CA125 monoclonal antibodies – epitope specificity and matched pairs for immunoassays

Study of epitope specificity
Epitope specificity of XEMA CA125 monoclonal antibodies was determined by competitive binding to enzyme-labelled human single patient CA125 antigen. Antigen: natural human CA125 (lot CA23) isolated from single patient ovarian tumour ascitic fluid; purification method did not include affinity binding to monoclonal antibodies. Conjugate of human CA125 lot CA23 with HRPO enzyme (Faizyme, Lansdowne, RSA) was performed by perjodate method.
Antibodies:
1) workshop ISOBM TD1 (1996) monoclonals kindly provided by K.Nustad (University in Oslo, Norway);
2) purified mAbs of XEMA;
Purity of all antibody preparations estimated as >90% by SDS-PAGE. Preparations of diluted antibodies were made from stock solutions in equal concentrations – 20 ug/ml in EIA buffer (0.1M PBS containing Tween-20 and casein).
Preparation of antibody-coated microwells was made in alkaline carbonate buffer; the microwells were post-coated by casein, dried and stored for up to one month before use.
HRPO-labelled CA125 preparation (lot CA23-HRPO) was diluted 1:500 by EIA buffer.
Cross-inhibition of natural CA125 binding was performed as follows: 50 ul of EIA buffer (in control well) or diluted antibodies were placed onto each antibody-coated microplate followed by an equal volume of diluted CA23-HRPO. The microplates were incubated for 45’ at room temperature on shaking platform. After 5xfold washing with saline-Tween, 100 ul of substrate were added to each well. The enzymatic reaction was stopped by addition of 5% sulphuric acid. The optical density was measured in microplate reader; inhibition percentages (see table 1) were calculated by dividing the average OD in actual wells by the average OD in control wells.

	K95 (A1)	K93 (A2)	K90 (B1)	X52	X75	X306	X325	Epitope group
X52	0	2	28	85	50	0	41	B2
X75	0	6	82	85	87	0	78	B1
X306	88	78	5	3	5	91	5	A1
X325	0	0	40	41	66	3	89	B1

Table 1. Cross-inhibition of XEMA CA125 mAbs and reference mAbs from TD1 workshop.
Rows – liquid phase antibodies; columns – immobilized antibodies.
See explanation in text.

Recommended pairs for immunoassays The results show that mAb X306 clearly belongs to A1 (OC125 like) epitope group; mAb X75 shows high homology to B1 (M11 like) group.
Recommended pairs for immunoassays are:
X306 solid X52-HRPO conjugate – HIGHEST signal
X325 solid X52-HRPO conjugate
X306 solid X325-HRPO conjugate.
Please note that mAb X306 may suffer from perjodate and thus may not be recommended for HRPO-labelling.

Application of XEMA CA125 for immunohistochemistry
Anti-CA125 mAbs can be used for IHC staining of cryostat or paraffin-embedded tissues mAb X325 is recommended for this purpose (see the example picture below – mammary carcinoma)

Development of rapid test for CA125 (lateral flow)
Rapid test for mass screening for ovarian carcinoma may be constructed from XEMA anti-CA125 mAbs
In a table below, there is a summary of the co-operative development performed with HBI Inc., Republic of Korea

	GOLD/TEST	X52	X75	X306	X325
Antibody	X52			+++	+++
	X75	+		+	+
	X306	+	+		+
	X325	+	+	+
	AS226	+	+	+	+

Gold conjugation(X52) – Test dispense(X325) Pair is best