Immune test paper for quantitatively measuring concentration of serum amyloid protein and application
By setting two detection lines and antigen lines, combining specific fluorescein and chromatographic membrane materials, the narrow linear range and inaccurate detection of serum amyloid concentration quantitative determination are solved, and higher linear correlation is achieved Coefficient and detection precision.
Patent Information
- Application Number
- CN202310683622.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-09
- Publication Date
- 2024-12-10
AI Technical Summary
In the quantitative determination of serum amyloid concentrations, the linear range is narrow, the linear fit is low, and the linear correlation coefficient is low, resulting in inaccurate detection, especially at high concentrations, false low values and false negative results are prone to occur.
Set two detection lines, use antigen lines as quality control lines, and use the ratio of (T1+T2)/A for detection, combine specific fluorescein such as Cy5 labeling to improve linear correlation and linear upper limit, and optimize chromatography membrane materials. and flow velocity to control immune response.
The linear range is significantly expanded, the linear correlation coefficient is improved to 0.9995, the accuracy and precision of detection are enhanced, and the problem of inaccurate detection at high concentrations is solved, making quantitative detection more accurate.
Smart Images
Figure CN119104727A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of fluorescent immunoassay, and in particular to a kit for quantitatively measuring the concentration of serum amyloid protein. Background Art
[0002] Based on immunofluorescence technology, the double antibody sandwich method is used to mix the sample to be tested with the detection buffer. The fluorescently labeled detection antibody in the buffer will bind to the serum amyloid A antigen. After the sample mixture is added to the sample well of the strip, it diffuses to the nitrocellulose membrane by capillary action and is captured by the paired antibody fixed on the membrane. Therefore, the amount of complex accumulated on the test strip is proportional to the amount of antigen contained in the sample. The intensity of the fluorescent antibody signal is proportional to the amount of antigen captured by the reaction. After analysis by an immunoassay analyzer or a dry fluorescent immunoassay analyzer, the concentration of the antigen in the sample can be calculated. The value displayed in the quality control area is the basis for determining whether there is enough sample and whether the chromatography process is normal. It also serves as the internal control standard of the reagent.
[0003] However, when the above technology is used to quantitatively determine the concentration of serum amyloid protein (SAA), it is susceptible to interference due to the complexity of the sample to be tested and the immune response process: the influence of sample fluidity during the chromatography process, the influence of the sample matrix, the difference in protein coating capture, the uniformity of the nitrocellulose membrane, the difference in capillary traction, etc., resulting in the reaction intensity of the test line (T line) may change significantly due to the difference in the chromatography process, while the quality control line (C line) will have the same influence as the T line. Based on the experimental concept of controlling variables, the C line can correct the test results, so the T / C value is used to reduce the gap between test strips. In theory, the conjugate signal should be positively correlated with the sample concentration, but due to the high dose of the analyte in the sample and the limitations of the instrument's detection range of fluorescence signal intensity, the fluorescence signal intensity of the T line reaches saturation and is difficult to continue to rise. The linear back-end curve of the test signal T / C tends to be flat, and even appears to bend downward, resulting in false low values or even false negative results. This phenomenon is a unique abnormal phenomenon of the solid phase determination method. The occurrence of this phenomenon will make the linear range of the test sample concentration too narrow and the detection value of high-concentration samples inaccurate.
[0004] In the face of this problem, existing studies such as patent CN115951046A have proposed a solution, that is, setting two T lines. However, there are still problems such as narrow linear range, low linear fit, and low linear correlation coefficient, which makes it difficult for existing test strips and kits to accurately and quantitatively determine the concentration of serum amyloid protein. Therefore, there is an urgent need for a technology that can expand the linear range, improve the linear fit and linear correlation coefficient, and thus accurately and quantitatively determine serum amyloid protein. Summary of the invention
[0005] To solve the above problems, the present invention provides a test strip and application for quantitative determination of serum amyloid concentration. Two detection lines are set, and the antigen line is used as the quality control line, so as to achieve a wider linear range, which is increased from the existing linear upper limit of 100 mg / L to 300 mg / L; the linear fit within the linear range is improved, and the linear correlation coefficient of the existing serum amyloid concentration immunoassay strip or kit is up to 0.9985, and the immunoassay strip and kit for quantitative determination of serum amyloid concentration provided by the present invention can reach 0.9995; in actual operation, the immunoassay strip or kit for quantitative determination of serum amyloid concentration provided by the present invention has accurate quantitative determination, and the high-end measurement precision is higher.
