u/Wild_Bat9844

Hello everyone, I'm using ELISA kits from Invitrogen (Thermo Fisher Scientific) to measure IgG in the release medium from a hydrogel system. The standard curve range is 1.6-100 ng/mL. I don't know the approximate IgG concentration in each release sample, but the maximum possible concentration should be around 100 µg/mL, based on the amount of IgG encapsulated in the hydrogel.

Should I prepare a series of dilutions for every sample? How can I determine which dilution gives the most reliable results within the standard curve range? Any recommendations would be appreciated!

Hello everyone,

I'm running an ELISA to quantify IgG released from a hydrogel system. This is my first time performing an ELISA, so I wanted to start by testing the assay with the standards only.

I'm using the Invitrogen Human IgG Total Uncoated ELISA Kit:
https://www.thermofisher.com/elisa/product/Human-IgG-Total-Uncoated-ELISA-Kit/88-50550-88

I followed the protocol as described:

• 100 µL/well capture antibody solution, followed by overnight incubation at 4°C
• Washed 4 times, then added 250 µL/well blocking buffer and incubated for 2 hours at room temperature
• Washed 4 times, then added 100 µL standard + 100 µL assay buffer and incubated for 2 hours at room temperature with shaking
• Washed 4 times, then added 100 µL/well detection antibody solution and incubated for 1 hour at room temperature with shaking
• Washed 4 times, then added 100 µL/well substrate solution, with the plan to add stop solution once the color developed

However, no color developed, even after a couple of hours!

I'm wondering what might have gone wrong. Any suggestions or recommendations would be greatly appreciated.

Thank you!

Hello everyone,

I'm running an ELISA to quantify IgG released from a hydrogel system. This is my first time performing an ELISA, so I wanted to start by testing the assay with the standards only.

I'm using the Invitrogen Human IgG Total Uncoated ELISA Kit:
https://www.thermofisher.com/elisa/product/Human-IgG-Total-Uncoated-ELISA-Kit/88-50550-88

I followed the protocol as described:

• 100 µL/well capture antibody solution, followed by overnight incubation at 4°C
• Washed 4 times, then added 250 µL/well blocking buffer and incubated for 2 hours at room temperature
• Washed 4 times, then added 100 µL standard + 100 µL assay buffer and incubated for 2 hours at room temperature with shaking
• Washed 4 times, then added 100 µL/well detection antibody solution and incubated for 1 hour at room temperature with shaking
• Washed 4 times, then added 100 µL/well substrate solution, with the plan to add stop solution once the color developed

However, no color developed, even after a couple of hours!

I'm wondering what might have gone wrong. Any suggestions or recommendations would be greatly appreciated.

Thank you!