Cystatin C fluorescent microsphere immunochromatographic quantitative test strip, and test card thereof
A fluorescent microsphere, quantitative detection technology, applied in the field of immunodetection, to reduce non-specific binding, improve performance, clear detection lines and quality control lines
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Embodiment 1
[0045] Example 1. Preparation of Cystatin C Monoclonal Antibody Labeled with Fluorescent Microspheres (EDC Method)
[0046] Take one 1.5ml centrifuge tube, mark it well, add 500μl, 0.02M, PH=7.5 Tris-HCl, and then add 10μl carboxyl fluorescent microspheres (lanthanide europium (Eu) with a particle size of 180nm 3+ ) chelate) and 60ul 2mg / ml EDC activator, shake and mix, place on a constant temperature shaker for 20min, centrifuge at 15000r / min for 10min, discard the supernatant, add 1000ul 0.02M, PH=7.5Tris-HCl Redissolve the precipitate; add 40 μg of mouse cystatin C monoclonal antibody, mix well, put it on a constant temperature shaker to oscillate and couple, centrifuge at 15000rpm / min for 10min, remove the supernatant, add 500ul reconstitution solution to redissolve the precipitate, add Block with 50 μl of blocking solution containing 10% BSA for 1 hour, and store at 4°C for later use.
Embodiment 2
[0047] Embodiment 2, the preparation of sample pad
[0048] Take a commercially available glass fiber membrane as the sample pad substrate, and divide it into three groups, 6 in each group:
[0049] The first group: without treatment, spray the fluorescent microsphere-labeled cystatin C monoclonal antibody prepared in Example 1 onto the glass fiber membrane in an amount of 10 ul / ml with a three-dimensional gold spray film apparatus, and heat Dry for 2 hours;
[0050] The second group: use the treatment liquid for soaking treatment, then place it in a constant temperature drying oven at 37°C to dry for 2 hours, and then perform the same spraying operation, wherein the treatment liquid composition is: Tris with a working concentration of 0.05M and a pH of 7.5-8.0 – HCl buffer, BSA at a mass concentration of 0.05-0.5%, and 1% sucrose and 0.05% NaN 3 ;
[0051] The third group: use the treatment liquid for soaking treatment, then place it in a constant temperature drying oven a...
Embodiment 3
[0054] Embodiment 3, the preparation of nitrocellulose membrane (NC membrane)
[0055] Use 0.02M pH7.5 PBS buffer to dilute the rabbit-derived Cystatin C polyclonal antibody to a concentration of 2mg / mL, and use 0.02M pH7.5 PBS buffer to dilute the goat anti-mouse IgG polyclonal antibody to a concentration of 1mg / mL , The resulting diluted solution was streaked on the NC membrane to form a test line (T line) and a quality control line (C line), the two lines were separated by 5mm, dried overnight at 37°C, and stored in a dry environment at room temperature for later use.
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