[0006] The test strip and test kit provided by the present invention are provided with two test lines in the test area and are provided with different antibody concentrations, which can effectively reduce the influence of the high dose of the analyte in the sample and the limitation of the intensity range of the fluorescence signal detected by the instrument, and avoid the problem of inaccurate testing under high concentration.
[0007] Due to the complexity of the sample and the test environment, the influence of sample fluidity, sample matrix, protein coating capture differences, uniformity of nitrocellulose membrane, left and right differences in capillary traction, etc. during the chromatography process, the reaction intensity of the T line may change significantly due to differences in the chromatography process, while the quality control line A line will have the same influence as the T line. Based on the experimental concept of controlling variables, the A line can correct the test results; in the test paper and the test kit provided by the present invention, (T1+T2) / A is used for detection: the fluorescence intensity on the three lines is measured using a fluorescent immunoassay analyzer, and then (T1+T2) / A is calculated, and the result is substituted into the standard curve to convert it into a concentration value.
[0008] The antigen line is used as the quality control line, which is combined with the t antibody that is not combined with the previous two T lines. The higher the SAA concentration in the sample, the lower the fluorescence intensity value of the antigen line, which can significantly improve the linear correlation and the linear upper limit. The corresponding buffer does not require other labeled antibodies, only the corresponding SAA labeled antibodies, which makes the process more convenient.
[0009] On the one hand, the present invention provides a fluorescent immunochromatographic test paper for serum amyloid protein, comprising: a T1 detection line, a T2 detection line, and an A-line quality control line; the A-line quality control line is an antigen line coated with serum amyloid protein.
[0010] Furthermore, the T1 detection line and the T2 detection line are coated with mouse anti-human SAA monoclonal antibody.
[0011] Furthermore, it also includes a sample pad, a conjugation pad, and a chromatography membrane; the conjugation pad contains a fluorescein-labeled antibody.
[0012] Furthermore, the fluorescein used for labeling includes but is not limited to: 5-aminofluorescein, tetraethylrhodamine, tetramethylrhodamine isothiocyanate, tetrachlorofluorescein, hexachlorofluorescein, carbonyltetramethylrhodamine, phycoerythrin, and peridinium chlorophyll protein.
[0013] Preferably, the fluorescent dye used for labeling is Cy5.
[0014] Selecting Cy5 as the fluorescent dye used for labeling can improve the linear correlation coefficient of the test results and make the test results more accurate; in principle: specific fluorescent dyes used for labeling can correctly reflect the relationship between fluorescence intensity and concentration at high concentrations, thereby increasing the linear fit of the result curve.
[0015] Furthermore, the T1 detection line, the T2 detection line, and the A-line quality control line are all located on the chromatographic membrane.
[0016] In some embodiments, the chromatography membrane is a nitrocellulose membrane.
[0017] In some embodiments, the bonding pad is made of glass fiber.
[0018] The membrane material we selected can achieve accurate test results. In principle, the selection of a specific membrane can control the flow rate of the liquid to be tested on the membrane and affect the binding rate of the immune response.
[0019] Furthermore, along the liquid flow direction, the liquid to be tested contacts the T1 test line, the T2 test line, and the A line quality control line in sequence. The time it takes for the liquid to be tested to flow on the chromatographic membrane and pass through each line will also affect the test results.
[0020] In some embodiments, the distance between the T1 detection line, the T2 detection line, and the A-line quality control line is 5.5 mm.
[0021] On the other hand, the present invention provides a detection kit, which includes the above-mentioned fluorescent immunochromatographic test paper for serum amyloid protein.
[0022] On the other hand, the present invention also provides the use of serum amyloid protein for preparing an immune test paper for quantitatively determining the concentration of serum amyloid protein, wherein the test paper comprises a T1 detection line, a T2 detection line, and an A-line quality control line arranged in sequence; and the A-line quality control line is coated with serum amyloid protein.
