Abx150871-96tests | Human Membrane Attack Complex C5b-9 (C5b-9) ELISA

Human Membrane Attack Complex C5b-9 (C5b-9) ELISA Kit

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Human Membrane Attack Complex C5b-9 (MAC) ELISA Kit is an ELISA Kit for the in vitro quantitative measurement of Human Membrane Attack Complex C5b-9 (MAC) concentrations in serum, plasma and other biological fluids.

Introduction	The Membrane Attack Complex (MAC, also known as the Terminal Component Complex, TCC) is a protein complex formed late in the complement cascade. Its construction is initiated by C5b’s adherence to the cell membrane, which recruits C6, C7, and C8 (in that order). The resulting C5b678 complex recruits several C9 monomers to form a pore in the cell membrane, causing water to enter and many solutes to leave. CD59 protects self cells by binding to the C5b678 complex and preventing C9 from polymerising; mutations in this gene can cause paroxysmal nocturnal hemoglobinuria.
Target	Membrane Attack Complex C5b-9 (C5b-9)
Reactivity	Human
Tested Applications	ELISA
Recommended dilutions	Optimal dilutions/concentrations should be determined by the end user.
Storage	Shipped at 4 °C. Upon receipt, store the kit according to the storage instruction in the kit's manual.
Validity	The validity for this kit is at least 6 months. Up to 12 months validity can be provided on request.
Stability	The stability of the kit is determined by the rate of activity loss. The loss rate is less than 5% within the expiration date under appropriate storage conditions. To minimize performance fluctuations, operation procedures and lab conditions should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same user throughout.
Test Range	1.25 ng/ml - 80 ng/ml
Sensitivity	0.75 ng/ml
Standard Form	Lyophilized
Detection Method	Colorimetric
Assay Type	Sandwich
Assay Data	Quantitative
Sample Type	Serum, plasma and other biological fluids.
Assay Principle	This kit is based on sandwich enzyme-linked immuno-sorbent assay technology. An antibody is pre-coated onto a 96-well plate. Standards, test samples, and biotin-conjugated reagent are added to the wells and incubated. The HRP-conjugated reagent is then added, and the whole plate is incubated. Unbound conjugates are removed using wash buffer at each stage. TMB substrate is used to quantify the HRP enzymatic reaction. After TMB substrate is added, only wells that contain sufficient MAC will produce a blue coloured product, which then changes to yellow after adding the acidic stop solution. The intensity of the yellow colour is proportional to the MAC amount bound on the plate. The Optical Density (OD) is measured spectrophotometrically at 450 nm in a microplate reader, from which the concentration of MAC can be calculated.
Kit Components	The kit components listed are for reference only. The product manual may differ slightly. The product should be used as stated on the product manual included and delivered together with the product. Pre-coated 96-Well Microplate Standard Standard Diluent Buffer Wash Buffer Detection Reagent A Detection Reagent B Diluent A Diluent B TMB Substrate Stop Solution Plate Sealer
Material Required But Not Provided	37°C incubator Multi and single channel pipettes and sterile pipette tips Squirt bottle or automated microplate washer 1.5 ml tubes Distilled water Absorbent filter papers 100 ml and 1 liter graduated cylinders Microplate reader (wavelength: 450 nm) ELISA Shaker
Reagent Preparation	This procedure is provided for reference only. The product manual may differ slightly. The product should be used as stated on the product manual included and delivered together with the product. 1) Standard: Prepare the standard with the recommended volume of Standard Diluent Buffer, to make the standard solution. Then use the Standard Diluent buffer to carry out serial dilutions of the standard solution, as instructed in the Protocol. 2) Wash Buffer: Dilute the concentrated Wash Buffer with distilled water, as instructed in the Protocol. 3) Detection Reagent Preparation: Calculate the total volume of working solution required. Dilute Detection Reagent A and Detection Reagent B with Diluent A and Diluent B, respectively, at 1:100.
Assay Procedure	This procedure is provided for reference only. The product manual may differ slightly. The product should be used as stated on the product manual included and delivered together with the product. 1) Set standard, test samples and control wells. 2) Aliquot 100 µl of diluted standard into the standard wells. 3) Aliquot 100 µl of Standard Diluent buffer into control (zero) well. 4) Aliquot 100 µl of diluted samples into the sample wells. Incubate for 90 mins at 37 °C. 5) Aliquot 100 µl of Detection Reagent A to each well. Incubate for 1 hr at 37 °C. 6) Wash 3 times. 7) Aliquot 100 µl of Detection Reagent B to each well. Incubate for 30 mins at 37 °C. 8) Wash 5 times. 9) Aliquot 90 µl of TMB Substrate to each well. Incubate for 10-20 mins at 37 °C. 10) Aliquot 50 µl of Stop Solution. 11) Measure the OD at 450 nm.
Protocol	This procedure is provided for reference only. The product manual may differ slightly. The product should be used as stated on the product manual included and delivered together with the product. Equilibrate the kit components and samples to room temperature (18 - 25 °C) before use. It is recommended to plot a standard curve for each test. 1. Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample at least in duplicate. 2. Add 100 µL of each standard, control and sample into the appropriate wells. Seal the plate with a cover and incubate for 1 h at 37°C. 3. Remove the cover and discard the liquid. 4. Add 100 µl of the detection Reagent A working solution to each well. Seal the plate with a cover and incubate for 1 h at 37°C. 5. Remove the cover and discard the solution. Wash the plate 3 times with 1X Wash Buffer. 6. Add 100 µL of Detection Reagent B working solution into each well, seal and incubate at 37°C for 30 min. 7. Discard the solution and wash the plate 5 times with wash buffer as explained in previous step. 8. Aliquot 90 µl of TMB Substrate into each well. Seal the plate with a cover and incubate at 37°C for 10-20 min. Avoid exposure to light. The incubation time is for reference use only, the optimal time should be determined by end user. Do not exceed 30 min. 9. Add 50 µL of Stop Solution to each well. Read at 450 nm immediately.
Results Calculation	For calculation, average the O.D.450 duplicate readings for each reference standard and each sample and substract the average control (zero) O.D.450 reading. The standard curve can be plotted as the relative O.D.450 of each reference standard solution (Y) vs. the respective concentration of each standard solution (X). The MAC concentration of the samples can be interpolated from the standard curve.
Assay Precision	Intra-assay Precision (Precision within an assay): 3 samples with low, medium and high levels of Terminal Complement Complex C5b-9 (MAC) were were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, medium and high levels of Terminal Complement Complex C5b-9 (MAC) were tested on 3 different plates, 8 replicates in each plate. CV (%) = (Standard Deviation / mean) × 100 Intra-Assay: CV<10% Inter-Assay: CV<10%
Availability	Shipped within 5-7 working days.
Note	This product is for research use only. The range and sensitivity is subject to change. Please contact us for the latest product information. For accurate results, sample concentrations must be diluted to mid-range of the kit. If you require a specific range, please contact us in advance or write your request in your order comments. Please note that our ELISA and CLIA kits are optimised for detection of native samples, rather than recombinant proteins. We are unable to guarantee detection of recombinant proteins, as they may have different sequences or tertiary structures to the native protein.
Standard	160 ng/ml

Research Articles on Membrane Attack Complex C5b-9 (C5b-9)

Human Membrane Attack Complex C5b-9 (C5b-9) ELISA Kit

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