Background/Purpose:   Despite numerous efforts aimed at the standardization of assays for detection of anti-β₂Glycoprotein I (aβ₂GPI)antibodies, there are still concerns, including the type and source of properly validated calibrant and reference material and the lack of universal units of measurement.  Based on recommendations of an international task force at the 13^th Congress on Antiphospholipid Antibodies , a project was started with the goals of establishing a reference preparation (RP) and  international consensus units (IU) for the measurement for IgG aβ₂GPI antibodies. 

Methods: Whole IgG fractions were affinity-purified (AP) from sera of 2 primary Antiphospholipid syndrome patients with high IgG ab₂GPI levels using a Protein G Sepharose column. Pooled IgG fractions were further AP using an affinity column coupled to β₂GPI, and the protein concentration of the AP material was determined using two methods, and a value based on the definition that 1 IU/ml equates to 1µg/ml of AP aβ₂GPI, was assigned.  A reference preparation (RP) serum created from sera taken from the 2 original PAPS patients was then assigned an IU value based on repeated testing using original AP material as a calibrator. RP was sent to six commercial companies for testing in their respective kits (eight total) according to an approved protocol to enable evaluation of linearity and unit equivalency, along with a set of 30 samples (APS samples and healthy controls) to allow for commutability studies to be done. Companies (and kits) included INOVA Diagnostics (QUANTA Lite ^® β₂GPI IgG ELISA, QUANTA Flash ^® β₂GPI IgG chemiluminescent assay), Bio-Rad Laboratories (BioPlex ^® 2200 APLS IgG Kit, Anti-β₂Glycoprotein I IgG EIA Test Kit) TheraTest (EL-b₂GPI IgG kit), Corgenix (IgG Anti-Beta 2 Glycoprotein I Test Kit), Phadia (EliA ß2-Glycoprotein I IgG on Phadia® 250) and Instrumentation Laboratory (HemosIL^® AcuStar anti-β₂Glycoprotein-I IgG).

Results: The pooled AP material had a protein concentration of 103.1 µg/ml (OD280nm) and 108.8 µg/ml (Bradford) and was assigned a value of 100 IgG aβ₂GPI IU/ml. RP had a value of 270 IgG aβ₂GPI IU/ml. The R²  values of the regression lines of the RP for all kits were >0.95. The value of the RP in the various  kit units ranged from 115 to 9993.1. Results of correlations between kits with commutability samples are shown in Table 1 (kit units and international units).

Conclusion: Establishment of equivalency between kit units and IU value of RP was successful. The RP demonstrated excellent linearity and was commutable in the various aβ₂GPI IgG assays and is therefore adequate to be used as a reference material. Expressing results in in the new IU instead of arbitrary kit units illustrated the comparability of the results obtained in the different kits.  These studies contribute significantly to the much-needed standardization of aβ₂GPI immunoassays.

Table 1. Correlation of commutability sample values among various ab₂GPI assays

CORRELATIONS OF COMMUTABILTY SAMPLES IN INTERNATIONAL UNITS		IL AcuStar	INOVA BIO-FLASH	INOVA ELISA	Bio-Rad ELISA	Bio-Rad BioPlex	Theratest	Corgenix	Phadia	CORRELATIONS OF COMMUTABILITY SAMPLES IN KIT UNITS
	IL AcuStar		.996	.961	.940	.990	.978	.966	.950
	INOVA BIO-FLASH	0.996		.959	.935	.988	.971	.961	.939
	INOVA ELISA	0.938	0.933		.896	.942	.987	.925	.956
	BioRad ELISA	0.972	0.963	0.897		.929	.914	.915	.846
	Bio-Rad BioPlex	0.948	0.945	0.810	0.920		.960	.963	.933
	Theratest	0.964	0.958	0.978	0.936	0.891		.949	.958
	Corgenix	0.974	0.968	0.911	0.993	0.907	0.939		.905
	Phadia	0.953	0.944	0.944	0.898	0.878	0.958	0.903

For values in table: p<0.001

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