DRG TM-CA 72-4 ELISA Instruction Manual

**DRG TM-CA 72-4 ELISA Instruction Manual

**

INTRODUCTION

Intended Use
The DRG TM-CA 72-4 ELISA is an enzyme immunoassay for the quantitative in vitro diagnostic measurement of CA 72-4 (TAG-72) in serum and plasma.

Summary and Explanation
CA 72-4 (Cancer antigen 72-4) was originally described as an antigenic determinant recognized by B 72.3, a murine monoclonal antibody raised against a membrane extract of mammary carcinoma metastases (1). CA 72-4 was identified as a 1 MDa mucine-like Glycoprotein complex termed TAG-72 (tumor associated antigen 72) (2). The molecular weight of the TAG-72 protein is 48 kD. Elevated CA 72-4 levels in serum and plasma have been reported in various malignant diseases including carcinomas of pancreas, stomach, gall, colon, breast, ovaries, cervix and endometrium (3). The highest diagnostic sensitivities are found for carcinomas of the gastrointestinal tract and ovaries. Although some benign diseases such as rheumatic diseases or ovary cysts may also result in elevated levels of CA 72-4, clinical studies demonstrated diagnostic specificities of more than 95% for gastrointestinal and ovarian malignancies (4). There is a good correlation between CA 72-4 levels and tumor stage and size (3). CA 72-4 is the marker of choice for the therapeutic monitoring and follow-up care of gastrointestinal cancer patients. Suitable second markers are CA 19-9 or CEA. Additionally, CA 72-4 has been used as an independent marker for the therapeutic monitoring and follow-up care of ovarian cancer patients, in particular in CA 125 negative patients (3, 5).

PRINCIPLE OF THE TEST*

The DRG TM-CA 72-4 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 (Clone CC49) directed towards a unique antigenic site on a CA 72-4 molecule. An aliquot of patient sample containing endogenous CA 72-4 is incubated in the coated well with enzyme conjugate, which is an anti-CA 72-4 antibody (Clone B72.3) conjugated with horseradish peroxidase. After incubation the unbound conjugate is washed off.
The amount of bound peroxidase is proportional to the concentration of CA 72-4 in the sample.
Having added the substrate solution, the intensity of colour developed is proportional to the concentration of CA 72-4 in the patient sample.

• The antibodies used in this assay are patented by:

  1. U.S. Patent No. 5,512,443, issued April 4, 1996 entitled “Second generation monoclonal antibodies having binding specificity to TAG-72 and human carcinomas and methods for employing the same”
     (HHS Reference No. E-160-1987/0-US-18)

  2. Canadian Patent No. 1339980, issued August 4, 1998 entitled “Second generation monoclonal antibodies having binding specificity to TAG-72 and human carcinomas and methods for employing the same” (HHS Reference No. E-160-1987/0-CA-04)

  3. U.S. Patent No. 4,522,918, issued June 11, 1985 (now expired) entitled “Process for Producing Monoclonal Antibodies Reactive with Human Breast Cancer” (HHS Reference No. E-185-1981/0-US-01)

WARNINGS AND PRECAUTIONS

1. This kit is for in vitro diagnostic use only. For professional 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 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 each step.
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 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 °C to 26 °C) before starting the test. Temperature will affect the absorbance readings of the assay. However, values for the patient 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 microtiter plate readers.
16. Do not mix or use components from kits with different lot numbers. It is advised not to exchange wells of 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.
17. Avoid contact with Stop Solution containing 0.5 M H2SO4. It may cause skin irritation and burns.
18. Some reagents contain Proclin 300, BND and/or MIT as preservatives. In case of contact with eyes or skin, flush immediately with water.
19. TMB substrate has an irritant effect on skin and mucosa. In case of possible contact, wash eyes with an abundant 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 DRG.
22. REAGENTS

Reagents provided

1. Microtiterwells, 12 x 8 (break apart) strips, 96 wells;
   Wells coated with anti-CA 72-4 antibody (monoclonal).

2. Standard (Standard 0-4), 5 vials, 0.5 mL, ready to use Concentration: 0, 3, 20, 50, 100 U/mL Contain non-mercury preservative.

3. Control Low & High, 2 vials, (lyophilized) 0.5 mL each, see „Reagent Preparation“
   Control values and ranges please refer to vial label or QC-Datasheet.
   Contains non-mercury preservative.

4. Sample Diluent, 1 vial, 3 mL, ready to use, Contains non-mercury preservative.

5. Enzyme Conjugate 10X concentrate, 1 vial, 1.4 mL, anti-CA 72-4 antibody conjugated to horseradish peroxidase; see „Reagent Preparation“
   Contains non-mercury preservative.

