The below protocol is a sample protocol for Monkey GAD-Ab(Anti-Glutamic Acid Decarboxylase antibody) ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Monkey GAD-Ab(Anti-Glutamic Acid Decarboxylase antibody) present in their sample.
Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37 °C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.
Serum, Plasma, Cell Culture Supernatant, Cell or Tissue Lysate, Other Liquid Samples
Storage:
2-8°C(Sealed), Don't cryopreserve.
Linearity:
Sample
1:2
1:4
1:8
Serum (n = 5)
89-102%
87-98%
83-97%
EDTA Plasma (n = 5)
87-103%
86-99%
80-95%
Heparin Plasma (n = 5)
88-102%
84-101%
80-99%
Recovery:
Sample
Recovery Range (%)
Average (%)
Serum (n = 5)
86-96
91
EDTA Plasma (n = 5)
86-103
95
Heparin Plasma (n = 5)
90-103
95
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Step
Procedure
1
Reagent & Plate Preparation: Equilibrate reagents and TMB substrate to room temperature. Set standard, test sample and control (zero) wells on the pre-coated plate and record their positions.
2
Primary Incubation: Prepare standards, samples, blanks and load into designated wells. Incubate plate at 37°C for 90 minutes to allow antigen binding.
3
Biotin-Labeled Antigen Binding: Add biotin-labeled antigen working solution and incubate at 37°C for 60 minutes.
4
HRP-Streptavidin Binding: Add SABC working solution and incubate at 37°C for 30 minutes.
5
Color Development: Add TMB substrate and incubate in the dark for 10–20 minutes until color develops.
6
Stop Reaction & Reading: Add stop solution and read absorbance at 450 nm immediately.
Sample Type
Protocol
Serum
Allow blood to clot, centrifuge at 1000 × g for 20 minutes and collect supernatant.
Plasma
Collect in anticoagulant tubes, centrifuge at 1000 × g for 15 minutes at 2–8°C and collect plasma.
Tissue Homogenate
Homogenize tissue with PBS or lysis buffer, centrifuge and collect supernatant.
Cell Culture Supernatant
Centrifuge at 2500 rpm for 5 minutes and collect clarified supernatant.
Cell Lysate
Lyse cells on ice using lysis buffer, centrifuge and collect supernatant.
Other Sample Types
For more information about how to process other sample types, (e.g., body fluids, breast milk & more), please contact our Tech Support Team at techsupport@assaygenie.com.
Component
Quantity
Storage
48T
96T
ELISA Microplate (Dismountable)
8×6
8×12
Place the test strips into a sealed foil bag with the desiccant. Store for 1 month at 2-8°C; Store for 12 months at -20°C.
Lyophilized Standard
1 vial
2 vial
Place the standards into a sealed foil bag with the desiccant. Store for 1 month at 2-8°C; Store for 12 months at -20°C.
