Instructions for use

CEA ELISATM E-4100R

CEA ELISA
1. INTRODUCTION
1.1 Intended Use
The CEA ELISA is an enzyme immunoassay for the quantitative measurement of Carcinoembryonic Antigen(CEA) in serum or lithium heparin plasma.
2. PRINCIPLE OF THE TESTThe CEA ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich
principle.
The microtiter wells are coated with a monoclonal antibody directed towards a unique antigenic site of theCEA molecule.
An aliquot of sample containing endogenous CEA is incubated in the coated well with enzyme conjugate,
which is an anti-CEA antibody conjugated with horseradish peroxidase. After incubation the unboundconjugate is washed off.
The amount of bound peroxidase conjugate is proportional to the concentration of CEA in the sample.
Having added the substrate solution, the intensity of colour developed is proportional to the concentration ofCEA in the sample.
3. WARNINGS AND PRECAUTIONS
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This kit is for research use only.
All reagents of this test kit which contain human serum or plasma have been tested and confirmed
negative for HIV I/II, HBsAg and HCV by FDA approved procedures. All reagents, however, should betreated as potential biohazards in use and for disposal.
Before starting the assay, read the instructions completely and carefully. Use the valid version ofinstructions for use provided with the kit. Be sure that everything is understood.
The microplate contains snap-off strips. Unused wells must be stored at 2 °C to 8 °C in the sealed foil
pouch and used in the frame provided.
Pipetting of samples and reagents must be done as quickly as possible and in the same sequence foreach step.
Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using areservoir for dispensing a substrate solution that had previously been used for the conjugate solution
may turn solution colored. Do not pour reagents back into vials as reagent contamination may occur.
Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reusemicrowells.
Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
Allow the reagents to reach room temperature (21 °C to 26 °C) before starting the test. Temperature
will affect the absorbance readings of the assay. However, values for the samples will not be affected.
Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
Wear disposable latex gloves when handling specimens and reagents. Microbial contamination ofreagents or specimens may give false results.
Handling should be done in accordance with the procedures defined by an appropriate national
biohazard safety guideline or regulation.
Do not use reagents beyond expiry date as shown on the kit labels.
All indicated volumes have to be performed according to the protocol. Optimal test results are only
obtained when using calibrated pipettes and microtiterplate readers.
Do not mix or use components from kits with different lot numbers. It is advised not to exchange wellsof different plates even of the same lot. The kits may have been shipped or stored under different
conditions and the binding characteristics of the plates may result slightly different.
Avoid contact with Stop Solution containing 0.5 M H2SO4. It may cause skin irritation and burns.
Some reagents contain Proclin 300, BND and/or MIT as preservatives. In case of contact with eyes orskin, flush immediately with water.
TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with anabundant volume of water and skin with soap and abundant water. Wash contaminated objects before
reusing them. If inhaled, take the person to open air.
Chemicals and prepared or used reagents have to be treated as hazardous waste according to thenational biohazard safety guideline or regulation.
For information on hazardous substances included in the kit please refer to Safety Data Sheets.
Safety Data Sheets for this product are available upon request directly from the manufacturer.

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4. REAGENTS
4.1 Reagents provided
Microtiterwells

TM E-4131
Content:

12x8 (break apart) strips, 96 wells; Wells coated with anti-CEA antibody (monoclonal)

Standards and Controls -Standards: ready to use
contain non-mercury preservative
StandardConcentration

Volume/Vial

TM E-4101

Cat. no.

SymbolStandard A

0 ng/ml

3 ml

TM E-4102

Standard B

5 ng/ml

1 ml

TM E-4103

Standard C

10 ng/ml

1 ml

TM E-4104

Standard D

25 ng/ml

1 ml

TM E-4105

Standard E

50 ng/ml

1 ml

TM E-4106

Standard F

1 ml

TM E-4151

Control 1

TM E-4152

Control 2

100 ng/ml
For control values andranges please refer tovial label or QC Report!

