Method for rapidly detecting serum amyloid protein A

Abstract

The invention relates to the technical field of detection methods, in particular to a method for detecting serum amyloid protein A. The specific technical scheme is as follows: after a serum amyloid protein A secondary antibody donor bead complex and a serum amyloid protein A primary antibody receptor bead complex are mixed with an antigen to be detected, a light-emitting signal value is detected. The method for detecting the serum amyloid protein A disclosed by the invention has the advantages of wide detection limit, homogeneous phase, no washing, simplicity and convenience in operation, good specificity, good repeatability and good anti-interference performance, can be used for rapidly detecting infection markers, and has clinical application potential.

Description

Technical Field

[0001] The present invention relates to the technical field of detection methods, and in particular to a method for rapidly detecting serum amyloid A. Background Art

[0002] Serum amyloid A (SAA) is a precursor of tissue amyloid A produced by liver cells. It is an acute phase protein produced during tissue damage and inflammation. While its level in normal serum is low (<10 mg / L), it can rise (>10 mg / mL) during acute inflammation and infection, typically within 5-6 hours of infection. With a half-life of 50 minutes, SAA is a relatively sensitive biomarker for diagnosing bacterial or viral infections. Currently, elevated SAA levels are detected in several diagnostic fields, such as viral infections, atherosclerosis, and tumors. Its rise occurs earlier than C-reactive protein (CRP), thus providing a more rapid indicator of disease progression. However, because SAA has a half-life of only 50 minutes, routine clinical testing is time-consuming and reagents for SAA detection are significantly more expensive than those for CRP. Therefore, CRP is often used as a surrogate marker for SAA in clinical practice. Although there are relevant literatures showing that there are different degrees of correlation between SAA and CRP tests, in fact, SAA and CRP tests cannot replace each other, because SAA can be detected in the early stages of infection, can reflect changes in the disease earlier, and guide clinical treatment, especially for the diagnosis of some critical and acute diseases.

[0003] Currently, common clinical methods for detecting SAA include enzyme-linked immunosorbent assay (ELISA), immunoturbidimetry, and traditional chemiluminescence. Turbidimetry is commonly used by medical institutions at all levels for SAA detection due to its advantages such as ease of operation, high sensitivity, suitability for automated detection, and fast detection speed. However, its sensitivity is limited and it is easily interfered with by sample turbidity, resulting in a high false-negative rate during the detection process. Although ELISA has high specificity, it is complex to operate and takes a long time to detect, making it difficult to achieve rapid bedside detection. While the traditional chemiluminescence method has improved in terms of automation, its enzymatic signal amplification system is easily affected by ambient temperature, and multi-index detection requires multiple independent experiments, resulting in an exponential increase in sample consumption. Therefore, it is important to develop a method that can quickly, instantly, and accurately detect SAA. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the present invention provides a method for detecting serum amyloid A.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0006] The invention discloses a method for rapidly detecting serum amyloid A. After a serum amyloid A secondary antibody donor bead complex and a serum amyloid A primary antibody acceptor bead complex are mixed with an antigen to be detected, a luminescent signal value is detected.

[0007] Preferably, the acceptor beads and the donor beads are activated separately, and the activation process is as follows: MES is added to the acceptor beads and the donor beads respectively, and after mixing, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added respectively, and after mixing, N-hydroxysuccinimide is immediately added, and after mixing, incubation is carried out.

[0008] Preferably, after the incubation is completed, the acceptor beads and donor beads are centrifuged, MES is added to the precipitate, and the mixture is mixed, centrifuged, and washed to complete the washing of the acceptor beads and donor beads.

[0009] Preferably, the preparation process of the complex is: adding serum amyloid A primary antibody to the washed acceptor beads to form a serum amyloid A primary antibody acceptor bead complex; adding serum amyloid A secondary antibody to the washed donor beads to form a serum amyloid A secondary antibody donor bead complex.

