Materials and components Assay procedures

Materials provided with the test kits: 1.Secure the desired number of coated wells in the holder.
Antibody-coated microtiter plate,96 wells 2.Dispense 50µL of standard, specimens, and controls into
CARCINOEMBRYONIC ANTIGEN Enzyme Conjugate Reagent 12 mL appropriate wells.
TMB Substrate 12 mL 3.Dispense 100µLof enzyme conjugate reagent to each well.
(CEA) Stop Solution 12 mL 4.Thoroughly mix for 10 seconds. It is very important to have a
Reference standards, containing 0, 3, 12, 30, 60, and 120ng/mL complete mixing in this step.
ENZYME IMMUNOASSAY TEST KIT of CEA, in liquid form (ready to use) or lyophilized Form 5.Incubate at room temperature (18-22°C) for 60 minutes.
Catalog Number: 10106 Wash Buffer Concentrate(50X,15mL) 6.Remove the incubation mixture by emptying plate content into a
waste container.
Materials required but not provided: 7.Rinse and empty the microtiter wells 5 times with washing
Enzyme Immunoassay for the Quantitative Precision pipettes: 40µL-200µL, 200-1000µL Buffer(1X).
Determination of Carcinoembryonic Antigen Disposable pipette tips 8.Strike the wells sharply onto absorbent paper or paper towels to
(CEA) in Human Serum Distilled water remove all residual water droplets.
Vortex mixer or equivalent 9.Dispense 100µL of TMB substrate into each well. Gently mix for 5
Absorbent paper or paper towel seconds.
Graph paper 10.Incubate at room temperature for 20 minutes.
Microtiter plate reader 11.Stop the reaction by adding100µL of Stop Solution to each well.
Intended use 12.Gently mix for 30 seconds to ensure that the blue color completely
Specimen collection and preparation changes to yellow.
CEA enzyme immunoassay test kit is intended for the quantitative 13.Read the optical density at 450nm with a microtiter plate Reader
determination of CEA concentration in human serum. 1.Blood should be drawn using standard venipuncture techniques within 15 minutes.
and the serum should be separated from the red blood cells as
Introduction soon as possible. Avoid grossly hemolytic, lipemic or turbid Important Note
samples.
Carcinoembroyonic antigen (CEA) is a cell-surface 200-kd glycoprotein. 2.Plasma samples collected in tubes containing EDTA, heparin, or 1.The wash procedure is critical. Insufficient washing will result in
In 1969, it was reported that plasma CEA was elevated in 35 of 36 oxalate may interfere with the test procedures and should be poor precision and falsely elevated absorbance readings.
patients with adenocarcinoma of the colon and that CEA titers avoided. 2.It is recommended that no more than 32 wells be used for each
decreased after successful surgery. Normal levels were observed in all 3.Specimens should be capped and may be stored up to 48 hours assay run if manual pipetting is used since pipetting of all standard,
patients with other forms of cancer or benign diseases. Subsequent at 2-8°C,prior to assaying. Specimens held for a longer time can be specimens and controls should be competed within 3 minutes. A
studies have not confirmed these initial findings, and it is now frozen at -20°C.Thawed samples must be mixed prior to testing. full plate of 96 well may be used if automated pipetting is
understood that elevated levels of CEA are found in many cancers. available.
Increased levels of CEA are observed in more than 30% of patients Storage of test kits and instrumentation 3.Duplication of all standards and specimens, although not required
with cancer of the lung, liver, pancreas, breast, colon, head or neck, is recommended.
bladder, cervix, and prostate. Elevated plasma levels are related to the Unopened test kits should be stored at 2-8°C upon receipt. The
stage and extent of the disease, the degree of differentiation of the microtiter plate should be stored at 2-8°C, in a sealed bag with
tumor, and the site of metastasis. CEA is also found in normal tissue. desiccants, to minimize exposure to damp air. Opened test kits will Calculation of results
remain stable until the expiration date, provided they are stored as
described above. A microtiter plate reader with a bandwidth of 10nm or Calculate the mean absorbance value (A450) for each set of reference
Principle of the test less and an optical density range of 0-2 OD or greater, at 450nm standards, controls and patient samples. Construct a standard curve
wavelength, is acceptable for use in the absorbance measurement. by plotting the mean absorbance obtained from each reference
The CEA Quantitative Test Kit is based on a solid phase enzyme-linked standard against its concentration in ng/mL on graph paper, with
immunosorbent assay. The assay system utilizes one monoclonal anti- absorbance values on the vertical or Y-axis and concentrations on the
CEA antibody for solid phase (microtiter wells) immobilization and Reagent preparation horizontal or X-axis. Use the mean absorbance values for each
another mouse monoclonal anti-CEA antibody in the antibody-enzyme specimen to determine the corresponding concentration of CEA in ng/
(horseradish peroxidase) conjugate solution. The standards and test 1.All reagents should be brought to room temperature(18-22°C) and mL from the standard curve.
specimen (serum) are added to the CEA antibody coated microtiter mixed by gently inverting or swirling prior to use. Do not induce
wells. Then CEA antibody labeled with horseradish peroxidase foaming.
