CN103777026B - A kind of for diagnosing the kit of hepatocellular carcinoma - Google Patents

Abstract

The invention provides and a kind ofly to it is characterized in that for diagnosing the kit of hepatocellular carcinoma, comprise Fuc-PON1 half-quantitative detection elisa plate, described Fuc-PON1 half-quantitative detection elisa plate is made up of AAL-ELISA plate and Protein-ELISA plate.Kit of the present invention has high flux property, comparatively high specific, detects and quick and precisely waits outstanding advantages, have important clinical value to the diagnosis of liver cancer.

Description

A kit for diagnosing hepatocellular carcinoma

technical field

The invention relates to the field of biomedicine and is connected with applied basic medical research and clinical research; covering oncology, diagnostics and glycobiology.

Background technique

HCC is a common malignant tumor with multiple genes, multiple factors and complex pathological mechanisms. In my country, hepatitis B virus carriers account for 7.18% of the population, which is 2/3 of the world's hepatitis B carriers. The number of HCC patients related to this is huge, accounting for about half of the world. The vast majority of patients have insidious onset, and they are already at an advanced stage when they seek medical treatment after the appearance of clinical symptoms, losing the opportunity for treatment. Therefore, early diagnosis is very important to improve the survival rate of patients. An ideal liver cancer marker needs to have high specificity and be able to distinguish liver cancer from liver cirrhosis, hepatitis, liver regeneration nodules and other diseases; at the same time, it needs high sensitivity to be able to prompt the diagnosis of liver cancer at an early stage, and at the same time It should have the characteristics of easy detection, repeatability, and non-invasive operation.

Glycoprotein glycan has macroscopic and microscopic heterogeneity, and its structure and function changes are closely related to the occurrence and development of diseases. Glycans play important roles in many key biological processes, such as cell adhesion, molecular transport and clearance, receptor activation, signal transduction, and endocytosis. During the development of organisms, the structure of glycans is also changing, and glycans are expressed differently at different differentiation stages. During the occurrence of cancer, changes in glycosylation are ubiquitous. If the changes in glycosylation can be paid attention to, the specificity and sensitivity of diagnosis can be improved. Therefore, the use of detection of abnormal glycoprotein glycan structure to diagnose malignant tumors has attracted considerable attention. For example, the change of AFP-L3 has become an important glycan marker for detection of liver cancer. At present, the technical methods for detecting the structural changes of glycoprotein glycans include biological mass spectrometry, liquid chromatography, and lectin affinity imprinting, etc. Among them, lectins are a kind of glycoproteins that exist in most plants, and their biggest feature is that they can recognize sugars. Glycans in proteins and glycolipids, especially those with complex structures in cell membranes, are cell membrane surface determinants, and their binding to glycans is non-covalent and reversible. AAL is a plant lectin that is compatible with α-linked fucose (Terminal ^αFuc and ±Sia-Lex ). Fucose modification can endow glycans with many unique functional properties. It plays an important role in adhesion to endothelium, host-microbe interaction and ontogeny. In addition, fucose also participates in the glycan structure of some important adhesion molecules, which is closely related to tumor metastasis. Increased levels of sialylLewis ^x and sialylLewis ^a were found in cancers, and the loss of A and B blood group antigens was accompanied by increased expression of H blood group antigens and Lewis ^y in many tumors.

PON1 is a glycoprotein synthesized by the liver. It binds to high-density lipoprotein (HDL) and exists in the blood and liver. When chronic liver disease has active lesions, the liver cells continue to die, the synthesis decreases, and it enters the circulation accordingly. PON1 decreased, resulting in decreased serum PON1 activity. Post-hepatitic cirrhosis has both continuous necrosis of liver cells and liver fibrosis. The pathological process is very complicated, and the activity of serum PON1 is significantly reduced. Serum PON1 activity can be used as one of the biochemical indicators for judging the survival of implanted liver, and it can be used together with other biochemical indicators to monitor the liver function of liver transplantation patients. According to reports, HCV-induced inflammation followed by fibrosis, PON1 levels will continue to decline, as a combination of many oxidative and antioxidant molecules together as a clinically useful monitoring indicator. It was recently found that the abnormal protein content of HDL particles and some lipids, including the glycosylation of PON1, would lead to a decrease in the expression level of PON1 in the serum of patients with liver disease. In the previous work, it was found that the ability of the serum unit PON1 to bind to AAL in patients with liver cancer was significantly enhanced, indicating that the content of fucosylated glycans in the unit PON1 of patients with liver cancer increased. The present invention utilizes the high affinity of AAL to fucosylated glycans, and through the preparation and detection of AAL-ELISA and Protein-ELISA, respectively forms PON1 antibody-antigen-biotin-labeled AAL complex and PON1 antibody-antigen-biotin Mark the antibody complex, obtain the ratio of the absorbance of PON1 binding AAL to the absorbance of PON1 protein, Fuc-PON1, and establish a tumor discriminant diagnostic function through retrospective research on clinical data, obtain the ROC curve and Cutoff value, and compare AFP-L3 and Fuc at the same time - Differences in the accuracy of PON1 in the diagnosis of liver cancer. The invention is suitable for large-scale screening of high-incidence sites of liver cancer or people with liver diseases, and has very important significance for improving the diagnosis rate of liver cancer, realizing early intervention and treatment, and reducing the mortality rate of liver cancer.