1 ml (lyophilized)*
1 ml (lyophilized)** see „Reagent Preparation“
TM E-4140
Content:
Volume:
FR E-0055

Enzyme Conjugate - Ready to use
Monoclonal Anti-CEA antibody conjugated to horseradish peroxidase, contains nonmercury preservative
1 x 14 ml

Content:

Substrate Solution - Ready to use
Tetramethylbenzidine (TMB)

Volume:1 x 14 ml

FR E-0080

Stop Solution - Ready to use
contains 0.5M H2SO4

Content:
Volume:1 x 14 ml
Avoid contact with Stop Solution It may cause skin irritations and burns.

Hazardidentification:
H290 May be corrosive to metals.
H314 Causes severe skin burns and eye damage.
FR E-0030

Wash Solution - 40x concentrated

1 x 30 ml
Volume:see “Reagent Preparation”
Note: Additional Standard A for sample dilution is available upon request.
4.2 Materials required but not provided‐‐‐‐‐‐

A microtiter plate calibrated reader (450 nm, with reference wavelength at 620 nm – 630 nm)
Calibrated variable precision micropipettes
Absorbent paper
Distilled or deionized waterTimer
Linear graph paper or software for data reduction

4.3 Storage Conditions
When stored at 2 °C to 8 °C unopened reagents will retain reactivity until expiration date. Do not usereagents beyond this date.
Opened reagents must be stored at 2 °C to 8 °C. Microtiter wells must be stored at 2 °C to 8 °C. Once thefoil bag has been opened, care should be taken to close it tightly again.
Opened kits retain activity for two months if stored as described above.
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4.4 Reagent Preparation
Bring all reagents and required number of strips to room temperature prior to use.
Control
Reconstitute the lyophilized content with 1.0 ml deionized water and let stand for 10 minutes in minimum.
Mix the control several times before use.
Note: The reconstituted control should be apportioned and stored at -20 °C.
Wash Solution
Add deionized water to the 40X concentrated Wash Solution.
Dilute 30 ml of concentrated Wash Solution with 1170 ml deionized water to a final volume of 1200 ml.
The diluted Wash Solution is stable for 2 weeks at room temperature.
4.5 Disposal of the Kit
The disposal of the kit must be made according to the national regulations. Special information for thisproduct is given in the Safety Data Sheet.
4.6 Damaged Test Kits
In case of any severe damage to the test kit or components, the manufacturer has to be informed in writing,at the latest, one week after receiving the kit. Severely damaged single components should not be used fora test run. They have to be stored until a final solution has been found. After this, they should be disposed
according to the official regulations.
5. SPECIMEN COLLECTION AND PREPARATION
Serum or lithium heparin plasma can be used in this assay.
Do not use haemolytic, icteric or lipaemic specimens.
Note: Samples containing sodium azide should not be used in the assay.
5.1 Specimen Collection
Serum:
Collect blood by venipuncture (e.g. Sarstedt Monovette for serum), allow to clot, and separate serum bycentrifugation at room temperature. Do not centrifuge before complete clotting has occurred.
Plasma:Whole blood should be collected into centrifuge tubes containing anti-coagulant (e.g. Sarstedt Monovettewith the appropriate plasma preparation) and centrifuged immediately after collection.
5.2 Specimen Storage and Preparation
Specimens should be capped and may be stored for up to 48 hours at 2 °C to 8 °C prior to assaying.
Specimens held for a longer time (up to 12 months) should be frozen only once at -20 °C prior to assay.
Thawed samples should be inverted several times prior to testing.
5.3 Specimen Dilution
If in an initial assay, a specimen is found to contain more than the highest standard, the specimens can bediluted with Standard A and reassayed as described in Assay Procedure.
For the calculation of the concentrations this dilution factor has to be taken into account.
Example:a) dilution 1:10: 10 µl sample + 90 µl Standard A (mix thoroughly)b) dilution 1:100: 10 µl dilution a) 1:10 + 90 µl Standard A (mix thoroughly).
6. ASSAY PROCEDURE
6.1 General Remarks‐‐‐‐‐

All reagents and specimens must be allowed to come to room temperature before use. All reagents must
be mixed without foaming.
Once the test has been started, all steps should be completed without interruption.
Use new disposal plastic pipette tips for each standard, control or sample in order to avoid crosscontamination.
Absorbance is a function of the incubation time and temperature. Before starting the assay, it isrecommended that all reagents are ready, caps removed, all needed wells secured in holder, etc. This will
ensure equal elapsed time for each pipetting step without interruption.
As a general rule the enzymatic reaction is linearly proportional to time and temperature.

