CA-125 Ovarian Cancer

Description:
CA-125 Ovarian Cancer

Application:
ELISA for CA-125

Presentation:
PREPARATION FOR ASSAY

1.        Bring all reagents and samples to room temperature (20-25C) and mix gently before beginning the test.
2.        Have all reagents and samples ready before the start of the assay. Once the test has begun it must be performed without any interruptions to get the most reliable and consistent results.
3.        Use new disposable tips for each specimen.

ASSAY PROCEDURE

1.        Secure the desired number of coated wells in the holder. Mark data sheet with sample identification.
2.        Dispense 25 ul of Sample diluent (as blank), standards, samples or controls into the assigned wells. Immediately, dispense 100 ul of biotinylated capture antibody (blue color) the assigned wells.
3.        Incubate for 120 minutes at room temperature.
4.        Remove incubation mixture and rinse the wells five times with Washing Buffer.
5.        Dispense 100 ul Enzyme Conjugate into each well except the blank well.
6.        Incubate for 60 minutes at room temperature.
7.        Remove incubation mixture and wash five times with the Washing buffer.
8.        Dispense 100 ul of Solution A and 100 ul of Solution B into each well including the blank well.
9.        Incubate for 30 minutes at room temperature.
10.     Stop reaction by adding 50 ul of Stop Solution of 1 N H2SO4 to each well.
11. Zero microreader on the blank and measure the absorbance of each well at 450 nm.

Notes:
CALCULATION OF RESULTS

1.        Plot the concentration (X) of each Reference Standard against its absorbance (Y) on full logarithmic graph paper.
2.        Obtain the CA125 antigen value of patient by referring to the standard curve as followed (These data are for demonstration purpose only and must not be used in place of data generated for each assay):

Well Description Absorbance EOC-Ag
No. (U/mL) 450 nm (U/mL)
A1 0 0.025
A 10 0.087
A3 50 0.343
A4 100 0.644
A5 200 1.193
A6 500 2.683
A7 Patient A 0.66 104.4
(Serum)
A8 Patient B 1.272 212.9
(Serum)

References:
1.        Jacobs, I. Bast. RC J. 1989 The CA 125 tumor-associated Antigen: A Review of the Literature. Hum. Reprod. 4:1-12
2.        Bast, R.C., C.M. Boyer, G.J. Olt, A. et al 1990 Identification of Markers for Early Detection of Epitherlial Ovarian Cancer, p. 265-275
3.        Hawkins, R.E., K Roberts, E. Wiltshaw, J. Murdy, I.J. Fryatt, and V.R. McCready. 1990 The Prognostic Significance of the Half-life Serum CA 125 in Patients Responding to Chemotherapy for Epithelal Ovarian Cancer. Br. J. Obstet, Gynaecol. 96:1395-1399.
4.        Noel R. Rose et al. 1992 Manual of Clinical Laboratory Immunology 4th ed. ASM p. 812