Contents of the invention

The purpose of the present invention is to provide a kit for diagnosing hepatocellular carcinoma (HCC) with higher accuracy.

In order to achieve the above object, the present invention provides a kit for diagnosing hepatocellular carcinoma, which is characterized in that it comprises a Fuc-PON1 semi-quantitative detection ELISA plate, and the Fuc-PON1 semi-quantitative detection ELISA plate is composed of AAL- Composed of ELISA plate and Protein-ELISA plate.

Preferably, the kit for diagnosing hepatocellular carcinoma further comprises an HCC prediction model:

P=exp(-0.915+0.275*Fuc-PON1)／[1+exp(-0.915+0.275*Fuc-PON1)]

The calculation method of the Fuc-PON1 value is: the ratio of the actual OD value of PON1 combined with AAL in each serum sample to the actual OD value of PON1 protein in the same serum sample, the actual OD of PON1 combined with AAL in the serum sample The value is obtained by AAL-ELISA plate detection, and the actual OD value of PON1 protein in serum samples is obtained by Protein-ELISA plate detection. The cutoff value of this model for distinguishing liver cancer and liver cirrhosis is 2.2727.

Preferably, the kit for diagnosing hepatocellular carcinoma further comprises a PON1 capture antibody at a concentration of 1 μg/mL.

Preferably, the kit for diagnosing hepatocellular carcinoma further comprises a first blocking agent and a second blocking agent, the first blocking agent is 3% BSA solution for blocking the AAL-ELISA plate, the second blocking agent A solution containing 1% BSA and 0.05% NaN ₃ for blocking Protein-ELISA plates.

Preferably, the kit for diagnosing hepatocellular carcinoma further comprises a first oxidant, which is a pH 4.0 solution containing 100mM NaIO ₄ and 50mM citric acid for oxidation of the AAL-ELISA plate.

Preferably, the kit for diagnosing hepatocellular carcinoma further comprises biotin-labeled AAL and biotin-labeled antibody.

Preferably, the kit for diagnosing hepatocellular carcinoma further includes a blood sample collection device and operating instructions.

The PON1 protein mentioned in the present invention has significantly enhanced AAL-affinity ability of unit PON1 as determined by previous proteomics work, indicating that the content of fucosylated glycans of unit PON1 in patients with liver cancer increases.

The present invention mainly establishes and optimizes AAL-ELISA and Protein-ELISA methods, determines optimal capture antibody concentration, loading amount of serum sample, lectin concentration and detection antibody concentration; And selects suitable AAL-ELISA blocking agent and oxidizing agent . Exclude the cross-reaction of PON1 antibody with other antigens, establish the detection range and repeatability of AAL-ELISA and Protein-ELISA for PON1, and construct a Fuc-PON1 semi-quantitative detection system that can be used for clinical serum sample screening.

As shown in Figure 1, the present invention combines and parallel analyzes the expression levels of fucose (Fuc) and protein of paraoxonase (PON) 1 in serum through the preparation of AAL-ELISA and Protein-ELISA, and is used for liver cancer and liver cancer. Quantitative detection of fucose-conjugated content (Fuc-PON1) per unit of PON1 protein in serum of cirrhotic patients. And according to the retrospective study of Fuc-PON1 detection data and clinical data, the establishment of tumor discriminant diagnostic function can be used for the early diagnosis of primary hepatocellular carcinoma.

