DRG EIA-4256 Human Soluble Transferrin Receptor ELISA Instructions

DRG EIA-4256 Human Soluble Transferrin Receptor ELISA

Product Information

The Human Soluble Transferrin Receptor ELISA EIA-4256 is a sandwich enzyme immunoassay designed for the quantitative measurement of the human soluble transferrin receptors. It is intended for in vitro diagnostic use in the European Union and for research use only in the rest of the world. The assay time is less than 3 hours and it can measure total soluble transferrin receptors in serum and plasma (EDTA, citrate, heparin). The kit comes in a 96-well format and utilizes human serum-based quality controls, with no animal sera used. The standard is based on natural human blood isolated sTfR. The components of the kit are provided ready to use or concentrated. This ELISA is specifically designed for investigating iron metabolism.

Product Usage Instructions

1. This product is for professional use only.
2. When handling immunodiagnostic materials, wear gloves and laboratory coats.
3. Avoid drinking, eating, or smoking in areas where immunodiagnostic materials are being handled.
4. This kit contains components of human origin. While they have been found non-reactive for HBsAg, HCV antibody, and HIV 1/2 antigen and antibody, they should still be handled as potentially infectious.
5. Avoid contact with the acidic Stop Solution and Substrate Solution, which contain hydrogen peroxide and tetramethylbenzidine (TMB). Wear gloves and eye and clothing protection when handling these reagents. In case of contact, wash skin/eyes thoroughly with water and seek medical attention if necessary.
6. Do not pipette the materials by mouth.
7. Do not mix reagents with different lot numbers.
8. Use thoroughly clean glassware and deionized (distilled) water stored in clean containers.
9. Avoid contamination among samples and reagents by using disposable tips for each sample and reagent.
10. Keep the Substrate Solution protected from light, as it should remain colorless until added to the plate.
11. The Stop Solution should also remain colorless until added to the plate. The color developed in the wells will turn from blue to…

Please use only the valid version of the Instructions for Use provided with the kit.

INTENDED USE

The Human sTfR ELISA is a sandwich enzyme immunoassay for the quantitative measurement of human soluble transferrin receptor.

Features

• European Union: for in vitro diagnostic use.
  • Rest of the world: for research use only!
• The total assay time is less than 3 hours.
• The kit measures total soluble transferrin receptor in serum and plasma (EDTA, citrate, heparin).
• Assay format is 96 wells.
• Quality Controls are human serum based. No animal sera are used.
• Standard is natural human blood isolated sTfR based.
• Components of the kit are provided ready to use or concentrated.

STORAGE, EXPIRATION

• Store the complete kit at 2 °C – 8 °C. Under these conditions, the kit is stable until the expiration date (see label on the box).
• For stability of opened reagents see Chapter 9.

INTRODUCTION

• The transferrin receptor (TfR) is the gateway for transferrin-bound iron entering all body cells. TfR is abundant on the surface of many newly formed cells, but the erythroid marrow cells account for 70 to 80 % of the total body TfR content.
• The soluble (or serum) transferrin receptor (sTfR) is a circulating truncated form of the membrane receptor protein; it is an 85 kDa glycoprotein forming in serum a 320 kDa complex with diferric transferrin. The serum sTfR concentration reflects the total body mass of cellular transferrin receptor. Anaemias associated with enhanced erythropoiesis and iron deficiency result in an elevation in the sTfR values.
• Elevation of the soluble transferrin receptor may be also caused by haemolytic anaemia, polycythaemia and thalassemia while aplastic anemia and chronic renal failure may result in its decrease. The most important clinical use of the sTfR determination is in the differential diagnosis between iron deficiency anaemia and the anaemia of chronic disease.

