An auxiliary reagent for chemiluminescence detection of sCD14-ST and application thereof

Abstract

The application discloses an auxiliary reagent for chemiluminescence detection of sCD14-ST and application thereof, and relates to the technical field of sCD14-ST chemiluminescence detection. The pre-excitation agent comprises H2O2 0.5-1.5 wt%, methanol 1.2-1.8 wt%, 1-hydroxyethyl-2-undecyl-1-carboxymethyl imidazoline 0.1-0.5 wt%, and further comprises sodium lauroyl glutamate or sodium cocoyl glutamate or sodium myristoyl glutamate 0.05-0.1 wt%, with the rest being water. The excitation agent is NaOH 0.05-1 mol / L, methanol 1.2-1.8 wt%, 1-hydroxyethyl-2-undecyl-1-carboxymethyl imidazoline 0.1-0.5 wt%, and further comprises sodium lauroyl glutamate or sodium cocoyl glutamate or sodium myristoyl glutamate 0.05-0.1 wt%, with the rest being water. The auxiliary reagent is used in combination with 10,10'-dimethyl-9,9'-bisacridine-labeled detection antibody and capture antibody coupled on magnetic beads, and the chemiluminescence effect is obviously enhanced, and the sensitivity and accuracy are obviously improved.

Description

Technical Field

[0001] This invention relates to the field of chemiluminescence detection technology for sCD14-ST, and particularly to auxiliary reagents for chemiluminescence detection of sCD14-ST. Background Technology

[0002] Sepsis is an infectious systemic inflammatory response syndrome (SIRS) with diverse clinical manifestations and rapid disease progression. It not only reduces patients' quality of life and incurs high treatment costs, but also has a persistently high mortality rate. Currently, the consensus for treating this disease is to provide comprehensive treatment, primarily effective anti-infective therapy, in the early stages. However, since many non-infectious critically ill patients also have SIRS, how to identify infectious diseases early and differentiate between SIRS and sepsis has been a topic of interdisciplinary discussion and research direction for many years. In 2004, Japanese researchers discovered a new biomarker, soluble CD14 subtype (sCD14-ST, also known as Presepsin), which has shown good clinical significance in the diagnosis, assessment, and prognostic evaluation of sepsis.

[0003] CD14 is the receptor for the lipopolysaccharide (LPS)-lipopolysaccharide-binding protein (LBP) complex. Once it binds to the LPS-LBP complex, it participates in activating a series of signal transduction pathways, including the Toll-like receptor 4 (TLR4) / MD2 complex, thereby activating the inflammatory cascade and leading to a systemic inflammatory response. CD14 is anchored on the surface of monocytes and macrophages in membrane-bound form (mCD14) or distributed in plasma in soluble form (sCD14).

[0004] Chemiluminescent immunoassay is an immunoassay method that uses chemiluminescent reagents to directly label antigens / antibodies. Commonly used chemiluminescent substances include acridine esters. Large molecular antigens are generally analyzed using a sandwich method. For example, an acridine ester-labeled antibody / antigen is used as the labeling antibody / antigen, and another antibody / antigen is used as the capture antibody / antigen. The labeled antibody / antigen, the capture antibody / antigen, and the corresponding antigen / antibody form a sandwich complex. After adsorption, separation, and washing using magnetic beads, pre-excitation and excitation solutions are added before chemiluminescent detection. Alternatively, avidin can be used to label the capture antibody / antigen to improve its affinity with magnetic beads.

[0005] In chemiluminescence analysis, the pre-excitation solution cleaves the label from the complex and provides an acidic environment to prevent premature reaction. The excitation solution provides an alkaline environment to activate the chemiluminescence reaction. Commonly used pre-excitation solutions are H₂O₂ solutions, some with the addition of HNO₃; excitation solutions are NaOH solutions, some with the addition of Triton X-100. However, these pre-excitation and excitation solutions have certain effects on the chemiluminescence reaction process and luminescence intensity, thus affecting the detection sensitivity. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, this invention provides an auxiliary reagent for chemiluminescence detection of sCD14-ST and its application, specifically achieved through the following techniques.

[0007] An auxiliary reagent for chemiluminescence detection of sCD14-ST includes a pre-activator and an activator; the pre-activator comprises 0.5-1.5 wt% H2O2, 1.2-1.8 wt% methanol, 0.1-0.5 wt% 1-hydroxyethyl-2-undecyl-1-carboxymethylimidazoline, and further comprises 0.05-0.1 wt% sodium lauroyl glutamate, sodium cocoyl glutamate, or sodium myristoyl glutamate, with the remainder being water;

[0008] The activator consists of 0.05-1 mol / L NaOH, 1.2-1.8 wt% methanol, 0.1-0.5 wt% 1-hydroxyethyl-2-undecyl-1-carboxymethylimidazoline, and also includes 0.05-0.1 wt% sodium lauroyl glutamate, sodium cocoyl glutamate, or sodium myristoyl glutamate, with the remainder being water.

