CALBIOTECH CA125 ELISA Elisa Kit Instructions

CALBIOTECH CA125 ELISA Elisa Kit

Specifications

• Catalog Number: CA239T (96 Tests)
• Intended Use: Quantitative determination of the Cancer Antigen CA125 concentration in human serum

Product Usage Instructions

Materials Provided

• Microwells coated Streptavidin
• CA125 reference standards: 6 vials (ready to use)
• Enzyme Conjugate Reagent
• TMB Reagent (One-Step)
• Stop Solution
• Wash Concentrate 20x: 1 Bottle

Materials Not Provided

• Distilled or deionized water
• Precision pipettes
• Disposable pipette tips
• ELISA reader capable of reading absorbance at 450nm
• Absorbance paper or paper towel
• Graph paper

Storage and Stability

• Store the kit at 2 – 8°C
• Keep microwells sealed in a dry bag with desiccants
• The reagents are stable until expiration of the kit
• Avoid exposing reagents to heat, sun, or strong light

Warnings and Precautions

• For Research Use Only. Not for use in diagnostic procedures.
• For Laboratory use only.
• Avoid internal or external use in humans or animals.
• No eating or drinking within the work area.
• Always wear gloves and a protective lab coat.
• Avoid pipetting by mouth. Handle all specimens and reagents as potentially infectious and biohazardous.
• Avoid adding sodium azide to samples as a preservative.
• Avoid using external controls containing sodium azide.
• Use disposable pipette tips to avoid contaminating chromogenic substrate reagent. Discard.

INTENDED USE
The Calbiotech CA125 ELISA Kit is intended for the quantitative determination of the Cancer Antigen CA125 concentration in human serum.

SUMMARY AND EXPLANATION
Cancer Antigen 125 (CA125) is a surface antigen associated with epithelial ovarian cancer. In serum, CA125 is associated with a high molecular weight glycoprotein. Published studies have indicated that elevated serum CA125 levels can be found in individuals with serious endometroid, clear-cell and undifferentiated ovarian carcinoma. The serum CA125 concentration is greater than 35 units per ml in 60% of women with ovarian cancer and >80% of patients with disseminated ovarian cancer. The serum CA125 is elevated in 1% of normal healthy women, 3% of normal healthy women with benign ovarian diseases, 6% of patients with non-neoplastic conditions (including but not limited to first trimester pregnancy, menstruation, endometriosis, uterine fibrosis, acute salphingitis, hepatic diseases and inflammation of peritoneum, pericardium or pleura). Serial determinations of serum CA125 as well as pelvic examination increase the test specificity. Serum CA125 concentration may be useful in monitoring treatment and distinguishing between good response to treatment and progressive malignant disease with poor therapeutic response. To date, CA125 is the most sensitive marker for residual epithelial ovarian cancer. CA125 may also be elevated in patients with lung, cervical, fallopian tube, and uterine cancer and endometriosis.

PRINCIPLE OF THE TEST
The CA125 ELISA test is an adapted solid phase 1-step sandwich ELISA. Samples, a biotinylated mouse anti-CA-125 capture antibody, and mouse anti-CA-125-HRP conjugate are all added to wells coated with streptavidin. CA-125 in the patient sample binds to the biotinylated capture antibody. The biotinylated capture antibody simultaneously binds to the streptavidin coated plate. Anti- CA-125–HRP enzyme conjugate forms a sandwich around captured CA-125. Unbound antibodies are washed off. TMB substrate is added resulting in the development of a blue color. The concentration of CA-125 is directly proportional to the color intensity developed. A standard curve is generated relating color intensity to CA-125 concentration.

MATERIALS NOT PROVIDED

1. Distilled or deionized water
2. Precision pipettes
3. Disposable pipette tips
4. ELISA reader capable of reading absorbance at 450nm
5. Absorbance paper or paper towel
6. Graph paper

STORAGE AND STABILITY

1. Store the kit at 2 – 8 C.
2. Keep microwells sealed in a dry bag with desiccants.
3. The reagents are stable until expiration of the kit.
4. Do not expose reagents to heat, sun, or strong light.

WARNINGS AND PRECAUTIONS

1. For Research Use Only. Not for use in diagnostic procedures.
2. For Laboratory use.
3. Not for Internal or External Use in Humans or Animals.
4. There should be no eating or drinking within work area.
5. Always wear gloves and a protective lab coat.
6. No pipetting should be done by mouth. Handle all specimens and reagents as potentially infectious and biohazardous.
7. Do not add sodium azide to samples as preservative.
8. Do not use external controls containing sodium azide.
9. Use disposable pipette tips to avoid contaminating chromogenic substrate reagent. Discard reagent if it turns blue.
10. Do not pour chromogenic substrate back into container after use.
11. Do not freeze reagents.
12. Do not mix reagents from different kit lot numbers.
13. Keep reagents out of direct sunlight.
14. Handle stop reagent with care, since it is corrosive.
15. Bring all reagents to room temperature.
16. Viscous forensic samples should always be diluted in phosphate buffered saline or distilled water prior to pipetting.
17. Ensure the bag containing the micro-plate strips and desiccant is sealed well, if only a few strips are used.

