CN115684042A - Detection method of anti-DCDC 2 antibody in blood plasma - Google Patents
Detection method of anti-DCDC 2 antibody in blood plasma Download PDFInfo
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Abstract
The invention provides a method for detecting an anti-DCDC 2 antibody in blood plasma, which comprises the following steps: (1) Diluting human DCDC2 recombinant protein by the coating solution to obtain a recombinant protein diluent, adding the recombinant protein diluent into an ELISA plate, standing overnight at low temperature, and performing antigen coating; (2) Washing the enzyme label plate by using PBST buffer solution, adding a sealing agent, and sealing at room temperature; (3) Washing the ELISA plate by using a PBST buffer solution, adding to-be-detected plasma, and incubating at room temperature; (4) Washing the enzyme label plate with PBST buffer solution, adding horseradish peroxidase labeled goat anti-human IgG, and incubating at room temperature; (5) And then washing the ELISA plate by using the PBST buffer solution, adding the TMB developing solution, incubating, adding the TMB developing stop solution, and measuring the absorbance at 450nm by using an ELISA reader. The method of the invention improves the accuracy and reliability of the diagnosis of the bile duct cancer by detecting the anti-DCDC 2 antibody in the plasma, and provides a method with better specificity and sensitivity for the diagnosis of the bile duct cancer.
Description
Technical Field
The invention relates to the technical field of G01N33/574, and in particular relates to a detection method of an anti-DCDC 2 antibody in blood plasma.
Background
Cholangiocarcinoma is a malignant tumor originated from epithelial cells lining the biliary system, is second to liver cancer in incidence of hepatobiliary malignant tumor, and shows an increasing trend year by year. The bile duct cancer is high in concealment, disease positions are located in the liver, and certain symptoms are similar to liver cancer in clinical aspects, so that the bile duct cancer is usually diagnosed in middle and late stages, patients are treated radically, and the survival rate of the patients is greatly reduced. At present, the diagnosis of cholangiocarcinoma relies mainly on clinical manifestations, imaging, laboratory and pathological examinations. However, patients with early stage cholangiocarcinoma have no obvious clinical symptoms, and only a few patients are fortunately discovered by physical examination at the early stage of cholangiocarcinoma. The method has obvious negative effects on diagnosis and treatment of the bile duct cancer and improvement of the survival rate of patients. With the progress and wide application of medical equipment and medical technology, the diagnosis rate of bile duct cancer is improved to some extent, but the diagnosis rate of bile duct cancer is still insufficient on the imaging of early tiny lesions.
The detection of the tumor marker is the main method for diagnosing and evaluating the tumor, and the enzyme-linked immunosorbent assay is the commonly used detection method of the tumor marker. Chinese patent CN107271661A discloses a method for preparing a liver cancer early detection kit, which is prepared by applying the principle of enzyme-linked immunosorbent assay. However, the kit has detection specificity and pertinence. The most studied of the biliary duct cancer-related tumor markers at present are CA19-9, CA242, CEA, CA125, CA211, AFP and the like, but the related tumor markers lack specificity and sensitivity for diagnosis of biliary duct cancer. Based on the detection, the invention provides a detection method of the anti-DCDC 2 antibody in the plasma, and the accuracy and reliability of early diagnosis of the cholangiocarcinoma are improved by detecting the anti-DCDC 2 antibody in the plasma.
Disclosure of Invention
The invention provides a detection method of an anti-DCDC 2 antibody in blood plasma, which comprises the following steps:
(1) Antigen coating: diluting human DCDC2 recombinant protein by the coating solution to obtain recombinant protein diluent, adding the recombinant protein diluent into an ELISA plate, and carrying out antigen coating at low temperature overnight;
(2) Washing the enzyme label plate by using PBST buffer solution, adding a sealing agent, and sealing at room temperature;
(3) Washing the ELISA plate by using a PBST buffer solution, adding to-be-detected plasma, and incubating at room temperature;
(4) Washing the enzyme label plate with PBST buffer solution, adding horseradish peroxidase labeled goat anti-human IgG, and incubating at room temperature;
(5) And then washing the ELISA plate by using the PBST buffer solution, adding the TMB developing solution, incubating, adding the TMB developing stop solution, and measuring the absorbance at 450nm by using an ELISA reader.
