CN114859062A - Chemiluminescence immunoassay method based on double-enzyme amplification system - Google Patents
Chemiluminescence immunoassay method based on double-enzyme amplification system Download PDFInfo
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Abstract
The invention relates to a chemiluminescent immunoassay method based on a double-enzyme amplification system, belonging to the field of immunoassay. Forming a complex between the detection antibody and catalase by a streptavidin-biotin signal amplification system, and using the enzymeReaction realization of H 2 O 2 To H 2 O and O 2 The conversion efficiency is high, horseradish peroxidase in the substrate is used as a high-efficiency catalyst, 4-bromophenol is used as a reinforcing agent of the horseradish peroxidase cycle transition, and a strong and durable chemiluminescent signal is generated. The invention improves the specificity and the accuracy of the method by a double-antibody sandwich strategy, the detection range of the IP-10 is 0.71-125,000pg/mL, the minimum detection limit is 0.63pg/mL, the coefficient of variation is 1.49%, the recovery rates in serum and plasma are 87.80-105.42% and 89.25-100.04% respectively, the detection method has simple operation, high sensitivity, wide detection range and strong stability, and can detect a plurality of samples simultaneously.
Description
Technical Field
The invention belongs to the field of immunological detection, and relates to a chemiluminescence immunoassay method based on a double-enzyme amplification system, which is used for detecting IP-10 in serum and plasma.
Background
Cancer is a major public health problem worldwide, and cancer markers are a class of bioactive substances generated and released in the processes of cancer occurrence and proliferation, mainly exist in blood, cells and tissues, and have important values on diagnosis, curative effect, recurrence monitoring and prognosis judgment of tumors.
IP-10 is a novel marker indicative of cancer occurrence and metastasis, and was originally used to detect M.tuberculosis infection. The performance of the molecule in the aspect of detecting latent tuberculosis can be compared with that of an interferon gamma release experiment, and the molecule also has extremely high sensitivity to active tuberculosis, so the molecule becomes an effective tuberculosis detection biomarker. In recent years, IP-10 has shown good diagnostic value in determining the occurrence and metastasis of cancer. Many studies have shown that IP-10 is highly correlated with the severity of cancer, the molecule in bloodThe expression of (b) is an excellent prediction index of the progress of tumors such as colorectal cancer and melanoma. In addition, IP-10 acts on CD4 + And CD8 + T cells regulate tumor immune response and have important diagnostic and therapeutic significance.
The existing detection methods of IP-10 comprise a conventional ELISA method, an mRNA release experiment and an electrochemical method. Compared with immunological methods, the sensitivity of mRNA release experiments is lower. Although the electrochemical method has high sensitivity, the method has high requirements on experimental operation and low detection flux, and is not favorable for clinical detection and practical application. The conventional ELISA method is one of the most commonly used methods for detecting blood markers, can be applied to samples with low purity of the detected object, has high data reproducibility, and has the advantages of low method sensitivity and nonlinear characteristics of enzyme amplification.
At present, ELISA detection kits for IP-10 are commercially available. The foreign detection kit has relatively high sensitivity, the minimum detection limit can reach 5.7pg/mL, the development of domestic related kits is less, and the sensitivity is generally low. The main advantages of chemiluminescent immunoassays compared to conventional ELISA methods are the strong signal and the absence of light source illumination, which provides higher sensitivity and signal-to-noise ratio for the establishment of the detection method.
Although the chemiluminescence immunoassay method has high sensitivity, because the serum components are very complicated, reducing the addition amount of serum or plasma is a common way to remove the nonspecific adsorption of blood components. Therefore, detection of protein molecules in serum or plasma generally requires higher sensitivity and greater specificity.
Furthermore, chemiluminescence has a luminous intensity of up to 10 5 Linear range relative to the light emitting unit, but generally exhibits a flicker type signal. The defect causes the traditional chemiluminescence immunoassay method to have high detection difficulty, low detection flux and great influence on detection sensitivity, thereby bringing challenges to practical application. Therefore, improving strategies to achieve higher sensitivity, wider detection range and greater stability is an important direction for chemiluminescent immunoassay methods.
Disclosure of Invention
The invention provides a chemiluminescence immunoassay method based on a double-enzyme amplification system, which aims to solve the problems of low sensitivity and generally low sensitivity of the existing detection method.
The technical scheme adopted by the invention is that the method comprises the following steps:
(1) adding 100 mu L of rabbit anti-IP-10 polyclonal antibody with the concentration of 5 mu g/mL to coat a white 96-hole Immuno plate, and incubating for 1h at 37 ℃;
(2) washing with PBST buffer solution for 3 times, each for 1 min;
(3) adding 200 μ L of 5% skimmed milk blocking solution into each well, and incubating at 37 deg.C for 1 h;
(4) adding 100 mu L of IP-10 fusion protein with different concentrations or 100 mu L of diluted sample to be detected into each hole, and incubating for 1h at 37 ℃;
(5) washing with PBST buffer solution for 3 times, each for 1 min;
(6) adding 100 mu L of detection antibody-streptavidin-biotin-catalase compound with the concentration of 5 mu g/mL into each hole, and incubating for 1h at 37 ℃;
(7) washing with PBST buffer solution for 3 times, each for 1 min;
(8) add 50. mu.L of substrate 1 per well, incubate for 5min at room temperature; subsequently 150 μ L of substrate 2 was added and incubated for 4min at room temperature;
(9) chemiluminescence intensity was measured using a Tecan Infinite 200 plate reader and IP-10 levels in serum or plasma were calculated from a standard curve.
The coating solution in the step (1) is 50-100mmol/L carbonate buffer solution, and the pH value is 9.6.
The formula of the PBST buffer solution in the step (2) comprises 8.1mmol/L disodium hydrogen phosphate, 1.9mmol/L sodium dihydrogen phosphate, 18mmol/L sodium chloride and 0.05% (v/v) Tween-20.
