This kit is based on a double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) detection method for the quantitative measurement of CysLTs (Cysteinyl leukotrienes LTC4/LTD4/LTE4) in the following samples: Serum, Plasma, Cell Culture Supernatant, Cell or Tissue Lysate, Other Liquid Samples. The microplate provided in this kit has been pre-coated with a specific antigen. Standards and appropriately diluted samples are added to the wells and incubated, allowing target antibodies in the sample to bind to the immobilized antigen. After incubation and washing to remove unbound components, a biotin-labeled antigen is added, forming a sandwich complex. HRP-conjugated Streptavidin is then added, followed by TMB substrate solution to produce a colorimetric reaction. The reaction is stopped with an acidic solution, and absorbance is measured at 450 nm using a microplate reader. The signal intensity is directly proportional to the concentration of the target analyte and is determined using a standard curve.
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 = 10)
87-101%
86-101%
86-97%
EDTA Plasma (n = 10)
90-105%
85-97%
90-100%
Heparin Plasma (n = 10)
90-103%
87-98%
92-99%
Recovery:
Sample
Recovery Range (%)
Average (%)
Serum (n = 10)
85-100
95
EDTA Plasma (n = 10)
85-105
99
Heparin Plasma (n = 10)
90-101
94
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 TMB substrate for 30 minutes at room temperature. Prepare standards, samples (minimum 1:2 dilution), blanks, assign wells, and pre-wash the plate twice.
2
Sample & Biotin-Antibody Binding: Add 50 µL standard or sample followed by 50 µL biotin-labeled antibody to each well. Mix gently and incubate at 37°C for 45 minutes.
3
Washing: Wash the plate 5 times with wash buffer, allowing 1 minute soak time per wash.
4
Color Development: Add TMB substrate and incubate in the dark at 37°C for 10-20 minutes until color develops.
5
Stop Reaction: Add stop solution to terminate the reaction. The color changes from blue to yellow immediately.
6
Reading: Measure absorbance at 450 nm using a microplate reader.
Sample Type
Protocol
Serum
Allow blood to clot, centrifuge at 1000 × g for 20 minutes, collect supernatant and store appropriately.
Plasma
Collect using EDTA anticoagulant, centrifuge at 1000 × g for 15 minutes at 2–8°C and collect plasma.
Cell Culture Supernatant
Centrifuge at 1000 × g for 20 minutes at 4°C and collect clarified supernatant.
Cell Lysate
Lyse cells using recommended lysis buffer with protease inhibitors, centrifuge at 10,000 rpm for 10 minutes, and collect protein supernatant.
Tissue Homogenate
Homogenize tissue in PBS with protease inhibitors, centrifuge at 5000 × g for 5 minutes, and collect supernatant.
Other Sample Types
Centrifuge samples at 1000 × g for 15 minutes at 2–8°C and collect supernatant. For additional guidance, please contact 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.
Biotin-labeled Antibody (Lyophilized)
1 vial
1 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.
