Complement C3d ELISA Kit

Catalog No. ABIN5564626

Product Details Complement C3d ELISA Kit

Target: Complement C3d (C3d) (Complement Fragment 3d (C3d))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 9.063-580 ng/mL

Minimum Detection Limit: 9.063 ng/mL

Application: ELISA

Purpose: The AssayMax™ Human Complement C3d ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of human complement C3d in plasma, serum, urine, milk, saliva, CSF, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures human complement C3d in 5 hours. A polyclonal antibody specific for human complement C3d has been pre-coated onto a 96-well microplate with removable strips. Complement C3d in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for complement C3d, which is recognized by a streptavidin-peroxidase conjugate. All unbound material is washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.

Brand: AssayMax™

Sample Type: Cell Culture Cells, Cerebrospinal Fluid, Milk, Plasma, Saliva, Serum, Urine

Analytical Method: Quantitative

Components: Human Complement C3d Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human complement C3d. Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay. Human Complement C3d Standard: Human complement C3d in a buffered protein base (580 ng, lyophilized). Biotinylated Human Complement C3d Antibody (40x): A 40-fold concentrated biotinylated polyclonal antibody against complement C3d (150 l). MIX Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml, 2 bottles). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (80 l). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).

Material not included: Microplate reader capable of measuring absorbance at 405 nm. Pipettes (1-20 µL, 20-200 µL, and multiple channel). Deionized or distilled reagent grade water Incubator (37 °C)

Application Details

Assay Time: 4 h

Plate: Pre-coated

Protocol:

• Step 1. Add 50 μL of Standard or Sample per well. Incubate 2 hours.
• Step 2. Wash, then add 50 μL of Biotinylated Antibody per well. Incubate 2 hours.
• Step 3. Wash, then add 50 μL of SP Conjugate per well. Incubate 30 minutes.
• Step 4. Wash, then add 50 μL of Chromogen Substrate per well. Incubate 30 minutes.
• Step 5. Add 50 μL of Stop Solution per well. Read at 450 nm immediately.

Reagent Preparation:

Freshly dilute all reagents and bring all reagents to room temperature before use. MIX Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the MIX Diluent Concentrate 1:10 with reagent grade water. Store for up to 30 days at 2-8 °C.

Sample Collection: Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 3000 x g for 10 minutes. Dilute samples 1:20000 into MIX Diluent and assay. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles (EDTA or Heparin can also be used as an anticoagulant). Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 3000 x g for 10 minutes, and remove serum. Dilute samples 1:20000 into MIX Diluent and assay. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Urine: Collect urine using sample pot. Centrifuge samples at 800 x g for 10 minutes and assay. Samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Saliva: Collect saliva using sample pot. Centrifuge samples at 800 x g for 10 minutes. Dilute samples 1:10 into MIX Diluent and assay. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Milk: Collect milk using sample tube. Centrifuge samples at 800 x g for 10 minutes. Dilute milk sample 1:200 into MIX Diluent and assay. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. CSF: Collect cerebrospinal fluid (CSF) using sample pot. Centrifuge samples at 800 x g for 10 minutes. Dilute CSF sample 1:20 into MIX Diluent and assay. The undiluted samples can be stored at -20 °C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Cell Culture Supernatants: Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris. Collect supernatants and assay. Store samples at -20 °C or below. Avoid repeated freeze-thaw cycles. Refer to Sample Dilution Guidelines below for further instruction. 4 Guidelines for Dilutions of 1:100 or Greater (for reference only, please follow the insert for specific dilution suggested) 1:100 1:10000 A) 4 μL sample: 396 μL buffer(100x) = 100 fold dilution Assuming the needed volume is less than or equal to 400 μL. A) 4 μL sample : 396 μL buffer (100x) B) 4 μL of A : 396 μL buffer (100x) = 10000 fold dilution Assuming the needed volume is less than or equal to 400 μL. 1:1000 1:100000 A) 4 μL sample : 396 μL buffer (100x) B) 24 μL of A : 216 μL buffer (10x) = 1000 fold dilution Assuming the needed volume is less than or equal to 240 μL. A) 4 μL sample : 396 μL buffer (100x) B) 4 μL of A : 396 μL buffer (100x) C) 24 μL of B : 216 μL buffer (10x) = 100000 fold dilution Assuming the needed volume is less than or equal to 240 μL.