Areas of investigation

• Iron metabolism

TEST PRINCIPLE

In the Human sTfR ELISA, standards, quality controls and samples are incubated in microplate wells pre-coated with monoclonal anti-human sTfR antibody. After 60 minutes incubation and washing, monoclonal anti-human sTfR antibody, conjugated with horseradish peroxidase (HRP) is added to the wells and incubated for 60 minutes with captured sTfR. Following another washing step, the remaining HRP conjugate is allowed to react with the substrate solution (TMB). The reaction is stopped by addition of acidic solution and absorbance of the resulting yellow product is measured. The absorbance is proportional to the concentration of sTfR. A standard curve is constructed by plotting absorbance values against concentrations of standards, and concentrations of unknown samples are determined using this standard curve.

PRECAUTIONS

• For professional use only.
• Wear gloves and laboratory coats when handling immunodiagnostic materials.
• Do not drink, eat or smoke in the areas where immunodiagnostic materials are being handled.
• This kit contains components of human origin. These materials were found non-reactive for HBsAg, HCV antibody and for HIV 1/2 antigen and antibody. These materials should be handled as potentially infectious, as no test can guarantee the complete absence of infectious agents.
• Avoid contact with the acidic Stop Solution and Substrate Solution, which contains hydrogen peroxide and tetramethylbenzidine (TMB). Wear gloves and eye and clothing protection when handling these reagents. Stop and/or Substrate Solutions may cause skin/eyes irritation. In case of contact with the Stop Solution and the Substrate Solution wash skin/eyes thoroughly with water and seek medical attention, when necessary.
• The materials must not be pipetted by mouth.

TECHNICAL HINTS

• Reagents with different lot numbers should not be mixed.
• Use thoroughly clean glassware.
• Use deionized (distilled) water, stored in clean containers.
• Avoid any contamination among samples and reagents. For this purpose, disposable tips should be used for each sample and reagent.
• Substrate Solution should remain colourless until added to the plate. Keep Substrate Solution protected from light.
• Stop Solution should remain colourless until added to the plate. The colour developed in the wells will turn from blue to yellow immediately after the addition of the Stop Solution. Wells that are green in colour indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution.
• Dispose consumable materials and unused contents in accordance with applicable national regulatory requirements.

REAGENT SUPPLIED

MATERIAL REQUIRED BUT NOT SUPPLIED

• Deionized (distilled) water
• Test tubes for diluting samples
• Glassware (graduated cylinder and bottle) for Wash Solution
• Precise pipettes to deliver 5 -1000 μL with disposable tips
• Multichannel pipette to deliver 100 μL with disposable tips
• Absorbent material (e.g. paper towels) for blotting the microplate after washing
• Vortex mixer
• Orbital microplate shaker capable of approximately 300 rpm
• Microplate washer (optional). [Manual washing is possible but not preferable.]
• Microplate reader with 450 ± 10 nm filter, preferably with reference wavelength 630 nm (alternatively another one from the interval 550 – 650 nm)
• Software package facilitating data generation and analysis (optional)

PREPARATION OF REAGENTS

• All reagents need to be brought to room temperature prior to use.
• Warm-up the Dilution Buffer to 25 °C – 30 °C prior to use.
• Centrifuge liquid containing microtube vials before opening.
• Always prepare only the appropriate quantity of reagents for your test.
• Do not use components after the expiration date marked on their label.

Assay reagents supplied ready to use

Antibody Coated Microtiter Strips

Stability and storage:

Return the unused strips to the provided aluminium zip-sealed bag with desiccant and seal carefully. Remaining Microtiter Strips are stable 3 months stored at 2 °C – 8 °C and protected from the moisture.

• Conjugate Solution
• Dilution Buffer
• Substrate Solution
• Stop Solution
• Stability and storage: Opened reagents are stable 3 months when stored at 2 °C – 8 °C.

Assay reagents supplied concentrated

Human sTfR Standards

• Dilute each concentration of Standard 10x with Dilution Buffer just prior to the assay, e.g. 30 μL of Standard + 270 μL of Dilution Buffer for duplicates.
• Mix Standards well (we recommend vortex) before taking the desired amount from the tube as well as after adding it to the Dilution Buffer (not to foam).
• Stability and storage: Opened Standards are stable 3 months when stored at 2 °C – 8 °C. Do not store the diluted Standard solutions.

