Rapid heat-mediated method for enzyme-linked immunosorbent assay procedure
An enzyme-linked immunosorbent, fast technology, applied in the direction of analysis materials, biochemical equipment and methods, microbial measurement/testing, etc., can solve the problems of long-term incubation, biomolecular shedding, low sensitivity, etc., to save time and time , reduce time consumption, simple method
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Embodiment 1
[0085] Example 1 Activation of polycarbonate and polystyrene surfaces
[0086] Add 50 μl methanol-dissolved 6.0 μmol of 1-fluoro-2-nitro-4-azidobenzene (FNAB) per well to the small wells of a PCR plate (polycarbonate plate, Greiner, Germany), and completely in the dark dry. The wells coated with FNAB were then placed in a UV Stratalinker 2400 ( , USA) in the instrument with 365nm UV radiation for 7.5 minutes, or in bright sunlight for 15 minutes. Similarly, polystyrene pores were activated with 50 μl of methanol-dissolved 10 μmol of FNAB, irradiated at 365 nm for 10 minutes, or kept in sunlight for 15 minutes.
[0087] The wells were washed several times with methanol to remove unbound linkers and dried at room temperature. These activated pores can be used for immobilization of antigen or antibody in the step of the present invention.
Embodiment 2
[0088] Example 2 Determination of Goat Anti-Human IgG Concentration for Preparation of Solid Phase on Activated Polycarbonate Surface
[0089] Goat anti-human IgG (dissolved in 0.1M carbonic acid / dicarbonate buffer, pH 9.6) in a doubling dilution series (2000-0.061g / ml) was added to three parallels of activated and untreated polycarbonate plates Wells (90 μl / well for each dilution). The plates were incubated in a thermal cycler at 50°C for 1 hour. It was then washed six times with washing buffer (0.05% Tween 20 containing 0.01M PBS) and incubated with 2% BSA solution (100 μl / well) at 37° C. for 1 hour to complete the blocking step. After washing, 90 µl of a human IgG solution at a concentration of 250 ng / ml (dissolved in 0.01 M PBS, pH 7.4) was added to each well, and the plates were incubated at 37°C for 3 hours. These plates were washed six times again, and 90 μl of 1 / 5000 (v / v) goat anti-human IgG-peroxidase conjugate solution (dissolved in 0.01M PBS, pH 7.4) was added to...
Embodiment 3
[0090] Example 3 Optimization of Goat Anti-Human IgG Curing Temperature on Activation and Untreated Polycarbonate Plates (Table 1)
[0091] Add goat-anti-human IgG solution (250ng / ml solution, 90μl / well) to three parallel wells of 8 activated polycarbonate plates, and heat them in a thermal cycler at 35°C, 40°C, and 45°C respectively. , 50°C, 55°C, 60°C, 65°C and 70°C for 60 minutes. Goat-anti-human IgG was similarly immobilized on untreated polycarbonate plates. The remaining steps of the ELISA were then carried out on these solid phases in the conventional ELISA method described in Example 2.
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