[0010] Preferably, the concentration of the serum amyloid A primary antibody is 1-5 mg / mL, the concentration of the serum amyloid A secondary antibody is 2-8 mg / mL, and the concentrations of the acceptor beads and donor beads are 0.2-3 mg / mL, respectively.

[0011] Preferably, the volume ratio of the acceptor beads to the primary antibody against serum amyloid A is 20 to 60:1, and the volume ratio of the donor beads to the secondary antibody against serum amyloid A is 20 to 60:1.

[0012] Preferably, the serum amyloid A primary antibody acceptor bead complex and the serum amyloid A secondary antibody donor bead complex are incubated in the dark. After the incubation is completed, the blocking solution is added, mixed, incubated, blocked, and centrifuged. Tween-20 is added to the precipitate, mixed, centrifuged, and then a homogeneous luminescent diluent is added and stored.

[0013] Preferably, the serum amyloid A secondary antibody donor bead complex and the serum amyloid A primary antibody acceptor bead complex are diluted with a homogeneous luminescent diluent and then mixed with the antigen to be tested, with a dilution ratio of 1:80.

[0014] Preferably, the concentration of the antigen to be detected is 0.000625-0.025 mg / mL, and the detection limit is 0.025 mg / L.

[0015] The present invention has the following beneficial effects:

[0016] 1. In the present invention, the secondary antibody against serum amyloid A is coated on donor beads, and the primary antibody against serum amyloid A is coated on acceptor beads. In the system, in the presence of the serum amyloid A antigen to be tested, an antibody-antigen-antibody sandwich complex is formed. This complex brings the photosensitive beads and the luminescent beads closer together, with the distance being less than 200 nm. When the photosensitive beads are excited by 680 nm excitation light, the donor beads produce singlet oxygen, which acts as a medium for energy transfer and transfers energy to the acceptor beads, thereby generating emitted light, and the instrument detects the light signal.

[0017] 2. The present invention provides a detection method based on homogeneous luminescence technology for detecting serum amyloid A, which has a wide detection limit, is homogeneous and wash-free, is easy to operate, has good specificity and repeatability, can be used for rapid detection of infection markers, and has clinical application value.

[0018] 3. Light Initiated Chemiluminescent Assay (LICA), as a new type of homogeneous immunoassay technology, provides an ideal solution for the present invention. Its core principle is to stimulate the luminescent substance in the chemical reaction by light, causing it to enter an excited state, and then release photons when returning to the ground state to generate a detectable light signal. Compared with traditional methods, this method has the following advantages: first, the homogeneous reaction avoids washing and enables faster detection; second, the high specific surface area and specific modification of the nanoparticles improve the sensitivity and linear range. Finally, since the detection relies on a 96-well plate, it is possible to quickly detect samples in batches. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the detection method of the present invention;

[0020] Figure 2 The concentrations of serum amyloid A antigen added at different concentrations and the standard curve;

[0021] Figure 3 The repeatability of the detection method of the present invention;

[0022] Figure 4 is the selectivity of the detection method of the present invention;

[0023] Figure 5 The stability of the detection method of the present invention;

[0024] Figure 6 It is the anti-interference property of the detection method of the present invention. DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0027] The invention discloses a method for rapidly detecting serum amyloid A. The process comprises the following steps: mixing a serum amyloid A secondary antibody donor bead complex and a serum amyloid A primary antibody acceptor bead complex with an antigen to be detected, and then detecting a luminescent signal value.

[0028] The acceptor and donor beads are activated separately beforehand. The activation process is as follows: MES is added to each of the acceptor and donor beads, mixed, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added to each of the beads. After mixing, N-hydroxysuccinimide is immediately added, mixed, and then incubated. After the incubation, the acceptor and donor beads are centrifuged, and MES is added to the precipitate. After mixing, centrifugation, and washing, the acceptor and donor beads are washed.

[0029] Furthermore, the preparation process of the complex is: adding serum amyloid A primary antibody to the washed acceptor beads to form a serum amyloid A primary antibody acceptor bead complex; adding serum amyloid A secondary antibody to the washed donor beads to form a serum amyloid A secondary antibody donor bead complex.