Quality Controls HIGH, LOW

• Refer to the Certificate of Analysis for current Quality Control concentration!!!
• Dilute Quality Control (HIGH and LOW) 50x with Dilution Buffer just prior to the assay, e.g. 5 μL of Quality Control + 245 μL of Dilution Buffer for duplicates.
• Mix Controls well (we recommend vortex) before taking the desired amount from the tube as well as after adding it to the Dilution Buffer (not to foam).

Stability and storage

• Opened Quality Control s are stable 3 months when stored at 2 °C – 8 °C.
• Do not store the diluted Quality Controls.

Note: Concentration of analyte in Quality Controls need not be anyhow associated with normal and/or pathological concentrations in serum or another body fluid. Quality Controls serve just for control that the kit works in accordance the with user’s manual and Certificate of Analysis and that ELISA test was carried out properly.

Wash Solution Conc. (10x)

• Dilute Wash Solution Concentrate (10x) ten-fold in distilled water to prepare a 1x working solution. Example: 100 mL of Wash Solution Concentrate (10x) + 900 mL of distilled water for use of all 96-wells.
• Stability and storage: The diluted Wash Solution is stable 1 month when stored at 2 °C – 8 °C. Opened Wash Solution Concentrate (10x) is stable 3 months when stored at 2 °C – 8 °C.

PREPARATION OF SAMPLES

• The kit measures sTfR in serum and plasma (EDTA, citrate, heparin).
• Samples should be assayed immediately after collection or should be stored at -20 °C. Mix thoroughly thawed samples just prior to the assay and avoid repeated freeze/thaw cycles, which may cause erroneous results. Avoid using hemolyzed or lipemic samples.
• Dilute samples 50x with Dilution Buffer just prior to the assay, e.g. 5 μL of sample + 245 μL of Dilution Buffer for duplicates. Mix well (not to foam). Vortex is recommended

Stability and storage

• Samples should be stored at -20 °C, or preferably at -70 °C for long-term storage. Avoid repeated freeze/ thaw cycles.
• Do not store the diluted samples.
• See Chapter 13 for effect of sample matrix (serum/plasma) on the concentration of sTfR.

Note: It is recommended to use a precise pipette and a careful technique to perform the dilution in order to get precise results.

ASSAY PROCEDURE

1. Pipet 100 μL of diluted Standards, Quality Controls, Dilution Buffer (=Blank) and samples, preferably in duplicates, into the appropriate wells. See Figure 1 for example of work sheet.

2. Incubate the plate at 25 °C – 30 °C for 1 hour, shaking at ca. 300 rpm on an orbital microplate shaker. Performing the incubation at the appropriate temperature is crucial in order to obtain valuable results!

3. Wash the wells 3-times with Wash Solution (0.35 mL per well). After final wash, invert and tap the plate strongly against paper towel.

4. Add 100 μL of Conjugate Solution into each well.

5. Incubate the plate at 25 °C – 30 °C for 1 hour, shaking at ca. 300 rpm on an orbital microplate shaker. Performing the incubation at the appropriate temperature is crucial in order to obtain valuable results!

6. Wash the wells 3-times with Wash Solution (0.35 mL per well). After final wash, invert and tap the plate strongly against paper towel. 7. Add 100 μL of Substrate Solution into each well. Avoid exposing the microtiter plate to direct sunlight. Covering the
   plate with e.g. aluminium foil is recommended.

7. Incubate the plate for 10 minutes at room temperature (20 °C – 30 °C). Do not shake the plate during the incubation.

8. Stop the colour development by adding 100 μL of Stop Solution.

9. Determine the absorbance of each well using a microplate reader set to 450 nm, preferably with the reference wavelength set to 630 nm (acceptable range: 550 – 650 nm). Subtract readings at 630 nm (550 – 650 nm) from the readings at 450 nm. The absorbance should be read within 5 minutes following step 9.