6. Conjugate Diluent, 1 vial, 14 mL, ready to use Contains non-mercury preservative.

7.  Substrate Solution, 1 vial, 14 mL, ready to use, Tetramethylbenzidine (TMB).

8. Stop Solution, 1 vial, 14 mL, ready to use, contains 0.5M H2SO4,
   Avoid contact with the stop solution. It may cause skin irritations and burns.

9. Wash Solution, 1 vial, 30 mL (40X concentrated), see „Preparation of Reagents“.

Note: Additional Sample Diluent for sample dilution is available upon request.

Materials required but not provided

• A microtiter plate calibrated reader (450 ± 10 nm) (e.g. the DRG Instruments Microtiter Plate Reader).
• Calibrated variable precision micropipettes.
• Absorbent paper.
• Distilled or deionized water
• Timer
• Graph 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 two months if stored as described above.

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 the controls 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 1 weeks at room temperature (20 °C to 26 °C).

Enzyme Conjugate
Dilute Enzyme Conjugate concentrate 1:10 in Conjugate Diluent.
Stability of the prepared Enzyme-Conjugate: 1 week at 2 °C to 8 °C in a sealed container.
Example:
If the whole plate is used, dilute 1.2 mL Enzyme Conjugate with 10.8 mL Conjugate Diluent to a total volume of 12 mL.
If the whole plate is not used at once prepare the required quantity of Enzyme Conjugate by mixing 100 µL of Enzyme Conjugate 10X conc. with 0.9 mL of Conjugate Diluent per strip (see table below):

No. of strips| Enzyme Conjugate 10X conc. (µL)| Conjugate Diluent (mL)
---|---|---
1| 100| 0.9
2| 200| 1.8
3| 300| 2.7
4| 400| 3.6
5| 500| 4.5
6| 600| 5.4
7| 700| 6.3
8| 800| 7.2
9| 900| 8.1
10| 1000| 9.0
11| 1100| 9.9
12| 1200| 10.8

Disposal of the Kit
The disposal of the kit must be made according to the national regulations. Special information for this product is given in the Safety Data Sheets (see chapter 13).

Damaged Test Kits
In case of any severe damage to the test kit or components, DRG has to be informed in writing, at the latest, 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 the official regulations.

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.

Specimen Collection
Serum:
Collect blood by venipuncture (e.g. Sarstedt Monovette for serum), allow to clot, and separate serum by centrifugation at room temperature. Do not centrifuge before complete clotting has occurred. Patients receiving anticoagulant therapy may require increased clotting time.
Plasma:
Whole blood should be collected into centrifuge tubes containing anti- coagulant (e.g. Sarstedt Monovette with the appropriate plasma preparation) and centrifuged immediately after collection.

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 12 months) should be frozen 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 be diluted with Sample Diluent 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 Sample Diluent (mix thoroughly)
b) dilution 1:100: 10 µL dilution a) 1:10 + 90 µL Sample Diluent (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 cross contamination
• Absorbance is a function of the incubation time and temperature. Before starting the assay, it is recommended 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.

Test Procedure
Each run must include a standard curve.

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

2. Pipette 20 µL of each Standard, Control and samples with new disposable tips into appropriate wells.

3. Add 100 µL freshly diluted Enzyme Conjugate (see “Reagent Preparation”) into each well.
   Thoroughly mix for 10 seconds. It is important to have a complete mixing in this step.

4. Incubate for 120 minutes at room temperature.

5. Wash the wells as follows:
   If the wash step is performed manually:
   Briskly shake out the contents of the wells.
   Rinse the wells 5 times with 300 µL diluted Wash Solution per well.
   If an automated plate washer is used:
   Rinse the wells 5 times with 400 µL diluted Wash Solution per well.
   At the end of the washing step, always strike the wells sharply on absorbent paper to remove residual droplet.

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. Measure the optical density of the solution in each well at 450 nm filter (reading) and at 620 nm to 630 nm (background subtraction, recommended).
   It is recommended that the wells be read within 10 minutes after adding the Stop Solution.

Calculation of Results

1. Calculate the average absorbance values for each set of standards, controls and patient samples.
2. Manual method: Using linear graph paper, construct a standard curve by plotting the mean absorbance obtained from each standard against its concentration with absorbance value on the vertical (Y) axis and concentration on the horizontal (X) axis.
3. Using the mean absorbance value for each sample determine the corresponding concentration from the standard curve.
4. Automated method: The results in the Instructions for Use have been calculated automatically using a
5. Parameter curve fit. (4 Parameter Rodbard or 4 Parameter Marquardt are the preferred methods.) Other data reduction functions may give slightly different results.
6. 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 > 100 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 of assay.