(conjugate) is added. If human CEA is present in the specimen, it will 2.If reference standards are lyophilized , reconstitute each standard Example of standard curve
combine with the antibody on the well and the enzyme conjugate with 0.5mL distilled water. Allow the reconstituted material to stand
resulting in the CEA molecules being sandwiched between the solid for at least 20 minutes. Reconstituted Standards should be sealed Results of a typical standard run with optical density reading at 450nm
phase and enzyme-linked antibodies. After a 1 hour incubation at room and stored at 2-8°C. shown in the Y-axis against CEA concentrations shown in the X-axis.
temperature, the wells are washed to remove unbound labeled 3.Dilute 1 volume of Wash Buffer (50x) with 49 volumes of distilled
antibodies. A solution of TMB is added and incubated for 20 minutes, water. For example, dilute 15 mL of Wash Buffer (50x) into 735
resulting in the development of a blue color. The color development is mL of distilled water to prepare 750 mL of Washing buffer (1x). Mix
stopped with the addition of 2N HCl. The color is changed to yellow and well before use.
measured spectrophotometrically at 450 nm. The concentration of CEA
is directly proportional to the color intensity of the test sample.
 CEA (ng/mL) Absorbance (450nm) Concentrations Replicates Mean S.D. % CV Limitations of the Procedure
0 0.019 Level 20 4.94 0.243 4.9
Level I 20 19.82 1.24 6.3 There are some limitation of the assay. We should let our customers
3 0.105 LevelII 20 35.36 1.33 3.8 know about that.
1)As with all diagnostic tests, a definite clinical diagnosis should not be
12 0.362 3.Linearity based on the results of a single test, but should only be made by the
A patient serum were serially diluted with 0 ng/mL standard physician after all clinical and laboratory findings have been
30 0.814 In a linearity study. The average recovery was 99.7 %. evaluated.
60 1.390 Sample 2)Studies have implicated possible interference in immunoassay
results in some patients with known rheumatoid factor and
120 2.032 Dilution Expected Observed % Recov. antinuclear antibodies. Serum samples from patients who have
Undiluted 103.50 103.50 received infusions containing mouse monoclonal antibodies for
2.5
diagnostic or therapeutic purposes, may contain antibody to mouse
2x 51.75 50.89 98.3 protein (HAMA). Although we have added some agents to avoid the
2
4x 25.88 26.26 101.5 interferences, we cannot guarantee to eliminate all the effects of
1.5 that.
OD 8x 12.94 13.22 102.2 3)The wash procedure (steps 6-8) is critical. Insufficient washing will
1 16x 6.47 6.25 96.6 result in poor precision and falsely elevated absorbance. The use of
tap water for washing could result in a higher background
0.5 32x 3.23 3.51 108.5 absorbance.
0
Average Recovery: 99.7 %
0 50 100 150 4.Recovery References
Conc. of CEA (ng/m L) Various patient serum samples of known CEA levels were
mixed and assayed in duplicate. The average recovery was 1.Gold P, Freedman S O. Demonstration of tumor specific Antigen
This standard curve is for the purpose of illustration only, and should 100.7 %. in human colonic carcinomata by immunologic tolerance and
not be used to calculate unknowns. Each user should obtain his or her Absorption techniques. J Exp Med 1965;127:439-462.
own standard curve and data.
Expected Observed % Recovery 2.Thompson D P M,Krupey J, Freedman S O,et al. The
Concentration Concentration radioimmunoassay of circulating carcinoembryonic antigen Of The
53.21 human digestive ystem. Proc Natl Acad Sci USA 1969;64:161-167.
Expected values and sensitivity 54.28 98.0
3.Schwartz M K. Tumor markers in diagnosis and screening. In:
The most complete study of CEA is a compilation of collaborative 28.42 29.13 102.5 TingS W, Chen J S, Schwartz M K, eds. Human tumor markers,
studies in which CEA values in 35,000 samples from more than 10,000 Amsterdam: Elsevier Science, 1987;3-16.
23.85 22.98 96.4 4.Zamcheck N. and Martin E.W. Sequential Carcinoembryonic Anigen
patients and controls were analyzed. Of 1425 normal persons who did
not smoke, 98.7% had values less than 5.0 ng/mL. It is recommended 16.53 15.99 96.7 Levels in Pancreatic Cancer: Some Clinical Correlations . Cancer
that each laboratory establish its own normal range. The minimum 1981;47: 1620-1627.
20.90 21.36 102.2 5.Mughal A.W., Hortobagyi G. N., Fritsche H.A., Buzdar A.U. ap H-
detectable concentration of CEA by this assay is estimated to be 1.0
ng/mL. 18.62 19.65 105.5 Y.,AndBlumenschein G.R.Serial Plasma Carcinoembryonic Antigen
Measurements During Treatment of Metastatic Breast Cancer.
28.77 29.78 103.5 JAMA 1983; 259:1881-1886.
Performance characteristics Average Recovery: 100.7 %
1.Accuracy: Comparison between Our kits and commercial 5.Sensitivity 7/2014
available kits provide the following data The minimum detectable concentration of this assay is
N = 46 estimated to be 1.0 ng/mL.
Correlation Coefficient = 0.946
Slope = 0.788 6.Cross-reactivity
Intercept = 0.74 The following human materials were tested for crossreactivity
Mean (Ours) = 7.59 Of the assay:
Mean (Abbott) = 6.03 Antigens Concentration Equivalent CEA % Cross-reactivity
2.Precision. Chemux Bioscience,Inc.
1].Intra-Assay: HCG 400 IU/mL 0.00 0.00
Website: www.chemux.com
PAP 1,000 ng/mL 0.00 0.00
385 Oyster Point Blvd Suite5-6.,South San Francisco,CA94080
Concentrations Replicates Mean S.D. % CV PSA 1,000 ng/mL 0.00 0.00 Tel:+1-650-872-1800
LeveI 24 5.07 0.173 3.4 AFP 1,000 ng/mL 0.00 0.00
Level I 24 20.3 0.744 3.7
LevelII 24 35.44 1.09 3.1 7.Hook Effect
No hook effect was observed up to 40,000 ng/mL CEA in this
2].Inter-Assay: Assay.