Human Complement Component 4b (C4b) ELISA Kit

Principle of the Assay

The microtiter plate provided in this kit has been pre-coated with an antibody specific to C4b. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific to C4b. 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 C4b, 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 C4b 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

Non-enzymatic component of the C3 and C5 convertases and thus essential for the propagation of the classical complement pathway. Covalently binds to immunoglobulins and immune complexes and enhances the solubilization of immune aggregates and the clearance of IC through CR1 on erythrocytes. C4A isotype is responsible for effective binding to form amide bonds with immune aggregates or protein antigens, while C4B isotype catalyzes the transacylation of the thioester carbonyl group to form ester bonds with carbohydrate antigens.

GENE ID	721
SWISS PROT	P0C0L5
SYNONYMS	CO4; CPAMD3; Basic complement C4; C3 and PZP-like alpha-2-macroglobulin domain-containing protein 3

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 C4b were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle, and high-level C4b 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 1.8ng/mL.
ASSAY RANGE	4.68-300ng/mL
SPECIFICITY	This assay has high sensitivity and excellent specificity for the detection of C4b. No significant cross-reactivity or interference between C4b and analogs was observed. Note: Limited by current skills and knowledge, it is impossible to perform all possible cross-reactivity detection tests between C4b and all analogs, therefore, cross reactivity may still exist.