A chimeric antibody with the human gamma1 constant region as a putative standard for assays to detect IgG beta2-glycoprotein I-dependent anticardiolipin and anti-beta2-glycoprotein I antibodies.
OBJECTIVE
Thromboembolic manifestations or thrombocytopenia in association with anticardiolipin antibodies (aCL) or lupus anticoagulant are known as the antiphospholipid syndrome (APS). Efforts have been made to elucidate precise clinical features and adequate therapeutic options for treating patients with APS. However, the lack of a proper international standard for measurement of aCL makes it difficult to compare data derived from different laboratories. We attempted to design a chimeric antibody with human gamma constant regions and variable regions of WBCAL-1, a monoclonal antibody established from an APS-prone mouse which has a specificity similar to that of aCL in sera from humans with APS.


METHODS
Variable-region genes of WBCAL-1, which were cloned using reverse transcription-polymerase chain reaction, were inserted into plasmids containing human gamma1 and kappa constant-region genes. The construct was transfected to a mouse myeloma cell line. Stable transfectants that secreted a chimeric antibody, HCAL, into the culture supernatant were obtained. The reactivity of HCAL to cardiolipin and to beta2-glycoprotein I (beta2GPI) was studied using a solid-phase enzyme immunoassay. The binding of HCAL was compared with the binding of standards for IgG aCL and anti-beta2GPI antibody assays done in 18 independent laboratories.


RESULTS
In the presence of beta2GPI, HCAL bound to the wells of cardiolipin-coated microtiter plates in a dose-dependent manner and reacted with beta2GPI on oxygenated polystyrene plates. The aCL activity of HCAL can be converted into GPL units (IgG phospholipid units), which is widely used to quantify IgG aCL activity, using the following formula: 1 GPL unit = 32.9 x (concentration of HCAL [in microg/ml])(0.503). The reactivity of HCAL to cardiolipin or beta2GPI was similar to the reactivity of standards for IgG aCL or anti-beta2GPI antibody assays done in collaborative laboratories.


CONCLUSIONS
Because the reactivity of HCAL is similar to that of aCL in sera from humans with APS, HCAL will be useful as a standard for human IgG aCL and anti-beta2GPI antibody assays.
Ichikawa
K
K
Department of Medicine II, Hokkaido University School of Medicine, Sapporo, Japan.
Tsutsumi
A
A
Atsumi
T
T
Matsuura
E
E
Kobayashi
S
S
Hughes
G R
GR
Khamashta
M A
MA
Koike
T
T
D000595:Amino Acid Sequence; D000818:Animals; D017153:Antibodies, Anticardiolipin / Q000032:analysis* / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D016736:Antiphospholipid Syndrome / Q000276:immunology; D001483:Base Sequence; D006023:Glycoproteins / Q000276:immunology*; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000235:genetics / Q000276:immunology; D007074:Immunoglobulin G / Q000032:analysis* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D007142:Immunoglobulin gamma-Chains / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000276:immunology; D053482:beta 2-Glycoprotein I
D017153:Antibodies, Anticardiolipin; D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D007142:Immunoglobulin gamma-Chains; D011993:Recombinant Fusion Proteins; D053482:beta 2-Glycoprotein I