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6.2 Test Procedure
Each run must include a standard curve.
All standards, samples, and controls should be run in duplicate. All standards, samples, and controls should
be run concurrently so that all conditions of testing are the same.
1.

Secure the desired number of Microtiter wells in the frame holder.

2.

Dispense 50 µl of each Standard, Control and samples with new disposable tips into appropriatewells.

3.

Dispense 100 µl Enzyme Conjugate into each well.
Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.

4.

Incubate for 60 minutes at room temperature.

5.

Briskly shake out the contents of the wells.
Rinse the wells 3 times with 400 µl diluted Wash Solution per well, if a plate washer is used - or rinse the wells 3 times with 300 µl diluted Wash Solution per well for manual washing.
Strike the wells sharply on absorbent paper to remove residual droplets.
Important note:
The sensitivity and precision of this assay is markedly influenced by the correct performance of thewashing procedure!

6.

Add 100 µl of Substrate Solution to each well.

7.

Incubate for 30 minutes at room temperature.

8.

Stop the enzymatic reaction by adding 100 µl of Stop Solution to each well.

9.

Determine the absorbance (OD) of each well at 450 nm (reading) and at 620 – 630 nm(background substraction, recommended) with a microtiter plate reader.
It is recommended that the wells be read within 10 minutes after adding the Stop Solution.

6.3 Calculation of Results
1. Calculate the average absorbance values for each set of standards, controls and samples.
2. Using linear graph paper, construct a standard curve by plotting the mean absorbance obtained fromeach standard against its concentration with absorbance value on the vertical (Y) axis and concentrationon the horizontal (X) axis.
3. Using the mean absorbance value for each sample determine the corresponding concentration from thestandard curve.
4. Automated method: The results in the Instructions for Use have been calculated automatically using a
4- Parameter curve fit. (4 Parameter Rodbard or 4 Parameter Marquardt are the preferred methods.)
Other data reduction functions may give slightly different results.
5. The concentration of the samples can be read directly from this standard curve. Samples withconcentrations higher than that of the highest standard have to be further diluted or reported as> 100 ng/ml. For the calculation of the concentrations this dilution factor has to be taken into account.
6.3.1 Example of Typical Standard Curve
The following data is for demonstration only and cannot be used in place of data generations at the time ofassay.
Standard
Standard A (0 ng/ml)
Standard B (5 ng/ml)
Standard C (10 ng/ml)
Standard D (25 ng/ml)
Standard E (50 ng/ml)
Standard F (100 ng/ml)

Opitcal Units (450 nm)
0.06
0.20
0.34
0.62
1.12
2.04

7. EXPECTED NORMAL VALUES
It is strongly recommended that each laboratory should determine its own normal and abnormal values.
The literature cut-off is 5 ng/ml for non-smokers and 10 ng/ml for smokers.

