CA 15-3 Ag All materials contaminated with patient specimens or [CAL] should be

inactivated by validated procedures (autoclaving or chemical treatment)

ELISA for the Quantitative Determination of in accordance with applicable regulations.

Cancer Antigen 15-3 (CA 15-3 Ag) in Human [STOP] irritates eyes, skin and mucous membranes. Upon contact, rinse
thoroughly with copious amounts of water and consult a doctor.
Serum
Stability
Package Size
The reagents are stable up to the stated expiry dates on the individual
[REF] 52070 96 Tests Complete Test Kit labels when stored at 2...8°C.
[IVD] After opening reagents have to be stored at 2...8°C and used within 60
days (see also "Note").
Intended Use
Cancer Antigen 15-3 has been characterised as sialylated high molecular [MIC]
weight glycoprotein of about 400 kDa. Serum level of CA 15-3 provides Sealed in an aluminium bag with desiccant
information about the clinical stage of the breast disease.
Must be at room temperature before opening
CA 15-3 is more sensitive than CEA in early detection of breast cancer,
however recent reports indicate a clear increase in diagnostic sensitivity unused: return to the zip-lock bag with the desiccant. Strips stored in
for combined use of both tumour markers. this way at 2...8°C can be used until the expiration date (see also
"Note").
High levels of CA 15-3 occur in patients suffering from breast cancer,
while elevated levels are also observed in a range of benign and Do not touch the upper rim or the bottom of the wells with fingers.
malignant diseases (see Expected Values). Although specificity and
Reagent Preparation
sensitivity of CA 15-3 Ag is limited, it is the first marker for diagnosis of
breast cancer and monitoring of therapy and patient progress. Higher Bring all reagents to room temperature (15...25°C) before use.
tumour stages are associated with higher CA 15-3 levels. Reagents not in use should always be stored at 2...8°C.

Principle Working Wash Solution [WASH]

The HUMAN CA 15-3 Ag ELISA intended for professional use, is an ELISA - dilute [WS] to 1000 ml with fresh, deionised water in a suitable
for direct antigen detection. Monoclonal anti-CA 15-3 antibodies are container. Rinse vial several times.
immobilised on the solid phase, the surface of microwells. In the first - Stability: up to 60 days at 15...25°C.
incubation step the CA 15-3 present in patient specimens, calibrators or
Specimen
controls binds to the immobilised antibodies. After removing and
Serum
washing out of unbound components the enzyme conjugate (enzyme-
labelled monoclonal antibodies) is added. In the second incubation step Do not use highly lipemic, hemolysed specimen or specimen containing
the enzyme conjugate binds to the immobilised antibody-antigen sodium azide.
complex forming the final sandwich complex. After incubation excess Specimens may be stored for 5 days at 2...8°C or up to 30 days at -20°C.
conjugate is washed out and TMB Substrate added. A blue colour Freeze and thaw twice only. Thawed specimen must be homogenised.
develops changing to yellow after stopping the reaction. The intensity of Eliminate particulate matter by centrifugation or filtration.
the colours is directly proportional to the CA 15-3 concentration in the
specimen. Procedure
The absorbance of calibrators and specimen is determined by using ELISA Follow the procedure exactly as described.
microplate readers or automated ELISA systems (like HUMAN's
HumaReader or ELISYS line) at 450 nm. The concentration for patient Procedural Notes
samples are obtained by means of a calibration curve which is P1: Do not mix caps of vials (risk of contamination). Do not use reagents
established from the calibrators supplied with the kit. after their expiration date.
P2: Do not use reagents that could be contaminated or look or smell
Reagents and Contents different than usual.
[MIC] 12 Microtiter Strips (in strip holder)
P3: Record specimens and controls carefully on the spread sheet supplied
breakable 8-well strips coated with monoclonal
with the kit.
anti-CA 15-3 Ag antibody (mouse)
P4: [MIC] - select the required number of Microtiter Strips.
[CAL] A-F Calibrators (imprinted, red cap)
6x2 ml ready for use, human P5: Run duplicates or triplicates for positive resp. negative control.
CA 15-3 level: (A) 0 U/ml, (B) 15 U/ml, (C) 30 U/ml, (D) Pipette controls and specimen on the bottom in the microwells.
60 U/ml, (E) 120 U/ml, (F) 240 U/ml P6: Always add reagents in the same order and timing to minimise
[DIL] 100 ml Dilution Buffer (white cap) reaction time differences between wells. This is important for
Ready for use, coloured yellow reproducible results. Pipetting of specimens should not exceed
Phosphate buffer 5 minutes. Otherwise pipette the controls in the indicated positions
BSA 1 g/l at half way time of the series. If more than 1 plate is used, repeat the
controls for each plate.
[CON] 25 ml Enzyme Conjugate (white cap)
Ready for use, coloured red P7: Avoid/remove air bubbles prior to incubations and reading of
Anti-CA 15-3 Ag monoclonal antibody (mouse) HRP absorbance.
labelled P8: [SUB] – incubate in the dark. [SUB] initiates a kinetic reaction, which is
[WS] 50 ml Wash Solution (white cap) terminated by [STOP].
concentrate for about 1000 ml
NaCl 1.8 % Wash Procedure
Tween 20 2.0 % The wash procedure is critical. Insufficient washing will result in poor
precision or falsely high absorbance.
[SUB] 13 ml Substrate Reagent (yellow cap, ready for use)
3,3', 5,5'-tetramethylbenzidin (TMB) 0.25 g/l W1: Remove Adhesive Strips, aspirate off the contents and add [WASH] to
Urea hydrogen peroxide 0.03 % each well, aspirate off after 30 sec. soak time and repeat washing
4 times.
[STOP] 13 ml Stop Solution (red cap)
sulphuric acid, ready for use 0.15 mol/l W2: In case of automatic washers fill and prime with [WASH].
Subsequently wash strips 5 times. Ensure the washer fills all wells
2 Adhesive Strips
completely and aspirates off efficiently after 30 sec. (remaining
Preservatives: Total concentration < 0.1% liquid: < 15 µl).

Safety Notes W3: After washing, remove remaining liquid by tapping the plate upside
down on tissue paper.
Do not swallow the reagents. Avoid contact with eyes, skin and mucous
membranes. All patient specimens and controls should be handled as
potentially infectious. [CAL] have been checked on donor level for HCV
and HIV-1/2 antibodies and HBsAg and found negative. Wear protective
clothing and disposable gloves according to Good Laboratory Practices.
Pipetting Scheme Expected values
Reagents and specimens should be at room temperature before use. CA 15-3 is elevated in case of malignant diseases. It may however be also
elevated in benign diseases like benign breast diseases, hepatic,
Sample Preparation:
pancreatic or rheumatic diseases, and tuberculosis.
Dilute the patient’s sera 1 + 50 with [DIL], e.g. 20 µl serum + 1 ml [DIL],
Expected values for the CA 15-3 Ag ELISA:
mix thoroughly.
Healthy donors: 35 U/ml
Diluted samples can be stored up to 4 h at 2...8°C before testing.
Each laboratory should establish its own expected values to its own
[CAL] A-F are ready for use.
patient population utilizing instrumentation, blood collection methods
Step 1 Well [µl] and testing techniques commonly used in that laboratory.
A1 B1...E2 F2...
Blank Calibrators Specimen
Performance Characteristics
Typical performance data can be found in the Verification Report,
[CAL] A-F in duplicate -- 200 -- accessible via
Diluted samples in duplicate -- -- 200 www.human.de/data/gb/vr/el-ca153.pdf or
Mix carefully www.human-de.com/data/gb/vr/el-ca153.pdf
[MIC] cover with Adhesive Strips
Note
Incubate 60 min. at 37°C The handling should always be in compliance with common GLP
Wash 5 times as described (see W1 - W3) requirements (*)! The validation criteria must be met!
[WASH] 300 300 300 (*This includes: Proper caps being replaced on the vials and firmly tightened / Remove only
reagents required for a run from stock solutions if they could come into contact with other
Step 2
contaminating solutions like patient specimens etc. / Stock solutions always returned to
[CON] -- 200 200 2...8°C when not in use.)
[MIC] cover with Adhesive Strips
References
Incubate 60 min. at 37°C 1. Sekine H. et al., J. Immunology, 135(5), 3610-3615 (1985)
Wash 5 times as described (see W1 - W3) 2. Stieber P. et al., In: New tumour markers and their monoclonal anti-
[WASH] 300 300 300 bodies, Klapdor, R. (ed). Stuttgart: Thieme 45-50 (1988)
Step 3 3. Hayes D.F., Hematol. Oncol. Clin. North. Am. 8(3), 485-506 (1994)
[SUB] 100 100 100 4. Banfi G. et al., Clin. Chem. 43, 2430-2431 (1997)
[MIC] cover with Adhesive Strips 5. Wild D. (ed), The Immunoassay Handbook, Stockton Press, 646 (2001)
Incubate 15 min. at 20...25°C (see P8) 6. Kumpulainen E.J. et al., Breast Cancer Research and Treatment 76, 95-
102 (2002)
[STOP] 100 100 100
7. De la Lande B. et al., Int. J. Biol Markers 17, 231-238 (2002)
Mix carefully
8. Molina R., In: Tumour markers, physiology, pathology and clinical
Measure the absorbance at 450 nm as soon as possible or within applications, Diamandis, Ep, et al., (eds), Washington DC; AACC Press;
30 min. after terminating of the reaction, using a reference wavelength 165-179 (2002)
of 620-630 nm (if available).

Validation of the Test EL-CA153 INF 5207001 GB 07-2010-07 |

The test results are valid provided the following criteria are met:
[CAL] Accepted range [OD]
A 0.10
B 2.00 x absorbance [CAL] A
C 1.50 x absorbance [CAL] B
D 1.40 x absorbance [CAL] C
E 1.30 x absorbance [CAL] D
F 1.20 x absorbance [CAL] E
F 1.00

The differences between the duplicates of [CAL] should not exceed 10%.

Calculation
Plot measured absorbances against [CAL] concentrations in a lin-lin graph.
Appropriate interpolation of plotted measuring points results in a
calibration curve, from which the analyte concentration in the sample can
be determined.
For calculation of analyte concentrations select an appropriate and
validated curve fitting option (e.g. point to point).

Interpretation of Results
The level of CA 15-3 cannot be used as absolute evidence for the presence
or absence of malignant disease due to low clinical sensitivity and
specificity of this tumour marker.
Determination of CA 15-3 is intended for monitoring of therapy and
patient progress. The results should only be used in conjunction with
other clinical investigations and procedures in the diagnosis and
prognosis of disease.
The laboratory finding must always include a statement on the CA 15-3
method used due to potential variations in the result depending on the
test procedure used. Therefore CA 15-3 values determined with different
test procedures cannot be directly compared with one another. Change of
assay procedure during monitoring of patients must be confirmed by
parallel measurements with both procedures.

Human Gesellschaft für Biochemica und Diagnostica mbH

Max-Planck-Ring 21 · 65205 Wiesbaden · Germany
Telefon +49 6122-9988-0 · Telefax +49 6122-9988-100 · e-Mail human@human.de