Pichia Pastoris Hcp Elisa Assay Kit

1.Preparation of Reagents:

• Take the reagent kit out of the refrigerator and allow it to equilibrate to room temperature for at least 30 minutes, ensuring all required reagents for the experiment are ready. Mark the opening date on the reagent kit and prioritize using the oldest opened kit. Kits from the same batch number can be used together, but kits from different batch numbers should not be mixed. Return any unused strip plates back to their sealed bag and store at 2–8°C for future use.

• Prepare the Wash Solution (1×): Dilute an appropriate amount of 20× Wash Buffer with deionized water to achieve a 1× solution. If crystals are present in the 20× Wash Buffer, place it at room temperature or in a 37°C water bath, gently shaking until the crystals are fully dissolved before dilution.

  
  

2.Standard Curve Preparation:

• The Pichia HCP calibration standard does not require dilution and can be used directly.

Sample Solution Preparation:

• Dilute the sample using Sample Diluent Buffer to bring it within the range of the standard curve, and mix thoroughly.

Spiked QC Solution Preparation:

• Take 120 µL of the sample solution and add 120 µL of the 25 ng/mL standard, mix thoroughly.

Washing the Plate:

• Wash the plate with 1× Wash Solution, adding 300 µL per well. Dry any residual liquid from the wells by inverting and tapping the plate gently. Repeat this process three times.

Sample Incubation:

• Add 100 µL of the standard, sample, or spiked QC to the corresponding wells. Seal the plate with a sealing film and incubate at 37°C with shaking at 300 rpm for 1 hour. (Note: Ensure the plate is properly sealed during incubation; incomplete sealing or lack of sealing can lead to evaporation, causing errors in the results.)

  
  

3.Washing the Plate Again:

• After incubation, let the plate sit at room temperature for 3-5 minutes. Carefully remove the sealing film and discard the liquid in the wells. Wash the plate with 1× Wash Solution, adding 300 µL per well. Dry any residual liquid by inverting and tapping the plate. Repeat this process three times.
  (Note: If washing manually, dispense the wash buffer above the wells without touching the well walls with the pipette tips. After each wash, let the plate sit for 30 seconds and gently shake before inverting the plate to dry the wells. Use new absorbent paper or a clean section of the same paper each time.)

Preparation of HRP-conjugated Antibody:

• Dilute the 100× Anti-Pichia HCP-HRP antibody using Antibody Dilution Buffer to achieve a 1× working solution. For example, add 100 µL of 100× Anti-Pichia HCP-HRP to 9.9 mL of Antibody Dilution Buffer.

  
  

4.Antibody Incubation:

• Add 100 µL of the diluted HRP-conjugated antibody to each well, seal the plate, and incubate at 37°C with shaking at 300 rpm for 1 hour. Take Color Reagent A and B out of the 4°C refrigerator and allow them to equilibrate to room temperature.

Washing the Plate Again:

• After incubation, let the plate sit at room temperature for 3-5 minutes. Carefully remove the sealing film and discard the liquid. Wash the plate five times with 1× Wash Solution, adding 300 µL per well each time. Dry any residual liquid from the wells.

  
  

5.Color Development:

• Mix Color Reagent A and B in a 1:1 ratio and add 100 µL of the mixture to each well. Seal the plate and incubate at room temperature, protected from light, for 20 minutes.

  
  

6.Termination:

• Add 50 µL of Stop Solution to each well to terminate the reaction. After stopping the reaction, read the plate as soon as possible. (Note: It is recommended to set the microplate reader to perform a 5-10 second shaking before reading.)

Reading the Plate:

• Within 20 minutes, measure the absorbance at 450 nm with a reference wavelength at 630 nm using a microplate reader.