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8. QUALITY CONTROLGood laboratory practice requires that controls be run with each standard curve. A statistically significant
number of controls should be assayed to establish mean values and acceptable ranges to assure proper
performance.
It is recommended to use control samples according to state and federal regulations. The use of control
samples is advised to assure the day to day validity of results. Use controls at both normal and pathologicallevels.
The controls and the corresponding results of the QC-Laboratory are stated in the QC - Report added to thekit. The values and ranges stated on the QC sheet always refer to the current kit lot and should be used for
direct comparison of the results.
It is also recommended to make use of national or international Quality Assessment programs in order toensure the accuracy of the results.
Employ appropriate statistical methods for analysing control values and trends. If the results of the assay do
not fit to the established acceptable ranges of control materials results should be considered invalid.
In this case, please check the following technical areas: Pipetting and timing devices; photometer, expiration
dates of reagents, storage and incubation conditions, aspiration and washing methods.
After checking the above mentioned items without finding any error contact your distributor or themanufacturer directly.
9. PERFORMANCE CHARACTERISTICS
9.1 Assay Dynamic Range
The range of the assay is between 0.596 ng/ml – 100 ng/ml.
9.2 Specificity of Antibodies (Cross Reactivity)
Cross-reactivities are not known.
9.3 Sensitivity
The analytical sensitivity of the CEA ELISA was calculated by adding 2 standard deviations to the mean of
20 replicate analyses of the Standard A and was found to be < 0.596 ng/ml.
9.4 Reproducibility
Intra-Assays of 3.2 % - 4.9 % were observed.
Inter-Assays of 5.6 % – 6.3 % were observed.
9.5 Recovery
Samples have been spiked by adding CEA solutions with known concentrations in a 1:1 ratio.
The % recovery has been calculated by multiplication of the ratio of the measurements and the expectedvalues with 100 (expected value = (endogenous CEA + added CEA) / 2; because of a 1:2 dilution of serumwith spike material).

Concentration (ng/ml)
Average Recovery (%)
Range of Recovery (%)

from
to

Sample 1
3.3
90.3
87.6
95.6

Sample 2
18.3
89.4
85.8
95.2

Sample 3
47.1
101.1
92.5
107.0

from
to

Sample 1
17.7
109.3
105.7
114.6

Sample 2
44.6
96.7
89.9
100.4

Sample 3
85.3
99.9
93.1
106.3

9.6 Linearity
Concentration (ng/ml)
Average Recovery (%)
Range of Recovery (%)

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10. LIMITATIONS OF USE
Reliable and reproducible results will be obtained when the assay procedure is performed with a complete
understanding of the package insert instruction and with adherence to good laboratory practice.
Any improper handling of samples or modification of this test might influence the results.
10.1 Interfering Substances
Haemolytic, icteric and lipemic sera should be avoided.
The assay contains reagents to minimize interference of HAMA and heterophilic antibodies. However,extremely high titers of HAMA or heterophilic antibodies may interfere with the test results.
10.2 Drug Interferences
Smokers have been reported to exhibit increased levels of CEA (see expected values)
10.3 High-Dose-Hook EffectNo hook effect was observed in this test up to 8000 ng/ml of CEA.
11. REFERENCES/LITERATURE
1.
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7.

Engall, E. Methods in Enzymology, Volume 70, Van Vunakis, H. and Langone, J.J. (eds.), Academic Press,New York, 419-492 (1980).
Uotila, M. Ruoslahti, E. and Engvall, E., J. Immunol. Methods, 42, 11-15 (1981).
Gold, P., Freedman S.O: Demonstration of tumor specific antigen in human colonic carcinoma byimmunologic tolerance and absorption techniques. J Exp. Med. 1965; 127:439-462.
Thompson, D.P.M. Krupey J, Freedman S.O., et al. The radioimmunoassay of circulating
carcinoembryonic antigen of the human digestive system. Proc Natl Acad Sci USA 1969, 64:161-167.
Schwartz M.K. Tumor markers in diagnosis and screening. In: Ting S.W., Chen J.S., Schwartz M.K: , eds.
Human tumor markers, Amsterdam: Elsevier Science, 1987; 3-16.
Zamchek N, and Martin E.W. Sequential Carcinoembryonic Antigen Levels in Pancreatic Cancer: Some
Clinical Correlations. Cancer 1981; 47:1620-1627.
Mughal A.W., Hortobagyi G.N:, Fritsche H.A., Buzdar A.U. Yap H-Y. and Blumenschein G.R. Serial Plasma
Carcinoembryonic Antigen Measurements During Treatment of Metastatic Breast Cancer. JAMA 1983;
259; 1881-1886.

Symbols:

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For research use
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