EXPECTED NORMAL VALUES

The reference values of the TM-CA 72-4 ELISA for healthy individuals were determined by measuring the values of apparently healthy subjects. 121 male and 119 female samples were measured. q-q plot (quantil-quantil plot) was performed in order to test normal distribution of values and give the chance to identify and exclude potentially false healthy subjects. Final calculation of 2.5th to 97.5th percentile was done with a data set which was cleared by q-q-plot
analysis. It is strongly recommended, that each laboratory should determine its own reference values.

| Male| Female| Total
---|---|---|---
Number| 121| 119| 240
97.5 th Percentile U/mL| 3.84| 6.56| 5.55
2.5 th Percentile U/mL| < 0.6| < 0.6| < 0.6
MEAN U/mL| 0.72| 0.99| 0.86
MEDIAN U/mL| < 0.6| < 0.6| < 0.6
Min Value U/mL| < 0.6| < 0.6| < 0.6
Max Value U/mL| 30.51| 26.78| 30.51

The normal range was established and corresponds well to data obtained from the literature.
The results alone should not be the only reason for any therapeutic consequences. The results should be correlated to other clinical observations and diagnostic tests.

QUALITY CONTROL

Good 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 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 direct comparison 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 patient 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 DRG directly.

PERFORMANCE CHARACTERISTICS

1. Assay Dynamic Range
   The range of the assay is between 0.60 U/mL – 100 U/mL.

2. Specificity of Antibodies (Cross Reactivity)
   No cross reactivity was observed with related proteins.

3. Sensitivity (LoB, LoD, LoQ)
   The Limit of Blank (LoB) is 0.44 U/mL.
   The Limit of Detection (LoD) is 0.60 U/mL.
   The Limit of Quantification (LoQ) is 0.82 U/mL.

4. Reproducible

5. Intra Assay
   The within assay variability is shown below: Sample| n| Mean (U/mL)| CV (%)| Status
   ---|---|---|---|---
   Sample 1| 40| 3.79| 4.7| passed
   Sample 2| 40| 6.35| 3.7| passed
   Sample 3| 40| 15.89| 3.0| passed
   Sample 4| 40| 63.44| 4.1| passed

6. Inter Assay
   The between assay variability is shown below: Sample| n| Mean (U/mL)| CV (%)| Status
   ---|---|---|---|---
   Sample 1| 80| 3.79| 10.1| passed
   Sample 2| 80| 6.35| 8.4| passed
   Sample 3| 80| 15.89| 6.1| passed
   Sample 4| 80| 63.44| 6.0| passed

7. Recovery
   Samples have been spiked by adding CA 72-4 solutions with known concentrations in a 1:1 ratio.
   The expected values were calculated by addition of half of the values determined for the undiluted samples and half of the values of the known solutions. The % Recovery has been calculated by multiplication of the ratio of the measurements and the expected values with 100.| Sample 1| Sample 2| Sample 3
   ---|---|---|---
   Concentration [U/m L]| 3.6| 8.1| 9.4
   Average Recovery [%]| 99.3| 98.2| 98.8
   Range of Recovery [%]| from| 96.6| 92.5| 88.2
   to| 102.1| 105.8| 106.8

8. Linearity
   | Sample 1| Sample 2| Sample 3
   ---|---|---|---
   Concentration [U/mL]| 51.0| 94.0| 10.0
   Average Recovery [%]| 91.2| 108.5| 97.8
   **Range of Recovery [%]**| from| 86.3| 106.4| 86.0
   to| 99.6| 112.3| 112.0

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.

1. Interfering Substances
   Haemoglobin (up to 4 mg/mL), Bilirubin (up to 0.5 mg/mL) and Triglyceride (up to 7.5 mg/mL) have no influence on the assay results.
   Triglycerides > 7.5 mg/mL will result in decreased values.
   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.

2. Drug Interferences
   Until today no substances (drugs) are known to us, which have an influence to the measurement of CA 72-4 in a
   sample.

