Serum amyloid protein A determination test strip
By introducing a dual-detection-line design and fluorescent microsphere-labeled antibodies into the fluorescence immunochromatographic assay kit, the problems of low repeatability and narrow linear range in existing technologies have been solved, enabling accurate detection and high-sensitivity determination of high-value samples.
Patent Information
- Application Number
- CN202422460549.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-11
- Publication Date
- 2025-11-18
- Estimated Expiration
- 2034-10-11
AI Technical Summary
Existing fluorescence immunochromatographic assay kits have problems with low repeatability and narrow linear range when detecting serum amyloid A, especially in high-value samples.
The system employs a dual-detection-line design, with first and second SAA antibodies coated onto a nitrocellulose membrane, and a third SAA antibody labeled with fluorescent microspheres, combined with a control line, to form a multilayer detection system that improves detection sensitivity and linear range.
It enables accurate detection of high-value samples, improves the repeatability and linear range of measurements, and ensures the accuracy and reliability of detection in high-concentration samples.
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Figure CN223565713U_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present disclosure relates to the field of medicine, immunology and in vitro diagnostics, in particular to a determination test strip for detecting serum amyloid A by fluorescent immunochromatography. BACKGROUND
[0002] Serum amyloid A (SAA) is an acute phase protein, belonging to a highly heterogeneous class of proteins in the apolipoprotein family. Since SAA is the serum precursor of amyloid A protein in amyloidosis, it is clinically referred to as SAA. The human SAA gene contains SAA1, SAA2, SAA3 and SAA4, which are located on the short arm of human chromosome 11. The composition of the above SAA genes all have 3 introns and 4 exons. According to the expression, SAA can be divided into two categories: constitutive SAA (C-SAA) and acute phase SAA (A-SAA). Under normal circumstances, C-SAA accounts for >90% of the total amount of SAA in the human body, accounting for about 1%-2% of apolipoprotein. Most of the C-SAA is combined with high-density lipoprotein 3 subtype HDL3, but does not transfer cholesterol. The remaining about 5% of C-SAA is combined with very low-density lipoprotein VLDL. When acute phase response occurs, the body releases a large amount of pro-inflammatory factors, which makes A-SAA synthesize a large amount of combination with a large amount of apolipoprotein on HDL3, thereby increasing HDL particles.
[0003] SAA is a commonly used inflammatory indicator in clinical detection, and is induced to express in large quantities in various inflammatory diseases, and the concentration in serum and inflammatory tissues can be as high as 1000 times the background. When bacterial or viral infection occurs, the SAA level of the body increases significantly. Unlike CRP, SAA increases significantly in a short time after viral or bacterial infection, while CRP is only more sensitive to bacterial infection, and the short-term increase level after infection is not obvious. Therefore, SAA has higher sensitivity than CRP as an inflammatory detection indicator.
[0004] At present, there are various SAA detection methods at home and abroad, such as fluorescence immunochromatography, enzyme-linked immunosorbent assay, radioimmunoassay, latex-enhanced turbidimetry and other methods for SAA detection. Fluorescence immunochromatography is suitable for bedside and timely diagnosis due to its high sensitivity, good stability, simple operation, good portability and other advantages, but the existing fluorescence immunochromatography kit uses a single detection line, the repeatability of the measured value is not high, the linear range is also narrow, and the linear range of most kits is between 1-300 mg / L. Therefore, a fluorescence immunochromatography kit with high linear range and high sensitivity is needed to solve the above problems in clinical detection. CONTENT OF THE INVENTION
[0005] In view of the aforementioned needs in the art, the present disclosure provides a serum amyloid A (hereinafter referred to as SAA) assay test strip; the test strip comprises (see Figure 1 ):
[0006] - a base plate;
[0007] - a sample pad, a conjugate pad, a nitrocellulose membrane and a water absorption pad successively lapped on the base plate.
[0008] In some embodiments, on the test strip described in the present disclosure, in the direction from the sample pad to the water absorption pad, the nitrocellulose membrane is successively coated with:
[0009] - a first detection line;
[0010] - a second detection line; and
[0011] - a quality control line.
[0012] In some embodiments, on the test strip described in the present disclosure, the first detection line is coated with a first SAA antibody.
