Quadruple test method for inflammatory factor markers CRP, SAA, PCT and IL-6

Abstract

The invention provides a quadruple test method for inflammatory factor markers CRP, SAA, PCT and IL-6, and belongs to the technical field of biological detection. According to the present invention, the digital fluorescence immunoassay technology is adopted, the antibodies are immobilized on the detection plate, and after the antigen in the sample is combined with the immobilized antibodies, the fluorescein-labeled antibody is combined with the other antigen determinant on the antigen so as to detect the antigen in the sample; therefore, a sandwich type compound structure is constructed between the immobilized antibody and the fluorescein labeled antibody, the higher the antigen concentration is, the more the fluorescent particles are, and the content of the antigen is quantified through the number of the fluorescent particles. By means of the unique design, high-sensitivity and high-precision detection of antigens can be achieved, the content of the antigens can be accurately quantified by accurately counting the number of the fluorescent particles, the influence of lipemia / hemolysis on the detection result can be avoided, the detection range is wide, sensitivity is high, accuracy is high, and detection is automatic, convenient and rapid.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a four-in-one test method for inflammatory markers CRP, SAA, PCT, and IL-6. Background Technology

[0002] C-reactive protein (CRP) is a protein that rapidly increases in plasma when the body experiences infection or tissue damage. As a classic acute-reactive protein, it is synthesized by the liver and has the ability to activate complement and enhance phagocytic cell function, helping to clear damaged, necrotic tissue and invading pathogens. CRP is considered an important inflammatory factor. When the body encounters various inflammatory responses, such as infections by viruses, bacteria, and mycoplasma, its level specifically increases, with large fluctuations and high sensitivity. The increase in CRP is positively correlated with the severity of inflammation and is not affected by genetic, individual, or racial differences. Once the infection is controlled and the inflammation subsides, CRP levels decrease rapidly. In particular, CRP rises significantly within 6 to 12 hours after bacterial infection, while the increase is not significant in viral infections. This characteristic makes CRP an important indicator for differentiating between bacterial and viral infections.

[0003] Serum amyloid A (SAA) is an acute-phase protein produced by hepatocytes. Its concentration rises rapidly during inflammation, increasing 1000-fold within 4 to 6 hours after infection and peaking within 12 hours. Its sensitivity is superior to other inflammatory markers, such as CRP. SAA not only rises rapidly but also falls quickly, returning to normal levels rapidly after pathogen clearance, thus reflecting the body's inflammatory state in real time. SAA is of great significance in the diagnosis, treatment, and prognostic assessment of infectious diseases, especially in the differential diagnosis of bacterial and viral pneumonia. Notably, SAA and CRP are often used together to differentiate between bacterial and viral infections. Elevated levels of both suggest a possible bacterial infection; while elevated SAA without elevated CRP suggests a viral infection.

[0004] Procalcitonin (PCT) is a precursor protein secreted solely by thyroid C cells. It possesses high diagnostic specificity and sensitivity for bacterial infections, helping physicians differentiate between bacterial and non-bacterial infections and assess infection severity. Under normal circumstances, PCT levels in the human body are extremely low, but blood PCT concentrations significantly increase when infection, inflammation, or tissue damage is present. PCT levels are closely related to the activity of systemic inflammation; higher values ​​indicate more severe inflammation. PCT is an important clinical marker for diagnosing bacterial infections, as it is rapidly released into the bloodstream within 2 to 3 hours after bacterial infection. It shows good correlation with infection and sepsis and is valuable for monitoring antibiotic treatment. Due to its high stability and specificity, PCT is currently recommended by numerous scholars both domestically and internationally as a specific biomarker for bacterial infections.

[0005] Interleukin-6 (IL-6) is a pleiotropic cytokine, a small glycoprotein composed of 184 amino acids, often existing in monomeric form. IL-6 can be produced by various cell types, including lymphoid cells, non-lymphoid cells, and fibroblasts, and is closely related to the pathophysiological processes of many clinical diseases. It is an inflammatory mediator produced earlier than CRP and PCT and has a longer duration of action, thus being considered a sensitive early "warning" biomarker for SIRS sepsis caused by common bacterial infections or systemic inflammatory responses. Its advantage lies in its ability to detect acute infections early, and dynamic monitoring helps assess the severity and progression of infectious diseases. In acute inflammatory responses, IL-6 plays a central role, rapidly produced within 1 to 2 hours after injury or infection, exhibiting extremely high sensitivity.