In a preferred embodiment, the method comprises the following steps:
(1) Antigen coating: diluting human DCDC2 recombinant protein by the coating solution to obtain a recombinant protein diluent, adding the recombinant protein diluent into an ELISA plate, standing overnight at low temperature, and performing antigen coating;
(2) Washing the ELISA plate with PBST buffer solution for three times, standing for 2min each time, adding a sealing agent, and sealing at room temperature;
(3) Washing the ELISA plate with PBST buffer solution for three times, standing for 2min each time, adding plasma to be detected, and incubating at room temperature;
(4) Washing the ELISA plate by using PBST buffer solution for five times, standing for 2min each time, adding horseradish peroxidase labeled goat anti-human IgG, and incubating at room temperature;
(5) And washing the ELISA plate by using the PBST buffer solution for three times, standing for 2min each time, adding the TMB developing solution, incubating, adding the TMB developing termination solution, and measuring the absorbance at 450nm by using an ELISA reader.
In a preferred embodiment, the coating solution in step (1) is an ELISA coating solution, and the concentration of the recombinant protein in the recombinant protein dilution in step (1) is 0.1-25. Mu.g/mL.
In a preferred embodiment, the concentration of the recombinant protein in the recombinant protein diluent in step (1) is 0.1 to 5. Mu.g/mL. More preferably, the concentration of the recombinant protein in the recombinant protein diluent in the step (1) is 1. Mu.g/mL.
In the present application, the applicant determined from the preliminary experiments of plasma antibody concentration that the optimal concentration of recombinant protein in the recombinant protein dilution in step (1) was 1 μ g/mL, with the best experimental results and the least background interference.
In a preferred embodiment, the ELISA coating is purchased from bio ss (BIOSS) in boaoson biotechnology, beijing.
In a preferred embodiment, the volume of the recombinant protein diluent added to the microplate in step (1) is 50. Mu.L/well, and the overnight low temperature in step (1) is 0-4 ℃ and not 0 ℃. More preferably, the low temperature overnight temperature is 4 ℃.
In a preferred embodiment, the blocking agent in step (2) is a PBST solution containing skim milk.
In a preferred embodiment, the skim milk-containing PBST solution has a skim milk mass concentration of 5%.
In a preferred embodiment, the blocking time for the room temperature blocking operation in step (2) is 1 to 8 hours. More preferably, the blocking time for the room temperature blocking operation in the step (2) is 1h.
In a preferred embodiment, the plasma is added in a volume of 50. Mu.L/well in step (3).
In a preferred embodiment, the incubation time for the incubation at room temperature in step (3) is 1-3h. More preferably, the incubation time for the room temperature incubation operation in the step (3) is 1.5h.
The anti-DCDC 2 antibody finds that the expression of the anti-DCDC 2 antibody in the plasma of a biliary duct cancer patient is abnormal when the applicant detects the plasma of the biliary duct cancer patient and the single-knife stone patient through a proteome chip, and the applicant also verifies that the titer of the anti-DCDC 2 antibody in the biliary duct cancer patient is higher than that of a healthy population through ELISA. Therefore, the applicant believes that the anti-DCDC 2 antibody can be used as a marker with specificity and sensitivity for detecting, diagnosing and prognosing bile duct cancer.
In the conventional detection process, serum is usually used as a primary antibody, and in the application, the applicant finds that the detection accuracy of the anti-DCDC 2 antibody can be improved by using plasma as the primary antibody, and the detection of the anti-DCDC 2 antibody is taken as one of the methods for early diagnosis of the bile duct cancer. The applicant speculates that the possible reasons are that more protein components in blood are not coagulated and settled by the blood coagulation function in the plasma compared with the serum, the loss of the anti-DCDC 2 antibody is less in the preparation process of the plasma compared with the loss of the serum, and the accuracy of the bile duct cancer diagnosis through judging the content of the anti-DCDC 2 antibody in clinic can be improved by detecting the content of the anti-DCDC 2 antibody in the plasma preparation process. In addition, the applicant finds that the dilution concentration of the DCDC2 recombinant protein in the step (1) has a large influence on the absorbance value of the anti-DCDC 2 antibody in the plasma, and only when the dilution concentration of the DCDC2 recombinant protein in the step (1) is 1 mu g/mL, the background value is small, and the absorbance value of the anti-DCDC 2 antibody is large and the interference is small.
In a preferred embodiment, in the step (4), the horseradish peroxidase-labeled goat anti-human IgG is diluted by the coating solution, and the volume ratio of the horseradish peroxidase-labeled goat anti-human IgG to the coating solution is 1: (2000-5000).
In a preferred embodiment, the volume ratio of the horseradish peroxidase-labeled goat anti-human IgG to the coating solution is 1:3000.
in a preferred embodiment, the incubation time for the room temperature incubation operation in step (4) is 0.5-2h.