3. High-Dose-Hook Effect
   No hook effect was observed in this test up to 6,400 U/mL of CA 72-4.

4. Method Comparison
   A comparison of DRG TM-CA 72-4 ELISA EIA-5071 (y) and reference method (x) using clinical samples gave the following correlation:
   n = 130
   r = 0.95

The DRG ELISA data perfectly correlate with the data of the reference method.
LEGAL ASPECTS

5. Reliability of Results
   The test must be performed exactly as per the manufacturer’s instructions for use. Moreover the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable national standards and/or laws. This is especially relevant for the use of control reagents. It is important to always include, within the test procedure, a sufficient number of controls for validating the accuracy and precision of the test.
   The test results are valid only if all controls are within the specified ranges and if all other test parameters are also within the given assay specifications. In case of any doubt or concern please contact DRG.

6. Therapeutic Consequences
   Therapeutic consequences should never be based on laboratory results alone even if all test results are in agreement with the items as stated under point 11.1. Any laboratory result is only a part of the total clinical picture of a patient.
   Only in cases where the laboratory results are in acceptable agreement with the overall clinical picture of the patient should therapeutic consequences be derived.
   The test result itself should never be the sole determinant for deriving any therapeutic consequences.

7. Liability
   Any modification of the test kit and/or exchange or mixture of any components of different lots from one test kit to another could negatively affect the intended results and validity of the overall test. Such modification and/or exchanges invalidate any claim for replacement.
   Claims submitted due to customer misinterpretation of laboratory results subject to point 11.2. are also invalid.
   Regardless, in the event of any claim, the manufacturer’s liability is not to exceed the value of the test kit. Any damage caused to the test kit during transportation is not subject to the liability of the manufacturer.

REFERENCES / LITERATURE

1. Colcher D., Horand Hand P., Nuti M., Schlom J. A spectrum of monoclonal antibodies reactive with human mammary tumor cells.
   Proc. Natl. Acad. Sci. 1981, 78:3199- 3208.

2. Johnson VG, Schlom J., Paterson AJ, Bennett J, Magnani JL, Colcher D. Analysis of a human tumor associated glycoprotein (TAG-72) identified by monoclonal antibody 72.3.
   Cancer Res. 1986; 46: 850-857.

3. Lamerz R. in Thomas L. (editor) Labor und Diagnose 6. edition,
   TH-Books Verlagsgesellschaft mbH, Frankfurt/Main 2005, 1310-13..

4. Guadagni F., Roselli M., Cosimelli M., Ferroni P., Spila A., Cavaliere F., Casaldi V., Wappner G., Abbolito M.R.,
   Greiner J.W., Schlom J. CA 72-4 serum marker – a new tool in the management of carcinoma patients.
   Cancer Invest. 1995; 13(2): 227 – 238.

5. Hasholzner U., Baumgartner L., Stieber P., Meier W., Hofmann K. Fateh-Moghadam A. Significance of the tumour markers CA 125 II, CA 72-4, CASA and CYFRA 21-1 in ovarian carcinoma.
   Anticancer Res. 1994 Nov-Dec; 14 (6B):2743-6.

6. Marrelli D., Pinto E., De Stefano A., Farnetani M., Garosi L., Roviello F. Clinical utility of CEA, CA 19-9, and CA 72-4 in the follow-up of patients with resectable gastric cancer. Am J Surg. 2001, 181(1):16-9.

Symbol

| European Conformity
| Consult instructions for use
| In vitro diagnostic device
| | For research use only
| Catalogue number
| Lot. No. / Batch code
| Contains sufficient for tests/
| Storage Temperature
| Expiration Date
| Legal Manufacturer
          | Distributor
Content| Content
Volume/No.| Volume / No.
Micrometer wells| Micrometer wells
Antiserum| Antiserum
Enzyme Conjugate| Enzyme Conjugate
Enzyme Complex| Enzyme Complex
Substrate Solution| Substrate Solution
Stop Solution| Stop Solution
Zero Standard| Zero Standard
Standard| Standard
Control| Control
Assay Buffer| Assay Buffer
Wash Solution| Wash Solution
1N NaOH| 1N NaOH
1 N HCl| 1 N HCl
Sample Diluent| Sample Diluent
Conjugate Diluent| **** Conjugate Diluent

DRG Instruments GmbH,
Germany Frauenbergstraße. 18, D-35039 Marburg
Phone: +49 (0)6421-17000,
Fax: +49 (0)6421-1700 50
Website: www.drg-diagnostics.de
E-mail: drg@drg-diagnostics.de

DRG International, Inc.,
USA 841 Mountain Ave., Springfield, NJ 07081
Phone: 973-564-7555, Fax: 973-564-7556
Website: www.drg-international.com
E-mail: corp@drg-international.com

References

• Analyticon® Biotechnologies GmbH
• It's Uptime | International® Trucks

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