[0006] While CRP is frequently tested in outpatient and emergency departments for inflammation, its ability to differentiate between bacterial and viral infections is not particularly strong in clinical interpretation. PCT, on the other hand, exhibits a strong correlation with disease severity, with significant differences in results, especially demonstrating high specificity in the diagnosis of sepsis. SAA, as an extremely sensitive inflammatory marker, can effectively rule out inflammatory infections with a negative result, while a positive result can provide strong support for monitoring the course of viral infections. IL-6, as a traditional inflammatory marker, has excellent sensitivity but relatively weak specificity. Combining the testing of these four markers can fully leverage their respective advantages, compensate for each other's shortcomings, and achieve a complementary and mutually beneficial effect.

[0007] Currently, there are various methods for detecting inflammatory markers, including enzyme-linked immunosorbent assay (ELISA), colloidal gold assay, immunoturbidimetry, chemiluminescence immunoassay, and fluorescence immunochromatography. ELISA boasts high sensitivity and specificity, accurately detecting multiple inflammatory markers; however, it can be affected by various factors leading to false positives or false negatives, such as improper sample processing or reagent quality variations. Furthermore, it is relatively complex to operate, requiring specialized technical expertise and equipment. Colloidal gold assays are simple, rapid, and intuitive, suitable for rapid on-site testing. However, its sensitivity is relatively low, potentially failing to detect low concentrations of inflammatory markers; and the stability of colloidal gold particles may be affected by environmental factors. While immunoturbidimetry provides fast results and is suitable for point-of-care testing, it can be interfered with by other components in the sample (such as lipemia), leading to false positives. While chemiluminescence immunoassays offer high sensitivity and reliable results, they typically require specialized equipment and automated instruments, resulting in higher costs. Fluorescence immunochromatography combines the speed of immunochromatography with the sensitivity of fluorescence detection, making it suitable for detecting various inflammatory markers. However, its accuracy can be affected by factors such as the stability of the fluorescent substance, sample processing, and the chromatographic medium. Currently, most inflammatory marker detection techniques target single indicators, while fluorescence immunochromatography is commonly used for the combined detection of multiple indicators. Patents involving multiple combined assays include: a serum amyloid A / procalcitonin / C-reactive protein triple assay kit and preparation method (patent application number: 201611165670.4) / a combined detection kit (patent application number: 202023195109.5) / combined assay test strips and detection kits (patent application number: 202023195109.5) / a method and kit for simultaneously detecting four inflammatory markers (patent application number: 201910933208.1), etc. All of the aforementioned patents employ fluorescence immunochromatography for their combined assays. Therefore, developing a four-in-one assay kit and method with a wide detection range, high sensitivity, rapid determination of inflammatory mediator levels, and high accuracy is currently an urgent market and clinical need. Summary of the Invention

[0008] The purpose of this invention is to provide a quadruple test method for inflammatory markers CRP, SAA, PCT, and IL-6, which can avoid the influence of lipemia / hemolysis on the test results, and has a wide detection range, high sensitivity, high accuracy, and convenient and fast automated detection.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides a quadruple test for inflammatory markers CRP, SAA, PCT, and IL-6, comprising the following steps:

[0011] (1) Antibody coating:

[0012] CRP, SAA, PCT, and IL-6 antibodies were added to the detection wells along with the coating solution for coating, and then blocking solution was added for blocking.

[0013] (2) Activation of fluorescent microspheres and conjugation with antibodies:

[0014] Add coupling buffer to the microsphere suspension, mix well, centrifuge and discard the supernatant, add more coupling buffer, mix well, centrifuge and discard the supernatant, take the precipitate and add coupling buffer to mix well; add EDC solution and mix well, then add NHS solution and mix well, activate in the dark.

[0015] After activation, centrifuge and discard the supernatant. Mix the precipitate with conjugation buffer, centrifuge and discard the supernatant, add more conjugation buffer, mix well, centrifuge and discard the supernatant, take the precipitate and add it to conjugation buffer, mix well, add antibody for light-protected conjugation, add blocking solution for blocking, and then dilute with fluorescent microsphere preservation solution to obtain antibody-conjugated fluorescent microspheres.