In a preferred embodiment, the TMB color developing solution in step (5) is incubated under ambient temperature and dark conditions for 10min.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a detection method of an anti-DCDC 2 antibody in plasma, which adopts DCDC2 recombinant protein as a detection antigen and proposes a set of ELISA detection method of the anti-DCDC 2 antibody aiming at the DCDC2 recombinant protein, adopts specific DCDC2 recombinant protein concentration and also adopts plasma as a primary antibody, improves the expression quantity of the anti-DCDC 2 antibody to improve the absorbance by reducing the background interference in the detection process, improves the detection accuracy of the anti-DCDC 2 antibody, improves the accuracy of diagnosing cholangiocarcinoma clinically by judging the content of the anti-DCDC 2 antibody, provides a detection marker with high specificity and high sensitivity for clinically diagnosing cholangiocarcinoma at the early stage.
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FIG. 1 shows the results of the detection of anti-DCDC 2 antibodies in the plasma of healthy volunteers, patients with gallstones, patients with cholangiocarcinoma, according to the method of example 2.
Detailed Description
Example 1
This example presents a method for determining the optimal concentration of the recombinant protein diluent of step (1) in a method for detecting anti-DCDC 2 antibodies in plasma:
(1) Diluting human DCDC2 recombinant protein by using ELISA (enzyme-linked immuno sorbent assay) coating solution to prepare recombinant protein gradient diluent with DCDC2 recombinant protein concentration of 0ng/mL, 100ng/mL, 500ng/mL, 1 mu g/mL, 5 mu g/mL and 25 mu g/mL, adding the recombinant protein gradient diluent into an ELISA plate, adding the recombinant protein gradient diluent into the ELISA plate with the volume of 50 mu L/hole, and carrying out antigen coating at 4 ℃ overnight;
(2) Washing the ELISA plate with PBST buffer solution for three times, each time washing the ELISA plate with 100 mu L, each time standing for 2min, adding skimmed milk PBST solution with skimmed milk mass concentration of 5%, and sealing for 1h at room temperature;
(3) Washing the ELISA plate with PBST buffer solution for three times, each time washing with 100 μ L, each time standing for 2min, adding to-be-detected blood plasma and reference substance, adding volume of 50 μ L/hole, and incubating at room temperature for 1.5h; the reference was an outsourcing standard purchased from Proteintech.
(4) Washing the ELISA plate with PBST buffer solution for five times, washing 100 mu L each time, standing for 2min each time, adding 50 mu L/hole of horseradish peroxidase labeled goat anti-human IgG, and incubating for 1h at room temperature; the volume ratio of the horseradish peroxidase labeled goat anti-human IgG to the coating solution is 1:3000;
(5) And washing the ELISA plate by using PBST buffer solution for three times, washing for 100 mu L each time, standing for 2min each time, adding 100 mu L of TMB developing solution, endowing for 10min at room temperature in a dark place, adding 100 mu L of TMB developing termination solution, and measuring the absorbance at 450nm by using an ELISA reader.
The results of the absorbance at 450nm are shown in the following table:
| coating density | 0ng/mL | 100ng/mL | 500ng/mL | 1μg/mL | 5μg/mL | 25μg/ |
| Plasma | ||||||
| 1 | 0.059 | 1.455 | 1.762 | 1.911 | 1.916 | 0.755 |
| |
0.068 | 1.258 | 1.906 | 2.178 | 2.202 | 0.591 |
| Plasma 3 | 0.067 | 1.312 | 1.733 | 1.725 | 1.966 | 0.758 |
| Control of | 0.063 | 0.067 | 0.063 | 0.063 | 0.081 | 0.147 |
The results show that at a coating concentration of 1. Mu.g/mL, the experimental results are optimal and the background interference is minimal.
Wherein the human DCDC2 recombinant protein was purchased from Proteintech.
The ELISA coating was purchased from bio technologies limited, boaosen, beijing.
TMB color development liquid is purchased from Biyuntian biotechnology, and is P0209-100mL; TMB chromogenic stop solution is purchased from Biyunnan biotechnology, P0215-100mL.
Goat anti-human IgG was labeled with horseradish peroxidase and purchased in Biyuntian biotechnology.
PBST buffer was prepared by mixing PBS buffer purchased from Gibco with Tween20 at a mass concentration of 0.05%.