Note 1: If some samples and standard/s have absorbances above the upper limit of your microplate reader, perform a second reading at 405 nm. A new standard curve, constructed using the values measured at 405 nm, is used to determine sTfR concentration of off-scale standards and samples. The readings at 405 nm should not replace the readings for samples that were “in range” at 450 nm.

Note 2: Manual washing: Aspirate wells and pipet 0.35 mL Wash Solution into each well. Aspirate wells and repeat twice. After final wash, invert and tap the plate strongly against paper towel. Make certain that Wash Solution has been removed entirely.

Figure 1: Example of a work sheet.

 | strip 1+2| strip 3+4| strip 5+6| strip 7+8| strip 9+10| strip 11+12
---|---|---|---|---|---|---
  A| Standard 2| Blank| Sample 8| Sample 16| Sample 24| Sample 32
  B| Standard 1| Sample 1| Sample 9| Sample 17| Sample 25| Sample 33
C| Standard 0.5| Sample 2| Sample 10| Sample 18| Sample 26| Sample 34
D| Standard 0.2| Sample 3| Sample 11| Sample 19| Sample 27| Sample 35
E| Standard 0.1| Sample 4| Sample 12| Sample 20| Sample 28| Sample 36
  F| Standard 0.05| Sample 5| Sample 13| Sample 21| Sample 29| Sample 37
G| QC HIGH| Sample 6| Sample 14| Sample 22| Sample 30| Sample 38
H| QC LOW| Sample 7| Sample 15| Sample 23| Sample 31| Sample 39
---|---|---|---|---|---|---

CALCULATIONS

Most microplate readers perform automatic calculations of analyte concentration. The standard curve is constructed by plotting the mean absorbance (Y) of Standards against the known concentration (X) of Standards in logarithmic scale, using the four-parameter algorithm. Results are reported as concentration of sTfR μg/mL in samples.

Alternatively, the logit log function can be used to linearize the standard curve i.e. logit of the mean absorbance (Y) is plotted against log of the known concentration (X) of Standards. The measured concentrations of samples and Quality Controls calculated from the standard curve have to be multiplied by their respective dilution factor. Since samples and Quality Controls are diluted 50x while standards are diluted 10x, the ratio 50/10 = 5 have to be used as the dilution factor.

Example: 0.4 μg/mL (from standard curve) x 5 (dilution factor) = 2.0 μg/mL (real concentration in sample).

PERFORMANCE CHARACTERISTICS

Typical analytical data of Human sTfR ELISA are presented in this chapter.

Sensitivity

• Limit of Detection (LOD) (defined as concentration of analyte giving absorbance higher than mean absorbance of blank* plus three standard deviations of the absorbance of blank: Ablank + 3xSDblank) is calculated from the real sTfR values in wells and is 2 ng/mL.
• Dilution Buffer is pipetted into blank wells.

Limit of assay

Results exceeding sTfR level 10 μg/mL should be repeated with more diluted samples (e.g. 100x). Dilution factor needs to be taken into consideration when calculating the sTfR concentration.

Specificity

The antibodies used in this ELISA are specific for human sTfR. The sTfR ELISA exhibits no interference with hemoglobin (0.1 mg/mL), bilirubin (170 μmol/L), or triglycerides (5.0 mmol/L). However, higher levels of hemoglobin can interfere with performance of this ELISA, therefore, we discourage the customers from using hemolyzed samples.

Sera of several mammalian species were measured in the assay. See results below.
For details please contact DRG.

Presented results are multiplied by respective dilution factor

Precision

Intra-assay (Within-Run) (n=8)

Sample| Mean (µg/mL)| SD

(µg/mL)

| CV (%)
---|---|---|---
1| 1.27| 0.04| 3.4
2| 5.22| 0.23| 4.3

Inter-assay (Run-to-Run) (n=8)

Sample| Mean (µg/mL)| SD

(µg/mL)

| CV (%)
---|---|---|---
1| 1.51| 0.11| 7.0
2| 6.12| 0.33| 5.5

Spiking Recovery

Serum samples were spiked with different amounts of human sTfR and assayed.