[0016] (4) Add the sample to be tested into the detection well after sealing in step (1) and incubate. Add the antibody-conjugated fluorescent microspheres from step (2) and incubate. Detect fluorescence under a microscope.

[0017] Preferably, in step (1), the concentrations of CRP, SAA, PCT, and IL-6 antibodies are 0.1-0.5 μg / T, the coating temperature is 2-8℃, and the coating time is 15-17 h.

[0018] Preferably, the light-protected activation temperature in step (2) is 35-37°C and the time is 15-30 min.

[0019] Preferably, the temperature for light-shielding coupling in step (2) is 35-37°C and the time is 1-2 hours.

[0020] Preferably, in step (3), the incubation time of the sample to be tested in the detection well after being sealed in step (1) is 30 to 70 minutes; the incubation time of the antibody-coupled fluorescent microspheres added in step (2) is 30 to 70 minutes.

[0021] The beneficial effects of this invention compared to the prior art are as follows:

[0022] (1) This invention employs digital fluorescence immunoassay technology, immobilizing antibodies on a detection plate. When antigens in the sample bind to these immobilized antibodies, the fluorescein-labeled antibody binds to another antigenic determinant on the antigen, thus constructing a "sandwich" complex structure between the immobilized antibody and the fluorescein-labeled antibody. The higher the antigen concentration, the more fluorescent particles there are, and the antigen content is quantified by the number of fluorescent particles. This unique design enables this invention to achieve high sensitivity and high precision detection of antigens, and also allows for accurate quantification of antigen content by precisely counting the number of fluorescent particles.

[0023] (2) The method of judging the antigen concentration by the number of fluorescent particles in this invention has the characteristics of strong stability, low cost and easy operation. It has important clinical value for the early diagnosis of inflammation, guiding treatment and monitoring the treatment effect. The method integrates sample addition, incubation, washing and detection, reducing operation error. Digital fluorescence counting accurately detects antigen concentration, avoids the influence of lipemia / hemolysis on the detection results, and the cost is much lower than that of chemiluminescence method, saving patient expenses and laboratory resources. In addition, it has a wide detection range, high sensitivity, high accuracy and convenient and fast automated detection. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The 96-well plate used in Example 1, wherein S0-S5 is the calibration area; Q1-Q2 is the quality control area; and P1-P8 is the sample area;

[0026] Figure 2 The correlation between the CRP, SAA, PCT, and IL-6 test results in Example 1 and clinical relevance of R;

[0027] Figure 3 The result is the linearity of PCT in Experiment Example 1. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] Example 1

[0034] Example 1 of this invention provides a quadruple test method for inflammatory markers CRP, SAA, PCT, and IL-6, with the specific steps as follows:

[0035] (1) Solution preparation:

[0036] Coupling buffer: 10 mM MES (pH 6.2 ± 0.05), containing 0.05% ProClin 300;

[0037] EDC solution: 10 mg / mL, prepared with coupling buffer, and used immediately;

[0038] NHS solution: 10 mg / mL, prepared with coupling buffer, and used immediately;

[0039] Fluorescent microsphere blocking solution: borate buffer (5mM boric acid, 11.2mM sodium tetraborate decahydrate, 0.05% Tween-20, pH 9.0±0.05), 1% BSA, 0.24% ethanolamine;

[0040] Fluorescent microsphere washing solution: 50 mM Tris (pH 8.0±0.05), 0.5% BSA, 0.05% Tween-20, 0.03% ProClin 300;

[0041] Preservative solution for fluorescent microspheres: 25 mM Tris (pH 7.2 ± 0.05), 150 mM NaCl, 0.05% Tween-20, 1% BSA, 5% trehalose, 0.1% ProClin 300.

[0042] (2) Activation of fluorescent microspheres

[0043] A. Take 0.05 mL of fluorescent microsphere (1% solid content) suspension into a 2 mL centrifuge tube containing 1 mL of coupling buffer, sonicate to mix, centrifuge at 15℃ and 20000g for 10 min, and remove the supernatant; add another 1 mL of coupling buffer, sonicate to mix, centrifuge at 15℃ and 20000g for 10 min, and remove the supernatant.

