Test strip for simultaneously and quantitatively detecting NSE and S100B in blood and preparation method thereof

[0029] Embodiment 1. This embodiment provides a magnetic immunochromatographic test strip for simultaneous quantitative detection of NSE and S100B in blood and a preparation method thereof.

[0030] The NSE paired antibody and S100B paired antibody used in this example are both monoclonal antibodies prepared by monoclonal antibody technology. Use the principle of double-antibody sandwich detection of NSE and S100B antigens to detect specimens. When the specimen to be tested contains NSE and S100 antigens, the antigens will first be combined with the magnetic beads coupled to the NSE antibody and the magnetic beads coupled to the S100B antibody on the binding pad. As the chromatography progresses, the conjugate moves forward to reach the S100B antibody detection line T2, the S100B antigen will combine with the coated antibody again to form a double antibody sandwich complex and gather at T2, and the conjugate moves forward to reach the NSE antibody At the detection line T1, the NSE antigen will combine with the coated antibody again to form a double-antibody sandwich complex and gather at T1. In addition, the unbound magnetic beads coupled with the NSE antibody and S100B antibody will continue to move forward and reach the quality control line At C, it will bind to the rabbit anti-mouse IgG antibody, so magnetic beads will also aggregate at C. The entire reaction can be completed within 30 minutes. Generally, it can be tested in a magnetic signal detector after 20 minutes of reaction. The T1, T2 and C lines will have corresponding magnetic signal values. Then, the detection value is brought into the fitting curve to calculate the quantitative result.

[0031] Preparation method of magnetic immunochromatographic test strip capable of simultaneously quantitatively detecting NSE and S100B in blood. The preparation method of test strip in this embodiment includes the following steps:

[0032] 1. Preparation of immunomagnetic beads:

[0033] Preparation of 2-morpholineethanesulfonic acid (MES) activation buffer: 0.01 M pH=5.5 (pH=5.0-pH=6.0 are applicable) MES buffer is the activation buffer, add Tween 20, the final concentration is 0.05 wt%, 0.22 μm microporous filter membrane is filtered and sterilized and stored at 4℃ for later use. The validity period is two weeks.

[0034] 1) Preparation of boric acid-borax (BS) coupling buffer: prepare 0.2 M pH=9.0 BS buffer with 0.05 M borax and 0.2 M boric acid in a 4:1 (v:v) ratio, and dilute with ultrapure water to 0.005 M, add Tween 20, the final concentration is 0.05%, 0.22 μm microporous membrane filter sterilizes and store at 4 ℃ for later use, the validity period is two weeks.

[0035] 2) Preparation of boric acid-borax (BS) blocking buffer: prepare 0.2 M pH=9.0 BS buffer with 0.05 M borax and 0.2 M boric acid in a 4:1 (v:v) ratio, and dilute to 0.005 with ultrapure water M, add BSA, Tween 20, the final concentration is 1wt%, 0.05 wt%, 0.22 μm microporous membrane filter sterilization, store at 4 ℃, the validity period is one week.

[0036] 3) Preparation of boric acid-borax (BS) storage buffer: prepare 0.2 M pH=9.0 BS buffer with 0.05 M borax and 0.2 M boric acid in a 4:1 (v:v) ratio, and dilute with ultrapure water To 0.005 M, add BSA, Tween 20, NaN 3 , The final concentration is 1 wt%, 0.05 wt%, 0.05 wt%, 0.22 μm microporous filter membrane is filtered and sterilized and stored at 4 ℃ for later use. The validity period is one week.

[0037] 4) Preparation of anti-NSE immunomagnetic beads: Wash the beads with 0.01 M (pH=5.0-pH=6.0 are applicable) pH=5.5 MES buffer containing 0.05 wt% Tween 20, and both EDC and NHS are added. It is twice the molar amount of the magnetic bead carboxylation degree. React at room temperature for 1-2 hours. Wash the magnetic beads with 0.005 M pH=9.0 BS buffer containing 0.05 wt% Tween 20. Add an appropriate amount of anti-S100B-mAb to -40 μg anti-NSE-mAb/mg magnetic beads, and react at room temperature for 4-6 hours. Use 0.005 M pH=9.0 containing 0.05 wt% Tween 20 and 1 wt% BSA. Wash the magnetic beads with BS buffer solution. After fully washing the magnetic beads, block them at 37°C for 30 minutes. Finally, use 0.05 wt% Tween 20, 1 wt% BSA, 0.05 wt% NaN 3 Resuspend the magnetic beads in 0.005 M pH=9.0 BS buffer and store at 4°C for later use.