Sample| Observed (µg/mL)| Expected (µg/mL)| Recovery O/E

(%)

---|---|---|---
1| 0.28| –| –
 | 2.18| 2.28| 96
 | 1.39| 1.28| 109
 | 0.78| 0.78| 100
2| 0.21| –| –
 | 2.12| 2.21| 96
 | 1.26| 1.21| 104
 | 0.68| 0.71| 96

Linearity

Serum samples (50x diluted) were serially diluted with Dilution Buffer and assayed.

Sample| Dilution| Observed (µg/mL)| Expected (µg/mL)| Recovery

O/E (%)

---|---|---|---|---
1| –| 5.13| –| –
 | 2x| 2.43| 2.57| 95
 | 4x| 1.25| 1.28| 98
 | 8x| 0.66| 0.64| 103
2| –| 5.90| –| –
 | 2x| 2.53| 2.95| 86
 | 4x| 1.39| 1.48| 94
 | 8x| 0.65| 0.74| 88

Effect of sample matrix

EDTA, citrate and heparin plasmas were compared to respective serum samples from the same 10 individuals. Results are shown below:

Volunteer

No.

| Serum

(µg/mL)

| Plasma (µg/mL)
---|---|---
EDTA| Citrate| Heparin
1| 0,89| 0,61| 0,66| 0,84
2| 1,43| 1,15| 1,07| 1,39
3| 1,33| 0,80| 0,92| 1,25
4| 1,24| 1,06| 0,93| 1,19
5| 1,19| 1,03| 0,90| 1,38
6| 1,18| 0,78| 0,76| 1,09
7| 1,54| 1,16| 1,14| 1,54
8| 2,19| 1,78| 1,70| 2,33
9| 1,35| 0,84| 1,08| 1,34
10| 0,94| 0,90| 0,72| 1,01
Mean (µg/mL)| 1,3| 1,0| 1,0| 1,3
Mean Plasma/Serum (%)| –| 76,1| 74,4| 100,6
Coefficient of determination R2| –| 0,82| 0,97| 0,95

DEFINITION OF THE STANDARD

The Standard used in this kit is a natural sTfR isolated from human blood.

Concentration Unit Conversions

(calculated from the sTfR molar mass):

• 1 nM = 0.075 μg/mL
• 1 μg/mL = 13.33 nM

REFERENCE RANGE

The following results were obtained when serum samples from 153 blood donors (84 men + 69 women, 20 – 65 years old, Caucasian population) were assayed with the Human sTfR ELISA (EIA-4256) in our laboratory:

BMI

(kg/m 2)

| N| Mean (µg/ml)| Median (µg/ml)| Max. (µg/ml)| Min. (µg/ml)| SD

(µg/ml)

| SEM

(µg/ml)

| 2.5 th – 97.5th percentile (µg/ml)
---|---|---|---|---|---|---|---|---
18-43| 153| 0.868| 0.854| 1.699| 0.0720| 0.307| 0.0248| 0.378 – 1.513
31-43| 35| 1.010| 0.928| 1.575| 0.631| 0.261| 0.0441| 0.649 – 1.558
26-30| 56| 0.836| 0.871| 1.464| 0.0720| 0.300| 0.0401| 0.303 – 1.550
18-25| 62| 0.817| 0.790| 1.699| 0.253| 0.316| 0.0401| 0.401 – 1.448

The data quoted in these instructions should be used for guidance only. Each laboratory should establish its own normal and pathological ranges for sTfR levels with the assay. Each laboratory should establish a quality control program to monitor the quality of the assay.

METHOD COMPARISON

The Human sTfR ELISA was compared to a commercial Immunoturbidimetry (IT). The following correlation graph was obtained.