[0044] B. Take the precipitate from A, add 1 mL of coupling buffer, sonicate to mix, then add 3.5 μL of EDC solution, vortex to mix, then add 33 μL of NHS solution, sonicate to mix; place the centrifuge tube on a turntable, activate at 37℃, 40 r / min, in the dark for 15-30 min; after activation, centrifuge at 15℃, 20000g for 10 min, and remove the supernatant;

[0045] C. Take the precipitate from B, add 1.5 mL of coupling buffer, sonicate to mix, centrifuge at 15℃ and 20000g for 10 min, remove the supernatant, add 1.5 mL of coupling buffer, and repeat the washing step once.

[0046] (3) Coupling of fluorescent microspheres with antibodies

[0047] Add 0.75 mL of conjugation buffer and sonicate to mix. Add 50 μg of the antibodies to be labeled CRP, SAA, PCT, and IL-6 to 0.25 mL of conjugation buffer to prepare the conjugation solution. Add the antibody-containing conjugation solution to the mixed fluorescent microspheres and vortex to mix. Place the centrifuge tube on a turntable and conjugate at 37 °C, 40 r / min, in the dark for 2 h.

[0048] (4) Blocking and preservation of fluorescent microspheres

[0049] Add 0.5 mL of fluorescent microsphere blocking solution to the centrifuge tube and vortex to mix. Place the centrifuge tube on a turntable and seal at 37℃, 40 r / min, and in the dark for 1 h. Centrifuge at 15℃ and 20000 g for 10 min and remove the supernatant. Add 1.5 mL of fluorescent microsphere washing solution, sonicate to mix, and centrifuge at 15℃ and 20000 g for 10 min to remove the supernatant. Add 1.5 mL of fluorescent microsphere washing solution and repeat the washing step once. Finally, add 0.5 mL of fluorescent microsphere preservation solution (final concentration of fluorescent microspheres 1 mg / mL) to the centrifuge tube, sonicate to mix, and store at 2-8℃ in the dark for later use.

[0050] (5) Antibody-coated solid-phase detection plate

[0051] A. Solution preparation

[0052] Coating solution: Sodium carbonate (Na2CO3) 0.1M, sodium bicarbonate (NaHCO3) 0.1M, pH 9.6.

[0053] Blocking solution: 0.05-0.1M PBS (pH 7.4) + 1-2% BSA + 0.1% ProClin 300 + 0.1% protein protectant.

[0054] Calibrator dilution solution: 0.1M PBS buffer, 1% protein (bovine serum albumin), 0.1% protein protectant and 0.2% ProClin 300.

[0055] Preparation of quality control / calibrators: Prepare CRP, SAA, PCT, and IL-6 calibrators using the above calibrator diluents according to the table below.

[0056] Table 1. Concentration of Quality Control / Calibration Samples

[0057]

[0058] B. Antibody coating of the detection plate.

[0059] According to the antibody concentration of 0.5 μg / T, CRP, SAA, PCT, and IL-6 antibodies were taken respectively, and then... Figure 1 The sections were coated with 100 μL / T coating solution and left to stand at 8°C for 16 h. The sections were then washed 5 times with PBS + TWEEN (0.05% wt) and patted dry.

[0060] Add 150 μL / well blocking solution, block at 37°C for 2 h, wash 5 times with PBS + TWEEN (0.05% wt), pat dry, place in desiccant, seal in foil bag and set aside for later use.

[0061] (6) Add the quality control products, calibrators and body fluid samples containing CRP, SAA, PCT and IL-6 into the corresponding wells of the coated ELISA plate, place it on a shaker, react at room temperature for 1 hour, wash 5 times with PBS + TWEEN (0.05% wt) and pat dry.

[0062] (7) Dilute the corresponding sealed fluorescent microspheres 25 times with microsphere preservation solution, take 50 μL and add it to the wells of the ELISA plate containing CRP, SAA, PCT, IL-6 quality control, calibrator and body fluid samples, place on a shaker and react at room temperature for 1 hour, then wash 5 times with PBS + TWEEN (0.05% wt) and pat dry.

[0063] (8) Place the above-mentioned ELISA plate onto the FX100 digital fluorescence immunoassay analyzer, a self-developed instrument by the Institute of Physics, Henan Academy of Sciences. The instrument will automatically calibrate according to the information on the reagent calibration card. Only after successful calibration and quality control testing within the acceptable range can subsequent clinical sample testing be performed. The results are as follows: Figure 2 As shown.