SPECIMEN COLLECTION AND PREPARATION
Serum should be prepared from a whole blood specimen obtained by acceptable medical techniques. This kit is for use with serum samples without additives only.

REAGENT PREPARATION

1. Prepare 1X Wash buffer by adding the contents of the bottle (25 ml, 20X) to 475 ml of distilled or deionized water. Store at room temperature (20-25 C).

ASSAY PROCEDURE

Bring all specimens and kit reagents to room temperature (20-25 C) and gently mix.

1. Secure the desired number of coated wells in the holder. Dispense 50l of CA125 standards, specimens, and controls into the appropriate wells.
2. Dispense 100l Enzyme Conjugate Reagent into each well.
3. Mix gently for 30 seconds. It is very important to have complete mixing in this setup.
4. Incubate for 60 minutes.
5. Remove the incubation mixture by emptying the plate content into a waste container.
6. Remove liquid from all wells. Wash wells three times with 300 L of 1X wash buffer. Blot on absorbance paper or paper towel.
7. Strike the microtiter plate sharply onto absorbent paper or paper towels to remove all residual liquid droplets.
8. Dispense 100l of TMB Reagent into each well. Gently mix for 10 seconds. Incubate at room temperature, in the dark, for 15 minutes.
9. Stop the reaction by adding 50l of Stop Solution to each well.
10. Read the absorbance at 450nm (using a reference wavelength of 630nm) with a microtiter plate absorbance reader within 15 minutes.

CALCULATION RESULTS

1. Calculate the average absorbance values (A450) for each set of reference standards, control, and samples.
2. Construct a standard curve by plotting the mean absorbance obtained for each reference standard against its concentration in U/ml on linear graph paper, with absorbance 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 of CA125 in U/ml from the standard curve.

Example of Standard Curve
Results of a typical standard run with OD readings at 450nm shown in the Y axis against CA125 concentrations shown in the X axis. This standard curve is for the purpose of illustration only, and should not be used to calculate unknowns. Each user should obtain his or her own data and standard curve in each experiment.

LIMITATIONS OF THE PROCEDURE

1. Reliable and reproducible results will be obtained when the assay procedure is carried out with a complete understanding of the package insert instructions and with adherence to good laboratory practice.
2. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.

REFRENCES

1.  all, E., Methods in Enzymology, Volume 70, Van Vunakis, H. and Langone, J. J. (eds.),Academic Press, New York, 419- 492 (1980).

2. tila, M., Ruoslahti, E. and Engvall, E., J. lmmunol. Methods, 42, 11-15 (1981).

3. Kenemans P, Yedema CA, Bon GG, von Mensdorff-Pouilly S. Ca125 in gynecological pathology a review. Eur J Obstet Gynecol 1993;49:115-124.

4. Saksela F. Prognostic markers in epithelial ovarian cancer. Intl J Gynecol Pathol 1993;12:156-161.

5. Farghaly SA. Tumor markers in gynecologic cancer. Gynecol & Obstet Invest 1992;34:65-72.

6. Welander CE. What do CA 125 and other antigens tell us about ovarian cancer biology.
   Acta Obstet Gynecol Scand Sup 1992;155:85-93.

7. McGowan L. Pathology of the ovary. Curr Opin on Obstet Gynecol 1991;3:580-586.

8. Niloff JM. Ovarian malignancy. Curr Opin on Obstet Gynecol 1991;3:66-72.

9. Olt G, Berchuck A, Bast RC. The role of tumor markers in gynecologic oncology. Obstet Gynecol Survey 1990;45-:570-577.

10. Diez M, Cerdan FJ, Ortega MD, Torres A, Picardo A, Balibrea JL. Evaluation of serum CA125 as a tumor marker in non-small cell lung cancer. Cancer 1991;67:150-154.

11. Niloff JM, Klug TL, Schaetzl E. Elevation of serum CA125 in carcinomas of the fallopian tube, endometrium, and endocervix. AM J. Obstet Gynecol 1984; 148:1057

Cat#: CA239T (96 Tests)
For Order and Inquiries, please contact
Calbiotech Inc.,
1935 Cordell Ct., El Cajon, CA 92020
Tel 619-660-6162, Fax 619-660-6970,
www.calbiotech.com

Frequently Asked Questions (FAQ)

Can this kit be used for diagnostic purposes?

No, this kit is for research use only and not intended for diagnostic procedures.

How should I store the kit?

The kit should be stored at 2 - 8°C, and microwells should be kept sealed in a dry bag with desiccants.

What precautions should I take while using this kit?

Always wear gloves and a lab coat, avoid pipetting by mouth,and handle all specimens and reagents as potentially infectious and biohazardous.

References

• Calbiotech - Contract Development and Manufacturing for Life Science
• Calbiotech - Contract Development and Manufacturing for Life Science

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