Aidis 25-hydroxyvitamin D enzyme-linked immunosorbent assay kit (25-OH-VD) detection steps | Â Â Â Â Â Â Â Â Key points and explanation | |
All reagents were equilibrated to room temperature before testing and carefully mixed. Reconstitute or prepare the relevant reagent as described in “Reagent Preparationâ€. 1. Prepare a labeled glass test tube or polypropylene tube for dilution of standards, controls, and samples. 2. Add 25 μl of the standard, control, and sample to the corresponding labeled tubes. 3. Add 1 ml of 25D biotin to all tubes. Mix with a vortex mixer for 10 seconds. 4. Take 200 μl of diluted standard, control or sample into the microwells of the corresponding microplates of the coated antibody to make double wells. The plate was sealed with a parafilm and incubated at 18-25 ° C for 2 hours. 5. Wash the plate 3 times with diluted solution. a) Automatic washing: set the washing machine to dispense at least 300μl of lotion per well. After the filling, take the time, so cycle 3 times. b) Manually wash the plate: quickly invert the contents of the hole. 250 μl of wash solution was added to each well. The plate was washed twice more. Before going to the next step, the inverted plate was vigorously tapped on the absorbent paper to remove excess wash solution. 6. Add 200 μl of enzyme conjugate to all microwells using a multichannel pipettor. The plate was sealed with a parafilm and incubated at 18-25 ° C for 30 minutes. 7. Repeat step 5. 8. Add 200 μl of TMB substrate to all wells using a multichannel pipette, seal the plate with a parafilm, and incubate at 18-25 ° C for 30 minutes. 9. Add 100μ stop solution to all microwells using a multichannel pipettor. 10. The absorbance was measured at a wavelength of 450 nm (reference 650 nm) of the microplate reader within 30 minutes after the addition of the stop solution. |
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