[0064] Test results: The correlation R2 between the CRP, SAA, PCT, and IL-6 test results and the test results of different well-known listed companies is greater than 0.95, indicating that the results are accurate and highly reliable.

[0065] Experimental Example 1

[0066] Test Example 1 of this invention tested the effectiveness of the detection method in Example 1. The specific steps are as follows:

[0067] (1) High sensitivity:

[0068] Assessment methods:

[0069] Blank limit (LOB): Take 5 samples with 0 values ​​(samples without the analyte), and test them according to the steps in Example 1. Repeat the test 3 times and evaluate for 4 days.

[0070] Limit of detection (LOD): Take 5 samples with 1 to 4 times the LoB and test them according to the steps in Example 1. Repeat the test 3 times and evaluate for 4 days.

[0071] Taking the PCT project as an example for verification, the test data is shown in Table 1 below.

[0072] As shown in Table 1, the detection limit of the present invention is significantly lower than that of commercially available products (0.02 ng / mL).

[0073] Table 2 PCT Project Sensitivity

[0074]

[0075] (2) Wide linear range:

[0076] The linear range is determined by selecting samples close to 100%–130% of the upper limit of the calibrator, along with one sample close to zero, mixed in different proportions, with at least 4–6 gradients (the number of points can be increased appropriately depending on the width of the linear range). The correlation between the measured and theoretical values ​​should be r > 0.99. Using PCT as an example for verification, the detection data are shown in Table 2 below. The upper limit of linearity can be achieved at 119.801 ng / mL, while the upper limit of linearity for commercially available products is 100 ng / mL. The PCT dominant results are as follows... Figure 3 As shown.

[0077] Table 3 shows the PCT project linear range verification data. The above description is merely a preferred embodiment of the present invention. It should be noted that for general knowledge in this technical field...

[0078]

[0079] For those skilled in the art, several improvements and modifications can be made without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention.

Claims

1. A method for testing the four inflammatory markers CRP, SAA, PCT, and IL-6, characterized in that: The steps include: (1) Antibody coating: Four antibodies, namely CRP, SAA, PCT and IL-6, and coating solution were added to the detection wells for coating, and then blocking solution was added for blocking; (2) Activation of fluorescent microspheres and coupling with antibodies: Add coupling buffer to the microsphere suspension, mix well, centrifuge and discard the supernatant, continue to add coupling buffer, mix well, centrifuge and discard the supernatant, take the precipitate and add coupling buffer to mix well; add EDC solution and mix well, then add NHS solution and mix well, protect from light for activation; After activation, centrifuge and discard the supernatant, mix the precipitate with coupling buffer, centrifuge and discard the supernatant, continue to add coupling buffer, mix, centrifuge and discard the supernatant, take the precipitate and add coupling buffer to mix evenly, add antibody for light-protected coupling, continue to add blocking solution for blocking, after blocking, fix and dissolve the fluorescent microsphere preservation solution to obtain antibody-coupled fluorescent microspheres; (3) adding the sample to be tested to the detection well blocked in step (1) and incubating, adding the antibody-coupled fluorescent microspheres in step (2) and incubating, and detecting fluorescence under a microscope.

2. The method for detecting the inflammatory markers CRP, SAA, PCT and IL-6 according to claim 1, characterized in that: In step (1), the concentrations of CRP, SAA, PCT and IL-6 antibodies are 0.1-0.5 μg / T respectively, the coating temperature is 2-8° C., and the coating time is 15-17 h.

3. The method for the four-in-one test of inflammatory factor markers CRP, SAA, PCT, and IL-6 according to claim 1, characterized in that: In step (2), the light-protected activation temperature is 35-37° C. and the time is 15-30 min.

4. The method for detecting the inflammatory factor markers CRP, SAA, PCT and IL-6 according to claim 1, characterized in that: In step (2), the light-shielded coupling temperature is 35-37° C. and the time is 1-2 h.

5. The method for detecting the inflammatory factor markers CRP, SAA, PCT and IL-6 according to claim 1, characterized in that: In step (3), the sample to be tested is added to the detection well blocked in step (1) and incubated for 30 to 70 minutes; the antibody-coupled fluorescent microspheres in step (2) are added and incubated for 30 to 70 minutes.

Images