Leukotriene C4 (LTC4) is a potent mediator of immediate hypersensitivity, bronchoconstriction, smooth muscle contraction, increased vascular permeability and epithelial mucous secretion. It is derived from arachidonic acid through the 5-lipoxygenase pathway. Arachidonic acid is first converted to 5-hydroperoxy derivative which is then transformed to Leukotriene A4 (LTA4). Enzymatic addition of glutathione to LTA4 produces LTC4.
Principle of the Assay:
This is an ELISA (Enzyme-Linked Immunosorbent Assay) for the quantitative analysis of Leukotriene C4 levels in biological fluid. This test kit operates on the basis of competition between the enzyme conjugate and the Leukotriene C4 in the sample for a limited number of binding sites. First, the sample or standard solution is added to the microplate. Next, the diluted enzyme conjugate is added and the mixture is shaken and incubated at room temperature for one hour. During the incubation, competition for binding sites is taking place. The plate is then washed, removing all the unbound material. The bound enzyme conjugate is detected by the addition of substrate, which generates an optimal color after 30 minutes. Quantitative test results may be obtained by measuring and comparing the absorbance reading of the wells of the samples against the standards with a microplate reader at 650nm. The extent of color development is inversely proportional to the amount of Leukotriene C4 in the sample or standard. For example, the absence of Leukotriene C4 in the sample will result in a bright blue color, whereas the presence of Leukotriene C4 will result in decreased or no color development.
L2047-10A ELISA Buffer: 1x30ml. To be used to dilute enzyme conjugate and Leukotriene C4 standards
L2047-10B Wash Buffer 10X: 1x20ml. To be diluted 10X with deionized water. This is used to wash all unbound enzyme conjugate, samples and standards from the plate after the one hour incubation.
L2047-10C Substrate: 1x20ml. Stabilized 3,3', 5,5' Tetramethylbenzidine (TMB) plus hydrogen peroxide (H2O2) in a single bottle. It is used to develop the color in the wells after they have been washed.
L2047-10D Extraction Buffer 5X: 1x30ml. To be diluted 5X with deionized water. This is used for diluting extracted and non-extracted samples.
*L2047-10E Leukotriene C4 (HRP): 2x75ul. Lyophilized LTC4 HRP conjugate. Reconstitute one vial with 75ul of deionized water for a 50X stock solution.
L2047-10F Leukotriene Standard C4: 1x100ul Leukotriene C4 standard at the concentration of 1ug/ml.
L2047-10G Leukotriene C4 Microtiter Plate: 1x96 well MaxiSorp(TM) Nunc microplate with anti-LTC4 rabbit antibody precoated on each well. The plate is ready to use as is. Do not wash.
Storage and Stability:
Store *L2047-10E powder at --20 degrees C liquid at 4 degrees C. Do not freeze.. Store other components at 4 degrees C. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.