Compared with prior art, the beneficial effect of the present invention is:

The present invention forms antibody-antigen-biotin-labeled AAL complex and antibody-antigen-biotin-labeled antibody complex respectively through the preparation and detection of AAL (poplar agglutinin)-ELISA and Protein-ELISA, and the absorbance of PON1 combined with AAL (OD value) and the absorbance ratio of PON1 protein as the unit of measure, the content of fucose-bound fucose in PON1 protein (Fuc-PON1), through retrospective research on clinical data, a tumor discriminant diagnostic function was established, and the receiver operating curve ( ROC) and discrimination (Cutoff) value, the diagnostic sensitivity and specificity of this function for HCC were 60.3% and 71.4%, respectively, and the AUC was 0.736, which was better than alpha-fetoprotein N-glycan core fucose (AFP-L3). The method and kit of the invention have outstanding advantages such as high throughput, high specificity, fast and accurate detection, and the like, and have important clinical value for the diagnosis of liver cancer.

Description of drawings

Figure 1 is a schematic diagram of the principle of the high-throughput Fuc-PON1 detection system;

Figure 2a is a graph of the linear detection range of AAL-ELISA

Figure 2b is a graph of the linear detection range of Protein-ELISA.

Figure 3 is the ROC curves constructed by the Fuc-PON1 and AFP-L3 detection systems respectively;

detailed description

The present invention will be described in detail below in conjunction with the examples.

Example:

1. A test kit for diagnosing hepatocellular carcinoma, comprising the following components:

1. Fuc-PON1 semi-quantitative detection ELISA plate, the Fuc-PON1 semi-quantitative detection ELISA plate is composed of AAL-ELISA plate and Protein-ELISA plate.

2. HCC prediction model:

P=exp(-0.915+0.275*Fuc-PON1)／[1+exp(-0.915+0.275*Fuc-PON1)]

The calculation method of the Fuc-PON1 value is: the ratio of the actual OD value of PON1 combined with AAL in each serum sample to the actual OD value of PON1 protein in the same serum sample, the actual OD of PON1 combined with AAL in the serum sample The value is obtained by AAL-ELISA plate detection, and the actual OD value of PON1 protein in serum samples is obtained by Protein-ELISA plate detection. The cutoff value of this model for distinguishing liver cancer and liver cirrhosis is 2.2727.

3. PON1 capture antibody, the concentration of which is 1 μg/mL. (R&D Systems, Catalog#DYC5816, Part#843480)

4. The first blocking agent (3% BSA in PBS, pH7.2-7.4).

5. The second blocking agent (1% BSA, 0.05% NaN ₃ inPBS, pH7.2-7.4).

6. The first oxidizing agent (100 mM NaIO ₄ , 50 mM Citric acid, pH 4.0).

7. Biotin-labeled AAL.

8. Biotin-labeled antibody (1% BSAinPBS, diluted at pH 7.2-7.4).

9. HRP-streptavidin. (Thermo Scientific, Product #N100).

10. Blood sample collection device and operating instructions.

2. Detection method:

(1) Pretreatment method of AAL-ELISA plate and Protein-ELISA plate:

1. Take two ELISA plates and use them as AAL-ELISA plate and Protein-ELISA plate respectively;

2. Capture antibody: add PON1 capture antibody to AAL-ELISA plate and Protein-ELISA plate, the concentration of PON1 capture antibody in AAL-ELISA is 1 μg/mL, 100 μL/well; the concentration of PON1 capture antibody in Protein-ELISA is 1 μg/mL , 100 μL/well. Incubate overnight at room temperature.

3. Plate washing: Wash the plate 4 times with PBST (0.05% Tween20inPBS, pH7.2-7.4), soak for 2 minutes each time, 300 μL/well, spin dry, and pat dry the liquid in the wells on absorbent paper.

4. Blocking: AAL-ELISA plate uses the first blocking agent (3%BSAinPBS, pH7.2-7.4); Protein-ELISA uses the second blocking agent (1%BSA, 0.05%NaN ₃ inPBS, pH7.2-7.4) Block at room temperature for 1 hour, 200 μL/well.

5. Plate washing: Wash the plate 4 times with PBST (0.05% Tween20inPBS, pH7.2-7.4), soak for 2 minutes each time, 300 μL/well, spin dry, and pat dry the liquid in the wells on absorbent paper.

6. Oxidation: AAL-ELISA plates use the first oxidant (100mMNaIO ₄ , 50mM Citricacid, pH4.0); Protein-ELISA uses the second blocking agent (1%BSA, 0.05%NaN ₃ inPBS, pH7.2-7.4) for oxidation , 4 ℃ dark reaction 1h, 200μL/well.