[0013] In some embodiments, on the test strip described in the present disclosure, the second detection line is coated with a second SAA antibody.
[0014] In some embodiments, on the test strip described in the present disclosure, the conjugate pad is coated with a third SAA antibody-labeled fluorescent microsphere.
[0015] "SAA antibody" refers to an antibody that specifically binds to human SAA or an epitope thereof. The term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies); full-length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, single-domain antibody, single-chain Fab (scFab), diabody, linear antibody, single-chain antibody (e.g., scFv); and multispecific antibodies formed from antibody fragments.
[0016] The term "specifically binds" refers to an antibody employed in the present disclosure that is capable of binding to a SAA antigen or an epitope thereof with higher affinity than to other antigens other than SAA. Typically, the antibody binds to the SAA antigen or an epitope thereof with an affinity of about 1 x 10 -7 M or less (e.g., about 1 x 10 -8 M, 1 x 10 -9 M, 1 x 10-10 M, 1×10 -11 An antibody binds to an antigen or its epitope using an equilibrium dissociation constant (KD) of M or less. In some embodiments, the KD of the antibody binding to the antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a nonspecific antigen. KD can be measured using known methods, such as by... The surface plasmon resonance assay is used to measure this. However, antibodies that specifically bind to an antigen or its epitope do not preclude cross-reactivity with other related antigens, such as cross-reactivity with corresponding antigens from other species (homologous) (e.g., humans or monkeys, such as the cynomolgus (cyno), the chimpanzee (chimp), or the common marmoset (marmoset)).
[0017] Those skilled in the art should understand that the SAA antibodies used in this disclosure can be commercially available or laboratory-made, as long as they can specifically bind to SAA, and there are no particular restrictions on the antibody strain.
[0018] The ordinal numbers “first,” “second,” “third,” etc. in this disclosure (such as “first SAA antibody,” “second detection line”) are used only to distinguish different features, elements, components, or steps and are not intended to limit the number, order, or level.
[0019] Therefore, the first SAA antibody, the second SAA antibody, and the third SAA antibody are each independently a monoclonal antibody or a polyclonal antibody; the first SAA antibody and the second SAA antibody are different from the third SAA antibody; the first SAA antibody and the second SAA antibody are the same or different.
[0020] In this context, the "difference" of the antibodies refers to the fact that while the first SAA antibody, the second SAA antibody, and the third SAA antibody are all antibodies that use human SAA as an antigen, they allow targeting different epitopes of SAA. The reason why "the first SAA antibody and the second SAA antibody are different from the third SAA antibody" is that when encountering SAA in a sample, the third SAA antibody labeled on the fluorescent microspheres binds to the first SAA antibody (or the second SAA antibody) at different sites on the SAA.
[0021] In some embodiments, on the test strip of the present disclosure, the surface of the fluorescent microspheres is fixed with a fluorescent label selected from the group consisting of fluorescein isothiocyanate, rhodamine, Cy3, Cy5, carmine protein, quantum dots; any detectable label common in the art is suitable as long as the purpose of visualization is achieved.
[0022] In some specific embodiments, the rhodamine is tetramethyl rhodamine isothiocyanate.
[0023] In some embodiments, the test strip of the present disclosure is loaded in a card shell, which comprises an upper shell and a lower shell, and the upper shell and the lower shell are fastened to form the card shell.
[0024] In some embodiments, the upper shell is provided with a sample port corresponding to the sample pad. Therefore, there is no special restriction on the shape and size of the sample port, and it is only necessary to facilitate the dropwise addition of the sample.
[0025] In some embodiments, the upper shell is provided with an observation window corresponding to the first detection line, the second detection line, and the quality control line; so as to achieve the observation of the detection results. Therefore, there is no special restriction on the shape and size of the observation window, as long as it can wrap and fix the test strip and can observe the results.
[0026] In some embodiments, the average particle size of the fluorescent microspheres is 20 nm to 600 nm, preferably 300 nm; and the emission wavelength of the fluorescent microspheres is 200 nm to 700 nm, preferably 365 nm.
[0027] In some embodiments, the fourth antibody is coated on the quality control line.
