Human ATP Binding Cassette Transporter A4 (ABCA4) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to ABCA4. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to ABCA4. 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 ABCA4, 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 ABCA4 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

Flippase that catalyzes in an ATP-dependent manner the transport of retinal-phosphatidylethanolamine conjugates like the 11-cis and all-trans isomers of N-retinylidene-phosphatidylethanolamine from the lumen to the cytoplasmic leaflet of photoreceptor outer segment disk membranes, where N-cis-retinylidene-phosphatidylethanolamine (N-cis-R-PE) is then isomerized to its all-trans isomer (N-trans-R-PE) and reduced by RDH8 to produce all-trans-retinol (all-trans-rol) and therefore prevents the accumulation of excess of 11-cis-retinal and its schiff-base conjugate and the formation of toxic bisretinoid (PubMed:24097981, PubMed:22735453, PubMed:23144455, PubMed:20404325, PubMed:10075733, PubMed:29847635, PubMed:33375396). May display both ATPase and GTPase activity that is strongly influenced by the lipid environment and the presence of retinoid compounds (PubMed:22735453). Binds the unprotonated form of N-retinylidene-phosphatidylethanolamine with high affinity in the absence of ATP, and ATP binding and hydrolysis induce a protein conformational change that causes the dissociation of N-retinylidene-phosphatidylethanolamine (By similarity).

GENE ID	24
SWISS PROT	P78363
SYNONYMS	ABC-A4; ABC10; ABCR; ARMD2; CORD3; FFM; RMP; RP19; STGD; STGD1; Stargardt disease protein; Retinal-specific ATP-binding cassette transporter; RIM ABC transporter

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 ABCA4 were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level ABCA4 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 0.112ng/mL.
ASSAY RANGE	0.312-20ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of ABCA4. No significant cross-reactivity or interference between ABCA4 and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between ABCA4 and all analogs, therefore, cross reactivity may still exist.