[0023] On the other hand, the present invention also provides the use of SAA antibody for preparing an immune test paper for quantitatively determining the concentration of serum amyloid protein, wherein the test paper comprises a T1 test line, a T2 test line, and an A line quality control line arranged in sequence; the A line quality control line is coated with serum amyloid protein; and the T1 test line and the T2 test line are coated with SAA monoclonal antibodies.
[0024] The beneficial effects of the present invention include:
[0025] 1. The innovative selection of the antigen line as the A-line quality control line improves the precision and accuracy of the test, and provides a new research basis and direction for the development of test strips, test kits and even test methods.
[0026] 2. The achievable range of the linear upper limit has been significantly expanded, from the existing 100 mg / L detection upper limit to 300 mg / L, expanding the detection range of serum amyloid protein concentration.
[0027] 3. The linear correlation coefficient was improved to 0.9995, which solved the problem of inaccurate detection under high concentrations and significantly improved the accuracy and precision of overall quantitative detection. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 Appearance of the test kit
[0029] Figure 2 Linear graph of quality control product test results
[0030] Figure 3 Secondary antibody as quality control line linear spectrum
[0031] Figure 4 Antigen line as quality control line linear map DETAILED DESCRIPTION
[0032] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.
[0033] The main purchased reagents: nitrocellulose membrane, cellulose acetate membrane, glass fiber membrane: purchased from Sartorius; BSA: bovine serum albumin, purchased from Shanghai Xibao Technology and Sigma; TWEEN20: purchased from Sigma; trehalose: purchased from Sigma; polyvinyl pyrrolidone: purchased from Sigma; goat anti-mouse IgG antibody, purchased from Sigma; Cy5, 5-aminofluorescein, tetraethylrhodamine, tetrachlorofluorescein, hexachlorofluorescein, carbonyltetramethylrhodamine, phycoerythrin, and chlorophyll protein of Dinoflagellate: purchased from Bangs Laboratories; mouse anti-human SAA monoclonal antibody, SAA antigen: purchased from Hangzhou Boyin Biotechnology Co., Ltd.
[0034] Self-prepared reagents: 10mM pH7.4PB: phosphate buffer with a concentration of 10 mM, pH7.4; 10mM pH7.4PBS: phosphate buffer with a concentration of 10 mM, pH7.4, containing a final concentration of 0.9% sodium chloride; sample pad treatment solution PBS: phosphate buffer with a concentration of 10 mM, pH7.4.
[0035] Example 1: Preparation of test strips and test kits
[0036] 1.1 Preparation of sample pad
[0037] The glass fiber membrane was immersed in a sample pad treatment solution and then placed in a 45°C oven for drying for 8 hours. The sample pad treatment solution consisted of the following raw materials: 94.45% PBS, 5% trehalose, 0.5% polyvinyl pyrrolidone (PVP), 0.2% TWEEN20, 0.1% S9, and 0.75% BSA.
[0038] 1.2 Preparation of conjugate pad
[0039] 1) Prepare fluorescent solution: dilute Cy5 to 10 mM with DMSO;
[0040] 2) Add SAA monoclonal antibody to the fluorescent solution and rotate and mix at room temperature for 3 hours;
[0041] 3) The labeled antibody is purified and desalted using a Sephadex G-25 column;
[0042] 4) The treatment solution was diluted to 400ul and homogenized by ultrasound. The treatment solution consisted of the following raw materials: 94.45% PBS, 5% trehalose, 0.5% PVP, 0.2% TWEEN20, 0.1% S9, and 0.75% BSA.
[0043] 5) Spray the centrifuged fluorescent solution onto the glass fiber membrane and dry it in an oven at 37°C for 12 hours.
[0044] 1.3 Preparation of chromatographic membrane
[0045] Test line T1: Mouse anti-human SAA monoclonal antibody was diluted with 0.01M pH7.4 PBS and then coated. The streaking parameters were 1 μL / cm and the speed was 80 mm / s.
[0046] Test line T2: Mouse anti-human SAA monoclonal antibody was diluted with 0.01M PBS at pH 7.4 and then coated. The streaking parameters were 1 μL / cm and the speed was 80 mm / s.