[0009] Preferably, the pre-activator comprises 1 wt% H2O2, 1.6 wt% methanol, 0.4 wt% 1-hydroxyethyl-2-undecyl-1-carboxymethylimidazoline, and 0.08 wt% sodium lauroyl glutamate, sodium cocoyl glutamate, or sodium myristoyl glutamate, with the remainder being water;

[0010] The activator consists of 0.5 mol / L NaOH, 1.6 wt% methanol, 0.4 wt% 1-hydroxyethyl-2-undecyl-1-carboxymethylimidazoline, and also includes 0.08 wt% sodium lauroyl glutamate, sodium cocoyl glutamate, or sodium myristoyl glutamate, with the remainder being water.

[0011] Preferably, the PBS also includes a cleaning agent, which is a PBS buffer containing Tween-20.

[0012] An application of the above-mentioned auxiliary reagent for chemiluminescent detection of sCD14-ST is characterized in that the detection antibody for detecting sCD14-ST is a monoclonal antibody labeled with 10,10'-dimethyl-9,9'-bisacridine, and the capture antibody for capturing sCD14-ST is a biotin-labeled monoclonal antibody coupled to magnetic beads.

[0013] A kit for chemiluminescence detection of sCD14-ST, comprising any of the auxiliary reagents described above.

[0014] Preferably, the above-mentioned chemiluminescence detection kit also includes calibrators, wherein the standards are prepared by using sCD14-ST standards with PBS buffer at a series of concentration gradients.

[0015] Compared with the prior art, the advantages of the present invention are as follows: The present invention provides an auxiliary reagent for chemiluminescence detection of sCD14-ST, consisting of a pre-exciter and an exciter. This auxiliary reagent is used in combination with a detection antibody labeled with 10,10'-dimethyl-9,9'-bisacrididine and a capture antibody conjugated to magnetic beads. Compared with the use of conventional pre-exciter and exciter solutions, the chemiluminescence effect of the above reagent combination is significantly enhanced, and the sensitivity and accuracy are significantly improved. Detailed Implementation

[0016] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] The pre-activator raw materials used in the following examples and comparative examples are H2O2, methanol, 1-hydroxyethyl-2-undecyl-1-carboxymethylimidazoline, and sodium lauroyl glutamate / sodium cocoyl glutamate / sodium myristoyl glutamate, which are prepared with deionized water in a specific ratio.

[0018] The activators selected are NaOH, methanol, 1-hydroxyethyl-2-undecyl-1-carboxymethylimidazoline, and sodium lauroyl glutamate / sodium cocoyl glutamate / sodium myristoyl glutamate, which are prepared with deionized water in a specific ratio.

[0019] The cleaning agent used was a PBS buffer containing Tween-20, pH 7.0-7.2, with a Tween-20 volume fraction of 2.5%. The basal PBS buffer was commercially available.

[0020] The calibrators are sCD14-ST standards with concentrations of 0, 3.75, 7.5, 15, 37.5, 75, 150, 375, 750, 1500, 3750, 7500, and 15000 pg / mL, prepared using basic PBS buffer.

[0021] The detection antibodies used were prepared using conventional methods, as detailed in the following examples and comparative examples:

[0022] (1) Take 100 μg of sCD14-ST monoclonal antibody and add labeling buffer (0.01-0.15 mol / L Na2CO3-NaHCO3 buffer, pH=8.0-11.0) to 300 μl;

[0023] Weigh 2 mg of 10,10'-dimethyl-9,9'-bisacridine and dissolve it in 450 μL of dimethylformamide (DMF);

[0024] (2) Mix the solution prepared in step (1) evenly, shake, and react at room temperature in the dark for 1 hour; add 100 μL of 10 g / L lysine solution, let stand for 15 minutes to terminate the reaction;

[0025] (3) Analyze the product from step (2) using a Sephadex G-50 column, wash the column with basic PBS buffer, collect the eluent, add 1% BSA (blocking buffer), and freeze for storage.

[0026] The capture antibody used is a biotin-labeled monoclonal antibody, conjugated to magnetic beads, and prepared using conventional methods, as detailed in the following examples and comparative examples:

[0027] (1) Take 50 μl of biotin-labeled sCD14-ST monoclonal antibody and 50 μl of streptavidin-coated magnetic bead suspension, mix well, and incubate at 37°C for 10 min.