The preparation method of the IP-10 fusion protein in the step (4) of the invention comprises the following steps:
optimizing and synthesizing an IP-10CDS gene sequence added with a His label, subcloning the sequence to a pET-28a vector by using Nco I and Xho I enzyme cleavage sites and transforming the sequence into E.coli competence of E.coli Rosetta (DE3), wherein the nucleotide sequence of the IP-10 fusion protein is shown as SEQ ID NO.1, the amino acid sequence of the IP-10 fusion protein is shown as SEQ ID NO.2, the E.coli is induced at 28 ℃ to obtain the IP-10 fusion protein, and the pure protein can be obtained by a nickel affinity chromatography gravity column or a pre-packed column.
In the step (4), the range of the sample to be detected comprises serum and plasma, and 10 mu L of the sample to be detected and 90-200 mu L of diluent are added into each hole.
Wherein, the diluent in the step (4) is PBS buffer solution, and the formula is as follows: 9-36mmol/L sodium chloride, 20-60% (v/v) acetonitrile, 8.1mmol/L disodium hydrogen phosphate, 1.9mmol/L sodium dihydrogen phosphate, and pH value of 5.5-6.5;
preferably, the PBS formulation is: 18mmol/L sodium chloride, 40% (v/v) acetonitrile, 8.1mmol/L disodium hydrogen phosphate and 1.9mmol/L sodium dihydrogen phosphate, pH 6.0.
The preparation method of the detection antibody-streptavidin-biotin-catalase complex in the step (6) is as follows:
1) preparing a detection antibody-streptavidin conjugate based on a click chemistry coupling scheme of azide-alkyne cycloaddition reaction, adding 22.5 mu L of dibenzocyclooctyne-tetraethylene glycol-succinimidyl ester DBCO-PEG4-NHS with the concentration of 10mg/mL into 500 mu L of streptavidin with the concentration of 5mg/mL for overnight incubation, purifying by high performance liquid chromatography, then dissolving the IP-10 detection antibody in 0.01mol/L PBS buffer solution with the pH of 7.2, slowly adding the streptavidin-dibenzocyclooctyne SA-DBCO into the IP-10 detection antibody for overnight incubation, and purifying the streptavidin-antibody conjugate by size exclusion chromatography;
2) specifically reacting NHS-LC-LC-biotin with catalase primary amine to form a stable amido bond based on succinimidyl ester-long-arm biotin to prepare biotinylated catalase, dissolving the succinimidyl ester-long-arm biotin NHS-LC-biotin in DMF (dimethyl formamide) at the concentration of 10mg/mL, dissolving catalase in bicarbonate buffer solution with the pH value of 8.0-8.5 at the concentration of 5mg/mL, slowly dropping the solution of the succinimidyl ester-long-arm biotin NHS-LC-LC-biotin into the solution of the catalase, incubating at 4 ℃ for 12 hours, and removing unconjugated long-arm biotin from the reacted solution through a 10kDa ultrafiltration tube;
3) the detection antibody-streptavidin-biotin-catalase complex is incubated at room temperature based on the detection antibody-streptavidin conjugate and biotinylated catalase, and the detection antibody-streptavidin conjugate and the biotinylated catalase are incubated at room temperature for 1h according to a molar ratio of 1: 4.
Wherein the PBS buffer solution has the following formula: 9-36mmol/L sodium chloride, 20-60% (v/v) acetonitrile, 8.1mmol/L disodium hydrogen phosphate, 1.9mmol/L sodium dihydrogen phosphate, and pH of 5.5-6.5;
preferably, the PBS formulation is: 18mmol/L sodium chloride, 40% (v/v) acetonitrile, 8.1mmol/L disodium hydrogen phosphate and 1.9mmol/L sodium dihydrogen phosphate, pH 6.0.
In step (8) of the present invention, the substrate 1 was 0.005% H 2 O 2 The pH value of the solution is 7.0; the chemiluminescence enhancing agent in the substrate 2 comprises: 0.71 mu mol/L horseradish peroxidase and 35 mmol/L4-bromophenol, strong and durable luminescent signals, 11.5 pH value and 10 mu mol/L luminol concentration.
The invention has the beneficial effects that:
(1) the detection method captures the antigen through a double-antibody sandwich strategy, can be effectively distinguished from 6 common inflammatory factors in serum, and has the accuracy equivalent to that of a commercial ELISA kit.
(2) The detection method comprises the steps of enabling a detection antibody connected with streptavidin and catalase connected with long-arm biotin to form a compound, and improving the sensitivity of the method through a catalase and horseradish peroxidase double-enzyme enzymatic reaction, wherein the detection minimum by the method can reach 0.63pg/mL, and is 1-2 orders of magnitude higher than that of a commercially available ELISA kit.
(3) The chemiluminescent substrate takes horseradish peroxidase as a catalyst, 4-bromophenol as a horse radish peroxidase cyclic transition reinforcing agent, a strong and durable chemiluminescent signal is generated, the method sensitivity is high, the detection range is 3 orders of magnitude higher than that of a commercially available kit, the luminescence is stable, and the chemiluminescent substrate can be applied.
(4) The 4-bromophenol enhanced double-enzyme catalytic reaction can sensitively and stably detect the content of IP-10 in serum or plasma. The detection range is 0.71-125,000pg/mL, the lowest detection limit is 0.63pg/mL, the coefficient of variation is 1.49%, and the recovery rates in serum and plasma are 87.80-105.42% and 89.25-100.04% respectively. The method is simple and convenient to operate, high in sensitivity, wide in detection range and strong in stability, can be used for simultaneously detecting a plurality of samples, and has a relatively high application prospect.
