An in vitro enzyme immunoassay for quantifying
Human Transferrin Receptor
in serum or plasma as an aid in the diagnosis of iron deficiency anemia,
particularly in the presence of other disease states
Catalog Number TFC-94
For In Vitro Diagnostic Use Only
The Ramco TfR test kit is an enzyme immunoassay (EIA) for the quantitative determination of transferrin receptor concentration in human serum or plasma as an aid in the diagnosis of iron deficiency, particularly in the presence of other disease states.
SUMMARY AND EXPLANATION
The transferrin receptor (TfR) is a transmembrane protein present in all body cells which facilitates the uptake and internal transport of iron bound to circulating transferrin.1,2 The cellular content of TfR varies with the iron needs of the tissue and is therefore highest in human placentum and in the erythroid marrow.
The serum TfR is a truncated fragment of cellular TfR.3,4 Clinical studies have demonstrated that the concentration of serum TfR is proportional to the total body mass of tissue receptors, two-thirds of which are contained in erythroid marrow. Consequently, an elevation of serum TfR levels occurs whenever erythroid precursors are increased as in iron deficiency anemia.5,6,7 The levels of serum TfR in iron deficiency anemia are elevated in excess of red cell precursor mass due to increased density of TfR on red cell precursors. Measurements of serum transferrin receptor provide a useful clinical index of tissue iron deficiency.4,5,8,9
PRINCIPAL OF TEST
Ramco's TfR assay is an enzyme immunoassay based upon the double antibody sandwich method. Plasma or serum samples are diluted in buffer and pipetted into microwells pre-coated with polyclonal antibody to TfR. Horseradish peroxidase (HRP) conjugated murine monoclonal antibody specific for TfR is added to the wells and the wells are incubated for two hours at room temperature. During this incubation, the TfR binds to the polyclonal antibodies adsorbed to the wells and the HRP-conjugated second antibodies bind to the captured TfR.
Any unbound TfR and excess HRP conjugate are removed from the wells by washing. Enzyme substrate (chromogen TMB) is added to the wells and through the action of HRP forms a blue product. Upon the addition of an acid stop solution, the blue product is converted to a yellow color, the intensity of which is measured in a plate reader set at 450nm.
The optical density of the resulting solution is directly proportional to the concentration of the TfR in the standard samples. A standard curve is generated by plotting the absorbance versus concentration of the TfR standards provided in the kit. The concentration of the TfR in the sample is then determined by comparing the sample's optical density reading with the standard curve graph.
Microwell Strips - 12 (twelve) 1 x 8 microwell strips (96 total wells), pre-coated with polyclonal anti-TfR antibody and stabilized in dry form. The plate is provided in a resealable, ziploc foil pouch containing an inert gas and a drying agent. Store at 2-8 C.
Plate Sealing Tape - 1 sheet
Microwell Frame - 1
HRP-Conjugate - 1 vial containing 16 ml of Horseradish Peroxidase-conjugated anti-TfR monoclonal antibody and detergent with ProClin 300™ as a preservative. Store at 2-8 C.
Sample Diluent - 1 vial containing 45 ml of phosphate buffered saline, detergent, and an inert coloring agent with ProClin 300™ as a preservative. Store at 2-8 C.
Wash Solution 5X Concentrate - 1 bottle containing 50 ml of concentrated phosphate buffered saline and detergent with ProClin 300™ as a preservative. Store at room temperature.
Pre-diluted Standards - 7 vials each containing 0.5 ml of purified TfR protein diluted to concentrations of 0, 10, 20, 40, 100, 200 and 400 ng/ml in phosphate buffer containing BSA, normal rabbit serum, and inert coloring agents with ProClin 300™ as a preservative. Store at 2-8 C.
Control Sera - 2 vials containing 100 μl of undiluted serum, one having a normal level and one having a high level of human transferrin receptor. Store at 2-8 C.
TMB Substrate Solution A - 1 vial containing 12 ml of a stabilized solution of tetramethylbenzidine (TMB) substrate. Store at 2-8C.
TMB Substrate Solution B - 1 vial containing 12 ml of a TMB substrate component in a citric acid buffer containing hydrogen peroxide (H2O2). Store at 2-8 C.
Acid Stop Solution - 1 bottle containing 10 ml of 2.5 Molar sulfuric acid (H2SO4). Store at 2-8 C.
Caution: Caustic material. Wear eye, hand, face and clothing protection.
Graph Paper - 1 sheet of 3 cycle log × log graph paper.
This Kit is Intended for In-vitro Diagnostic Use Only
WARNING - The Acid Stop Solution is a solution of 2.5 Molar sulfuric acid (H2SO4). Wear appropriate eye, hand, face and clothing protection when using this material.
Human Specimens - Standards provided in this kit contain purified protein obtained from human donors. Each donor's serum was tested by FDA approved methods for the presence of antibodies to HIV-1 as well as for hepatitis B surface antigen and found to be negative. However, since no test method can absolutely guarantee the absence of these or other infectious agents, all standards, controls and human sera tested with this assay should be handled in accordance with NCCLS guidelines for preventing the transmission of blood-borne infections during laboratory procedures. Wear gloves and avoid contact with skin and mucous membranes.
Glassware - Disposable glassware is preferred; however, before use, all non-disposable glassware should be rinsed with 1N HCl or 1N sulfuric acid followed by at least six rinses with deionized water. There should be no acid or detergent residues.
• Kit reagents should not be used beyond the expiration date.
• Avoid exposure of reagents to strong light during incubation and storage.
• Samples or solutions containing sodium azide should not be used as the sodium azide will interfere with the assay performance.
• Reagents containing ProClin 300™ may be toxic if ingested.
• Perform the assay only as provided and described in this product insert.
• Substitution of kit reagents and/or alteration of incubation times or conditions may lead to erroneous results.
• Use only high quality (deionized and filtered or distilled) water for the dilution of the 5X Wash Solution.
STORAGE OF KIT
Reagents provided in this kit should be stored at 2-8 C for the life of the kit, except for the 5X Wash Solution which may be stored at room temperature.
Reagents should be returned to 2-8 C as soon as possible after the completion of the assay.
INDICATIONS OF REAGENT INSTABILITY OR DETERIORATION
Alll solutions should be clear and free of cloudiness and precipitates. The optical density of the color generated by the 200 ng/ml Pre-diluted Standard should be at least 0.4. If any of the above conditions are not met, the reagents in question should be discarded.
MATERIALS REQUIRED BUT NOT PROVIDED
• Purified water (deionized or distilled).
• Precision pipettes capable of delivering 2-20 μl, 20-200 μl, and 200-1000μl μl . Alternatively, single volume pipettors of 10 μl, 50 μl, 150 μl, 200 μl, and 1 ml volume
can be used.
• Glass, polypropylene, or polyethylene disposable test tubes. Do Not Use tubes made of Polystyrene.
• Measuring pipettes and cylinders.
• Microplate reader capable of reading at 450nm.
• Absorbent pad or paper towels.
• Microplate washing device or wash bottle.
• Laboratory horizontal rotator table (Optional).
• Statistical/Scientific calculator with regression program or a computer program for point to point or polynomial curve fitting (Optional).
Collect at least 2 ml of venouse blood aseptically. Allow the bood to coagulate and separate the clot from the serum by centrifugation as soon as possible. Plasma may also be used for TfR measurements. If the assay will be performed within 7 days, the serum/plasma may be stored at 2-8 C, otherwise store the serum/plasma frozen at 20 C for up to 6 months or 70 C for up to 2 years. Avoid repeated freezing and thawing.
Because less than 50 ul of plasma or serum is required to perform triplicate determinations of serum TfR, capilllary samples of 100 ul drawn into heparinized capillary tubes and centrifuged in a microhematocrit centrifuge to obtain plasma can also be used.
Samples must be diluted 1:100 in Sample Diluent prior to evaluating in the assay. DO NOT DILUTE STANDARDS.
Wash Solution - Prepare a 1X Wash Solution by pouring the entire contents of the Wash Solution 5X ConWcentrate into a clean 250 ml graduated cylinder. Bring the total volume to 250 ml with distilled water. Cover and invert to mix. Transfer to a clean wash bottle and store at room temperature.
