Instructions for use

CA 125 ELISATM E-4300R

CA 125 ELISAINTRODUCTION
Intended Use
The CA 125 ELISA is an enzyme immunoassay for the quantitative measurement of CA 125 in serum andplasma.
PRINCIPLE OF THE TESTThe CA 125 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 125 molecule. An aliquot of sample containing endogenous CA 125 is incubated in the coated well withenzyme conjugate, which is a monoclonal anti- CA 125 antibody conjugated with horseradish peroxidase. Afterincubation the unbound conjugate is washed off.
The amount of bound peroxidase is proportional to the concentration of CA 125 in the sample.
Having added the substrate solution, the intensity of colour developed is proportional to the concentration ofCA 125 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
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.
3. Before starting the assay, read the instructions completely and carefully. Use the valid version of thepackage 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 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.
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 (21-26°C) before starting the test. Temperature will affectthe 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 reagentsor 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 Material Safety Data Sheets.
Material Safety Data Sheets for this product are available upon request.

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REAGENTS
Reagents provided
TM E-4331 Microtiterwells
12x8 (break apart) strips, 96 wells: Wells coated with anti-CA 125 antibody (monoclonal).
Standards + Controls
Cat. no.
TM E-4301
TM E-4302TM E-4303
TM E-4304
TM E-4305
TM E-4306
TM E-4351

Standard
Zero Standard
Standard 1
Standard 2
Standard 3
Standard 4Standard 5
Control 1

TM E-4352

Control 2

Concentration
0 U/mL25 U/mL
75 U/mL
150 U/mL
300 U/mL
600 U/mL
For control values andranges please refer to viallabel or QC-Datasheet!

Volume/Vial
3 ml
0.5 ml
0.5 ml
0.5 ml
0.5 ml
0.5 ml
0.5 ml
0.5 mlcontain non-mercuryas preservative.
TM E-4340 Enzyme Conjugate
1 vial, 7 mL, ready to use, Anti-CA 125 antibody (monoclonal) conjugated to horseradish peroxidase;
Contains non-mercury preservative.
FR E-0055 Substrate Solution
1 vial, 14 mL, ready to use; TMB.
FR E-0080 Stop Solution
1 vial, 14 mL, ready to use; contains 0.5M H2SO4. Avoid contact with the stop solution. It may cause skinirritations and burns.
FR E-0030 Wash Solution
1 vial, 30 mL (40X concentrated) see „Preparation of Reagents“
Note: Additional Zero Standard for sample dilution is available on request.
Materials required but not provided
A microtiter plate calibrated reader (450 ± 10 nm)
Calibrated variable precision micropipettes.
Absorbent paper.
Distilled or deionized waterTimerGraph paper or software for data reduction
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.
Reagent Preparation
Bring all reagents and required number of strips to room temperature prior to use.
Wash Solution
Add deionized water to the 40X concentrated Wash Solution. Dilute 30 mL of concentrated Wash Solution with1170 mL deionized water to a final volume of 1200 mL.
The diluted Wash Solution is stable for 2 weeks at room temperature.
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Disposal of the Kit
The disposal of the kit must be made according to the national regulations. Special information for this productis given in the Material Safety Data Sheet.
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 tothe official regulations.

SPECIMEN COLLECTION AND PREPARATION
Serum and Plasma (EDTA, Heparin, Citrate) 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.
Specimen Collection
Serum:
Collect blood by venipuncture (e.g. Sarstedt Monovette # 02.1388.001), 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 and centrifuged immediately
after collection.
(E.g. for EDTA plasma
Sarstedt Monovette – red cap - # 02.166.001;
for Heparin plasma Sarstedt Monovette – orange cap - # 02.165.001;
for Citrate plasma
Sarstedt Monovette – green cap - # 02.167.001.)
Specimen Storage and Preparation
Specimens should be capped and may be stored for up to 2 days 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.
Specimen Dilution
If in an initial assay, a specimen is found to contain more than the highest standard, the specimens can bediluted 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 sample + 90 µL Zero Standard (mix thoroughly)b) dilution 1:100: 10 µL dilution a) 1:10 + 90 µL Zero Standard (mix thoroughly).

ASSAY PROCEDURE
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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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 appropriatewells.

3.

Dispense 50 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 diluted Wash Solution (300 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.

Add 100 µL of Substrate Solution to each well.

7.

Incubate for 15 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 ± 10 nm with a microtiter plate reader.
It is recommended that the wells be read within 10 minutes after adding the Stop Solution.

Calculation of Results
1.
2.
3.
4.
5.

Calculate the average absorbance values for each set of standards, controls and samples.
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.
Using the mean absorbance value for each sample determine the corresponding concentration from thestandard curve.
Automated method: The results in the IFU have been calculated automatically using a 4 PL (4 ParameterLogistics) curve fit. 4 Parameter Logistics is the preferred method. Other data reduction functions may
give slightly different results.
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> 600 U/mL. For the calculation of the concentrations this dilution factor has to be taken into account.

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

Optical Units (450 nm)

Standard 0 (0 U/mL)

0.02Standard 1 (25 U/mL)

0.16

Standard 2 (75 U/mL)

0.41

Standard 3 (150 U/mL)

0.75

Standard 4 (300 U/mL)

1.27

Standard 5 (600 U/mL)

1.76

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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 CA 125 ELISA the following
values are observed:
Population

Valid NMean(U/mL)

Median(U/mL)

5th Percentile(U/mL)

95th Percentile(U/mL)

Males

35

6.47

8.90

2.53

18.80

Females

35

3.51

5.30

1.67

13.09

QUALITY CONTROLGood laboratory practice requires that controls be run with each calibration 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 certificate 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, expiring
dates of reagents, storage and incubation conditions, aspiration and washing methods.
After checking the above mentioned items without finding any error, contact your distributor.

PERFORMANCE CHARACTERISTICS
Assay Dynamic Range
The range of the assay is between 0.25 U/mL – 600 U/mL.
Specificity of Antibodies (Cross Reactivity)
The following substances were tested for cross reactivity (in %) of the assay:
CA 19-9 (0%),
CEA(0%),CA 72-4 (0%).
Sensitivity
The analytical sensitivity of the CA 125 ELISA was calculated by adding 2 standard deviations to the mean of 20
replicate analyses of the Zero Standard and was found to be 0.25 U/mL.
Reproducibility
Intra Assay
The within assay variability is shown below:
SamplenMean (U/mL)

CV (%)

120

16.9

5.9

220

26.7

5.8

320

135.8

4.5

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Inter Assay
The between assay variability is shown below:
SamplenMean (U/mL)
CV (%)
140

19.5

13.8

240

33.8

10.6

340

82.7

6.5

Recovery
Samples have been spiked by adding CA 125 solutions with known concentrations in a 1:1 ratio.
The % recovery has been calculated by multiplication of the ratio of the measurements and the expected values
with 100 (expected value = (endogenous CA 125 + added CA 125) / 2; because of a 1:2 dilution of serumwith spike material).
Sample 1

Sample 2

Sample 3

Concentration [U/mL]

19.0

97.1

191.1

Average Recovery

89.4

94.4

94.9

from89.1

91.4

93.4

to89.799.8

96.3

Sample 1

Sample 2

Sample 3

Concentration [U/mL]

17.7

29.5

103.0Average Recovery

106.9

101.7

90.6

from95.8

90.7

88.7

to113.3

108.6

94.8

Range of Recovery [%]Linearity

Range of Recovery [%]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.
Interfering Substances
Haemoglobin (up to 4 mg/mL), Bilirubin (up to 0.5 mg/mL) and Triglyceride (up to 30 mg/mL) have noinfluence on 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.
Drug Interferences
Until today no substances (drugs) are known to us which have an influence to the measurement of CA 125 in asample.
High-Dose-Hook EffectNo hook effect was observed in this test up to 19,200 U/mL of CA 125.

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REFERENCES / LITERATURE
1. Bast R.C. et al.: Reactivity of a monoclonal antibody with human ovarian carcinoma.
J. Clin. Invest. 1981; 68:1331-1337.
2. Duffy M.J.: Role of tumor markers in patients with solid cancers: A critical review.
Eur. J. Intern. Med. 2007; 18(3):175-184.
3. Ataseven H. et al.: Cancer antigen 125 levels in inflammatory bowel diseases.
J. Clin. Lab. Anal. 2009; 23(4): 244-248.
4. Powell J.L. et al.: Preoperative serum CA-125 levels in treating endometrial cancer.
J. Reprod. Med. 2005; 50(8): 585-589.
5. Juretzka M.M. et al.: CA125 level as a predictor of progression-free survival and overall survival in ovariancancer patients with surgically defined disease status prior to the initiation of intraperitoneal consolidation
therapy.
Gynecol. Oncol. 2007; 104(1): 176-180.
6. Micha J.P. et al: Clinical utility of CA-125 for maintenance therapy in the treatment of advanced stage
ovarian carcinoma.
Int. J. Gynecol. Cancer. 2009; 19(2): 239-241.
7. Kang W.D., Choi H.S., Kim S.M.: Value of serum CA125 levels in patients with high-risk, early stageepithelial ovarian cancer.
Gyneco. Oncol. 2010; 116(1): 57-60.
8. Klug TL,et al.: Monoclonal antibody immunoradiometric assay for an antigenic determinant (CA 125)
associated with human epithelial ovarian carcinomas.
Cancer Res. 1984; 44(3):1048-53.
9. Kaesemann H. et al.: Monoclonal antibodies in the diagnosis and follow-up of ovarian cancer. CA 125 as atumor marker. A cooperative study of the Gynecologic Tumor Marker Group (GTMG).
Klin. Wochenschr. 1986, 64(17):781-5.

Symbols:

Vers. 2.0-r

Storagetemperature

Manufacturer

Contains sufficient for<n> tests

Expiry date

Batch code

For in-vitro diagnostic
use only!

Consult instructions
for useContent

CE labelled

CautionCatalogue
number

For research use
only!

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