Drawings
FIG. 1 is a graph of the absorption spectrum of streptavidin conjugated to anti-IP-10 antibody;
FIG. 2 is an absorption spectrum of catalase coupled with succinimidyl ester-long-arm biotin;
FIG. 3 is a diagram showing the expression and identification of IP-10 protein;
wherein: a is expression and purification of IP-10 fusion protein, 1, empty vector contrast 2, purification of protein expression 4.IP-10 protein of IP-10; b is Westernblot verification of IP-10, 1. Westernblot verification of IP-10, 2. negative control;
FIG. 4 is a graph of optimization of the conditions for binding of the capture antibody to IP-10;
wherein: a is the influence of pH on binding, B is the influence of sodium chloride concentration on binding, C is the influence of acetonitrile concentration on binding, and D is the influence of incubation time on binding;
FIG. 5 is a graph of optimization of the conditions for detecting binding of antibody to IP-10;
wherein: a is the influence of pH on binding, B is the influence of sodium chloride concentration on binding, C is the influence of acetonitrile concentration on binding, and D is the influence of incubation time on binding;
FIG. 6 is a condition optimization diagram for a chemiluminescent substrate, wherein:
a is the influence of the concentration of horseradish peroxidase on the chemiluminescence intensity, and the leftmost curve in the graph is as follows from top to bottom: 2.84. mu. mol/L, 1.42. mu. mol/L, 0.71. mu. mol/L, 0.36. mu. mol/L, 0.18. mu. mol/L;
b is the effect of the concentration of 4-bromophenol on the chemiluminescence intensity, and the leftmost curve in the graph is as follows: 20mmol/L, 25mmol/L, 30mmol/L, 35mmol/L, 40mmol/L, 45mmol/L, 50mmol/L, 55 mmol/L;
c is the influence of the luminol concentration on the chemiluminescence intensity, and the leftmost curve in the graph is as follows from top to bottom: 13. mu. mol/L, 12. mu. mol/L, 11. mu. mol/L, 10. mu. mol/L, 9. mu. mol/L, 8. mu. mol/L, 7. mu. mol/L;
d is H 2 O 2 The effect of concentration on the chemiluminescence intensity, # indicates the maximum chemiluminescence reading range outside the instrument, E is the effect of pH on the chemiluminescence intensity, F is H 2 O 2 Effect of incubation time on chemiluminescence intensity;
FIG. 7 is a kinetic graph of a chemiluminescent reaction;
wherein: a is the chemiluminescence kinetic curve without the addition of 4-bromophenol, B is the chemiluminescence kinetic curve with the addition of 4-bromophenol;
FIG. 8 is a graph showing the detection range and thus the sensitivity analysis result of the chemiluminescence immunoassay method of the present invention;
FIG. 9 is a graph showing the results of specific detection by the chemiluminescence immunoassay method of the present invention.
Detailed Description
Example 1
Comprises the following steps:
(1) adding 100 mu L of rabbit anti-IP-10 polyclonal antibody with the concentration of 5 mu g/mL to coat a white 96-hole Immuno plate, and incubating for 1h at 37 ℃; the coating solution is 50mmol/L carbonate buffer solution, and the pH value of the coating solution is 9.6;
(2) washing with PBST buffer solution for 3 times, each for 1 min; the formulation of PBST buffer solution comprises 8.1mmol/L disodium hydrogen phosphate, 1.9mmol/L sodium dihydrogen phosphate, 18mmol/L sodium chloride and 0.05% (v/v) Tween-20;
(3) adding 200 μ L of 5% skimmed milk blocking solution into each well, and incubating at 37 deg.C for 1 h;
(4) adding 100 mu L of IP-10 fusion protein with different concentrations or 100 mu L of diluted sample to be detected into each hole, and incubating for 1h at 37 ℃; the preparation method of the IP-10 fusion protein comprises the following steps:
optimizing and synthesizing an IP-10CDS gene sequence added with a His label, subcloning the sequence to a pET-28a vector by using Nco I and Xho I enzyme cleavage sites and transforming the sequence into E.coli competence of E.coli Rosetta (DE3), wherein the nucleotide sequence of the IP-10 fusion protein is shown as SEQ ID NO.1, the amino acid sequence of the IP-10 fusion protein is shown as SEQ ID NO.2, the E.coli is induced at 28 ℃ to obtain the IP-10 fusion protein, and the pure protein can be obtained by a nickel affinity chromatography gravity column or a pre-packed column;
the range of the sample to be detected comprises serum and plasma, and 10 mu L of the sample to be detected and 90 mu L of diluent are added into each hole;
the diluent is PBS buffer solution, and the formula comprises: 9mmol/L sodium chloride, 60% (v/v) acetonitrile, 8.1mmol/L disodium hydrogen phosphate, 1.9mmol/L sodium dihydrogen phosphate, pH 5.5;
(5) washing with PBST buffer solution for 3 times, each for 1 min;
(6) adding 100 mu L of detection antibody-streptavidin-biotin-catalase compound with the concentration of 5 mu g/mL into each hole, and incubating for 1h at 37 ℃; the preparation method of the detection antibody-streptavidin-biotin-catalase complex comprises the following steps:
1) preparing a detection antibody-streptavidin conjugate based on a click chemistry coupling scheme of azide-alkyne cycloaddition reaction, adding 22.5 mu L of dibenzocyclooctyne-tetraethylene glycol-succinimidyl ester DBCO-PEG4-NHS with the concentration of 10mg/mL into 500 mu L of streptavidin with the concentration of 5mg/mL for overnight incubation, purifying by high performance liquid chromatography, then dissolving the IP-10 detection antibody in 0.01mol/L PBS buffer solution with the pH of 7.2, slowly adding the streptavidin-dibenzocyclooctyne SA-DBCO into the IP-10 detection antibody for overnight incubation, and purifying the streptavidin-antibody conjugate by size exclusion chromatography;
2) specifically reacting NHS-LC-LC-biotin with catalase primary amine to form a stable amido bond based on succinimidyl ester-long-arm biotin to prepare biotinylated catalase, dissolving the succinimidyl ester-long-arm biotin NHS-LC-biotin in DMF (dimethyl formamide) at the concentration of 10mg/mL, dissolving catalase in bicarbonate buffer solution with the pH value of 8.0 at the concentration of 5mg/mL, slowly dropping the solution of the succinimidyl ester-long-arm biotin NHS-LC-LC-biotin into the solution of the catalase, incubating for 12h at 4 ℃, and removing unconjugated long-arm biotin from the reacted solution through a 10kDa ultrafiltration tube;
3) the detection antibody-streptavidin-biotin-catalase complex is incubated at room temperature based on the detection antibody-streptavidin conjugate and biotinylated catalase, and the detection antibody-streptavidin conjugate and the biotinylated catalase are incubated at room temperature for 1h according to a molar ratio of 1: 4;
(7) washing with PBST buffer solution for 3 times, each for 1 min;
(8) add 50. mu.L of substrate 1 per well and incubate for 5min at room temperature, followed by addition of 150. mu.L of substrate 2 and incubate for 4min at room temperature; wherein substrate 1 is 0.005% H 2 O 2 The pH value of the solution is 7.0; the chemiluminescence enhancing agent in the substrate 2 comprises: 0.71 mu mol/L horseradish peroxidase and 35 mmol/L4-bromophenol, strong and durable luminescent signals, the pH value of 11.5 and the luminol concentration of 10 mu mol/L;
(9) chemiluminescence intensity was measured using a Tecan Infinite 200 plate reader and IP-10 levels in serum or plasma were calculated from a standard curve.