[0030] The concentration of the primary antibody against serum amyloid A is 1-5 mg / mL, the concentration of the secondary antibody against serum amyloid A is 2-8 mg / mL, the concentrations of the acceptor beads and donor beads are 0.2-3 mg / mL, respectively. The volume ratio of the acceptor beads to the primary antibody against serum amyloid A is 20-60:1, and the volume ratio of the donor beads to the secondary antibody against serum amyloid A is 20-60:1.

[0031] Furthermore, the washing and preservation of the complex are as follows: incubating the serum amyloid A primary antibody acceptor bead complex and the serum amyloid A secondary antibody donor bead complex in the dark, adding blocking solution after the incubation is completed, mixing, incubating, blocking, centrifuging, adding Tween-20 to the precipitate, mixing, centrifuging, adding homogeneous luminescent diluent, and preserving.

[0032] The specific detection process is as follows: serum amyloid A secondary antibody donor bead complex and serum amyloid A primary antibody acceptor bead complex are diluted with homogeneous luminescent diluent at a dilution ratio of 1:80 and then mixed with the test antigen. The concentration of the test antigen is 0.000625-0.025 mg / mL, and the detection limit is 0.025 mg / L.

[0033] The present invention will be further described below with reference to specific embodiments.

[0034] Example 1

[0035] The test process for serum amyloid A is as follows:

[0036] 1) Cleaning of beads

[0037] Take 20 μL of 10 mg / mL donor beads (lisa donor beads (Weidu Biotechnology Co., Ltd., catalog number: 67500001)) (acceptor beads (lisa acceptor beads (Weidu Biotechnology Co., Ltd., catalog number: 67700100)) and add 1 mL of 0.05 mM MES (pH 6.2); sonicate for 10 seconds in a 2 mL transparent centrifuge tube and mix thoroughly, then centrifuge at 12000 rpm and 4°C for 40 minutes, and discard the supernatant.

[0038] 2) Activation of beads

[0039] Add 200 μL of 0.05 mM MES (pH 6.2) to the precipitate, mix by ultrasonication for 10 s, and then immediately add 6 μL of (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) solution (10 μg / μL) to the dispersed microspheres. After oscillation mixing, immediately add 66 μL of (N-hydroxysuccinimide) (10 μg / μL), oscillate and mix, then seal with a sealing film and incubate in a constant temperature shaking instrument at 50 r / min, 25°C, and incubate for 15 min.

[0040] 3) Cleaning of beads

[0041] ① After incubation, centrifuge at 12000 rpm and 4°C for 40 min and discard the supernatant.

[0042] ② Add 1 mL of 0.05 mM MES (pH 6.2) to the precipitate, mix by ultrasonication for 10 seconds, and then centrifuge at 12,000 rpm and 4°C for 40 minutes, and discard the supernatant.

[0043] ③ Add 1 mL of ultrapure water to the precipitate, mix thoroughly by ultrasonication, and centrifuge at 12,000 rpm and 4°C for 40 min. Discard the supernatant.

[0044] 4) Bead-coupled antibodies

[0045] ① Add 200 μL of phosphate buffer (1X) to the washed beads, mix thoroughly by ultrasonication, and then carefully add:

[0046] To 200 μL of acceptor beads (1 mg / mL), 5 μL of 1.88 mg / mL serum amyloid A primary antibody (SAAAb1) (Hangzhou Huakui Jinpei Biotechnology Co., Ltd., catalog number: M30041) was added to form a SAAAb1-acceptor bead complex;

[0047] 4 μL of 4.65 mg / mL serum amyloid A secondary antibody (SAA Ab2) (Hangzhou Huakui Jinpei Biotechnology Co., Ltd., catalog number: M30042) was added to 200 μL of donor beads (1 mg / mL) to form a SAA Ab2-donor bead complex.

[0048] ② After careful oscillation and mixing, seal with sealing film and incubate in a constant temperature oscillator in the dark at 50 r / min, 25°C, and activate for 200 min.