[0028] (2) After incubation, place the reaction tube on a magnetic separator and let it stand for 1 minute to remove the supernatant; add 300 μl of rapid PBS buffer, place it on a magnetic separator and let it stand for 1 minute to remove the supernatant, and repeat this cycle 3 times; for later use.

[0029] Specifically, when performing chemiluminescence detection of sCD14-ST, the above-mentioned calibrators, pre-activators, activators, and cleaning agents are assembled into a kit for use together. The specific detection method is as follows:

[0030] (1) Establishing a standard curve: Add calibrator solutions of different concentrations to the suspension of magnetic beads conjugated with sCD14-ST monoclonal antibody, and then add sCD14-ST monoclonal antibody labeled with 10,10'-dimethyl-9,9'-bisacridine. After centrifugation in a water bath, add washing buffer, shake and centrifuge, remove supernatant, and repeat this washing method 3-5 times. Then add pre-activator and activator to the reaction system in sequence, mix well, and detect on a chemiluminescence detector to obtain the luminescence values ​​of standard solutions of different concentrations. Plot a standard curve based on the luminescence values.

[0031] (2) Sample detection: The sample to be tested (sCD14-ST monoclonal antibody, from Pujian (Wuhan) Biotechnology Co., Ltd.) was added to a suspension of magnetic beads conjugated with sCD14-ST monoclonal antibody, and then 10,10'-dimethyl-9,9'-bisacridine-labeled sCD14-ST monoclonal antibody was added. The luminescence value of the sample to be tested was obtained by operating according to the method in step (1). The luminescence value was compared with the standard curve to calculate the content of sCD14-ST in the sample to be tested.

[0032] To enable those skilled in the art to better understand the technical solutions of this disclosure, the technical solutions of this disclosure will be described in detail below with reference to specific embodiments.

[0033] Example 1

[0034] In this embodiment, the auxiliary reagents used consist of 1 wt% H2O2, 1.6 wt% methanol, 0.4 wt% 1-hydroxyethyl-2-undecyl-1-carboxymethylimidazoline, 0.08 wt% sodium lauroyl glutamate, and the remainder being deionized water.

[0035] The activator consists of 0.5 mol / L NaOH, 1.6 wt% methanol, 0.4 wt% 1-hydroxyethyl-2-undecyl-1-carboxymethylimidazoline, 0.08 wt% sodium lauroyl glutamate, and the remainder is deionized water.

[0036] Example 2

[0037] In this embodiment, the auxiliary reagents used consist of 1 wt% H2O2, 1.6 wt% methanol, 0.1 wt% 1-hydroxyethyl-2-undecyl-1-carboxymethylimidazoline, 0.05 wt% sodium lauroyl glutamate, and the remainder being deionized water.

[0038] The activator consists of 0.5 mol / L NaOH, 1.6 wt% methanol, 0.1 wt% 1-hydroxyethyl-2-undecyl-1-carboxymethylimidazoline, 0.05 wt% sodium lauroyl glutamate, and the remainder is deionized water.

[0039] Example 3

[0040] In this embodiment, the auxiliary reagents used consist of 1 wt% H2O2, 1.6 wt% methanol, 0.5 wt% 1-hydroxyethyl-2-undecyl-1-carboxymethylimidazoline, 0.1 wt% sodium lauroyl glutamate, and the remainder being deionized water.

[0041] The activator consists of 0.5 mol / L NaOH, 1.6 wt% methanol, 0.5 wt% 1-hydroxyethyl-2-undecyl-1-carboxymethylimidazoline, 0.1 wt% sodium lauroyl glutamate, and the remainder is deionized water.

[0042] Comparative Example 1

[0043] The difference between this comparative example and Example 1 is that the detection antibody used is labeled with acridine ester NSP-DMAE-NHS.

[0044] Comparative Example 2

[0045] The difference between this comparative example and Example 1 is that the pre-activator used consists of 2.0 wt% H2O2, 1.0 mol / L HNO3 and deionized water; the activator consists of 1.0 wt% Triton X-100, 0.5 mol / L NaOH and deionized water.

[0046] Comparative Example 3

[0047] The difference between this comparative example and Example 1 is that the pre-activator used consists of 2.0 wt% H2O2, 1 wt% DMF, 0.05 wt% Tween-20, and deionized water. The chemiluminescent excitation solution consists of 0.5 mol / L NaOH, 1 wt% DMF, 0.05 wt% Tween-20, and deionized water.