Example 2
Comprises the following steps:
(1) adding 100 mu L of rabbit anti-IP-10 polyclonal antibody with the concentration of 5 mu g/mL to coat a white 96-hole Immuno plate, and incubating for 1h at 37 ℃; the coating solution is 75mmol/L carbonate buffer solution, and the pH value of the coating solution is 9.6;
(2) washing with PBST buffer solution for 3 times, each for 1 min; the formulation of PBST buffer solution comprises 8.1mmol/L disodium hydrogen phosphate, 1.9mmol/L sodium dihydrogen phosphate, 18mmol/L sodium chloride and 0.05% (v/v) Tween-20;
(3) adding 200 μ L of 5% skimmed milk blocking solution into each well, and incubating at 37 deg.C for 1 h;
(4) adding 100 mu L of IP-10 fusion protein with different concentrations or 100 mu L of diluted sample to be detected into each hole, and incubating for 1h at 37 ℃; the preparation method of the IP-10 fusion protein comprises the following steps:
optimizing and synthesizing an IP-10CDS gene sequence added with a His tag, subcloning the sequence to a pET-28a vector by using Nco I and Xho I enzyme cleavage sites and transforming the sequence to E.coli Rosetta (DE3) escherichia coli competence, wherein the nucleotide sequence of the IP-10 fusion protein is shown in SEQ ID NO.1, the amino acid sequence of the IP-10 fusion protein is shown in SEQ ID NO.2, the escherichia coli is induced at 28 ℃ to obtain IP-10 fusion protein, and pure protein can be obtained by a nickel affinity chromatography gravity column or a pre-packed column;
the range of the sample to be detected comprises serum and plasma, and 10 mu L of the sample to be detected and 100 mu L of diluent are added into each hole;
the diluent is PBS buffer solution, and the PBS formula is as follows: 18mmol/L sodium chloride, 40% (v/v) acetonitrile, 8.1mmol/L disodium hydrogen phosphate and 1.9mmol/L sodium dihydrogen phosphate, pH 6.0;
(5) washing with PBST buffer solution for 3 times, each for 1 min;
(6) adding 100 mu L of detection antibody-streptavidin-biotin-catalase compound with the concentration of 5 mu g/mL into each hole, and incubating for 1h at 37 ℃; the preparation method of the detection antibody-streptavidin-biotin-catalase complex comprises the following steps:
1) preparing a detection antibody-streptavidin conjugate based on a click chemistry coupling scheme of azide-alkyne cycloaddition reaction, adding 22.5 mu L of dibenzocyclooctyne-tetraethylene glycol-succinimidyl ester DBCO-PEG4-NHS with the concentration of 10mg/mL into 500 mu L of streptavidin with the concentration of 5mg/mL for overnight incubation, purifying by high performance liquid chromatography, then dissolving the IP-10 detection antibody in 0.01mol/L PBS buffer solution with the pH of 7.2, slowly adding the streptavidin-dibenzocyclooctyne SA-DBCO into the IP-10 detection antibody for overnight incubation, and purifying the streptavidin-antibody conjugate by size exclusion chromatography;
2) specifically reacting NHS-LC-LC-biotin with catalase primary amine to form a stable amido bond based on succinimidyl ester-long-arm biotin to prepare biotinylated catalase, dissolving the succinimidyl ester-long-arm biotin NHS-LC-biotin in DMF (dimethyl formamide) at the concentration of 10mg/mL, dissolving catalase in bicarbonate buffer solution with the pH value of 8.2 at the concentration of 5mg/mL, slowly dropping the solution of the succinimidyl ester-long-arm biotin NHS-LC-LC-biotin into the solution of the catalase, incubating for 12h at 4 ℃, and removing unconjugated long-arm biotin from the reacted solution through a 10kDa ultrafiltration tube;
3) the detection antibody-streptavidin-biotin-catalase complex is incubated at room temperature based on the detection antibody-streptavidin conjugate and biotinylated catalase, and the detection antibody-streptavidin conjugate and the biotinylated catalase are incubated at room temperature for 1h according to a molar ratio of 1: 4;
(7) washing with PBST buffer solution for 3 times, each for 1 min;
(8) add 50. mu.L of substrate 1 per well and incubate for 5min at room temperature, followed by addition of 150. mu.L of substrate 2 and incubate for 4min at room temperature; wherein substrate 1 is 0.005% H 2 O 2 The pH value of the solution is 7.0; the chemiluminescence enhancing agent in the substrate 2 comprises: 0.71 mu mol/L horseradish peroxidase and 35 mmol/L4-bromophenol, strong and durable luminescent signals, the pH value of 11.5 and the luminol concentration of 10 mu mol/L;
(9) chemiluminescence intensity was measured using a Tecan Infinite 200 plate reader and IP-10 levels in serum or plasma were calculated from a standard curve.
Example 3
Comprises the following steps:
(1) adding 100 mu L of rabbit anti-IP-10 polyclonal antibody with the concentration of 5 mu g/mL to coat a white 96-hole Immuno plate, and incubating for 1h at 37 ℃; the coating solution is 100mmol/L carbonate buffer solution, and the pH value of the coating solution is 9.6;
(2) washing with PBST buffer solution for 3 times, each for 1 min; the formulation of PBST buffer solution comprises 8.1mmol/L disodium hydrogen phosphate, 1.9mmol/L sodium dihydrogen phosphate, 18mmol/L sodium chloride and 0.05% (v/v) Tween-20;
(3) adding 200 μ L of 5% skimmed milk blocking solution into each well, and incubating at 37 deg.C for 1 h;
(4) adding 100 mu L of IP-10 fusion protein with different concentrations or 100 mu L of diluted sample to be detected into each hole, and incubating for 1h at 37 ℃; the preparation method of the IP-10 fusion protein comprises the following steps:
optimizing and synthesizing an IP-10CDS gene sequence added with a His label, subcloning the sequence to a pET-28a vector by using Nco I and Xho I enzyme cleavage sites and transforming the sequence into E.coli competence of E.coli Rosetta (DE3), wherein the nucleotide sequence of the IP-10 fusion protein is shown as SEQ ID NO.1, the amino acid sequence of the IP-10 fusion protein is shown as SEQ ID NO.2, the E.coli is induced at 28 ℃ to obtain the IP-10 fusion protein, and the pure protein can be obtained by a nickel affinity chromatography gravity column or a pre-packed column;
the range of the sample to be detected comprises serum and plasma, and 10 mu L of the sample to be detected and 200 mu L of diluent are added into each hole;
the diluent is PBS buffer solution, and the formula comprises: 36mmol/L sodium chloride, 20% (v/v) acetonitrile, 8.1mmol/L disodium hydrogen phosphate, 1.9mmol/L sodium dihydrogen phosphate, pH 6.5;
(5) washing with PBST buffer solution for 3 times, each for 1 min;
(6) adding 100 mu L of detection antibody-streptavidin-biotin-catalase compound with the concentration of 5 mu g/mL into each hole, and incubating for 1h at 37 ℃; the preparation method of the detection antibody-streptavidin-biotin-catalase complex comprises the following steps:
1) preparing a detection antibody-streptavidin conjugate based on a click chemistry coupling scheme of azide-alkyne cycloaddition reaction, adding 22.5 mu L of dibenzocyclooctyne-tetraethylene glycol-succinimidyl ester DBCO-PEG4-NHS with the concentration of 10mg/mL into 500 mu L of streptavidin with the concentration of 5mg/mL for overnight incubation, purifying by high performance liquid chromatography, then dissolving the IP-10 detection antibody in 0.01mol/L PBS buffer solution with the pH of 7.2, slowly adding the streptavidin-dibenzocyclooctyne SA-DBCO into the IP-10 detection antibody for overnight incubation, and purifying the streptavidin-antibody conjugate by size exclusion chromatography;
2) specifically reacting NHS-LC-LC-biotin with catalase primary amine to form a stable amido bond based on succinimidyl ester-long-arm biotin to prepare biotinylated catalase, dissolving the succinimidyl ester-long-arm biotin NHS-LC-biotin in DMF (dimethyl formamide) at the concentration of 10mg/mL, dissolving catalase in bicarbonate buffer solution with the pH value of 8.0-8.5 at the concentration of 5mg/mL, slowly dropping the solution of the succinimidyl ester-long-arm biotin NHS-LC-LC-biotin into the solution of the catalase, incubating at 4 ℃ for 12 hours, and removing unconjugated long-arm biotin from the reacted solution through a 10kDa ultrafiltration tube;
3) the detection antibody-streptavidin-biotin-catalase complex is incubated at room temperature based on the detection antibody-streptavidin conjugate and biotinylated catalase, and the detection antibody-streptavidin conjugate and the biotinylated catalase are incubated at room temperature for 1h according to a molar ratio of 1: 4;
(7) washing with PBST buffer solution for 3 times, each for 1 min;
(8) add 50. mu.L of substrate 1 per well and incubate for 5min at room temperature, followed by addition of 150. mu.L of substrate 2 and incubate for 4min at room temperature; wherein substrate 1 is 0.005% H 2 O 2 The pH value of the solution is 7.0; the chemiluminescence enhancing agent in the substrate 2 comprises: 0.71 mu mol/L horseradish peroxidase and 35 mmol/L4-bromophenol, strong and durable luminescent signals, the pH value of 11.5 and the luminol concentration of 10 mu mol/L;
(9) chemiluminescence intensity was measured using a Tecan Infinite 200 plate reader and IP-10 levels in serum or plasma were calculated from a standard curve.
The following describes the embodiments of the present invention with reference to the drawings and experimental examples. The following experimental examples are only used to illustrate the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
The experimental methods without specifying specific conditions in the following experimental examples were selected according to the methods and conditions which are conventional in the art, or according to the specifications of commercial products. Reagents and starting materials, the specific components of which are not indicated in the following examples, are commercially available. In the quantitative tests in the following examples, three replicates were set up and the results averaged.
The IP-10 chemiluminescence immunoassay method based on the double-enzyme amplification system provided by the invention has the following basic principles: capturing the IP-10 protein by a double antibody sandwich strategy; the introduction of streptavidin-linked detection antibody and long-armed biotin-linked catalase into the system to amplify the signal and efficiently deplete H in the substrate 2 O 2 (ii) a In a substrate of a chemiluminescence reaction, horseradish peroxidase is used as a high-efficiency catalyst, 4-bromophenol is used as an enhancer of horseradish peroxidase cyclic transition, and a strong and durable chemiluminescence signal is generated. The method is based on a double-antibody sandwich strategy, a streptavidin-biotin strategy, a catalase-horseradish peroxidase double-catalysis strategy and a 4-bromophenol-based substrate enhancement strategy, and can sensitively, specifically and stably detect the content of IP-10 in serum and plasma.
EXAMPLE 1 conjugation of streptavidin to detection antibodies
22.5 μ L of dibenzocyclooctyne-tetrapolyethylene glycol-succinimidyl ester DBCO-PEG4-NHS at a concentration of 10mg/mL was added to 500 μ L of streptavidin at a concentration of 5mg/mL and incubated overnight and purified by high performance liquid chromatography; then, the IP-10 detection antibody was dissolved in 0.01mol/L PBS buffer at pH 7.2, and streptavidin-dibenzocyclooctyne SA-DBCO was slowly added to the IP-10 detection antibody; after overnight incubation, the streptavidin-antibody conjugate was purified by size exclusion chromatography and the change in absorbance peaks before and after conjugation was detected by uv spectrophotometer. The results show that the prepared streptavidin-antibody conjugate has a significantly increased absorption peak at a 280nm absorption light, which indicates that the streptavidin and the detection antibody are successfully conjugated (fig. 1), and the conjugated protein complex is added with serum albumin BSA and proclin-300 to final concentrations of 0.5% and 0.05%, respectively, and is stored at 4 ℃.