[0028] The fluorescent microspheres coated with the third SAA antibody label that are not captured by the target to be tested (such as SAA) will continue to move forward, and when they reach the quality control line, they will bind to the fourth antibody, thereby appearing at the quality control line. Therefore, the purpose of the fourth antibody is to determine the availability of the test strip and as a control.
[0029] In some embodiments, when the first SAA antibody, the second SAA antibody, and the third SAA antibody are mouse-derived antibodies, the fourth antibody is an anti-mouse antibody.
[0030] In other embodiments, when the first SAA antibody, the second SAA antibody, and the third SAA antibody are rabbit-derived antibodies, the fourth antibody is an anti-rabbit antibody.
[0031] In other embodiments, when the first SAA antibody, the second SAA antibody, and the third SAA antibody are chicken-derived antibodies, the fourth antibody is an anti-chicken antibody.
[0032] In other embodiments, when the first, second, and third SAA antibodies are camelid-derived antibodies, the fourth antibody is an anti-camelid antibody.
[0033] In other embodiments, when the first, second, and third SAA antibodies are ovine-derived antibodies, the fourth antibody is an anti-ovine antibody.
[0034] In other embodiments, when the first, second, and third SAA antibodies are equine-derived antibodies, the fourth antibody is an anti-equine antibody.
[0035] As an example, when the first, second, and third SAA antibodies are murine-derived antibodies, the fourth antibody is an anti-murine antibody.
[0036] The present disclosure also provides a SAA test kit comprising the aforementioned SAA assay test strip.
[0037] In some embodiments, the SAA test kit further comprises a sample diluent, and optionally, a calibration information card.
[0038] In some embodiments, the sample diluent comprises:
[0039] 10 mM to 200 mM buffer;
[0040] 0.5 g / L to 50 g / L surfactant;
[0041] 0.5 g / L to 50 g / L electrolyte;
[0042] 0.5 g / L to 50 g / L preservative;
[0043] Optionally, 0.5 g / L to 50 g / L dispersing agent;
[0044] Optionally, 0.5 g / L to 100 g / L stabilizer;
[0045] 5.0 to 9.0 pH.
[0046] In some embodiments, the buffer is selected from one or a combination of glycine buffer, Tris buffer, MOPS buffer, MES buffer, Hepes buffer, PBS buffer.
[0047] In some embodiments, the surfactant is selected from one or a combination of fatty alcohol polyoxyethylene ether ammonium sulfate, Triton, Tween 20, Tween 80, NP40, thesit, Brij L23.
[0048] In some embodiments, the electrolyte is selected from one or a combination of: sodium chloride, potassium chloride, magnesium chloride, zinc chloride, calcium chloride, magnesium sulfate.
[0049] In some embodiments, the dispersing agent is selected from one or a combination of: potassium thiocyanate, choline chloride, PVP.
[0050] In some embodiments, the preservative is selected from one or a combination of: sodium azide, phenol, p-hydroxybenzoic acid, PC300.
[0051] In some embodiments, the stabilizer is selected from one or a combination of: mannose, glucose, chitosan, sorbitol, bovine serum albumin, trehalose, fructose, sucrose.
[0052] In some embodiments, the sample diluent comprises:
[0053] 50mM PBS buffer, 20g / L fatty alcohol polyoxyethylene ether ammonium sulfate, 10g / L sodium chloride, 10g / L Tween 20, 1g / L PC300, pH 7.20.
[0054] According to some embodiments, a method for preparing an SAA assay test strip is provided, comprising the steps of:
[0055] Step 1)
[0056] 1.1) contacting fluorescent microspheres and an activating agent to obtain activated fluorescent microspheres;
[0057] 1.2) contacting a third SAA antibody and the activated fluorescent microspheres to obtain antibody-coated fluorescent microspheres;
[0058] 1.3) blocking the antibody-coated fluorescent microspheres with a blocking agent;
[0059] 1.4) spraying the antibody-coated fluorescent microspheres on a conjugate pad to obtain a conjugate pad 3;
[0060] Step 2):
[0061] Spraying a first SAA antibody, a second SAA antibody, and a fourth antibody on a nitrocellulose membrane respectively to form a first detection line, a second detection line, and a quality control line respectively, to obtain a nitrocellulose membrane;
[0062] Step 3):
[0063] Lapping and fixing a sample pad, the conjugate pad, the nitrocellulose membrane, and a water absorption pad in sequence on a base plate.
