Rat MCH EIA

Cell Culture Supernatants
Serum

Colorimetric

96-well Microplate

Pre-
Coated 96-well Strip Microplate
Wash Buffer
Standard Peptide
Assay Diluent(s)
Biotinylated Peptide
H
R
P-
Streptavidin
T
M
B One-
Step Substrate
Stop Solution
Assay Diagram
Positive Control Sample
Capture Antibody
User Manual

Prepare all reagents, samples and standards as instructed.
Add 100 µl detection antibody to each well.
Incubate 1.5 h at R
T or O/
N at 4°
C.
Add 100 µl standard or sample to each well.
Incubate 2.5 h at R
T.
Add 100 µl prepared streptavidin solution.
Incubate 45 min at R
T.
Add 100 µl T
M
B One-
Step Substrate Reagent to each well.
Incubate 30 min at R
T.
Add 50 µl Stop Solution to each well.
Read plate at 450 nm immediately.

Melanin-concentrating hormone (MCH)

Melanin-concentrating hormone (MCH) is a cyclic 19-amino acid neuropeptide. It binds to two G-coupled protein receptors (GCPRs), MCHR1 and MCHR2. MCH is involved in the regulation of the sleep cycle, maternal behavior, eating behaviors, energy conservation, reproduction and skin pigmentation.The RayBio® MCH Enzyme Immunoassay (EIA) Kit is an in vitro quantitative assay for detecting MCH peptide based on the competitive enzyme immunoassay principle. In this assay, a biotinylated MCH peptide is spiked into the samples and standards. The samples and standards are then added to the plate, where the biotinylated MCH peptide competes with endogenous (unlabeled) MCH for binding to the anti-MCH antibody. After a wash step, any bound biotinylated MCH then interacts with horseradish peroxidase (HRP)-streptavidin, which catalyzes a color development reaction. The intensity of the colorimetric signal is directly proportional to the amount of captured biotinylated MCH peptide and inversely proportional to the amount of endogenous MCH in the standard or samples. A standard curve of known concentration of MCH peptide can be established and the concentration of MCH peptide in the samples can be calculated accordingly.