Bring all reagents to room temperature before beginning assay.
Determine the number of microwells needed for the assay (each sample, standard, and control should be assayed in duplicate).
1. Remove the necessary strips from the packet and place in Microwell Frame. NOTE: Once a foil packet is opened, any unused Microwell Strips should be immediately resealed in the ziploc foil packet, along with the packet containing desiccant, and returned to 2-8 C storage. These opened strips should be utilized within 6 weeks.
2. Prepare a 1:100 dilution of each patient sample and Control by dispensing 1 ml of Sample Diluent into a test tube and adding 10 μl of patient serum or plasma or Control. Mix thoroughly. DO NOT DILUTE THE STANDARDS.
3. Pipette 50 μl of each Pre-diluted Standard, in duplicate, into individual wells of the 8- well strips.
4. Pipette 50 μl of each diluted patient sample and Control, in duplicate, into individual wells of the 8-well strips.
A typical plate map might be as shown below:
5. Pipette 150 μl of HRP-Conjugate into all individual wells containing samples, controls, and Standards.
6. Seal the Microwell Strips with the self-adhesive plastic film and place on a laboratory horizontal rotating table and mix for 10 minutes at 190 rpm or rotate by hand, pressing the Frame firmly against the counter to avoid sloshing, to ensure mixing of the HRP- Conjugate with the samples, Controls, and Standards. Allow reaction to proceed for an additional two hours (no rotation) at room temperature upon completion of the mixing.
7. Prepare the Substrate Solution: Calculate the amount of Substrate Solution needed by multiplying 0.2 ml by the number of wells in the assay run plus 0.5 - 1.0ml dead volume. Mix equal volumes of TMB Substrate Solution A and TMB Substrate Solution B just prior to addition to the microwells. This solution must be used within 30 minutes of preparation. Discard any unused portion.
8. After the two hour incubation in #6 above, remove the plastic film, invert the plate, and dump the contents of the Microwell Strips. Tap the plate dry on an absorbent pad or paper towels. Wash the wells with the previously prepared Washing Solution using a plate washing device or by using the wash bottle to flood the wells and then inverting and dumping the contents of the wells. The wells should be washed a total of three times, tapping dry between each wash. After the final wash, make sure the wells are completely empty and free of bubbles.
9. Pipette 200 μl of Substrate Solution into each blank well and those containing samples, Controls, and Standards, mix for 1 minute at 190 rpm on the horizontal rotator, and incubate in the dark at room temperature for 30 minutes. A blue color should develop in those wells containing TfR.
10. Stop the color reaction by pipetting 50 μl of Acid Stop Solution into the wells and mix for 1 minute at 190 on the horizontal rotator to remove any air bubbles.
11. Read the absorbance (optical density (OD) of each well at 450nm using a microplate reade and, if available, use a background correction wavelength of 570 -650nm. It is recommended that the reader be zeroed using blanks prepared with 200μl of Substrate Solution and 50 μl of Acid Stop Solution. Alternatively, the reader can be zeroed on the 0 ng/ml Standard.
12. If automatic background correction is not available, it is suggested that the plate be read a second time at 570 - 650 nm and that these readings be manually subtracted from the initial 450 nm readings. Failure to compensate for the backgrouind absorbance may increase the variablility of the assay and result in potentially erroneous results.
13. Calculate the results by plotting a standard curve of absorbance vs. concentration of the Pre-diluted Standards. See below.
CALCULATION of RESULTS
Calculate the average OD for each set of Standards. If the microplate reader has not already done so, subtract the average OD of the 0 ng/ml Standard from the average OD of each of the other Standards (= Net OD). Plot the Net OD on the vertical (Y) axis and the corresponding TfR concentration on the horizontal (X) axis of the enclosed 3-cycle log x log Graph Paper. Construct the standard curve by connecting the points with a best fit line.