[0064] In some embodiments, in the aforementioned preparation method, the test strip can also be optionally packaged with a card shell.
[0065] In some embodiments, in the aforementioned preparation method, the average particle size of the fluorescent microspheres is 20 nm to 600 nm, preferably 300 nm; the emission wavelength of the fluorescent microspheres is 200 nm to 700 nm, preferably 365 nm.
[0066] In some embodiments, the first detection line is upstream of the second detection line; the quality control line is downstream of the second detection line; the upstream means the direction close to the sample pad.
[0067] In some embodiments, in the aforementioned preparation method, the first SAA antibody, the second SAA antibody, and the third SAA antibody are each independently monoclonal antibodies or polyclonal antibodies; the first SAA antibody and the second SAA antibody are different from the third SAA antibody; the first SAA antibody and the second SAA antibody are the same or different.
[0068] In some embodiments, in the aforementioned preparation method, the order of the step 1), the step 2) is interchangeable or parallel.
[0069] In some embodiments, in the aforementioned preparation method:
[0070] When the first SAA antibody, the second SAA antibody, and the third SAA antibody are murine antibodies, the fourth antibody is an anti-murine antibody; or
[0071] When the first SAA antibody, the second SAA antibody, and the third SAA antibody are rabbit-derived antibodies, the fourth antibody is an anti-rabbit antibody; or
[0072] When the first SAA antibody, the second SAA antibody, and the third SAA antibody are chicken-derived antibodies, the fourth antibody is an anti-chicken antibody; or
[0073] When the first SAA antibody, the second SAA antibody, and the third SAA antibody are camel-derived antibodies, the fourth antibody is an anti-camel antibody; or
[0074] When the first SAA antibody, the second SAA antibody, and the third SAA antibody are sheep-derived antibodies, the fourth antibody is an anti-sheep antibody; or
[0075] When the first SAA antibody, the second SAA antibody, and the third SAA antibody are horse-derived antibodies, the fourth antibody is an anti-horse antibody.
[0076] In some specific embodiments, the aforementioned preparation method comprises the steps of:
[0077] Step 1):
[0078] a) contacting 0.2 mg / mL of the fluorescent microspheres with 10 pg EDAC in 10 mol / L MES buffer solution at pH 6.0 for 30 min to obtain activated fluorescent microspheres;
[0079] b) contacting the third SAA antibody with the activated fluorescent microspheres at 37 °C for 2 hours to obtain antibody-coated fluorescent microspheres;
[0080] c) blocking the antibody-coated fluorescent microspheres with a blocking agent containing 10% BSA at 37 °C for 2 hours; optionally, washing the antibody-coated fluorescent microspheres;
[0081] d) spraying the antibody-coated fluorescent microspheres on a conjugate pad at 3 pL / cm; obtaining a conjugate pad;
[0082] Step 2):
[0083] diluting the first SAA antibody and the second SAA antibody to 2.0 mg / mL and the fourth antibody to 1.0 mg / mL with an antibody diluent, spraying on a nitrocellulose membrane at 1.0 pL / cm to form a first detection line, a second detection line and a quality control line respectively, and obtaining a nitrocellulose membrane after drying;
[0084] Step 3):
[0085] sequentially lapping and fixing the sample pad, the conjugate pad, the nitrocellulose membrane and the water absorption pad on a base plate; obtaining an SAA assay test strip;
[0086] Optionally, step 4): packaging the SAA assay test strip in a card shell.
[0087] In some specific embodiments, the antibody diluent comprises:
[0088] 10 mM to 200 mM buffer,
[0089] 0.5 g / L to 50 g / L electrolyte,
[0090] 0.5 g / L to 50 g / L preservative,
[0091] optionally 0.5 g / L to 100 g / L stabilizer,
[0092] 5.0 to 9.0 pH.
[0093] In some specific embodiments, the buffer is selected from one or a combination of the following: glycine buffer, Tris buffer, MOPS buffer, MES buffer, Hepes buffer, PBS buffer.