[0047] Quality control line A: SAA was diluted to 3.0 mg / mL with 0.01 M PBS at pH 7.4 for coating, with streaking parameters of 1 μL / cm and speed of 80 mm / s;
[0048] After streaking, the nitrocellulose membrane was placed in a 37°C oven to dry for 12 h.
[0049] 1.4 Test strip assembly
[0050] Assemble the sample pad, conjugate pad and chromatography membrane in sequence and cut into strips and mount them on the card.
[0051] 1.5 Kit Assembly
[0052] The test strips assembled in 1.4 are assembled into the shell of the test kit. The shell of the test kit is made of PVC and is manufactured by conventional methods. The appearance is as follows: Figure 1 shown.
[0053] 1.6 Detection
[0054] The quality control product was prepared by the fetal bovine serum dilution antigen method, and the SAA sample was diluted with serum to form quality control products of different concentrations; each quality control product was measured three times, and the measurement method was to use the above-mentioned immune test paper and a fluorescent immunoassay analyzer to measure the fluorescence values reflected by the T1, T2, and A lines. The measured value was (T1+T2) / A. The results are shown in Table 1. A linear graph was drawn based on the data in Table 1 as shown in Figure 2 shown.
[0055] Table 1: Quality control product test results
[0056] Concentration (mg / L) Test value 1 Test value 2 Test value 3 average value 3.0 3.3 2.9 3.1 3.1 28.2 25.6 25.8 26.8 26.1 53.4 52.9 51.7 50.9 51.8 78.6 75.3 75.6 76.1 75.7 103.7 102.6 101.5 104.2 102.8 128.9 129.0 129.0 128.9 129.0 154.1 157.2 158.5 159.1 158.3 179.3 179.3 179.4 179.4 179.3 204.5 202.5 197.6 200.5 200.2 229.6 226.3 225.1 227.4 226.3 254.8 253.2 253.9 256.1 254.4 300.0 299.0 297.5 298.7 298.4
[0057] The product was determined to have a linear range of 3.0 mg / L-300.0 mg / L, with a correlation coefficient (r) of 0.9995; the linear expression was y=0.9979x-0.7684.
[0058] Example 2: Precision Test
[0059] Serum amyloid protein solutions (quality control products from Example 1) with three concentrations (respectively 150 mg / L, 100 mg / L, and 10 mg / L) were taken, and the kit prepared in Example 1 was selected. The solution to be tested was added dropwise to the injection port. After the test was completed, the fluorescence intensity on the three lines was measured using a fluorescent immunoassay analyzer, and the ratio of the fluorescence intensity (T1+T2) / A was calculated, and the result was substituted into the standard curve and converted into a concentration value; the three groups were tested in parallel 10 times, and the means, variances, and coefficients of variation of the three groups were calculated, and the results are shown in Table 2.
[0060] Table 2: Precision test
[0061] Level 1 Level 2 Level 3 1 161.2 101.2 11.1 2 145.2 108.4 10.3 3 150.5 93.8 10.1 4 144.5 102.2 10.4 5 147.0 98.7 10.7 6 145.0 100.0 10.1 7 146.9 87.7 9.9 8 146.5 99.9 10.0 9 145.6 97.0 10.4 10 157.0 93.5 10.3 A 148.9 98.2 10.3 SD 5.70 5.66 0.38 CV 3.8 5.8 3.7
[0062] The results showed that the precision was very good at three concentrations, and when the concentration was 150 mg / L, the coefficient of variation was only 3.8, indicating that the test paper provided by the present invention can accurately and quantitatively detect high-concentration solutions, and the test results are stable and reliable.
[0063] Example 3: Linear comparison of antigen line and secondary antibody as quality control line
[0064] The prior art often uses goat anti-mouse IgG as a quality control line, but in the test strip and kit provided by the present invention, we introduce an antigen line as a quality control line to achieve improved linearity; in this embodiment, we use the test strip prepared in Example 1 in one group, and add goat anti-mouse IgG to the conjugate pad in another group, and select goat anti-mouse IgG as a quality control line. The preparation method is: dilute the goat anti-mouse IgG antibody to 2.0 mg / mL with 0.01M PBS at pH 7.4 for coating, the streaking parameters are 1 μL / cm, the speed is 80 mm / s, and the other preparation methods are the same as in Example 1. The linearity between the two groups is compared.