[0049] ③After activation, add 20 μL of QuickBlock to each of the beads (acceptor beads and donor beads). TM Western blocking solution was mixed by oscillation, sealed with sealing film, and incubated in a constant temperature shaking instrument at 50 r / min, 25°C, for 200 min.

[0050] 5) Washing and storage

[0051] After blocking, centrifuge the tube at 12,000 rpm and 4°C for 40 minutes, discarding the supernatant. Add 1 mL of 0.1% Tween-20 to the pellet, mix by sonication, and centrifuge at 12,000 rpm and 4°C for 40 minutes. Discard the supernatant. Add 200 μL of Homogeneous Luminescence Diluent (Weidu Biotechnology Co., Ltd., Catalog No. HI0101A-3) and store at 4°C in the dark.

[0052] 6) Detection process: Take out the coupled acceptor bead-coated serum amyloid A primary antibody acceptor bead complex and the donor bead-coated serum amyloid A secondary antibody donor bead complex from the refrigerator and let it stand to room temperature. Then, take appropriate amounts of the two complexes respectively and dilute them to a certain concentration with a homogeneous luminescent diluent (in this embodiment, the volume ratio of the diluent to the complex is 1:80); then dilute the antigen to be tested with 1xPBS to different concentrations, add 10 μL of different concentrations of the known concentration of the antigen to be tested to a 96-well plate, and immediately add 25 μL (1:80 diluted) of the acceptor bead-coated serum amyloid A primary antibody acceptor bead complex, place it in an incubator at 25°C and incubate for 8 minutes, then add 10 μL (1:80 diluted) of the donor bead-coated serum amyloid A secondary antibody donor bead complex, place it in an incubator at 25°C and incubate for 8 minutes, and immediately detect the luminescent signal value.

[0053] Test methods and results:

[0054] 1. Linear relationship

[0055] Serum amyloid A antigen at concentrations of 0.000625 mg / mL, 0.00156 mg / mL, 0.00313 mg / mL, 0.00625 mg / mL, 0.0125 mg / mL, and 0.025 mg / mL was added to a test tube. Luminescence values ​​were then measured using the same procedure as in step 6). Specifically, serum amyloid A antigen at different concentrations was measured at 25°C for 8 minutes. The results were as follows: Figure 2 As shown, the linear equation is y=32020+194.34, R 2 =0.9977, the linear range is 0.000625 mg / mL to 0.025 mg / mL, and the detection limit is 0.025 mg / L.

[0056] 2. Repeatability

[0057] Take 25 μL of the acceptor bead-coated serum amyloid A primary antibody acceptor bead complex in a 96-well test plate, add 10 μL of serum amyloid A antigen to it, place it in an incubator and shake for 8 minutes, then immediately add 10 μL of the donor bead-coated serum amyloid A secondary antibody donor bead complex, shake and incubate it in an incubator for 8 minutes. After the shaking is completed, the luminescence signal value is immediately measured. Under the conditions of 25 ° C and 8 minutes of incubation, the repeatability of 0.001 mg / mL serum amyloid A is tested. The measurement is repeated 16 times and the relative standard deviation RSD is calculated. Figure 3 As shown, the RSD was <2.53%.

[0058] 3. Selectivity

[0059] Some cytokines that may interfere were selected for selective determination, including CRP (C-reactive protein), MxA (myxovirus resistance protein A), IL6 (interleukin 6), P24 (human immunodeficiency virus p24 antigen), tPALC (tissue plasminogen activator-plasminogen activator inhibitor-1 complex), Ctni (cardiac troponin I), and PCT (procalcitonin). Their photoluminescence values ​​were determined according to the above detection step 6). The results are as follows. Figure 4 As shown in the figure, this detection method only has a signal for serum amyloid A antigen, so it has good specificity.

[0060] 4. Stability

[0061] The coupled beads were placed in a 4°C refrigerator away from light and tested for the same concentration of serum amyloid A antigen under the same conditions for 16 consecutive days to observe their stability. Figure 5 As shown, the RSD is 2.45%, indicating that the method has good stability.