[0094] In some specific embodiments, the electrolyte is selected from one or a combination of the following: sodium chloride, potassium chloride, magnesium chloride, magnesium sulfate, zinc chloride, calcium chloride.
[0095] In some specific embodiments, the preservative is selected from one or a combination of the following: sodium azide, phenol, p-hydroxybenzoic acid, PC300.
[0096] In some specific embodiments, the stabilizer is selected from one or a combination of the following: mannose, glucose, chitosan, sorbitol, bovine serum albumin, trehalose, fructose, sucrose.
[0097] In some specific embodiments, the antibody diluent comprises: 50mM PBS buffer, 10g / L sodium chloride, 30g / L trehalose, 1g / L PC300, pH 7.20.
[0098] In some embodiments, the surface of the fluorescent microspheres is fixed with a fluorescent label selected from the following: fluorescein isothiocyanate, rhodamine, Cy3, Cy5, erythrolabe, quantum dots.
[0099] In some specific embodiments, the rhodamine is tetramethyl rhodamine isothiocyanate. BRIEF DESCRIPTION OF DRAWINGS
[0100] Figure 1 : Structure of the test strip.
[0101] Figures 2 to 5 : Line test of the kit. DETAILED DESCRIPTION
[0102] Example 1. Preparation of test strip
[0103] 1. Activation of microspheres: Take fluorescent microspheres with a particle size of 300 nm (the specific average particle size used is 300 nm) and add them to a 10 mol / L MES buffer solution with a pH of 6.0, the concentration of the fluorescent microspheres being 0.2 mg / mL, then add 10 μg of EDAC and activate for 30 min, centrifuge and discard the supernatant, resuspend the precipitate in a 10 mol / L MES buffer solution with a pH of 6.0 using ultrasonic waves, and obtain an activated fluorescent microsphere solution.
[0104] 2. Antibody cross-linking: Add 20 μg of SAA antibody to the fluorescent microsphere solution prepared in step 1, mix well, and cross-link at a temperature of 37 degrees for 2 hours.
[0105] 3. Blocking: Block with 20 μl of a 10% BSA solution. The blocking temperature is 37 degrees, and the blocking time is 2 hours.
[0106] 4. Washing: Centrifuge at a speed of 16000 rpm for 15 minutes, and discard the supernatant.
[0107] 5. The mixture of fluorescent microspheres coated with antibodies is sprayed on the conjugate pad at 3 μL / cm and dried to obtain the finished conjugate pad 3.
[0108] 6. Coating: SAA antibodies (mouse monoclonal, commercially available or laboratory-prepared monoclonal, as long as the antibodies have sufficient affinity and specificity for human SAA according to the standards in the art) are diluted to 2.0 mg / mL with an antibody diluent (50 mM PBS buffer, 10 g / L sodium chloride, 30 g / L trehalose, 1 g / L PC300), and goat anti-mouse is diluted to 1.0 mg / mL, and sprayed on the nitrocellulose membrane at a spraying amount of 1.0 μL / cm to form the SAA first detection line 6, the SAA second detection line 7, and the quality control line 8, respectively, and dried to obtain the nitrocellulose membrane 4.
[0109] The antibodies of the first detection line and the second detection line are the same.
[0110] 7. Laminating: The sample pad 2, the conjugate pad 3 obtained in step 5, the nitrocellulose membrane 4 obtained in step 6, and the water absorption pad 5 are laminated and fixed on the base plate 1 in sequence, and cut into 4.00 mm using a slitting machine. The sample pad used is glass fiber or polyester fiber obtained after soaking in a buffer (containing a surfactant) and drying or without soaking and drying.
[0111] 8. Stripping: The test strip is loaded into a card shell to obtain an SAA assay test strip.
[0112] Example 2. Preparation of a control test strip
[0113] Compared with Example 1, the differences of the control test strip are as follows:
[0114] Table 1
[0115]
[0116] Example 3. Preparation of a kit
[0117] 1. Preparation of sample diluent:
[0118] 50 mM PBS buffer
[0119] 10 g / L sodium chloride
[0120] 20 g / L fatty alcohol polyoxyethylene ether ammonium sulfate
[0121] 10 g / L Tween 20
[0122] 1 g / L PC300
[0123] pH 7.20.