[0038] 5) Preparation of anti-S100B immunomagnetic beads: Use 0.01 M (pH=5.0-pH=6.0 are applicable) pH=5.5 MES buffer solution containing 0.05 wt% Tween 20 to wash the magnetic beads, EDC and NHS The added amount is twice the molar amount of the carboxylation degree of the magnetic beads. React at room temperature for 1-2 hours. Wash the magnetic beads with 0.005 M pH=9.0 BS buffer containing 0.05 wt% Tween 20. Then, add an appropriate amount of anti-S100B-mAb in the amount of 20-40 μg anti-S100B-mAb/mg magnetic beads, and react at room temperature for 4-6 hours. Use 0.005 M pH containing 0.05 wt% Tween 20 and 1 wt% BSA =9.0 BS buffer to wash the magnetic beads. After fully washing the magnetic beads, block them at 37°C for 30 minutes. Finally, use 0.05 wt% Tween 20, 1 wt% BSA, 0.05% NaN 3 Resuspend the magnetic beads in 0.005 M pH=9.0 BS buffer and store at 4°C for later use.

[0039] 2. Preparation of nitrocellulose membrane-coated antibody:

[0040] 1) Preparation of antibody diluent: 0.01 M, pH=7.4 phosphate (PBS) buffer is the coating buffer, sucrose is added, the final concentration is 1 wt%, and the 0.22 μm microporous membrane is filtered and sterilized Store at 4 ℃ for later use. The validity period is one week.

[0041] 2) Preparation of nitrocellulose membrane-coated antibody: Dilute the NSE antibody to 1.5 mg/mL and the S100B antibody to 1.5 mg/mL using 0.01 M pH=7.4 PBS buffer containing 1 wt% sucrose mL, the rabbit anti-mouse IgG antibody was diluted to 0.5 mg/mL, and the three were uniformly sprayed on a 2.5 cm wide nitrocellulose membrane at 0.4 cm intervals at a volume of 1 μL/cm using a streak machine, at room temperature Air dry, add desiccant to seal, store at 4°C for later use.

[0042] 3. Processing of sample pad:

[0043] The 15 mm long sample pad is soaked in the sample pad treatment solution for 2 hours, and then dried in an oven at 25 ℃-37 ℃ for 12 hours.

[0044] The sample pad treatment solution is a 0.005 M pH=9.0 BS buffer containing 2 wt% NaCl, 2 wt% TritonX-100, 0.5 wt% BSA, and 0.5 wt% PVP.

[0045] 4. Treatment of bonding pad

[0046] The 10 mm long sample pad was soaked in the sample pad treatment solution for 2 hours, and then dried in an oven at 30 ℃ for 12 hours.

[0047] The binding pad treatment solution is a 0.005 MpH=9.0 BS buffer containing 5 wt% sucrose, 2 wt% trehalose, and 0.05 wt% TritonX-100.

[0048] 5. Preparation of bonding pad

[0049] Spread the prepared anti-NSE immunomagnetic beads and anti-S100B immunomagnetic beads at a ratio of 1:1, 1.5 mg/mL, and 12 μL, using a micropipette, respectively, and evenly apply 30 μL/cm to the processed Combine it on the mat, then place it in a 30 ℃ oven to dry and add a desiccant to seal for later use.

[0050] 6. Assembly and cutting of test strips

[0051] Paste the nitrocellulose membrane (length 25 mm), bonding pad (length 10 mm), sample pad (length 15 mm) and absorbent paper (length 25 mm) on the backing plate in turn, and the adjacent pads are interlaced with each other Overlapping 2 mm, after assembly, use an automatic cutting machine to cut into finished test strips with a width of 3 mm, put them into the test card slot, put them into an aluminum foil bag and add a desiccant to seal and store for later use.

[0052] Example 2

[0053] Except for the preparation steps of immunomagnetic beads: the addition amount of EDC and NHS are both 5 times the molar amount of the carboxylation degree of the magnetic beads, the other steps are the same as in Example 1.

[0054] Example 3

[0055] Except for the bonding pad preparation step, the content of sucrose in the bonding pad treatment solution used is 10 wt%, and the other steps are the same as in Example 1.

[0056] Method of using the detection card of the present invention

[0057] 1. Add samples

[0058] Take out the single test card from the packaging box, tear open the aluminum foil packaging bag, place it on a flat table, and use a micropipette to draw 70 μL of the serum sample into the sample hole of the test card, and wait for the reaction to proceed for 20 minutes.