[0062] 5. Anti-interference

[0063] In order to evaluate the effect of other substances in serum samples on serum amyloid A antigen, 0.001 mg / mL of serum amyloid antigen was mixed with other interfering substances (ethylenediaminetetraacetic acid (EDTA), heparin, fibrinogen, triglyceride, cholesterol, hemoglobin, bilirubin, immunoglobulin) at a concentration ratio of (1:100, 1:1000, 1:10000) to form an antigen mixed solution. 10 μL of the mixed solution was added to a 96-well plate, and then the photoluminescence signal value was detected according to the detection step 6), and the spiked recovery rate of serum amyloid antigen was calculated. The results are shown in Figure 6. Figure 6 As shown in the figure, in the presence of other interfering substances, the recovery rate is still 90.66% to 106.03%, indicating that this method has good anti-interference ability.

[0064] 6. Testing of actual samples

[0065] This method was applied to the detection of serum amyloid A in serum samples. The measured luminescence signal values ​​were substituted into a linear equation to calculate the detection concentration. The results, shown in Table 1, show that the photochemiluminescence method has similar detection results to commonly used clinical methods and exhibits potential advantages in ease of use and detection speed, making it a promising alternative method for the detection of serum amyloid A antigen in serum samples.

[0066] Table 1 Comparison between clinical detection and detection by this method

[0067]

[0068] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for detecting serum amyloid A, characterized in that: After the serum amyloid A secondary antibody donor bead complex and the serum amyloid A primary antibody acceptor bead complex are mixed with the antigen to be tested, the luminescent signal value is detected.

2. The method for detecting serum amyloid A according to claim 1, wherein: The acceptor beads and donor beads were activated separately. The activation process was as follows: MES was added to the acceptor beads and donor beads respectively, and after mixing, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added respectively, and after mixing, N-hydroxysuccinimide was immediately added, mixed, and incubated.

3. The method for detecting serum amyloid A according to claim 2, wherein: After the incubation is completed, the acceptor beads and donor beads are centrifuged, and MES is added to the precipitate. After mixing, centrifugation, and washing, the acceptor beads and donor beads are washed.

4. The method for detecting serum amyloid A according to claim 3, wherein: The preparation process of the complex is as follows: adding serum amyloid A primary antibody to the washed acceptor beads to form a serum amyloid A primary antibody acceptor bead complex; adding serum amyloid A secondary antibody to the washed donor beads to form a serum amyloid A secondary antibody donor bead complex.

5. The method for detecting serum amyloid A according to claim 4, wherein: The concentration of the serum amyloid A primary antibody is 1-5 mg / mL, the concentration of the serum amyloid A secondary antibody is 2-8 mg / mL, and the concentrations of the acceptor beads and donor beads are 0.2-3 mg / mL, respectively.

6. The method for detecting serum amyloid A according to claim 5, wherein: The volume ratio of the acceptor beads to the serum amyloid A primary antibody is 20 to 60:1, and the volume ratio of the donor beads to the serum amyloid A secondary antibody is 20 to 60:

1.

7. The method for detecting serum amyloid A according to claim 4, wherein: The serum amyloid A primary antibody acceptor bead complex and the serum amyloid A secondary antibody donor bead complex were incubated in the dark. After the incubation was completed, blocking solution was added, mixed, incubated, blocked, and centrifuged. Tween-20 was added to the precipitate, mixed, centrifuged, and then homogeneous luminescence diluent was added and stored.

8. The method for detecting serum amyloid A according to claim 1, wherein: The serum amyloid A secondary antibody donor bead complex and the serum amyloid A primary antibody acceptor bead complex were diluted with homogeneous luminescent diluent and then mixed with the antigen to be tested at a dilution ratio of 1:

80.

9. The method for detecting serum amyloid A according to claim 8, wherein: The concentration of the antigen to be tested is 0.000625-0.025 mg / mL, and the detection limit is 0.025 mg / L.

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