Immunoassay for the Quantitative Determination of Human Ferritin
Concentration in Human Serum
In Vitro Diagnostic Use Only)
of the most prevalent disorders of man is the dietary deficiency
of iron and the resulting anemia.
Therefore, the assays of iron, total iron binding
capacity and other assessments of iron compounds in the body are
compounds in the body include hemoglobin, hemosiderin,
myoglobulin and the cytochromes.
In most tissues, ferrrtin is a major iron-storage
ferritin has a molecular weight of approximately 450,000 daltons,
and consists of a protein shell around an iron core; each
molecule of ferritin may contain as many as 4,000 iron atoms.
Under normal conditions, this may represent 25% of the
total iron found in the body.
In addition, ferrritin can be found in several isomers.
concentrations of ferritin are found in the cytoplasm of the
reticuloendothelial system, the liver, spleen and bone marrow.
Methods previously used to measure iron in such tissues
are invasive, cause patient trauma and lack adequate
measurement of ferritin in serum is useful in determining
changes in body iron storage, and is non-invasive with
relatively little patient discomfort.
Serum ferritin levels can be measured routinely and are
particularly useful in the early detection of iron-deficiency
anemia in apparently healthy people.
ferritin measurements arre also clinically significant in the
monitoring of the iron status of pregnant women, blood donors,
and renal dialysis patients.
High ferritin levels may indicate iron overload without
apparent liver damage, as may be noted in the early stages of
Ferritin levels in serum have also been used to evaluate
clinical conditions not related to iron storage, including
inflammation, chronic liver disease, and malignancy.
Immunoassay Test Kit provides a rapid, sensitive and reliable assay.
The antibodies developed for the test will determine a
minimal concentration of human ferritin of 5 ng/ml.
There is minimal cross-reativity with human serum
albumin, alpha-fetoprotein, human hemoglobin, human transferrin,
and ferric chloride.
of the test
Ferritin Quantitative Test Kit is based on a solid phase
enzyme-linked immunosorbent assay.
The assay system utilizes one anti-ferritin antibody for
solid phase (microtiter wells) immobilization and another mouse
monoclonal anti-ferritin antibody in the antibody-enzyme
(horseradish peroxidase) conjugate solution.
The test sample is allowed to react simultaneously with
the antibodies, resulting in the ferritin molecules being
sandwiched between the solid phase and enzyme-linked antibodies.
After a 60 minute incubation at room temperature, the
wells are washed with water to remove unbound labeled
solution of TMB is added and incubated for 20 minutes, resulting
in the development of a blue color.
The color development is stopped with the addition of 2N
HCl, and the color is changed to yellow and measured
spectrophotometrically at 450 nm. The concentration of ferritin is directly proportional to the
color intensity of the test sample.
the quantitative determination of Human Ferritin concentration
in human serum.
provided with the test kits:
Antibody-coated microtiter wells.
Reference standard set, contains 0, 10, 50, 100, 400, and
800 ng/ml, ready for use.
(NIBSC-WHO 80/602, human liver standard)
Enzyme conjugate reagent, 12 ml.
Color Reagent A, 12 ml.
Color Reagent B, 12 ml.
Stop Solution, 10 ml.
required but not provided:
0.05, 0.1, and 0.2,
Disposable pipette tips.
Glass tubes or flasks to mix Color Reagent A and
Color Reagent B.
Vortex mixer or equivalent.
Absorbent paper or paper towel.
Microtiter well reader.
collection and preparation
should be prepared from a whole blood specimen obtained by
acceptable medical techniques.
This kit is for use with serum samples without additives
of test kits and instrumentation
test kits should be stored at 2-8oC upon receipt and the microtiter
plate should be kept in a sealed bag with desiccants to minimize
exposure to damp air. Opened
test kits will remain stable until the expiring date shown,
provided it is stored as prescribed above.
A microtiter plate reader with a bandwidth of 10nm or
less and an optical density range of 0-2 OD or greater at 450nm
wavelength is acceptable for use in absorbance measurement.
All reagent should be brought to room temperature (18-25oC
) before use.
To prepare TMB solution, make an 1:1 mixing of Color
Reagent A with Color Reagent B right before use.
Mix gently to ensure complete mixing.
The prepared TMB substrate reagent is stable at room
temperature in the dark for up to 1 hours.
Discard the excess after use.
Secure the desired number of coated wells in the holder.
of standard, specimens, and controls into appropriate wells.
of Enzyme Conjugate
Reagent into each well.
Thoroughly mix for 30 seconds.
It is very important to have complete mixing in this
Incubate at room temperature (18-25oC) for 60 minutes.
Prepare TMB solution up to one hour before use.
Remove the incubation mixture by flicking plate content
into a waste container.
Rinse and flick the microtiter wells 5 times with running
tap or distilled water.
Strike the wells sharply onto absorbent paper or paper
towels to remove all residual water droplets.
of TMB solution
into each well.
Gently mix for 5
Incubate at room temperature in the dark for 20 minutes.
Stop the reaction by adding 50ml
of 2N HCl to each well.
Gently mix for 30 seconds.
It is important to make sure that
all the blue color changes to yellow color completely.
Read optical density at 450nm with a microtiter reader
within 30 minutes.
wash procedure is critical.Insufficient washing will result
in poor precision and falsely
elevated absorbances readings.
the mean absorbance value (A450) for each set of reference
standards, specimens, controls and patient samples. Constructed a standard curve by plotting the mean absorbance
obtained from each reference standard against its concentration
in ng/ml on graph paper, with absorbance values on the vertical
or Y axis and concentrations on the horizontal or X axis.
Use the mean absorbance values for each specimen to
determine the corresponding concentration of Ferritin in ng/ml
from the standard curve.
of standard curve
of typical standard run with optical density reading at 450nm
shown in the Y- axis against Ferritin concentrations shown in
the X-axis. This standard curve is for the purpose of illustration only,
and should not be used to calculate unknowns.
Each user should obtain his or her own data and standard
values and sensitivity
laboratory must establish its own normal ranges based on patient
results provided below are based on a limited number of healthy
adult blood specimens. The minimal sensitivity of the test is 5.0 ng/ml.
White, D. ; Kramer, D.; Johnson, G.; Dick, F.
and Hamilton, H.
A.m. J. Clin. Path.
Valberg, L. CMAJ.
Forman, D. and Parker, S.
Ann. Clin. Lab.
Hazard, J.T.; Yokota, M.; Arosio, P. and Drysdale, J.
Smimes, M.A.; Addiego, Jr.J.E. and Dallman,