Instructions for use

CA 19-9 ELISATM E-4500R

CA19-9 ELISA
1. INTRODUCTION
1.1 Intended Use
The CA 19-9 ELISA is an enzyme immunoassay for the quantitative measurement of CA 19-9 in serum or plasma(EDTA, heparin or citrate plasma)
2. PRICIPLE OF THE TESTThe CA 19-9 ELISA Kit is a solid phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich
principle.
The microtiter wells are coated with a monoclonal [mouse] antibody directed towards a unique antigenic site of
the CA 19-9 molecule. An aliquot of sample containing endogenous CA 19-9 is incubated in the coated well withassay buffer.
After a washing step a second incubation follows with enzyme conjugate, which is an anti-CA 19-9 antibody
conjugated with horseradish peroxidase. After incubation the unbound conjugate is washed off.
The amount of bound peroxidase is proportional to the concentration of CA 19-9 in the sample.
Having added the substrate solution, the intensity of colour developed is proportional to the concentration of
CA 19-9 in the sample.
WARNINGS AND PRECAUTIONS
1. This kit is for research use only.
2. All reagents of this test kit which contain human serum or plasma have been tested and confirmed negativefor HIV I/II, HBsAg and HCV by FDA approved procedures. All reagents, however, should be treated as
potential biohazards in use and for disposal.
3. Before starting the assay, read the instructions completely and carefully. Use the valid version of the
package insert provided with the kit. Be sure that everything is understood.
4. 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.
5. Pipetting of samples and reagents must be done as quickly as possible and in the same sequence for eachstep.
6. Use reservoirs only for single reagents. This especially applies to the substrate reservoirs. Using a reservoir
for dispensing a substrate solution that had previously been used for the conjugate solution may turnsolution colored. Do not pour reagents back into vials as reagent contamination may occur.
7. Mix the contents of the microplate wells thoroughly to ensure good test results. Do not reuse microwells.
8. Do not let wells dry during assay; add reagents immediately after completing the rinsing steps.
9. Allow the reagents to reach room temperature (21 °C - 26 °C) before starting the test. Temperature willaffect the absorbance readings of the assay. However, values for the samples will not be affected.
10. Never pipet by mouth and avoid contact of reagents and specimens with skin and mucous membranes.
11. Do not smoke, eat, drink or apply cosmetics in areas where specimens or kit reagents are handled.
12. Wear disposable latex gloves when handling specimens and reagents. Microbial contamination of reagents or
specimens may give false results.
13. Handling should be done in accordance with the procedures defined by an appropriate national biohazard
safety guideline or regulation.
14. Do not use reagents beyond expiry date as shown on the kit labels.
15. All indicated volumes have to be performed according to the protocol. Optimal test results are only obtained
when using calibrated pipettes and microtiterplate readers.
16. Do not mix or use components from kits with different lot numbers. It is advised not to exchange wells ofdifferent 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.
17. Avoid contact with Stop Solution containing 0.5 M H2SO4. It may cause skin irritation and burns.
18. Some reagents may contain Proclin 300, BND and/or MIT as preservatives. In case of contact with eyes orskin, flush immediately with water.
19. 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.
20. Chemicals and prepared or used reagents have to be treated as hazardous waste according to the national
biohazard safety guideline or regulation.
21. 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.
4. REAGENTS
4.1 Reagents provided
TM E-4531
Content:
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Microtiterwells
12x8 (break apart) strips, 96 wells; Wells coated with anti-CA 19-9 antibody(monoclonal).
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Standards and Controls -Standards: ready to use
contain non-mercury preservative
Cat. no.

SymbolStandard

Concentration

Volume/Vial

TM E-4501

Zero Standard

0 U/ml3 ml

TM E-4502

Standard 1

15 U/ml

0.5 mlTM E-4503

Standard 2

30 U/ml

0.5 mlTM E-4504

Standard 3

60 U/ml

0.5 mlTM E-4505

Standard 4

120 U/ml

0.5 mlTM E-4506

Standard 5

TM E-4551

Control low

0.5 ml (lyophilized)*

TM E-4552

Control high240 U/ml
For control values andranges please refer tovial label or QC Report!