[0048] Test examples: Sensitivity, accuracy, and repeatability tests

[0049] 1. Sensitivity Test

[0050] The zero-concentration calibrator was used as the test sample for detection, repeated 5 times, and the luminescence values ​​(RLU values) of the 5 measurements were obtained. The mean (M) and standard deviation (SD) were calculated, and the RLU value corresponding to M+2SD was obtained. According to the calibration curve equation of the calibrator used in the kit, the RLU value corresponding to M+2SD was substituted into the standard curve to find the corresponding concentration value, which is the limit of detection. Finally, it was found that the analytical sensitivity using the auxiliary reagents provided in Examples 1-3 was 0.05 ng / ml, while the analytical sensitivity using the auxiliary reagents in Comparative Examples 1-3 could only reach 1-5 ng / ml.

[0051] 2. Accuracy Test

[0052] A 5 ng / ml concentration of sCD14-ST standard was used for testing. The measurement was repeated three times, and the relative deviation of the measured concentration was calculated using the formula B = [(MT) / T] × 100%, where B is the relative deviation, M is the average of the three measurements, and T is the accuracy reference concentration. Finally, it was found that using the auxiliary reagents provided in Examples 1-3 resulted in high accuracy, with relative deviations not exceeding 2.35%; while the relative deviations using the auxiliary reagents in Comparative Examples 1-3 ranged from 3.34% to 5.62%.

[0053] 3. Repeatability test

[0054] A 5 ng / ml concentration of sCD14-ST standard was tested, with each test repeated 8 times. The mean M and standard deviation SD of the 8 concentration measurements were calculated. The coefficient of variation (CV) was calculated using the formula CV = (SD / M) × 100%. Where: CV is the coefficient of variation, SD is the standard deviation of the 8 measurements, and M is the mean of the 8 measurements. Finally, it was found that using the auxiliary reagents provided in Examples 1-3 resulted in high repeatability with a coefficient of variation not exceeding 2.94%; while using the auxiliary reagents in Comparative Examples 1-3 resulted in the highest coefficient of variation reaching 3.86%.

[0055] Therefore, compared with existing conventional pre-activators and activators, the auxiliary reagent provided by this invention has very high sensitivity and accuracy in detecting sCD14-ST, and also has better repeatability.

[0056] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. The application of an auxiliary reagent for the chemiluminescent detection of sCD14-ST, characterized in that, The detection antibody used to detect sCD14-ST is a monoclonal antibody labeled with 10,10'-dimethyl-9,9'-bisacridine, and the capture antibody used to capture sCD14-ST is a biotin-labeled monoclonal antibody coupled to magnetic beads. The auxiliary reagents include a pre-activator and an activator; the pre-activator includes 0.5-1.5 wt% H2O2, 1.2-1.8 wt% methanol, 0.1-0.5 wt% 1-hydroxyethyl-2-undecyl-1-carboxymethylimidazoline, and also includes 0.05-0.1 wt% sodium lauroyl glutamate, sodium cocoyl glutamate, or sodium myristoyl glutamate, with the remainder being water; The activator consists of 0.05-1 mol / L NaOH, 1.2-1.8 wt% methanol, 0.1-0.5 wt% 1-hydroxyethyl-2-undecyl-1-carboxymethylimidazoline, and also includes 0.05-0.1 wt% sodium lauroyl glutamate, sodium cocoyl glutamate, or sodium myristoyl glutamate, with the remainder being water; The method of using the pre-activator and activator is as follows: Add calibrator solutions of different concentrations to the suspension of magnetic beads conjugated with sCD14-ST monoclonal antibody, and then add 10,10'-dimethyl-9,9'-bisacridine-labeled sCD14-ST monoclonal antibody. After water bath centrifugation, add washing buffer, shake and centrifuge, remove supernatant, and repeat this washing method 3-5 times. Then add the calibrator solution to the reaction system in sequence, mix well, and detect on a chemiluminescence detector to obtain the luminescence values ​​of standard solutions of different concentrations.

2. The application of the auxiliary reagent for chemiluminescent detection of sCD14-ST according to claim 1, characterized in that, The pre-activator comprises 1 wt% H2O2, 1.6 wt% methanol, 0.4 wt% 1-hydroxyethyl-2-undecyl-1-carboxymethylimidazoline, and 0.08 wt% sodium lauroyl glutamate, sodium cocoyl glutamate, or sodium myristoyl glutamate, with the remainder being water. The activator consists of 0.5 mol / L NaOH, 1.6 wt% methanol, 0.4 wt% 1-hydroxyethyl-2-undecyl-1-carboxymethylimidazoline, and also includes 0.08 wt% sodium lauroyl glutamate, sodium cocoyl glutamate, or sodium myristoyl glutamate, with the remainder being water.

3. The application of the auxiliary reagent for chemiluminescent detection of sCD14-ST according to claim 1, characterized in that, It also includes a cleaning agent, which is a PBS buffer containing Tween-20.