Human Peptidylglycine Alpha Amidating Monooxygenase (PAM) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to PAM. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to PAM. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After the TMB substrate solution is added, only those wells that contain PAM, biotin-conjugated antibody, and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution, and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of PAM in the samples is then determined by comparing the O.D. of the samples to the standard curve.

For Use with serum, plasma, and cell culture supernatants. For Research Use Only. Not for use in diagnostic procedures.

Target Information

Bifunctional enzyme that catalyzes the post-translational modification of inactive peptidylglycine precursors to the corresponding bioactive alpha-amidated peptides, a terminal modification in biosynthesis of many neural and endocrine peptides (PubMed:12699694). Alpha-amidation involves two sequential reactions, both of which are catalyzed by separate catalytic domains of the enzyme. The first step, catalyzed by peptidyl alpha-hydroxylating monooxygenase (PHM) domain, is the copper-, ascorbate-, and O2- dependent stereospecific hydroxylation (with S stereochemistry) at the alpha-carbon (C-alpha) of the C-terminal glycine of the peptidylglycine substrate (PubMed:12699694). The second step, catalyzed by the peptidylglycine amidoglycolate lyase (PAL) domain, is the zinc-dependent cleavage of the N-C-alpha bond, producing the alpha-amidated peptide and glyoxylate (PubMed:12699694). Similarly, catalyzes the two-step conversion of an N-fatty acylglycine to a primary fatty acid amide and glyoxylate (By similarity).

GENE ID	5066
SWISS PROT	P19021
SYNONYMS	PAL; PHM; Peptidylamidoglycolate lyase; Peptidyl-Alpha-hydroxyglycine Alpha-amidating Lyase; Peptidylglycine Alpha-Hydroxylating Monooxygenase

Materials Supplied

Kit Components	96 Wells Quantity/Size
Pre-coated, ready-to-use 96-well strip plate	1 plate
Plate sealer for 96 wells	2
Standard	2 tubes
Diluent buffer	1 bottle
Detection Reagent A	1 bottle
Detection Reagent B	1 bottle
TMB Substrate	1 tube
Stop Solution	1 tube
Wash Buffer (30 ℅ concentrate)	1 tube
Product data sheet	1 copy

Storage

The TMB Substrate, Wash Buffer (30X concentrate), and the Stop Solution should be stored at 4°C upon receipt, while the other items should be stored at -20°C.

Performance Characteristics

REPEATABILITY	Intra-assay Precision (Precision within an assay): 3 samples with low, middle, and high-level PAM were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level PAM were tested on 3 different plates, with 8 replicates in each plate. CV(%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%
SENSITIVITY	The minimum detectable dose was 13.5pg/mL.
ASSAY RANGE	31.2-2000pg/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of PAM. No significant cross-reactivity or interference between PAM and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between PAM and all analogs, therefore, cross reactivity may still exist.