0.5 ml
0.5 ml (lyophilized)** see „Reagent Preparation“
Assay Buffer - Ready to use

TM E-4513
Content:

Contains non-mercury preservative.

Volume:1 x 7 ml

TM E-4540

Enzyme Conjugate - Ready to use
Anti-CA 19-9 antibody conjugated to horseradish peroxidase;
Contains non-mercury preservative.
1 x 14 ml

Content:
Volume:
FR E-0055

Substrate Solution - Ready to use

Content:

Tetramethylbenzidine (TMB)

Volume:1 x 14 ml

FR E-0080
Content:
Volume:Stop Solution - Ready to use
contains 0.5 M H2SO4
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.
Wash Solution - 40x concentrated

FR E-0030
Volume:1 x 30 ml

see “Reagent Preparation”
Note: Additional Zero Standard for sample dilution is available upon request.
4.2 Materials required but not provided




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

A microtiter plate calibrated reader (450 ± 10 nm)
Calibrated variable precision micropipettes.
Absorbent paper
Distilled or deionized water
Timer
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 use reagents
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 the foil
bag has been opened, care should be taken to close it tightly again.
Opened kits retain activity for 8 weeks 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 0.5 ml distilled water and let stand for 10 minutes in minimum. Mix thecontrols several times before use.
Note: The reconstituted controls 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 distilled 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 this product isgiven in the Safety Data Sheet.
4.6 Damaged Test Kits
In case of any severe damage to the test kit or components, the supplier has to be informed in writing, at thelatest, one week after receiving the kit. Severely damaged single components should not be used for a test run.
They have to be stored until a final solution has been found. After this, they should be disposed according to theofficial regulations.

5. SPECIMEN COLLECTION AND PREPARATION
Serum or plasma (EDTA, heparin or citrate plasma) can be used in this assay.
Do not use haemolytic, icteric or lipaemic specimens.
Please 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 Monovette with theappropriate plasma preparation) and centrifuged immediately after collection.
5.2 Specimen Storage and Preparation
Specimens should be capped and may be stored for up to 5 days at 2 °C to 8 °C prior to assaying.
Specimens held for a longer time (up to two 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 be
diluted with Zero Standard 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 Serum + 90 µl Zero Standard (mix thoroughly)b) dilution 1:100: 10 µl dilution a) 1:10 + 90 µl Zero Standard (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.

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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.

6.2 Test Procedure
Each run must include a standard curve.
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 appropriate
wells.

3.

Dispense 50 µl Assay Buffer into each well.

4.

Incubate for 60 minutes at room temperature.

5.

Briskly shake out the contents of the wells.
Rinse the wells 4 times with diluted Wash Solution (400 µl per well). 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.

Dispense 100 µl Enzyme Conjugate into each well.

7.

Incubate for 60 minutes at room temperature.

8.

Briskly shake out the contents of the wells.
Rinse the wells 4 times with diluted Wash Solution (400 µl per well). Strike the wells sharply on
absorbent paper to remove residual droplets.

9.

Add 100 µl of Substrate Solution to each well.

10.

Incubate for 30 minutes at room temperature.

11.

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

12.

Determine the absorbance (OD) of each well at 450 ± 10 nm 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. Manual method: Using linear graph paper, construct a standard curve by plotting the mean absorbanceobtained from each standard against its concentration with absorbance value on the vertical (Y) axis andconcentration on 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 4Parameter 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 with concentrations
higher than that of the highest standard have to be further diluted or reported as > 240 U/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 of
assay.
Standard

Optical Units (450 nm)

Standard 0 (0 U/ml)

0.04

Standard 1 (15 U/ml)

0.26

Standard 2 (30 U/ml)

0.44Standard 3 (60 U/ml)