[0124] 2. Package the sample diluent, test strip, and calibration curve card as a kit.
[0125] Test Example 1. Single line test strip (control test strip)
[0126] Table 2. Calibration results for the test strip of Example 2
[0127] Concentration mg / L T / C 0.0 0.003 6.3 0.221 12.6 0.433 25.2 0.854 50.4 1.526 100.7 4.257 201.4 7.392 402.8 11.525 805.6 12.503
[0128] Table 3. Linearity results for the test strip of Example 2 Figure 3 )
[0129]
[0130] From Table 2, Table 3 and Figure 3 As can be seen from the results, the test strip of Example 2 has only one SAA detection line, and the measured value of the high-value sample is low.
[0131] Test Example 2. Test of the test strip / kit of the present disclosure (Example 1)
[0132] 1. SAA assay kit determination steps of the present disclosure
[0133] - Insert the calibration curve card into the corresponding position of the fluorescence analyzer, read the curve information, and select the corresponding sample type before testing.
[0134] - Sampling: Take 5 μL of serum, plasma or whole blood sample with a pipette, add to 500 μL of sample buffer and mix thoroughly for 3-5 s.
[0135] - Sample addition: Take the test card from the aluminum foil bag, and use a pipette to take 80 μL of the diluted sample above and add it to the sample addition hole of the test card.
[0136] - Testing: After sample addition, incubate in the incubator (21°C) for 15 min, then place the test strip into the fluorescence analyzer to read the data. The reaction time is strictly controlled for 15 min.
[0137] - The fluorescence analyzer measures and analyzes the optical signal, and quantitatively obtains the concentration of the measured substance.
[0138] 2. Calibration:
[0139] Table 4A. Calibration results of Example 1
[0140] Concentration mg / L T / C value 0.0 0.003 6.3 0.462 12.6 0.935 25.2 1.732 50.4 3.698 100.7 6.258 201.4 12.527 402.8 24.369 805.6 50.213
[0141] 3. Reproducibility
[0142] The low-value sample and the high-value sample were detected by the reagent strip of Example 1 of the present disclosure, and the test was repeated for 20 times, and the mean value and the coefficient of variation were calculated. The results are as follows:
[0143] Table 4B. Reproducibility results of Example 1
[0144] Low value sample mg / L High value sample mg / L 1 10.2 50.6 2 11.5 48.5 3 11.3 49.7 4 10.6 46.2 5 10.7 50.2 6 9.8 51.0 7 10.5 49.5 8 9.6 47.5 9 8.7 48.5 10 8.5 46.7 11 11.2 51.3 12 10.5 49.6 13 10.6 46.5 14 10.3 45.4 15 9.8 52.1 16 11.2 44.6 17 8.6 52.3 18 8.7 43.5 19 8.9 45.2 20 10.2 46.0 Mean 10.1 48.2 SD 1.0 2.6 CV 9.6％ 5.5％
[0145] As can be seen from the results of Table 4B, the CVs of the test strips prepared in Example 1 for detecting low-concentration samples and high-concentration samples were 9.6% and 5.5%, respectively, indicating that the measurement reproducibility of the present disclosure was good.
[0146] 4. Linearity
[0147] Table 5. Linearity results of Example 1
[0148]
[0149]
[0150] As can be seen from the results of Table 3, Table 5, Figures 2 to 4 As can be seen from the results of Table 3, Table 5,
[0151] Test Example 3. Comparison of the kit of the present disclosure with other methodologies
[0152] The kit of Example 1 of the present disclosure was used to detect high and low value samples to investigate the correlation with a serum amyloid A determination kit (latex immunoturbidimetry):
[0153] Table 6. Correlation with latex immunoturbidimetry reagents (mg / L)
[0154]
[0155] As can be seen from the results of Table 6 and Figure 5 As can be seen from the results of Table 6 and 2 = 0.9921.