Example 2
This example provides a method for detecting anti-DCDC 2 antibodies in plasma, using the optimal coating concentration obtained in example 1, the specific steps are as follows:
(1) Antigen coating: diluting human DCDC2 recombinant protein by using ELISA coating solution to obtain recombinant protein dilution solution with DCDC2 recombinant protein concentration of 1 mug/mL, adding the diluted solution into an ELISA plate, adding the diluted solution into the ELISA plate, and carrying out antigen coating overnight at 4 ℃;
(2) Washing the ELISA plate with PBST buffer solution for three times, each time washing the ELISA plate with 100 mu L, each time standing for 2min, adding skimmed milk PBST solution with skimmed milk mass concentration of 5%, and sealing at room temperature for 1h;
(3) Washing the ELISA plate with PBST buffer solution for three times, each time washing the ELISA plate with 100 mu L, each time standing for 2min, adding blood plasma to be detected, wherein the adding volume of the blood plasma is 50 mu L/hole, and incubating for 1.5h at room temperature;
(4) Washing the ELISA plate with PBST buffer solution for five times, each time washing the ELISA plate with 100 mu L, each time standing for 2min, adding horseradish peroxidase labeled goat anti-human IgG with 50 mu L/hole, and incubating for 1h at room temperature; the volume ratio of the horseradish peroxidase labeled goat anti-human IgG to the coating solution is 1:3000;
(5) And washing the ELISA plate by using PBST buffer solution for three times, washing for 100 mu L each time, standing for 2min each time, adding 100 mu L of TMB developing solution, endowing for 10min at room temperature in a dark place, adding 100 mu L of TMB developing termination solution, and measuring the absorbance at 450nm by using an ELISA reader.
Wherein human DCDC2 recombinant protein was purchased from Proteitech.
The ELISA coating was purchased from bio technologies limited, boaosen, beijing.
TMB color development liquid is purchased from Biyuntian biotechnology, and is P0209-100mL; TMB chromogenic stop solution is purchased from Biyuntian biotechnology, P0215-100mL.
Goat anti-human IgG was labeled with horseradish peroxidase and purchased in Biyuntian biotechnology.
PBST buffer was prepared by mixing PBS buffer purchased from Gibco with Tween20 at a mass concentration of 0.05%.
According to the specific steps of the detection of this example, the anti-DCDC 2 antibody in plasma of healthy volunteers, patients with gallstone, and patients with cholangiocarcinoma was detected, and the results are shown in FIG. 1. The distribution content of the anti-DCDC 2 antibody in the plasma of the patient with the cholangiocarcinoma is obviously higher than that of healthy volunteers and patients with gallstone.
Claims (10)
1. A method for detecting an anti-DCDC 2 antibody in plasma, comprising the steps of:
(1) Antigen coating: diluting human DCDC2 recombinant protein by the coating solution to obtain a recombinant protein diluent, adding the recombinant protein diluent into an ELISA plate, standing overnight at low temperature, and performing antigen coating;
(2) Washing the enzyme label plate by using PBST buffer solution, adding a sealing agent, and sealing at room temperature;
(3) Washing the ELISA plate by using a PBST buffer solution, adding to-be-detected plasma, and incubating at room temperature;
(4) Washing the enzyme label plate by using a PBST buffer solution, adding horseradish peroxidase labeled goat anti-human IgG, and incubating at room temperature;
(5) And then washing the ELISA plate by using the PBST buffer solution, adding the TMB developing solution, incubating, adding the TMB developing stop solution, and measuring the absorbance at 450nm by using an ELISA reader.
2. The method according to claim 1, wherein the coating solution in step (1) is an ELISA coating solution, and the concentration of the recombinant protein in the recombinant protein dilution in step (1) is 0.1 to 25. Mu.g/mL.
3. The method for detecting an anti-DCDC 2 antibody in plasma according to claim 2, wherein the concentration of the recombinant protein in the recombinant protein dilution of step (1) is 0.1 to 5 μ g/mL.
4. The method for detecting anti-DCDC 2 antibody in blood plasma of claim 1, wherein said blocking agent in step (2) is PBST solution containing skim milk.
5. The method of claim 4, wherein the concentration of skim milk in the PBST solution containing skim milk is 5% by mass.
6. The method for detecting anti-DCDC 2 antibody in plasma according to claim 1, wherein the blocking time for the room temperature blocking operation in step (2) is 1-8h.
7. The method for detecting anti-DCDC 2 antibody in plasma according to claim 1, wherein the incubation time for the incubation at room temperature in step (3) is 1-3h.
8. The method for detecting the anti-DCDC 2 antibody in the plasma according to claim 1, wherein in the step (4), the horseradish peroxidase-labeled goat anti-human IgG is diluted by a coating solution, and the volume ratio of the horseradish peroxidase-labeled goat anti-human IgG to the coating solution is 1: (2000-5000).
9. The method for detecting anti-DCDC 2 antibody in plasma according to claim 8, wherein the incubation time of the incubation operation at room temperature in step (4) is 0.5-2h.
10. The method for detecting anti-DCDC 2 antibody in blood plasma according to claim 1, wherein the incubation condition of the TMB color developing solution in step (5) is room temperature and away from light, and the incubation time is 10min.
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