[0065] The test strip prepared in Example 1 was tested using the test and data analysis method described in Example 1; another group was tested using the same method, and data analysis was also performed using the T / A method. The samples used for testing were all from the quality control products in Example 1. The results are shown in FIG. Figure 3 , Figure 4 As shown, Figure 3 Make a linear plot of the quality control line for the secondary antibody. Figure 4 It is the linear spectrum of the antigen line as the quality control line. The results show that the standard curve with the secondary antibody as the quality control line has poor linearity and is only linear when the test solution is at a low concentration; the standard curve with the antigen line as the quality control line has very good linearity, a high upper limit of linearity, and a high linear correlation coefficient; it can be seen that the antigen line as a quality control line can significantly improve linearity, increase the concentration range of detection, and improve the precision and accuracy of detection.
[0066] And according to the above-mentioned linear ratio data of 0-300 mg / L, it can be concluded that the linear correlation coefficient of the test strip or kit provided by the present invention can reach 0.9995. Using the test strip or kit provided by the present invention, the precision and accuracy of the test are much higher than those of the existing test strip or kit.
[0067] Example 4: Effect of Setting Two T Lines on the Linear Upper Limit
[0068] This example studies the effect of the T line on the upper limit of linearity. The test strips were prepared using the method described in Example 1, in which only one T line was prepared in one group, and all the T lines in the other group were prepared according to Example 1.
[0069] The quality control samples in Example 1 were selected as the samples for testing. The detection and data analysis were carried out according to the method described in Example 1. The linear upper limits of the two groups were studied. The results showed that the linear upper limit of one T line was only 100 mg / L, while the linear upper limit of two T lines could reach 300 mg / L, which could solve the problem of inaccurate detection at high concentrations.
[0070] Example 5: Effect of different fluoresceins on linearity
[0071] In the method for using the test strip and the kit provided by the present invention, after the determination is completed, it is necessary to first use a fluorescent immunoassay analyzer to measure the fluorescence intensity on the test line and the quality control line, so selecting different fluoresceins will affect the fluorescence intensity, thereby affecting the linear correlation of the results. This embodiment selects different fluoresceins, prepares test strips according to the method of Example 1, and uses the method described in Example 2 to perform testing and data analysis. The results are shown in Table 3.
[0072] Table 3: Effects of different fluoresceins on linearity
[0073] Fluorescein Linear correlation coefficient 5-aminofluorescein 0.9920 Tetraethylrhodamine 0.9981 Cy5 0.9995 Tetrachlorofluorescein 0.9901 Hexachlorofluorescein 0.9873 Carbonyltetramethylrhodamine 0.9866 Phycoerythrin 0.9774 Peridinium chlorophyll protein 0.9853
[0074] The results showed that when Cy5 was used as the fluorescent dye, the upper limit of the linear detection was the highest, which could reach 300 mg / L, and the linear correlation coefficient was high, which was 0.9995. The detection precision and accuracy were high, making it the preferred choice.
[0075] Example 6: Effect of different membrane materials on linearity
[0076] In the method for using the test strip and the kit provided by the present invention, the liquid to be tested flows along the membrane during detection, and the flow rate of the liquid will also have a certain impact on the test results. In this embodiment, nitrocellulose membrane, glass fiber membrane, and cellulose acetate membrane are selected for different combinations on the conjugate pad and the chromatography pad, and the test strips are made according to the method of Example 1. The sample used for testing is the quality control product in Example 1, and the method described in Example 1 is used for testing and data analysis. The results are shown in Table 4.