[0156] In summary, it can be found that the test strip of the present disclosure uses two detection lines. When the antigen concentration is high, the first detection line intercepts part of the antigen-antibody fluorescent microsphere marker complex, and the excess antigen-antibody fluorescent microsphere marker complex flows further along the nitrocellulose membrane to the second detection line, thereby effectively solving the problem of insufficient linearity caused by the fact that the fluorescence signal intensity on a single detection line test strip cannot continue to rise when it reaches saturation. The test strip and kit of the present disclosure can improve linearity while also taking into account measurement reproducibility.
Claims
1. A serum amyloid A assay strip, comprising: -Base plate (1); -The sample pad (2), the bonding pad (3), the nitrocellulose membrane (4) and the absorbent pad (5) are sequentially overlapped on the base plate (1); Its features are: Following the direction from the sample pad (2) to the absorbent pad (5), the nitrocellulose membrane (4) is sequentially coated with: - First detection line (6); -Second detection line (7); and - Quality control line (8); The first detection line (6) is coated with a first serum amyloid A antibody; The second detection line (7) is coated with second serum amyloid A antibody; The conjugate pad (3) is coated with fluorescent microspheres labeled with third serum amyloid A antibody; The first serum amyloid A antibody, the second serum amyloid A antibody, and the third serum amyloid A antibody are each independently a monoclonal antibody or a polyclonal antibody; The first serum amyloid A antibody and the second serum amyloid A antibody are different from the third serum amyloid A antibody; The first serum amyloid A antibody and the second serum amyloid A antibody may be the same or different.
2. The serum amyloid A assay strip according to claim 1, characterized in that: The surface of the fluorescent microspheres is immobilized with fluorescent markers selected from the following: fluorescein isothiocyanate, rhodamine, Cy3, Cy5, jujube red protein, and quantum dots.
3. The serum amyloid A assay strip according to claim 2, characterized in that: The rhodamine is rhodamine tetramethylisothiocyanate.
4. The serum amyloid A assay strip according to claim 1, characterized in that: The serum amyloid A assay strip is loaded into a cartridge. The retainer includes an upper shell and a lower shell, which are fastened together to form the retainer.
5. The serum amyloid A assay strip according to claim 1, characterized in that... The base plate (1) is a polystyrene board or a polyethylene board.
6. The serum amyloid A assay strip according to claim 1, characterized in that: The average particle size of the fluorescent microspheres is 20 nm to 600 nm. The fluorescence microspheres emit wavelengths from 200 nm to 700 nm.
7. The serum amyloid A assay strip according to claim 6, characterized in that: The average particle size of the fluorescent microspheres is 300 nm. The fluorescence microspheres emit at a wavelength of 365 nm.
8. The serum amyloid A assay strip according to claim 1, characterized in that: The quality control line (8) is coated with a fourth antibody; When the first serum amyloid A antibody, the second serum amyloid A antibody, and the third serum amyloid A antibody are murine antibodies, the fourth antibody is an anti-mouse antibody; or When the first serum amyloid A antibody, the second serum amyloid A antibody, and the third serum amyloid A antibody are rabbit-derived antibodies, the fourth antibody is an anti-rabbit antibody; or When the first serum amyloid A antibody, the second serum amyloid A antibody, and the third serum amyloid A antibody are chicken-derived antibodies, the fourth antibody is an anti-chicken antibody; or When the first serum amyloid A antibody, the second serum amyloid A antibody, and the third serum amyloid A antibody are camel-derived antibodies, the fourth antibody is an anti-camel antibody; or When the first serum amyloid A antibody, the second serum amyloid A antibody, and the third serum amyloid A antibody are sheep-derived antibodies, the fourth antibody is an anti-sheep antibody; or When the first serum amyloid A antibody, the second serum amyloid A antibody, and the third serum amyloid A antibody are equine-derived antibodies, the fourth antibody is an anti-equine antibody.
9. The serum amyloid A assay strip according to claim 1, characterized in that: The first detection line (6) and the second detection line (7) are parallel and spaced 1 mm to 20 mm apart; The second detection line (7) and the quality control line (8) are parallel and spaced 1 mm to 20 mm apart.
10. The serum amyloid A assay strip according to claim 4, characterized in that: The upper shell is provided with a sample inlet corresponding to the sample pad (2); The upper shell is provided with observation windows at locations corresponding to the first detection line (6), the second detection line (7), and the quality control line (8).