Intended Use:The Transferrin (TF) BioAssay™ ELISA Kit (Human) is a sandwich assay intended for the quantitative determination of human Transferrin levels in serum or other biological fluids.
For Research Use Only (RUO). Not for diagnostic or therapeutic use in humans or animals.
Kit Components:This kit contains enough reagents for 10 x 96-well plates.
T8199-03H1: Capture Antibody, 1x1vialAffinity-purified chicken IgY against human TransferrinVolume: 4500ulConcentration: 1.0mg/mlWorking Dilution: 1:250
T8199-03H2: Calibrator, 1x1vialPurified human Transferrin antigenVolume: 260ulConcentration: 20ug/ml
T8199-03H3: Detection Antibody (HRP), 1x1vialAffinity-purified chicken IgY against human Transferrin - HRP ConjugateVolume: 30ulConcentration: 0.2mg/mlWorking Dilution: 1:4,000
* Buffers, Horseradish Peroxidase Substrate and Plates are not included
** Always centrifuge the reagent vials before opening.
Storage and Stability:Store kit components at -20°C. Stable for 6 months after receipt.
Detection Range:0.686-500ng/ml
Assay Condition:The kit performance has been optimized for the protocol and materials listed below using standard dilutions of human Transferrin in the 0.686- 500ng/ml range. The operator must determine appropriate dilutions of reagents for alternative assay conditions. ELISA assay reactivity is sensitive to variations in operator, pipetting and washing techniques, incubation time, temperature, composition of reagents, and other experimental variables. Assay optimization may be required to generate the standard curve and fit the samples in the specified detection range.
ELISA Protocol:Buffer Preparation:Prepare the following buffers:A. Coating Buffer: 0.05M Carbonate-Bicarbonate, pH 9.6B. Wash Solution: 0.05% Tween-20 in PBS, pH 7.4C. Blocking Solution: 1% Non-fat dry milk in PBSD. Sample/Conjugate Diluent: 50mM Tris, 0.14M sodium chloride, 1% BSA, 0.05% Tween-20, pH 8.0E. Enzyme Substrate, TMB (KPL Cat. #50-76-00)F. Stop Solution: 2M H2SO4 or other appropriate solution
Perform all steps at room temperature.
Coating with Capture Antibody:1. Determine the number of single wells needed. It is strongly suggested that standards, samples, blanks and/or controls be analyzed in duplicate. Insert the required number of microtiter well strips into a holder
2. Dilute 44ul of Capture Antibody with 11ml coating buffer to make a 1:250 dilution. Add 100ul to each well. 3 Incubate plate for 60 minutes. 4. After incubation, aspirate the Capture Antibody solution from each well
5. Wash each well with Wash Solution as follows:a. Fill each well with Wash Solutionb. Remove Wash Solution by aspirationc. Repeat for a total of 3 washes.
Blocking (Post-coat):1. Add 200ul of Blocking Solution to each well
2. Incubate for 60 minutes
3. After incubation, remove the Blocking Solution and wash each well three times as in step 5 of the Coating procedure.
Standards and Samples:1. Add 25ul of Calibrator to 975ul of Sample/Conjugate Diluent to prepare the highest concentration Protein Calibrator solution (500ng/ml) Prepare serial dilutions at 1:3 ratio (one part Calibrator to plus two parts diluent) until the lowest Calibrator concentration of 0.686 ng/ml is obtained. 2. Dilute the samples, based on the expected concentration, to fit within the concentration range of the standards
3. Transfer 100ul of Calibrator solutions and sample solutions to assigned wells
4. Incubate plate for 60 minutes
5. After incubation, remove the liquid and wash each well 5 times as in step 5 of the Coating Procedure.
Detection Antibody (HRP) Conjugate1. Dilute 2.75ul of the Detection Antibody (HRP) Conjugate in 11ml Conjugate Diluent to make a 1:4,000 dilution. Adjustments in dilution may be needed depending on substrate used, incubation time, and other experimental conditions
2. Transfer 100ul to each well
3. Incubate for 60 minutes
4. After incubation, remove the liquid and wash each well 5 times as in step 5 of the Coating Procedure.
Enzyme Substrate Reaction:1. Prepare the Substrate Solution according to the manufacturer’s recommendation
2. Transfer 100ul of Substrate Solution to each well
3. Incubate plate for 5-30 minutes
4. To stop the TMB reaction, apply 100ul of 2M H2SO4 to each well. When using another substrate, follow the manufacturer’s recommendations.
Plate Reading:1. Using a microtiter plate reader, read the plate at the wavelength that is appropriate for the substrate used (450nm for TMB, 490nm for OPD, 405nm for ABTS).
Calculation of Results:1. Average the duplicate readings for each standard, control and sample
2. Subtract the zero reading from each averaged value above
3. Create a standard curve by reducing the data using computer software capable of generating a four-parameter logistic (4-PL) curve fit. Other curve fits may also be used
4. A standard curve should be generated with every assay run
5. Determine the concentration of unknowns from the standard curve using absorbance readings for the samples