Experimental example 2 preparation of biotinylated Catalase
The biotinylation catalase is prepared from NHS-LC-LC-biotin and primary amine of catalase 2 Chemical reactions between the groups result. First, succinimidyl ester-long-armed biotin NHS-LC-LC-biotin was dissolved in dimethylformamide DMF at a concentration of 10mg/mL, and catalase was dissolved in bicarbonate buffer at pH 8.2 at a concentration of 5 mg/mL. According to the result of the preliminary experiment, the molar ratio of the succinimidyl ester-long-arm biotin NHS-LC-LC-biotin to the catalase is set to be 50:1, the succinimidyl ester-long-arm biotin NHS-LC-LC-biotin solution is slowly dripped into the catalase solution, and the mixture is incubated for 12 hours at 4 ℃. The reacted liquid was transferred to a 10kDa ultrafiltration tube and centrifuged at 12000g at 4 ℃ for 10min to remove the unconjugated succinimidyl ester-long-arm biotin. The conjugate was washed 2 times with PBST resuspension and the change in absorbance peak before and after conjugation was detected by uv spectrophotometer. The results showed that the prepared biotinylated catalase conjugate had a significantly increased absorption peak at light absorption at 410nm, indicating that the biotinylated catalase was successfully prepared (FIG. 2). The conjugates were added to serum albumin BSA and proclin-300 to final concentrations of 0.5% and 0.05%, respectively, and stored at 4 ℃.
EXAMPLE 3 preparation of IP-10 fusion protein
The IP-10CDS gene sequence with the 6 × His tag was optimized and synthesized, subcloned into pET-28a vector using Nco I and Xho I enzymatic cleavage sites and transformed into e.coli Rosetta (DE3) e. The nucleotide sequence of the IP-10 fusion protein is shown in SEQ ID NO.1, the Escherichia coli is induced at 28 ℃ to obtain the IP-10 fusion protein, pure protein is obtained by nickel column affinity chromatography, and the amino acid sequence is shown in SEQ ID NO. 2.
Restriction sites were added upstream and downstream of the target sequence, with NcoI CATGCCATGG upstream and XhoI CTCGAGCGG downstream. Human IP-10 gene sequence (BC010954.1) was queried according to GENBANK, and E.coli strain K12 preferred codon optimization was performed on the target sequence. And a His tag is added to the N end of the target sequence, and the optimized sequence is shown as DNA SEQID 1. The gene of interest was synthesized and the gene sequence was subcloned into the pET-28a vector. The pET-28a-IP-10 recombinant plasmid is transformed into E.coli competence of E.coli Rosetta (DE3), IPTG is added at 28 ℃ to induce the expression of the E.coli protein, and the expressed protein sequence is shown as DNA SEQID 2. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis verification is carried out on the expressed protein, the result shows that an obvious band can be seen at the position of about 11kDa, and the high-purity protein is obtained by nickel column affinity chromatography and is successfully expressed by Westernblot verification (figure 3).
Experimental example 4 determination of optimum reaction conditions for Capture antibody and antigen
The IP-10 protein was obtained according to the method described in example 3, and the optimized conditions included pH, sodium chloride concentration, acetonitrile concentration and incubation time of the antigen and capture antibody binding buffer. The pH values were set at 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0, the sodium chloride concentrations were set at 9mmol/L, 18mmol/L, 36mmol/L, 72mmol/L and 18mmol/L, the acetonitrile concentrations (v/v) were set at 5%, 10%, 20%, 40% and 60%, and the incubation times were set at 15min, 30min, 45min, 60min and 75 min. Detection was performed by indirect ELISA and the binding capacity between the capture antibody and IP-10 was assessed by measuring absorbance at 450nm with a microplate reader (FIG. 4). The results showed that the optimum pH of the capture antibody and the IP-10 binding buffer was 6.0, the optimum sodium chloride concentration was 18mmol/L, the optimum acetonitrile concentration (v/v) was 40%, and the optimum incubation time was 60 min.
Experimental example 5 determination of optimum reaction conditions for detecting antibody and antigen
The IP-10 protein was obtained according to the method described in example 3, and the optimized conditions included pH, sodium chloride concentration, acetonitrile concentration and incubation time of the antigen and detection antibody binding buffer. Wherein the pH values were set at 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0, the sodium chloride concentrations were set at 9mmol/L, 18mmol/L, 36mmol/L, 72mmol/L and 18mmol/L, the acetonitrile concentrations (v/v) were set at 5%, 10%, 20%, 40% and 60%, and the incubation times were set at 15min, 30min, 45min, 60min and 75 min. The optimal reaction conditions of the detection antibody and the antigen were analyzed by indirect chemical immunoassay, and the binding ability between the detection antibody and IP-10 was evaluated by chemiluminescence intensity (FIG. 5). The results showed that the optimum pH of the detection antibody and the IP-10 binding buffer was 6.0, the optimum sodium chloride concentration was 36mmol/L, the optimum acetonitrile concentration (v/v) was 40%, and the optimum incubation time was 60 min. The optimal conditions for binding of the detection antibody to the antigen are essentially the same as those for binding of the capture antibody to the antigen described in example 4, except that the concentration of sodium chloride is slightly different. Since the binding capacity between the detection antibody and the antigen is not significantly different between the sodium chloride concentration of 18mmol/L and 36mmol/L, for the convenience of operation, the immunoreaction of IP-10 and the two antibodies is set to be 0.01mol/L PBS buffer solution, and the formula is as follows: 18mmol/L sodium chloride, 40% (v/v) acetonitrile, 8.1mmol/L disodium hydrogen phosphate and 1.9mmol/L sodium dihydrogen phosphate, pH 6.0.
