Edison 25 hydroxy vitamin D enzyme-linked immunoassay kit operation points

November 10, 2020

Aidis 25-hydroxyvitamin D enzyme-linked immunosorbent assay kit (25-OH-VD) detection steps

         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 Preparation”.
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.
  • Leave at room temperature for at least 30 minutes until equilibrated to room temperature.
  • Gently and repeatedly invert and mix.

  • Make sure the reagent is completely reconstituted before use.
  • Make sure that the tube used is a borosilicate glass tube or a polypropylene tube. Do not use a polystyrene tube because it can cause low results.
  • Add standards, controls, and samples directly to the bottom of the tube without sticking to the side walls.
  • When using a pipette, add along the tube wall to avoid spillage.
  • Mix well for 10 seconds.
  • Make sure not to touch the bottom of the microplate of the microplate when loading.
  • Do not place the microwell too high when loading to avoid spillage.
  • Make sure the incubation time is just right.

  • Make sure to dispense the appropriate volume of lotion per well.
  • Avoid creating bubbles in the micropores.
  • After washing the plate, quickly tap the plate to remove residual washing solution.

  • When using a multi-channel pipette, make sure that the amount of reagents taken by each nozzle is the same.
  • Use a new weighing pan to place the reagent bottle to prevent contamination.
  • Make sure the amount of reagent dispensed in each well is accurate.
  • Make sure each hole is sealed.
  • Make sure the incubation time is just right.
  • TMB substrates are sensitive to light and susceptible to contamination. Only remove the amount required for the test, discard the unused TMB substrate, and do not pour it back into the bottle.
  • Make sure the microplate reader is set to the correct wavelength. .
  • Read within 30 minutes after the addition of the stop solution.

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