Assay Procedure:

Prepare all reagents, standard solutions, and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20-25 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccants inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 l of Human Complement C3d Standard or sample per well. Cover wells with a sealing tape and incubate for 2 hours. Start the timer after the last addition. Wash five times with 200 l of Wash Buffer manually. Invert the plate each time and decant the contents, hit 4-5 times on absorbent material to completely remove the liquid. If using a machine, wash six times with 300 l of Wash Buffer and then invert the plate, decanting the contents, hit 4-5 times on absorbent material to completely remove the liquid. Add 50 l of Biotinylated Human Complement C3d Antibody to each well and incubate for 2 hours. Wash the microplate as described above. Add 50 l of Streptavidin-Peroxidase Conjugate to each well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash the microplate as described above. Add 50 l of Chromogen Substrate per well and incubate for 30 minutes or till the optimal blue color density develops. Gently tap plate to ensure thorough mixing and break the bubbles in the well with pipette tip. Add 50 l of Stop Solution to each well. The color will change from blue to yellow. Read the absorbance on the microplate reader at a wavelength of 450 nm immediately. If wavelength correction is available, subtract readings at 570 nm from those at 450 nm to correct optical imperfections. Otherwise, read the plate at 450 nm only. Please note that some unstable black particles may be generated at high concentration points 6 after stopping the reaction for about 10 minutes, which will reduce the readings.

Calculation of Results:

• Calculate the mean value of the duplicate or triplicate readings for each standard and sample.
• To generate a standard curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance (OD) on the y-axis. The best-fit line can be determined by regression analysis using log-log or four-parameter logistic curve-fit.
• Determine the unknown sample concentration from the standard curve and multiply the value by the dilution factor.

Restrictions: For Research Use only

Handling

Handling Advice: This product is for Research Use Only and is not intended for use in diagnostic procedures. 2 Prepare all reagents (working diluent buffer, wash buffer, standard, biotinylated antibody, and SP conjugate) as instructed, prior to running the assay. Prepare all samples prior to running the assay. The dilution factors for the samples are suggested in this insert. However, the user should determine the optimal dilution factor. Spin down the SP conjugate vial and the biotinylated antibody vial before opening and using contents. The Stop Solution is an acidic solution. The kit should not be used beyond the expiration date.

Storage: 4 °C,-20 °C

Storage Comment: Upon arrival, immediately store components of the kit at recommended temperatures up to the expiration date. Store SP Conjugate and Biotinylated Antibody at -20°C. Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C. Unused microplate wells may be returned to the foil pouch with the desiccant packs and resealed. May be stored for up to 30 days in a vacuum desiccator. 3 Diluent (1x) may be stored for up to 30 days at 2-8°C. Store Standard at 2-8°C before reconstituting with Diluent and at -20°C after reconstituting with Diluent.

Target Details for Complement C3d

Target: Complement C3d (C3d) (Complement Fragment 3d (C3d))

Alternative Name: Complement C3d (C3d Products)

Synonyms: AI255234 ELISA Kit, ASP ELISA Kit, HSE-MSF ELISA Kit, Plp ELISA Kit, complement component 3 ELISA Kit, complement C3 ELISA Kit, C3 ELISA Kit

Background: Complement component 3 (C3) plays a central role in all three complement activation pathways. The C3 precursor contains 1663 amino acids and has a molecular weight of about 180 kDa (1). This protein consists of alpha and beta disulfide-bridged chains. C3 is cleaved by C3 convertase into two activated fragments C3a and C3b. The anaphylatoxin C3a is a vasoactive peptide and a mediator of local inflammatory process (2). The C3b in complex with receptor can bind covalently to pathogen surfaces to promote phagocytosis (3). By the concerted actions of complement regulatory enzyme factor I and cofactors factor H and CD35, C3b is broken down progressively to first inactivated iC3b, then C3c and C3dg, and finally 33 kDa C3d by a non-complement proteinase trimming. Fragment C3d occupies approximately positions 345-610 of the alpha chain. C3d is a ligand for the B lymphocytes cell-surface complement receptor 2 (CR2) and a molecular adjuvant of innate immunity that influences an acquired immune response profoundly (4-5).

Gene ID: 718

UniProt: P01031