Experimental example 6 determination of chemiluminescent substrates
4-bromophenol-enhanced horse radish peroxidase cycling through H-regulation in chemiluminescence reaction 2 O 2 The/horseradish peroxidase ratio promoted the persistence of the luminol reaction. The detection procedure of chemiluminescence immunoassay comprises 2 steps, firstly adding substrate 1 and catalase combined in a 96-well plate for full reaction, wherein the substrate 1 is H 2 O 2 Diluting the solution to pH 7.0; then through the substrate 2 and the rest of H in the system 2 O 2 The reaction produces chemiluminescence. The optimized conditions of the chemiluminescent substrate comprise horseradish peroxidase concentration, p-bromophenol concentration and luminol concentrationDegree, H 2 O 2 Concentration, pH and H 2 O 2 And (4) incubation time. Wherein horseradish peroxidase concentrations were set to 0.18. mu. mol/L, 0.36. mu. mol/L, 0.71. mu. mol/L, 1.42. mu. mol/L and 2.74. mu. mol/L, p-bromophenol concentrations were set to 20mmol/L, 25mmol/L, 30mmol/L, 35mmol/L, 40mmol/L, 45mmol/L, 50mmol/L and 55mmol/L, luminol concentrations were set to 7mmol/L, 8mmol/L, 9mmol/L, 10mmol/L, 11mmol/L, 12mmol/L and 13mmol/L, substrate pH values were set to 7.0, 8.0, 9.0, 10.0, 11.0, 12.0 and 13.0, H 2 O 2 The incubation time was set at 2min, 5min, 10min and 15 min. The results show that H in substrate 1 2 O 2 The optimal concentration is 0.005%, the optimal incubation time is 5min, and the optimal pH value is 7.0; the optimal concentration of horseradish peroxidase in the substrate 2 is 0.71 mu mol/L, the optimal concentration of 4-bromophenol is 35mmol/L, the optimal concentration of luminol is 10 mu mol/L, and the optimal pH value is 11.0 (figure 6). In the absence of the addition of 4-bromophenol, the chemiluminescence intensity decayed rapidly with time, whereas it increased rapidly within 4min after the addition of 4-bromophenol and then remained stable for the next 10min (FIG. 7). The components and the proportion of the substrate are determined according to the chemiluminescence intensity and the stabilization time, the chemiluminescence intensity after stabilization is increased by more than 7 times compared with the highest chemiluminescence intensity without adding 4-bromophenol, and a strong and durable chemiluminescence signal is provided
Experimental example 7 drawing of Standard Curve
Standard IP-10 was diluted in 125000pg/mL, 41666.67pg/mL, 13888.89pg/mL, 4629.63pg/mL, 1543.21pg/mL, 514.40pg/mL, 171.47pg/mL, 57.16pg/mL, 19.05pg/mL, 6.35pg/mL, 2.12pg/mL, 0.71pg/mL, 0.24pg/mL, 0pg/m, 3 replicate wells per concentration, as described in the chemiluminescence immunoassay described in example 7. The logarithm of the concentration of the standard was plotted as the abscissa and the corresponding chemiluminescence intensity value was plotted as the ordinate to form a standard curve (fig. 8). And taking the measured blank cavitation chemiluminescence intensity plus 3 times of standard deviation as the lowest detection limit of the method. The detection range of the method for IP-10 is 0.71-125000pg/mL, the linear range exceeds 6 orders of magnitude, and R is 2 The detection limit was 0.987, with a limit of 0.63 pg/mL. Change of repeat assayThe coefficient of variation was 1.49%. Compared with the existing IP-10 detection method, the method has lower detection limit and better stability.
Experimental example 8 sample addition and recovery assay
Different concentrations of IP-10 protein were added to serum and plasma samples and vortexed for 1 min. After mixing, the serum or plasma samples were diluted 1:10 and assayed according to the conditions of the chemiluminescent immunoassay described in example 7. The results are shown in Table 1, with the recovery of IP-10 in serum and plasma samples between 87.80-105.42% and 89.25-100.04%, respectively. The sample of the detection method is not affected by serum components after being diluted by 10 times, and has higher accuracy when being used for detecting an actual sample.
TABLE 1 recovery analysis of IP-10 in samples
RSD is relative standard deviation.
Experimental example 9 analysis of accuracy
The accuracy of the inventive method was analyzed by comparing a commercially available ELISA kit from Abcam with the method of the invention (Table 2). The method and the method of the invention are respectively used for detecting the content of IP-10 with different concentrations in serum, wherein the concentration of the IP-10 with 12.500ng/mL exceeds the detection range of the ELISA kit sold in the market, the detection result of the method is similar to the actual value, and the average recovery rate is 89.94%. 125pg/mL and 12.5pg/mL IP-10 can be effectively detected by the chemiluminescence immunoassay method and a commercially available ELISA kit, and the detection value of the method is basically consistent with that of the commercially available ELISA kit, so that the method is proved to have good accuracy. The detection range of the method is larger than that of a commercially available ELISA kit, and the method has wider application value.
TABLE 2 analysis of accuracy
# out of detection range.
Experimental example 10 specificity analysis
Since IP-10 is associated with a specific immune response during carcinogenesis, the specificity of the method of the present invention was analyzed using 6 inflammatory factors that significantly change during cancer. The changes in chemiluminescence intensity were detected using the method of the invention with the addition of 500pg/mL of each of the IP-10, IL-2, IL-4IL-Ra, INF-gamma, IL-10 and IL-1. beta. standard proteins. The results show that the method of the invention reacts only to the IP-10 protein and not to other proteins, indicating that the method has good specificity (figure 9).