7. Plate washing: Wash the plate 4 times with PBST (0.05% Tween20inPBS, pH7.2-7.4), soak for 2 minutes each time, 300 μL/well, spin dry, and pat dry the liquid in the wells on absorbent paper. The processed AAL-ELISA plate and Protein-ELISA plate were respectively obtained for the following steps.

(2) Fuc-PON1 semi-quantitative detection ELISA detection method:

1. Adding samples: add 50 μL/well of diluted serum sample to AAL-ELISA plate (diluted with PBS 1:30); pH7.2-7.4 diluted to 100μL), room temperature for 2h.

2. Plate washing: wash the plate 4 times with PBST (0.05% Tween20inPBS, pH7.2-7.4), soak for 2 minutes each time, 300 μL/well, spin dry, and pat dry the liquid in the wells on absorbent paper.

3. Detection reagent: add 1 μg/mL biotin-labeled AAL (diluted in PBST) to the AAL-ELISA plate; add 600 ng/mL biotin-labeled antibody (1% BSAinPBS, pH7.2-7.4 dilution) to the Protein-ELISA plate , 100μL/well, room temperature 2h.

4. Plate washing: Wash the plate 4 times with PBST (0.05% Tween20inPBS, pH7.2-7.4), soak for 2 minutes each time, 300 μL/well, spin dry, and pat dry the liquid in the wells on absorbent paper.

5. Enzyme conjugate working solution: add HRP-sztreptavidin to the AAL-ELISA plate (using blocking agent 1, 1:12500 dilution); add HRP-sztreptavidin to the Protein-ELISA plate (using 1% BSAinPBS, 1:12500 dilution), 100μL/well, 30min at room temperature.

6. Plate washing: Wash the plate 4 times with PBST (0.05% Tween20inPBS, pH7.2-7.4), soak for 2 minutes each time, 300 μL/well, spin dry, and pat dry the liquid in the wells on absorbent paper.

7. Add substrate solution: mix substrate solution according to TMB:H ₂ O ₂ =1:2, 100 μL/well, room temperature for 20 minutes.

8. Add stop solution: 100μL StopSolution/well, the order of adding the stop solution is the same as that of the substrate solution.

9. Detection: Immediately measure the OD value of each well with a NanoQuantinfinite M200 (TECAN) microplate reader at a wavelength of 450 nm. The power of the microplate reader should be turned on in advance, the instrument should be preheated, and the detection program should be set.

(3) Fuc-PON1 semi-quantitative detection ELISA analysis method:

1. Data: The OD value of each serum sample should be subtracted from the actual OD value of the blank well. The ratio of the actual OD value (AAL-ELISA) of each serum sample PON1 combined with AAL to the actual OD value (Protein-ELISA) of the same serum sample PON1 protein is the Fuc-PON1 value.

Calculate the P value according to the above HCC prediction model, compare the P value with the Cutoff value, >2.2727 is liver cancer, and <2.2727 is liver cirrhosis.

Using the kit and detection method in the examples, the expression levels of fucose and protein of PON1 in 86 cases of liver disease serum samples (43 cases of liver cirrhosis and 43 cases of liver cancer patients, all clinically diagnosed) were analyzed in parallel, and Fuc- PON1 content. The above-mentioned HCC prediction model established by SPSS software logistic regression analysis was used to detect and distinguish liver cancer and liver cirrhosis with a cutoff value of 2.2727. (AFP-L3 detection kit is purchased from Abnova company (Catalog#KA1187), detects AFP-L3 content in the above-mentioned 86 cases of liver disease serum samples according to kit operation steps, adopts SPSS software to obtain ROC curve.), compare kit of the present invention and The accuracy and specificity of AFP-L3 to the diagnosis of liver cancer, the results are shown in Figure 3, the sensitivity of the kit of the present invention is 60.3%, specificity 71.4%, accuracy is 64%, AUC is 0.736, and AFP-L3 is 0.736 to the The diagnostic sensitivity, specificity and accuracy of HCC were all 60.5%, and the AUC was 0.612. As shown in Table 1, the mean value of AFP-L3 in patients with liver cirrhosis was 0.8466, with a standard deviation of 0.32; the mean value of AFP-L3 in patients with liver cancer was 0.9779, with a standard deviation of 0.36; the p value was 0.0393. The mean value of Fuc-PON1 in patients with liver cirrhosis was 2.6315, with a standard deviation of 2.18; the mean value of Fuc-PON1 in patients with liver cancer was 4.3609, with a standard deviation of 3.08; the p value was 0.002.