• EIA-4256 = -0.128 + (0.869 * Orion Diag. (μg/mL))
• R2 = 0.885
• N = 62

TROUBLESHOOTING AND FAQS

Weak signal in all wells

Possible explanations:

• Omission of a reagent or a step
• Improper preparation or storage of a reagent
• Assay performed before reagents were allowed to come to room temperature
• Improper wavelength when reading absorbance

High signal and background in all wells

Possible explanations:

• Improper or inadequate washing
• Overdeveloping; incubation time with Substrate Solution should be decreased

High coefficient of variation (CV)

Possible explanation:

• Improper or inadequate washing
• Improper mixing of Standards, Quality Controls or samples

REFERENCES

1. Lee E. J. et al.: Soluble transferrin receptor (sTfR), ferritin, and sTfR/log ferritin index in anemic patients with nonhematologic malignancy and chronic inflammation. Clinical Chemistry 48, 1118-1121 (2002)
2. Raya G. et al.: Soluble transferrin receptor (sTfR): biological variations and reference limits. Clinical Chemistry and Laboratory Medicine, 39, 1162-1168 (2001)
3. Cotton F. et al.: Measurement of soluble transferrin receptor by immunoturbidimety and immunonefelometry. Clinical Biochemistry, 33, 263-267 (2000)
4. Cook J. D.: The measurement of serum transferrin receptor. The American Journal of the Medical Sciences 318, 269-276 (1999)
5. Olivares M. et al.: Usefulness of serum transferrin receptor and serum ferritin in diagnosis of iron deficiency in infancy. American Journal of Clinical Nutrition 72, 1191-1195 (2000)
6. Suominen P. et al.: Single values of serum transferrin receptor and transferrin receptor ferritin index can be used to detect true and functional iron deficiency in rheumatoid arthritis patients with anemia. Arthritis & Rheumatism 43, 1016-1020 (2000)
7. De Block C. E. M. et al.: Soluble transferrin receptor level. A new marker for iron deficiency anemia, a common manifestation of gastric autoimmunity in type 1 diabetes. Diabetes Care 23, 1384-1388 (2000)
8. Kolbe-Busch S. et al.: Determination of the soluble transferrin receptor in Serum: Evaluation of two enzyme immunoassays and a particle-enhanced immunonephelometric assay. Clinical Laborators 45, 295-304 (1999)
9. Hikawa A. et al.: Soluble transferrin receptor-transferrin complex in serum: measurement by latex agglutination nephelometric asssay. Clinica Chimica Acta 254, 159-172 (1996)
10. Flowers C. H. et al.: The clinical measurement of serum transferrin receptor. Journal of Laboratory and Clinical Medicine 114, 368-377 (1989)
11. Fernández-Real J. M. et al. : Circulating soluble transferrin receptor according to glucose tolerance status and insulin sensitivity. Diabetes Care 30, 604-608 (2007)

ASSAY PROCEDURE SUMMARY

SYMBOLS USED

• European Conformity
• Consult instructions for use*
• In vitro diagnostic medical device *
• Catalogue number*
• Batch code*
• Contains sufficient for tests *
• Temperature limit *
• Use-by date*
• Manufacturer *
• Caution*
• RUO: For research use only
• Distributed by
• Content
• Volume / No.

Contacts

DRG Instruments GmbH, Germany

• Frauenbergstraße 18, 35039 Marburg
• Phone: +49 (0)6421-1700 0, Fax: +49 (0)6421-1700 50
• Website: www.drg-diagnostics.de
• E-mail: [email protected]

Distributed by:

• DRG International, Inc., USA
• 841 Mountain Ave., Springfield, NJ 07081
• Phone: 973-564-7555, Fax: 973-564-7556
• Website: www.drg-international.com
• E-mail: [email protected]

References

• Analyticon® Biotechnologies GmbH
• It's Uptime | International® Trucks
• DRG Diagnostics GmbH | Home
• Medical Supplies & Diagnostic Products | DRG International, Inc.

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