Calculate the average OD for each Control and unknown sample and, if the plate reader has not already done so, subtract the average OD of the0 ng/ml Standard from each (= Net OD). Determine the location of the Net OD on the Y-axis. Follow this point horizontally until it intersects the standard curve. Follow this point of intersection with the curve vertically until it intersects the X-axis. This X-axis value, multiplied by 100 (due to the pre-dilution of the samples), will be the TfR concentration of the Control or unknown sample.
Alternatively, the TfR concentration can be determined by calculating a linear regression plot of the logarithms of the Net OD and TfR concentration values of the Standards using a statistical/scientific calculator or computer. Computer programs are commercially available for point to point and polynomial best-fitting standard curves.
Example Standard Curve
DILUTION OF SERA WITH HIGH TfR VALUES
If the calculated TfR value of the sample is greater than 40 μg/ml, it must be re-assayed at a higher dilution. For example, 50 μl of the 1:100 dilution would be added to a tube containing 450 μl of Sample Diluent. The measured TfR value would be multiplied by 1000 to determine the concentration of the original sample.
Control sera of known TfR values are included and should be run with each assay as quality control. If the measured values of the Controls are not within the expected ranges or the optical density of the 200 ng/ml Standard is less than 0.4, the assay may be considered invalid.
1. Moderate hemolysis of the sample has no effect on the reproducibility or accuracy of the procedure.
2. Anticoagulants do not influence the test results as long as there is no dilution of the sample. No other drugs have been tested for interference in this assay.
3. Some elevation in serum TfR can occur following a blood donation.
4. Strict adherence to precise laboratory procedure is essential for maximum accuracy of the final results.
5. Values lower than 1 μg/ml (the limit of this assay) should be reported as less than 1 μg/ml.
6. The optical density of the 200 ng/ml TfR standard should be greater than 0.4.
7. If the OD reading for any unknown sample assay is greater than the 400 ng/ml Standard, the sample should be diluted appropriately withSample Diluent and re-assayed.
Normal Range - The serum TfR values for 239 normal, healthy volunteers from the Kansas City area were measured and the average serum TfR was 5.55 ± 1.35 μg/ml. The normal range for serum TfR as measured by the Ramco TfR assay has been determined to be 2.9 - 8.3 μg/ml. There is no significant sex or age difference in serum TfR values. Variations in serum TfR values in healthy subjects are believed to reflect differences in the rate of red cell production9.
Iron Deficiency Anemia - In a composite group of 22 patients with uncomplicated iron deficiency anemia, the mean serum TfR value was 18.6 μg/ml with a range of 5.1 to 35 μg/ml. In 21 out of 22 patients, the TfR value was greater than the upper cut-off level of the normal range (8.3 μg/ml).
Unlike most conventional measurements of iron deficiency, the serum TfR is useful for distinguishing iron deficiency anemia from the anemia associated with chronic infection or inflammation11,12. In 31 patients with the latter, the mean serum TfR was 5.36 ±2.1 μg/ml and 29 of the 31 patients had serum TfR levels below the 8.3 μg/ml upper cut-off of the normal range.
Additionally, the serum TfR is useful for identifying iron deficiency anemia when it develops in patients with chronic disease. In one study of anemic patients with inflammatory joint disease, the serum TfR was elevated above 8.3 μg/ml in 9 out of 13 patients with absent iron on bone marrow examination but was normal in all 9 patients with iron stores. Similarly, the serum TfR concentration was elevated above 8.3 μg/ml in 8 out of 10 anemic patients with inflammatory bowel disease and absent marrow iron but was normal in 3 out of 4 patients with stainable marrow iron.
Analytical Sensitivity (limit of detection) - Sensitivity has been defined as the lowest measurable concentration of TfR that differs significantly from zero (p = 0.001). In two assays performed on separate days, the 0 Standard was run in quadruplicate along with the set of Standards andControls. The mean OD and standard deviation of the zero standard were calculated: 0.006 ± 0.0008. The mean OD of the zero standard plus three times the standard deviation is considered the lowest detectable TfR concentration. This converts to 0.7 ng/ml, which corresponds to a serum TfR concentration of 0.07 μg/ml.