Table 1: Fuc-PON1 and AFP-L3 detection results compared with each other

Utilize the kit of the present invention to analyze the Fuc-PON1 level in 40 cases of test set serum (serum samples are from inpatients in the Department of Liver Surgery and Gastroenterology of the First Affiliated Hospital of Guangxi Medical University), referring to the Cutoff value, the results show that 27 cases of patients have been successfully predicted, The accuracy rate is 67.5%. Therefore, the present invention has high specificity and accuracy, and has important clinical value for the prediction of liver cancer.

Get 1 case of mixed serum samples (10 cases are mixed, and the serum samples come from the inpatients of the Department of Liver Surgery and Gastroenterology of the First Affiliated Hospital of Guangxi Medical University) to detect the AAL-ELISA and Whether other proteins have non-specific cross-reactions, the results are shown in Table 2. The OD value of other proteins detected by AAL-ELISA in the kit of the present invention is consistent with that of PBS buffer, and the OD value of serum PON1 is much greater than other proteins.

Table 2: No cross-reaction in AAL-ELISA in Fuc-PON1 detection system:

Take 3 cases of mixed serum samples (each 1 case is mixed with 10 cases of serum samples, and the serum samples come from inpatients in the Department of Liver Surgery and Gastroenterology of the First Affiliated Hospital of Guangxi Medical University) to test the repeatability of the kit used for the diagnosis of hepatocellular carcinoma. The serum of each case was tested for 3 times, and the mean value, standard deviation and CV value of Fuc-PON1 in each case were calculated. The results are shown in Table 3.

Table 3: Three replicate detection results of Fuc-PON1 detection system

Adopt the serum of known PON1 protein concentration to carry out doubling dilution, determine the AAL-ELISA linear detection range in the kit of the present invention, wherein PON1 protein concentration in the serum is determined to adopt ELISA quantitative detection kit (R&DSystems, Catalog#DYC5816), the result is as shown in the figure As shown in 2a, the recombinantPON1 standard (R&DSystems, Catalog#DYC5816, Part#843482) was used for doubling dilution to determine the linear detection range of Protein-ELISA. The results are shown in Figure 2b. The linear detection range of Protein-ELISA is greater than that of AAL-ELISA, so the detection range of the Fuc-PON1 detection system is the linear detection range of AAL-ELISA. The quantitative linear detection range of the kit of the invention is 0.71-7.1 ng/mL.

Claims (5)

1. A test kit for diagnosing hepatocellular carcinoma, characterized in that it comprises a Fuc-PON1 semi-quantitative detection ELISA plate, and the Fuc-PON1 semi-quantitative detection ELISA plate consists of an AAL-ELISA plate and a Protein-ELISA plate Composition; Wherein, the calculation method of Fuc-PON1 value is: the ratio of the actual OD value of PON1 binding AAL in each serum sample and the actual OD value of PON1 protein in the same serum sample, the actual OD value of PON1 binding AAL in the described serum sample The OD value is obtained by AAL-ELISA plate detection, and the actual OD value of PON1 protein in serum samples is obtained by Protein-ELISA plate detection; it also includes the HCC prediction model: P=exp(-0.915+0.275*Fuc-PON1)/[1+exp(-0.915+0.275*Fuc-PON1)] The cutoff value of the model for distinguishing liver cancer from cirrhosis was 2.2727. 2. The kit for diagnosing hepatocellular carcinoma according to claim 1, further comprising a PON1 capture antibody at a concentration of 1 μg/mL. 3. The kit for diagnosing hepatocellular carcinoma as claimed in claim 1, characterized in that, the kit for diagnosing hepatocellular carcinoma also comprises a first blocking agent and a second blocking agent, the first blocking agent One blocking agent is 3% BSA solution for blocking AAL-ELISA plates, and the second blocking agent is a solution containing 1% BSA and 0.05% NaN ₃ for blocking Protein-ELISA plates. 4. The test kit for diagnosing hepatocellular carcinoma as claimed in claim 1, characterized in that, the test kit for diagnosing hepatocellular carcinoma also comprises a first oxidant, the first oxidant being pH 4.0 A solution containing 100 mM NaIO ₄ and 50 mM citric acid was used for the oxidation of the AAL-ELISA plate. 5. The test kit for diagnosing hepatocellular carcinoma as claimed in claim 1, wherein the test kit for diagnosing hepatocellular carcinoma also comprises biotin-labeled AAL and biotin-labeled antibody .