北京产品商标: 义翘神州(sinobiologiBrand,null 抗体货号： SEK11019Item#, Transferrin / TF ELISA Kit , Human ELISA kit<br>英文名称： Transferrin / TF ELISA Pair Set, Human ELISA Pair Set

官方网址：http://www.sinobiological.cn/Transferrin-ELISA-Pair-Set-g-6333.html
 Human Transferrin / TF ELISA Kit
     
  Order or Inquire for Human Transferrin / TF ELISA Kit  
   
  High affinity Human Transferrin / TF ELISA Kit  
   
  Detection limit - 15.6 pg/ml  
   
  Affordable price and 30%-80% cost saving for Bulk order  
 
 
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 Protocol
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   1. Materials provided
 Capture Antibody - 0.5 mg/mL of mouse anti-Transferrin / TF monoclonal antibody. Dilute to a working concentration of  2 μg/mL in CBS before coating
 Detection Antibody - 0.4 mg/mL mouse anti-Transferrin / TF monoclonal antibody conjugated to horseradish-peroxidase ( HRP ). Dilute to working concentration of 1 μg/mL in detection antibody diluteion buffer before use
 Standard - Each vial contains 70 ng of recombinant Transferrin. Reconstitute with 1 mL detection antibody dilution buffer. After reconstitution, store at -20℃ to -70℃ in a manual defrost freezer . A seven-point standard curve using 2-fold serial dilutions in sample dilution buffer, and a high standard of 1ng/mL is recommended
 2. Sensitivity
 The minimum detectable dose of human Transferrin / TF was determined to be approximately 15.6 pg/ml. This is defined as at least three times standard deviations above the mean optical density of 10 replicates of the zero standard
 3. Principle of the product
 The human Transferrin / TF ELISA kit is for the quantitative determination of human Transferrin
 This ELISA kit contains the basic components required for the development of sandwich ELISAs. Each kit contains sufficient materials to run ELISAs on five 96-well plates.
 The Sino Biological ELISA kit is a solid phase sandwich ELISA (Enzyme-Linked Immunosorbent Assay). It utilizes a monoclonal antibody specific for Transferrin / TF coated on a 96-well plate. Standards and samples are added to the wells, and any Transferrin / TF present binds to the immobilized antibody. The wells are washed and a horseradish peroxidase conjugated mouse anti- Transferrin / TF monoclonal antibody is then added, producing an antibody-antigen-antibody “sandwich”. The wells are again washed and TMB substrate solution is loaded, which produces color in proportion to the amount of Transferrin / TF present in the sample. To end the enzyme reaction, the stop solution is added and absorbances of the microwell are read at 450 nm.
 STORAGE - Detection Antibody should be protected from prolonged exposure to light. Aliquot the reagents and store at -20℃ to -70℃ in a manual defrost freezer.
 
  Plate Preparation
  Dilute the capture antibody to the working concentration in CBS. Immediately coat a 96-well microplate with 100μL per well of the diluted capture antibody. Seal the plate and incubate overnight at 4℃.
 Aspirate each well and wash with at least 300μl wash buffer, repeating the process two times for a total of three washes. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining wash buffer by inverting the plate and blotting it against clean paper towels.
 Block plates by adding 300 μL of blocking buffer to each well. Incubate at room temperature for a minimum of 1 hour.
 Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
 
  
 Assay Procedure
  Add 100 μL of sample or standards in sample dilution buffer per well. Seal the plate and incubate 2 hours at room temperature.
 Repeat the aspiration/wash as in step 2 of plate preparation.
 Add 100 μL of the detection antibody, diluted in antibody dilution buffer, to each well. Seal the plate and incubate 1 hour at room temperature.
 Repeat the aspiration/wash as in step 2 of plate preparation.
 Add 200 μL of substrate solution to each well. Incubate for 20 minutes at room temperature ( if substrate solution is not as requested, the incubation time should be optimized ). Avoid placing the plate in direct light.
 Add 50 μL of stop solution to each well. Gently tap the plate to ensure thorough mixing.
 Determine the optical density of each well immediately, using a microplate reader set to 450 nm.
 
  
 Calculation of results
  Calculate the mean absorbance for each set of duplicate standards, controls and samples. Subtract the mean zero standard absorbance from each.
 Construct a standard curve by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph.
 To determine the concentration of the unknowns, find the unknowns’ mean absorbance value on the y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the x-axis and read the concentration. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
 Alternatively, computer-based curve-fitting statistical software may also be employed to calculate the concentration of the sample.
 
  
 
              
    Problems 
    Possible Sources 
    Solutions 
  
    No signal 
   Incorrect or no Detection Antibody was added
   Add appropriate Detection Antibody and continue
  
   Substrate solution was not added
   Add substrate solution and continue
  
   Incorrect storage condition
   Check if the kit is stored at recommended condition and used before expiration date
  
           
   Poor Standard Curve
   Standard was incompletely reconstituted or was inappropriately stored
   Aliquot reconstituted standard and store at  -70 ℃
  
   Imprecise / inaccurate pipetting
   Check / calibrate pipettes
  
   Incubations done at inappropriate temperature, timing or agitation
   Follow the general ELISA protocol
  
   Background wells were contaminated
   Avoid cross contamination by using the sealer appropriately
  
           
   Poor detection value 
   The concentration of antigen in samples was too low
   Enriching samples to increase the concentration of antigen
  
   Samples were ineffective
   Check if the samples are stored at cold environment. Detect samples in timely manner
  
           
   High Background 
  Insufficient washes   Use multichannel pipettes without touching the reagents on the plate
  
   Increase cycles of washes and soaking time between washes
  
   TMB Substrate Solution was contaminated
   TMB Substrate Solution should be clear and colorless prior to addition to wells
  
   Materials were contaminated
   Use clean plates, tubes and pipettes tips
  
           
   Non-specificity 
   Samples were contaminated
   Avoid cross contamination of samples
  
   The concentration of samples was too high
   Try higher dilution rate of samples
  
 
 
 
  Human Transferrin / TF ELISA Kit Image
 
 This standard curve is only for demonstration purposes. A standard curve should be generated for each assay.
  
 Human Transferrin / TF ELISA Kit Flash
      
 
 
 This flash is a vivid summary of the assay procedure.
 
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 Background
  Transferrin, also known as Serotransferrin, Beta-1 metal-binding globulin, TF, is a secreted protein which belongs to the transferrin family. It is expressed by the liver and secreted in plasma and contains two transferrin-like domains. Transferrins are iron binding transport proteins which can bind two Fe3+ ions in association with the binding of an anion, usually bicarbonate. It is responsible for the transport of iron from sites of absorption and heme degradation to those of storage and utilization. Serum transferrin may also have a further role in stimulating cell proliferation. When a transferrin protein loaded with iron encounters a transferrin receptor on the surface of a cell, it binds to it and, as a consequence, is transported into the cell in a vesicle. Transferrin is a glycoprotein that binds iron very tightly but reversibly. Although iron bound to transferrin is less than 0.1% ( 4 mg ) of the total body iron, it is the most important iron pool, with the highest rate of turnover ( 25 mg/24 h ). Defects in transferrin are the cause of a transferrinemia (ATRAF) which is rare autosomal recessive disorder characterized by iron overload and hypochromic anemia. Transferrin is also associated with the innate immune system.