0.78

Standard 4 (120 U/ml)

1.30

Standard 5 (240 U/ml)

1.99

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7. EXPECTED NORMAL VALUES
It is strongly recommended that each laboratory should determine its own normal and abnormal values.
In a study conducted with apparently normal healthy adults, using the CA19-9 ELISA the following values areobserved:

Populationn

Mean(U/ml)

Males andfemales

71

8.08

Median(U/ml)

5th - 95th
Percentile(U/ml)

2.5th - 97.5th
Percentile(U/ml)

Range (min. max.)(U/ml)

5.70

1.15 - 20.90

0.27 - 26.15

0.04 - 28.70

8. QUALITY CONTROLGood laboratory practice requires that controls be run with each standard curve. A statistically significant numberof 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 pathological levels.
The controls and the corresponding results of the QC-Laboratory are stated in the QC certificate added to the kit.
The values and ranges stated on the QC sheet always refer to the current kit lot and should be used for directcomparison of the results.
It is also recommended to make use of national or international Quality Assessment programs in order to ensure
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.

9. PERFORMANCE CHARACTERISTICS
9.1 Assay Dynamic Range
The range of the assay is between 0.26 U/ml – 240 U/ml.
9.2 Specificity of Antibodies (Cross Reactivity)
Cross-reactivities of the assay are not known.
9.3 Sensitivity
The analytical sensitivity of the CA 19-9 ELISA was calculated by adding 2 standard deviations to the mean of 20
replicate analyses of the Zero Standard and was found to be 0.26 U/ml.
9.4 Reproducibility
9.4.1 Intra Assay
The within assay variability is shown below:
SamplenMean (U/ml)
CV (%)
1

20

27.7

8.4

2

20

47.7

9.2

3

20

74.0

9.6

9.4.2 Inter Assay
The between assay variability is shown below:
SamplenMean (U/ml)
CV (%)
1

39

5.7

12.2

2

40

10.7

8.2

3

40

11.4

10.5

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9.4.3 Inter-Lot
The inter-assay (between-lots) variation was determined by measuring each sample 6 times with 3 different kit
lots (n = 18):
SamplenMean (U/ml)
CV (%)
1

18

10.5

10.1

2

18

14.3

7.8

3

18

25.2

3.4

4

18

28.6

1.0

9.5 Recovery
Samples have been spiked by adding CA 19-9 solutions with known concentrations.
The % recovery was calculated by multiplying the ratio of the measured and expected values with 100.
Sample 1

Sample 2

Sample 3

Concentration [U/ml]

17.6

22.1

22.6

Average Recovery [%]

91.1

87.7

91.9

from

87.2

85.5

87.4

to

98.5

91.6

95.9

Range of Recovery [%]9.6 Linearity
Samples were measured undiluted and in serial dilutions from 1:2 to 1:16 with Zero Standard. The recovery (%)
was calculated by multiplying the ratio of expected and measured values with 100.
Sample 1

Sample 2

Sample 3

Concentration [U/ml]

25.8

41.7

82.7

Average Recovery [%]

109.7

102.6

100.8

from

103.9

97.8

93.1

to

114.7

104.6

108.5

Range of Recovery [%]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
Haemoglobin (up to 4 mg/ml), Bilirubin (up to 0.5 mg/ml) and Triglyceride (up to 30 mg/ml) have no influenceon the assay results.
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
Until today no substances (drugs) are known to us, which have an influence to the measurement of CA 19-9 in asample.
10.3 High-Dose-Hook EffectHook effect was not observed in this test up to a concentration of 15 360 U/ml of CA 19-9.

