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  • 产品名称：HumanAlbuminEFCIAKit

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  • 产品厂商：半导体
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HumanAlbuminEFCIAKit
详情介绍：
Purpose The AssayLite Human Albumin EFCIA (Endpoint Fluorescent Competitive Immunoassay) Kit employs a quantitative competitive fluorescent probe technique that measures albumin in human plasma and serum in less than an hour. A polyclonal antibody specific for human albumin has been pre-coated onto a 96-well opaque microplate with removable strips. Albumin in standards and samples is competed with a human albumin fluorescent probe. All unbound material is then washed away before the endpoint fluorescence is measured.
Brand AssayLite™
Sample Type Cell Culture Cells, Plasma, Serum
Analytical Method Quantitative
Detection Method Fluorometric
Characteristics Human Albumin EFCIA Kit
Components Human Albumin Microplate: A 96-well black polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human albumin.
Sealing Films: Each kit contains 3 precut, pressure sensitive aluminum sealing films that can be cut to fit the format of the individual assay.
Human Albumin Standard: Human albumin in a buffered protein base (50 ?g, lyophilized).
Red Fluorescent Human Albumin Probe: 3 vials, lyophilized.
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, 1 bottle).
Stabilizing Solution: A ready-to-use solution to stabilize the fluorescent component (8 mL, 1 bottle).
Material not included Fluorescent microplate reader (Molecular Device Gemini XPS, Biotek FLx 800 & Synergy H1 have been evaluated, other readers have not been evaluated for performance).
Pipettes (1-20 µL, 20-200 µL, 200-1000 µL and multiple channel).
Deionized or distilled reagent grade water.
An incubator set at 37°C.
Alternative Name Albumin (ALB ELISA Kit Abstract)
Background Albumin is serum hepatic protein, the most abundant protein in serum and contributes to the maintenance of oncotic pressure as well as to the transport of hydrophobic molecules. Serum albumin level has been linked in clinical practice to several diseases. Low albumin levels can suggest liver disease, kidney disease, inflammation, shock, and malnutrition. On the other hand, high albumin levels usually reflect dehydration.
Gene ID 213
UniProt P02768
Research Area Serum/Plasma Proteins
Application Notes Normal human albumin plasma levels range from 30 to 50 mg/mL
Comment

Assay Type: Singleplex
Assay Time 1 h
Plate Pre-coated
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.
Standard Curve: Reconstitute the 50 µg of Human Albumin Standard with 1 mL of MIX Diluent to generate a working solution of 50 µg/mL. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting the standard solution (50 µg/mL) 1:4 with MIX Diluent to generate 12.5, 3.125, and 0.781 µg/mL solutions. MIX Diluent serves as the zero standard (0 µg/mL). Any remaining solution should be frozen at - 20°C and used within 30 days. Fluorescent Human Albumin Probe (1x): Dilute Fluorescent Human Albumin with 1.8 mL MIX Diluent to produce a working solution. Allow the probe to sit for 10 minutes with gentle agitation prior to making dilutions. Any remaining solution should be refrigerated at 2-8°C and used within 30 days.
Wash Buffer Concentrate (20x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the Wash Buffer Concentrate 1:20 with reagent grade water.
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 1:10000 into MIX Diluent and assay (suggested dilution factor only, the correct dilution factor should be determined by user). 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 1:10000 into MIX Diluent and assay (suggested dilution factor only, the correct dilution factor should be determined by user). The undiluted serum can be stored at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles Refer to Sample Dilution Guidelines section of this booklet for further instruction
Assay Procedure

Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is incubated at 37°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 µL of Human Albumin Standard or sample per well, and immediately add 50 µL of Fluorescent Human Albumin to each well (on top of the standard or sample) and mix gently. Cover wells with a sealing tape and incubate for 20 minutes and incubate at 37°C. Start the timer after the last sample addition.
Wash five times with 200 µL 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 µL of Wash Buffer and then invert the plate, decanting the contents, hit 4-5 times on absorbent material to completely remove the liquid.
Immediately add 50 µL of Stabilizing Solution to each well.
Read the endpoint fluorescence on a microplate reader at an excitation wavelength of 488 nm and emission wavelength of 576 nm within 2 hours.
For the FLx800 reader, a sensitivity of 100 was used. For the Synergy H1, the Automatic Gain Adjustment setting was used and the option Scale to Low Wells was selected with a threshold of 500. For the Gemini XPS reader, PMT was set to High.
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 576 emitted fluorescence 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
Precaution of Use Prepare all reagents (working diluent buffer, wash buffer, standards, and fluorescent probe) 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 protocol. However, the user should determine the optimal dilution factor.
This kit is for research use only.
The kit should not be used beyond the expiration date.
Avoid direct light exposure to the assay.
Store the Fluorescent Probe in a dark place. Do not freeze.
Storage 4 °C
Storage Comment Store components of the kit at 2-8°C or -20°C upon arrival up to the expiration date.
Store Microplate, Diluent Concentrate (10x), Stabilizing Solution, and Wash Buffer 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.
Diluent (1x) may be stored for up to 30 days at 2-8°C.
Store the standard and at 2-8°C before reconstituting with diluent and at -20°C after reconstituting with diluent.
Store the fluorescent probe in a dark place at 2-8°C before and after reconstituting with diluent. Do not freeze.