody

Weigh 0.6 mg of HRP and dissolve in 120 μl of water; add 120 μl of NaIO ₄ (12.8mg/ml), 4°C for 30 minutes; add 120 μl of ethylene glycol (9 μl in 1ml) for 30 minutes at room temperature; mix 240 μl of 1 mg/ml antibody and put it into a dialysis bag Slowly stir and dialyze carbonate buffer (0.05M pH9.6) overnight to allow it to bind; add NaBH ₄ (5mg/ml) 24μl the next day, 4°C for 2h; PBS (pH7.2) was dialyzed overnight at 4°C and centrifuged, and the supernatant was taken to detect the titer.

Embodiment 6ELISA detects

1. ELISA with GPC3-Ag3 polyclonal antibody as primary antibody and HRP-labeled mouse anti-GPC3 monoclonal antibody (gp2#) as secondary antibody

Dilute the purified rabbit anti-GPC3 polyclonal anti-GPC3-Ag3 polyclonal antibody to 5 μg/ml with coating buffer ((0.01mol/L phosphate buffer pH7.6)), and coat the enzyme-labeled reaction plate, each well 100μl, incubate overnight at 4°C and wash once (washing buffer PBST: add 0.05ml Tween-20 to 100ml PBS (pH7.2)); add 200μl blocking solution (blocking solution: add 3g BSA to 100ml PBST) to block the enzyme labeling reaction Wash the plate once after incubating at 37°C for 2 hours; add the sample to be tested, 100 μl per well (serum:PBS=1:3) (0.02mol/L PBS buffer pH7.2), incubate at 22°C for 2 hours and wash 5 times; Add 1:150 dilution of horseradish peroxidase-labeled mouse anti-GPC3 monoclonal antibody (gp2#-HRP), 100 μl per well, incubate at 22°C for 30 min, and then wash 6 times; add substrate solution 100 μl per well (A 50 μl each of solution and solution B) (solution A: 0.2mol/L (19.2g) citric acid 24.3ml; 0.2mol/L Na ₂ HPO (28.4g) ₄ 25.7ml; H ₂ O ₂ 0.1%; add ddH ₂ O to 1200ml; liquid B: TMB3.9g; citric acid 10.52g; EDTA1.86g; glycerin 300ml; add distilled water to 10000ml after heating to dissolve). Color was developed at 37°C in the dark for 15 minutes, and stop solution was added to each well to terminate the reaction, and the OD value of each well was read with a microplate reader ELX800 from Bio-Tek Company (wavelength 450 nm).

As a result, the above mouse anti-GPC3 monoclonal antibody (gp2#) and GPC3-Ag3 polyclonal antibody were used to establish a sandwich ELISA method to detect GPC3 recombinant protein diluted in 25% normal human serum, and the minimum detection concentration was 2ng/ml , in the range of 5ng-125ng, the GPC3 content and the O.D. degree show a good linear relationship, as shown in Figure 8.

Furthermore, the present inventors used the above-mentioned ELISA method to detect GPC3 in the serum of 106 normal persons, 96 cases of hepatitis cirrhosis and 364 cases of liver cancer patients. It was 11.7±12.23ng/ml. Serum GPC3 concentration of patients with liver cancer showed a skewed distribution, with an average value of 87.22ng/ml, 3 of whom were greater than 2000ng/ml, and the highest was 5432ng/ml. The difference of GPC3 between normal people, hepatitis cirrhosis and liver cancer was extremely significant (P<0.000), but there was no significant difference between normal people and hepatitis cirrhosis (Figure 9).

According to the receiver operating curve (Fig. 10) done to two groups of patients with liver cancer and hepatitis cirrhosis, when the positive value was 36.8ng/ml, the sensitivity and specificity of diagnosis were 32% and 2% respectively; When taking 27.1ng/ml, the sensitivity and specificity of diagnosis are 45.1% and 8.4% respectively (Table 7).

Table 7

GPC3 value (ng/ml) sensitivity 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, monoclonal antibody and polyclonal antibody, polyclonal antibody and polyclonal antibody

The present inventors used the various monoclonal antibodies and polyclonal antibodies prepared above to establish a sandwich ELISA detection method using different monoclonal or polyclonal antibodies as the first antibody or the second antibody for the detection of antigen GPC3. The ELISA detection steps adopted are similar to the method described in 1.

It was found that when used to establish a sandwich ELISA method, the detection sensitivities of various combinations vary greatly. When two polyclonal antibodies other than the rabbit anti-human GPC3-Ag3 polyclonal antibody were used as the primary antibody and the secondary antibody respectively, the sensitivity and specificity were poor. For example, if the plate is coated with rabbit anti-human GPC3-Ag4 and detected with anti-human GPC3-Ag1, the OD450 reading of the sample with a concentration of 100ng/ml is 0.234, and the OD450 reading of the negative sample is also 0.204, which does not meet the requirements of the diagnostic kit.

When the monoclonal antibody is compatible with the monoclonal antibody (such as 1F4+gp2#), the sensitivity is high, and the minimum detection concentration reaches 0.5ng/ml. However, the specificity is very poor, and normal human serum is prone to false positive results, and the false positive rate is even as high as 18%.

When the monoclonal antibody is compatible with the polyclonal antibody, the compatibility detection result of the rabbit anti-human GPC3-Ag3 polyclonal antibody (primary antibody) + mouse anti-GPC3 monoclonal antibody (gp2#) (secondary antibody) is accurate, and the polyantibody rabbit anti-human The OD reading of the same sample detected by GPC3-Ag3 polyclonal antibody (primary antibody) + 3C6 monoclonal antibody (secondary antibody) was close to that of the previous group. When polyantibody rabbit anti-human GPC3-Ag4 (primary antibody) + monoclonal antibody 7C8 (secondary antibody) was used for detection, the OD reading of the same sample was higher than that of rabbit anti-human GPC3-Ag3 polyclonal antibody and mouse anti-GPC3 monoclonal antibody ( gp2#) compatibility is low by 10-15%, and the accuracy is poor. The accuracy is even worse when polyantibody rabbit anti-human GPC3-Ag1 is used for detection with various monoclonal antibodies or polyantibodies prepared above.

Embodiment 7 Complementary Diagnosis of GPC3 and AFP

The best compatibility rabbit anti-human GPC3-Ag3 polyclonal antibody and mouse anti-GPC3 monoclonal antibody (gp2#) obtained by the inventors were used to establish an ELISA double sandwich kit; and the AFP detection radioimmunoassay kit ( Purchased from Shanghai Institute of Biological Products), AFP and GPC3 were detected simultaneously on 151 cases of hepatocellular carcinoma with clear pathological diagnosis.

As a result, when the positive judgment values of AFP and GPC3 were set at 20 ng/ml and 30 ng/ml respectively, the positive diagnostic rate of the former was 49% (74/151), and the latter was 40% (61/151) (Table 8). Significantly, 34 (44%) of the 77 cases of AFP-negative liver cancer were positive for GPC3, indicating that GPC3 is a liver cancer marker complementary to AFP. Combined detection of AFP and GPC3 can increase the diagnostic sensitivity of the former from 49% to 72%.

Table 8

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

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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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