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11. REFERENCES / LITERATURE
1. Koprowski H, Steplewski Z, Mitchell K, Herlyn M, Herlyn D, Fuhrer P: Colorectal carcinoma antigens detectedby hybridoma antibodies. Somatic Cell Genetics 5:957-972, 1979.
2. Koprowski H, Herlyn M, Steplewski Z, Sears H: Specific antigen in serum of patients with colon carcinoma.
Science 212:53-55, 1981.
3. Magnani J, et al.: The Antigen of Tumor-Specific Monoclonal Antibody is a Ganglioside Containing Sialylated
Lacto-N-Fucopentaose II. Federation Proceedings, 41:898, 1982.
4. Del Villano B, Brennan S, Brock P, Bucher C, Liu V, McClure M, Rake B, Space S, Westrick B, Schoemaker H,
Zurawski V Jr: Radioimmunometric Assay for a Monoclonal Antibody-Defined Tumor Marker, CA 19-9. Clin
Chem 29:549-552, 1983.
5. Steinberg W, Gelfand R, Anderson K, Glenn J, Kurtzman SH, Sindelar W, Toskes P: Comparison of theSensitivity and Specificity of the CA 19-9 and Carcinoembryonic Antigen Assays in Detecting Cancer of the
Pancreas. Gastroenterology 90:343-349, 1986.
6. Ritts R Jr, Del Villano B, Go VLW, Herberman R, Klug T, Zurawski V Jr: Initial Clinical Evaluation of an
Immunoradiometeric Assay for CA 19-9 Using the NCI Serum Bank. Int J Cancer 33:339-345, 1984.
7. Safi F, Beger H, Bittner R, Buchler M, Krautzberger W: CA 19-9 and pancreatic adenocarcinoma. Cancer
57:779, 1986.
8. NCCLS Guideline EP7-P, entitled “Interference Testing in Clinical Chemistry; Proposed Guideline” August
1986.
9. NCCLS Guideline EP5-A, entitled “Evaluation of Precision Performance of Clinical Chemistry Devices;
Approved Guideline,” February 1999.
10. Del Villano B and Zurawski V Jr: The carbohydrate antigenic determinant 19-9 (CA 19-9): a monoclonal
antibody defined tumor marker. Immunodiagnostics – Laboratory and Research Methods in Biology andMedicine, Volume 8. Ed. By J. Hyun and R. Aloisi, Alan R. Liss, New York, pp. 269-282, 1983.
11. Dietel M, Arps H, Müller-Hagen S, Sieck M and Hoffmann L: Antigen detection by the monoclonal antibodies
CA 19-9 and CA 125 in normal and tumor tissue and patients´ sera – J Cancer Research Clinical Oncology
111:257-265, 1986
12. Fritz S, Hackert T, Hinz U, Hartwig W, Büchler MW, Werner J.: Role of serum carbohydrate antigen 19-9 andcarcinoembryonic antigen in distinguishing between benign and invasive intraductal papillary mucinous
neoplasm of the pancreas. Br J Surg. 98(1):104-10, 2011.
13. Bedi MM, Gandhi MD, Jacob G, Lekha V, Venugopal A, Ramesh H: CA 19-9 to differentiate benign andmalignant masses in chronic pancreatitis: is there any benefit? Indian J Gastroenterol 28(1):24-7, 2009.
14. Morris-Stiff G, Teli M, Jardine N, Puntis MC: CA19-9 antigen levels can distinguish between benign andmalignant pancreaticobiliary disease. Hepatobiliary Pancreat Dis Int 8(6):620-6, 2008.
15. Steinberg WM, Gelfand R, Anderson KK, Glenn J, Kurtzman SH, Sindelar WF, Toskes PP: Comparison of thesensitivity and specificity of the CA19-9 and carcinoembryonic antigen assays in detecting cancer of thepancreas. Gastroenterology 90(2):343-9, 1986.
16. Howaizi M, Abboura M, Krespine C, et al: A new cause for CA19.9 elevation: heavy tea consumption. Gut
2003; 52:913-914.

Symbols:

Version: 5.0-r

Storagetemperature

Manufacturer

Expiry date

Batch code

Consult instructions
for useContent

CautionCatalogue
numberEffective: 2019-10-08

Contains sufficient for<n> tests

For research use
only!
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