Latex microsphere and protein coupling method and kit
A latex microsphere, protein coupling technology, applied in instruments, measuring devices, scientific instruments, etc., can solve the problems of limiting the application and performance of latex microsphere tracers, deviation of test results, etc.
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Embodiment 1
[0045] In this example, a carboxyl latex microsphere is prepared, which is coupled with IL-6 antibody, and the coupling method is as follows:
[0046] (1) A solution replacement treatment will be performed for fluorescent latex microspheres (200-400 nm in diameter), and the replacement solution is the same as the activation solution. In this example, the replacement solution was MES, the concentration was 10 mM, and the pH was 5.5.
[0047] (2) According to the carboxyl group content on the surface of the latex microspheres, according to the molar ratio of EDC to carboxyl group is 30:1, and the ratio of EDC to Sulfo-NHS is 1:4, calculate the amount of EDC and Sulfo-NHS, dissolve in activation buffer and add Microspheres, activated for 30min.
[0048] (3) resuspending, and then dispersing with MES coupling solution to obtain a dispersed solution of activated carboxyl microspheres.
[0049] (4) Add an IL-6 mouse monoclonal antibody to be labeled into the activated fluorescent ...
experiment example 1
[0063] Prepare immunofluorescence chromatography test paper as follows:
[0064] Preparation of coating film: A mouse anti-IL-6 monoclonal monoclonal antibody and goat anti-mouse polyclonal antibody were diluted to 2 mg / mL and 2 mg / mL with a streak diluent, respectively, and sprayed onto a nitrocellulose membrane as a detection line. (T) and quality control line (C), after which the coated NC film was dried in a vacuum-like environment for 2 h.
[0065] Test card assembly: Adhere the GE absorbent pad, the coating film, and the V9 sample pad to the PVC bottom plate in turn, cut them into test strips with a width of 3.2mm, and put them into the cartridge case, and then an immunofluorescence assay for detecting IL-6 antigen is completed. Card. The specific diagram is as figure 1 .
[0066] (2) Detection of IL-6 signal:
[0067] The specific test method is as follows: in each test, add 0.2 μL of the antibody-microsphere complex and mix with the sample diluent, then add the cal...
experiment example 2
[0072] In this experimental example, particle size and dispersity were detected respectively.
[0073] The specific test method is: turn on the ZSU3200 particle size analyzer and preheat for 30 minutes, mix 2 μL of the labeled protein-microsphere complex with 1.5 mL of deionized water, put it into the cleaned plastic cuvette, and then put the plastic colorimetric Put the dish into the card slot of the instrument, set the number of instrument tests to 3, and test 5 cycles for each test. The average particle size and dispersion coefficient of the microspheres were obtained.
[0074] (1) The effect of different EDC contents on the particle size of latex microspheres during the activation process was detected during the labeling process of latex microspheres.
[0075] Table 2 Comparative Examples 1-2 and Example 1 Particle Size During Activation
[0076] Condition Description particle size dispersion coefficient Comparative Example 1 EDC:Carboxyl = 100:1 ...
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