Claims (10)
1. A chemiluminescence immunoassay method based on a double-enzyme amplification system is characterized by comprising the following steps:
(1) adding 100 mu L of rabbit anti-IP-10 polyclonal antibody with the concentration of 5 mu g/mL to coat a white 96-hole Immuno plate, and incubating for 1h at 37 ℃;
(2) washing with PBST buffer solution for 3 times, each for 1 min;
(3) adding 200 μ L of 5% skimmed milk blocking solution into each well, and incubating at 37 deg.C for 1 h;
(4) adding 100 mu L of IP-10 fusion protein with different concentrations or 100 mu L of diluted sample to be detected into each hole, and incubating for 1h at 37 ℃;
(5) washing with PBST buffer solution for 3 times, each for 1 min;
(6) adding 100 mu L of detection antibody-streptavidin-biotin-catalase compound with the concentration of 5 mu g/mL into each hole, and incubating for 1h at 37 ℃;
(7) washing with PBST buffer solution for 3 times, each for 1 min;
(8) add 50. mu.L of substrate 1 per well, incubate for 5min at room temperature; subsequently 150 μ L of substrate 2 was added and incubated for 4min at room temperature;
(9) chemiluminescence intensity was measured using a Tecan Infinite 200 plate reader and IP-10 levels in serum or plasma were calculated from a standard curve.
2. The chemiluminescent immunoassay method based on the dual-enzyme amplification system of claim 1, wherein: the coating solution in the step (1) is 50-100mmol/L carbonate buffer solution, and the pH value is 9.6.
3. The chemiluminescent immunoassay method based on the dual-enzyme amplification system of claim 1, wherein: the formula of the PBST buffer solution in the step (2) comprises 8.1mmol/L disodium hydrogen phosphate, 1.9mmol/L sodium dihydrogen phosphate, 18mmol/L sodium chloride and 0.05% (v/v) Tween-20.
4. The chemiluminescent immunoassay method based on the dual-enzyme amplification system of claim 1, wherein: the preparation method of the IP-10 fusion protein in the step (4) comprises the following steps:
optimizing and synthesizing an IP-10CDS gene sequence added with a His label, subcloning the sequence to a pET-28a vector by using Nco I and Xho I enzyme cleavage sites and transforming the sequence into E.coli competence of E.coli Rosetta (DE3), wherein the nucleotide sequence of the IP-10 fusion protein is shown as SEQ ID NO.1, the amino acid sequence of the IP-10 fusion protein is shown as SEQ ID NO.2, the E.coli is induced at 28 ℃ to obtain the IP-10 fusion protein, and the pure protein can be obtained by a nickel affinity chromatography gravity column or a pre-packed column.
5. The chemiluminescent immunoassay method based on the dual-enzyme amplification system of claim 1, wherein: in the step (4), the range of the sample to be detected comprises serum and plasma, and 10 mu L of the sample to be detected and 90-200 mu L of diluent are added into each hole.
6. The chemiluminescent immunoassay method based on the dual-enzyme amplification system of claim 5, wherein: the diluent is PBS buffer solution, and the formula is as follows: 9-36mmol/L sodium chloride, 20-60% (v/v) acetonitrile, 8.1mmol/L disodium hydrogen phosphate, 1.9mmol/L sodium dihydrogen phosphate, and pH value of 5.5-6.5.
7. The chemiluminescent immunoassay method based on the dual-enzyme amplification system of claim 6 wherein: the PBS formula is as follows: 18mmol/L sodium chloride, 40% (v/v) acetonitrile, 8.1mmol/L disodium hydrogen phosphate and 1.9mmol/L sodium dihydrogen phosphate, pH 6.0.
8. The chemiluminescent immunoassay method based on the dual-enzyme amplification system of claim 1, wherein: the preparation method of the detection antibody-streptavidin-biotin-catalase complex in the step (6) is as follows:
1) preparing a detection antibody-streptavidin conjugate based on a click chemistry coupling scheme of azide-alkyne cycloaddition reaction, adding 22.5 mu L of dibenzocyclooctyne-tetraethylene glycol-succinimidyl ester DBCO-PEG4-NHS with the concentration of 10mg/mL into 500 mu L of streptavidin with the concentration of 5mg/mL for overnight incubation, purifying by high performance liquid chromatography, then dissolving the IP-10 detection antibody in 0.01mol/L PBS buffer solution with the pH of 7.2, slowly adding the streptavidin-dibenzocyclooctyne SA-DBCO into the IP-10 detection antibody for overnight incubation, and purifying the streptavidin-antibody conjugate by size exclusion chromatography;
2) specifically reacting NHS-LC-LC-biotin with catalase primary amine to form a stable amido bond based on succinimidyl ester-long-arm biotin to prepare biotinylated catalase, dissolving the succinimidyl ester-long-arm biotin NHS-LC-biotin in DMF (dimethyl formamide) at the concentration of 10mg/mL, dissolving catalase in bicarbonate buffer solution with the pH value of 8.0-8.5 at the concentration of 5mg/mL, slowly dropping the solution of the succinimidyl ester-long-arm biotin NHS-LC-LC-biotin into the solution of the catalase, incubating at 4 ℃ for 12 hours, and removing unconjugated long-arm biotin from the reacted solution through a 10kDa ultrafiltration tube;
3) the detection antibody-streptavidin-biotin-catalase complex is incubated at room temperature based on the detection antibody-streptavidin conjugate and biotinylated catalase, and the detection antibody-streptavidin conjugate and the biotinylated catalase are incubated at room temperature for 1h according to a molar ratio of 1: 4.
9. The chemiluminescent immunoassay method based on the dual-enzyme amplification system of claim 8, wherein: the PBS buffer solution comprises the following formula: 9-36mmol/L sodium chloride, 20-60% (v/v) acetonitrile, 8.1mmol/L disodium hydrogen phosphate, 1.9mmol/L sodium dihydrogen phosphate, and pH value of 5.5-6.5.
10. The chemiluminescent immunoassay method based on the dual-enzyme amplification system of claim 1, wherein: the substrate 1 in the step (8) was 0.005% H 2 O 2 The pH value of the solution is 7.0; the chemiluminescence enhancing agent in the substrate 2 comprises: 0.71 mu mol/L horseradish peroxidase and 35 mmol/L4-bromophenol, the pH value is 11.5, and the luminol concentration is 10 mu mol/L.
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| DAN LIU ET AL: "Catalase-linked immunosorbent pressure assay for portable quantitative analysis", 《ANALYST》 * |
| VINCENT HUYNH ET AL: "Competitive Affinity Release for Long Term Delivery of Antibodies", 《ANGEWANDTE CHEMIE INTERNATIONAL EDITION》 * |
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| CN114859062B (en) | 2023-07-07 |
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