[0077] Table 4: Effect of different membrane materials on linearity
[0078] Bonding pad Chromatography membrane Linear correlation coefficient Nitrocellulose membrane Nitrocellulose membrane 0.9883 Nitrocellulose membrane Glass fiber membrane 0.9632 Nitrocellulose membrane Cellulose acetate membrane 0.9522 Glass fiber membrane Nitrocellulose membrane 0.9995 Glass fiber membrane Glass fiber membrane 0.9856 Glass fiber membrane Cellulose acetate membrane 0.9630 Cellulose acetate membrane Nitrocellulose membrane 0.9534 Cellulose acetate membrane Glass fiber membrane 0.9447 Cellulose acetate membrane Cellulose acetate membrane 0.9063
[0079] The results showed that when glass fiber membrane was used as the conjugate pad and nitrocellulose membrane was used as the chromatography membrane, the linear correlation coefficient was the highest, the linearity of the result was the best, the detection precision was high and the accuracy was good, which was the preferred method.
[0080] Example 7: Effect of the distance between T1 line, T2 line and A line on linearity
[0081] In the method for using the test strip and the kit provided by the present invention, the detection process is a chromatographic process, so the distances between the T1 line, the T2 line, and the A line will affect the detection results. In this embodiment, different distances are selected, and other conditions are the same. The test strips are prepared according to the method of Example 1, and the samples used for testing are the quality control products in Example 1. The method described in Example 1 is used for testing and data analysis. The results are shown in Table 5.
[0082] Table 5: Effect of the distance between T1 line, T2 line and A line on linearity
[0083] Distance(mm) Linear correlation coefficient 3.5 0.9788 4 0.9826 4.5 0.9856 5 0.9914 5.5 0.9995 6 0.9943 6.5 0.9839
[0084] The results showed that when the distances between the T1 line, T2 line and A line were all 5.5 mm, the linearity was the best, the linear correlation coefficient could reach 0.9995, the detection precision was high and the accuracy was good, which was the preferred method.
[0085] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.
Claims
1. A fluorescent immunochromatographic test paper for serum amyloid protein, characterized in that: It comprises a T1 detection line, a T2 detection line and an A-line quality control line which are arranged in sequence; the A-line quality control line is coated with serum amyloid protein.
2. The fluorescent immunochromatographic test paper according to claim 1, characterized in that: The T1 detection line and the T2 detection line are coated with mouse anti-human SAA monoclonal antibody.
3. The fluorescent immunochromatographic test paper according to claim 1, characterized in that: It also includes a sample pad, a conjugate pad, and a chromatographic membrane; the conjugate pad contains a mouse anti-human SAA monoclonal antibody labeled with fluorescein; the fluorescein used for labeling includes but is not limited to: 5-aminofluorescein, tetraethylrhodamine, tetramethylrhodamine isothiocyanate, tetrachlorofluorescein, hexachlorofluorescein, carbonyltetramethylrhodamine, phycoerythrin, chlorophyll protein of polydinoflagellates, and Cy5.
4. The fluorescent immunochromatographic test paper according to claim 3, characterized in that: The fluorescent substance used for labeling is Cy5.
5. The fluorescent immunochromatographic test paper according to claim 3, characterized in that: The T1 detection line, T2 detection line and A-line quality control line are all located on the chromatographic membrane; the chromatographic membrane is a nitrocellulose membrane.
6. The fluorescent immunochromatographic test paper for serum amyloid protein according to claim 3, characterized in that: The material of the bonding pad is glass fiber.
7. The fluorescent immunochromatographic test paper for serum amyloid protein according to claim 1, characterized in that: Along the liquid flow direction, the liquid to be tested contacts the T1 test line, the T2 test line, and the A-line quality control line in sequence; the distance between the T1 test line, the T2 test line, and the A-line quality control line is 5.5 mm.
8. A method for detecting serum amyloid protein, characterized in that: The detection is performed using the immunochromatographic test paper as described in any one of claims 1 to 7.
9. Use of serum amyloid protein for preparing an immunoassay paper for quantitatively determining the concentration of serum amyloid protein, characterized in that: The test paper comprises a T1 detection line, a T2 detection line, and an A-line quality control line which are arranged in sequence; and the A-line quality control line is coated with serum amyloid protein.
10. Use of SAA antibody for preparing an immunoassay paper for quantitatively determining serum amyloid protein concentration, characterized in that: The test paper comprises a T1 test line, a T2 test line and an A-line quality control line which are arranged in sequence; the A-line quality control line is coated with serum amyloid antigen; and the T1 test line and the T2 test line are coated with SAA monoclonal antibodies.