Linearity - As demonstrated below, serial dilutions of sera with high TfR levels gave a linear response when normal rabbit serum (NRS) was added to keep serum levels of all dilutions constant while a progressive increase in apparent recovery occurred when NRS was not added. Because of this serum effect, 1% NRS is added to all Standards to equalize serum concentrations between Standards and diluted samples.
Analytical Specificity - No assay interference occurred at normal serum levels of transferrin, serum albumin and bilirubin nor at concentrations 50% higher than normal. A slight increase in TfR measurements occur at twice the normal concentrations of transferrin and bilirubin. No change occurred in measured TfR levels when samples became highly hemolyzed. When the dilution of the blood is accounted for, there is no difference in the TfR levels of blood collected into vacutainer tubes containing EDTA, Sodium Citrate, Heparin or no additives.
Intra-Assay Variability - Performing 10 replicate measurements on six control sera with mean TfR levels between 5.0 and 15.0 μg/ml yielded within-assay variability of 2.3 to 5.8% with a mean of 4.2%.
Inter-Assay Variability - Performing duplicate measurements on twelve serum samples over a period of five weeks yielded between-assay variability of 3.6 to 8.1% with a mean C.V. of 6.0%.
Recovery - Studies in which purified TfR was added to normal serum samples showed recoveries of 92.8 to 103.2% with a mean of 97.4%. Purified TfR was added to control sera at three levels. Percent recovery of added TfR was calculated as the actual measured TfR divided by the expected TfR concentration.
1. Huebers HA, Finch CA: The physiology of transferrin and transferrin receptors. Physiol Rev 67:520, 1987.
2. Trowbridge IS: Transferrin receptor as a potential therapeutic target. Prog Allergy, 45:121, 1988.
3. Shih YJ, Baynes RD, Hudson BG, Flowers CH, Skikne BS, Cook JD: Serum transferrin receptor is a truncated form of tissue receptor. J Biol Chem 265:19077, 1990.
4. Baynes RD, Skikne BS, Cook JD: Circulating transferrin receptors and assessment of iron status. J Nutr Biochem 5:322, 1994.
5. Huebers HA, Bequin Y, Pootrakul P, Einspahr D, Finch CA: Intact transferrin receptors in human plasma and their relation to erythropoiesis. Blood 75:102, 1990.
6. Kohgo Y, Nishisato T, Kondo H, Tsushima N, Niitsu Y, Urushizaki I: Circulating transferrin receptor in human serum. Br J Haematol 64:277, 1986.
7. Flowers CH, Skikne BS, Covell AM, Cook JD: The clinical measurement of serum transferrin receptor. J Lab Clin Med 114:368, 1989.
8. Skikne BS, Flowers CH, Cook JD: Serum transferrin receptor: A quantitative measure of tissue iron deficiency. Blood 75:1870, 1990.
9. Cook JD, Skikne BS, Baynes RD: Serum transferrin receptor. Annu Rev Med 44:63, 1993.
10. Kohgo Y, Niitsu Y, Nishisato T, Dato J, Kondo H, Sasaki K, Urushizaki I: Quantitation and characterization of serum transferrin receptor in patients with anemias and polycythemias. Jpn J Med 27:64, 1988.
11. Ferguson BJ, Skikne BS, Simpson KM, Baynes RD, Cook JD: Serum transferrin receptor distinguishes the anemia of chronic disease from iron deficiency anemia. J Lab Clin Med 119:385, 1992.
12. Pettersson T, Kivivuori SM, Siimes MA: Is serum transferrin receptor useful for detecting iron-deficiency in anaemic patients with chronic inflammatory diseases? Br J Rheumatol 33:740, 1994.
13. Carriaga MT, Skikne BS, Finley B, Cutler B, Cook JD: Serum transferrin receptor for the detection of iron deficiency in pregnancy. Am J Clin Nutr 54:1077, 1991.
Ramco Laboratories, Inc.
4100 Greenbriar Drive Suite 200
Stafford, Texas 77477
MT Promedt Consulting GmbH
66386 St. Ingbert
+49 (0) 68 94 - 58 10 20
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Revised: 9/09/03; 12/18/03; 6/18/04; 7/23/07: 11/03/11; 2/12/14; 7/3/14; 9/14/16