Rat CD74 ELISA Kit, Lot 21AO-1230 [Cancer Immune Checkpoint Assay Kit] (CAT#: IOK-05-P1141)

Assay plate (12 × 8 coated Microwells)
Standard (freeze dried)
Biotin-antibody (100 × concentrate)
HRP-avidin (100 × concentrate)
Biotin-antibody Diluent
HRP-avidin Diluent
Sample Diluent
Wash Buffer (25 × concentrate)
TMB Substrate
Stop Solution
Adhesive Strip (for 96 wells)
Instruction manual

Microplate reader.
Pipettes and pipette tips.
EP tube Deionized or distilled water.

100 μL

3 - 5 h

Pre-coated

1.Assay diluent: Dilute the concentrated assay diluent 1:5 with distilled water. Wash buffer: Dilute the concentrated wash buffer 1:20 with distilled water.
2.Standard: Briefly spin standard vial before use. Add 300 μL 1x Assay Diluent to prepare a 100 ng/mL standard. Gently vortex to mix. Take 100 μL standard into a tube, then add 400 μL 1x Assay Diluent to prepare a 20,000pg/mL stock standard solution. Add 400 μL 1x Assay Diluent to 7 tubes.
3.Detection Ab: Add 100 μL of 1X Assay Diluent into the vial to prepare a detection antibody concentrate. The detection antibody concentrate should be diluted 80-fold with 1X Assay Diluent.
4.Streptavidin-HRP: HRP-Streptavidin concentrate should be diluted 200-fold with 1X Assay Diluent.
5.Sample: Optimal dilution factors for each sample must be determined by the investigator, the recommended dilution for serum and plasma is 1:2.

1.Add 100μL each of dilutions of standard, blank and samples into the appropriate wells, respectively. Cover with the Plate sealer. Incubate for 1 hour at 37 °C.
2.Remove the liquid of each well, don't wash.
3.Add 100μL of Detection Reagent A working solution to each well, cover the wells with the plate sealer and incubate for 1 hour at 37 °C.
4.Aspirate the solution and wash with 350μL of 1x Wash Solution to each well and let it sit for 1-2 minutes. Remove the remaining liquid from all wells completely by snapping the plate onto absorbent paper. Totally wash 3 times. Invert the plate and blot it against absorbent paper.
5.Add 100μL of Detection Reagent B working solution to each well, cover the wells with the plate sealer and incubate for 30 minutes at 37 °C.
6.Repeat the aspiration/wash process for total 5 times as conducted in step 4.
7.Add 90μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for 10 - 20 minutes at 37 °C. Protect from light.
8.Add 50μL of Stop Solution to each well. Mix the liquid by tapping the side of the plate.
9.Remove any drop of water and fingerprint on the bottom of the plate. Then, run the microplate reader and conduct measurement at 450nm immediately.

Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit.

The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material.

4 °C

The Assay Plate, Standard, Detection Reagent A and Detection Reagent B should be stored at -20°C upon being received. After receiving the kit ,Substrate should be always stored at 4°C.

6 months

The Assay Plate, Standard, Detection Reagent A and Detection Reagent B should be stored at -20°C upon being received. After receiving the kit ,Substrate should be always stored at 4°C.

For Research Use only

Cluster of Differentiation 74

DHLAG; HLADG; II; Ia-GAMMA; CLIP; Ii; INVG34; Iclp-1; iclp1; wu:fb11f09; wu:fb12a01; wu:fb13d06; wu:fk94e07; CD74 molecule; CD74 antigen (invariant polypeptide of major histocompatibility complex; class II antigen-associated); CD74 molecule; major histocompatibility complex; class II invariant chain a; CD74; Cd74; cd74a

Synonyms: H-2 class II histocompatibility antigen gamma chain, Ia antigen-associated invariant chain(Ii),MHC class II-associated invariant chain

25599

P10247

Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response, Negative Regulation of intrinsic apoptotic Signaling, Cancer Immune Checkpoints

The microtiter plate provided in this kit has been pre-coated with an antibody specific to H-2 class II histocompatibility antigen gamma chain. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific forH-2 class II histocompatibility antigen gamma chain and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. Only those wells that contain H-2 class II histocompatibility antigen gamma chain, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm.