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Clinical Chemistry 53: 1137-1143, 2007. First published April 13, 2007; 10.1373/clinchem.2006.079632
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(Clinical Chemistry. 2007;53:1137-1143.)
© 2007 American Association for Clinical Chemistry, Inc.

Clinical Immunology

Biosensor Analysis of ß2-Glycoprotein I–Reactive Autoantibodies: Evidence for Isotype-Specific Binding and Differentiation of Pathogenic from Infection-Induced Antibodies

Jochen Metzger1,2, Philipp von Landenberg2,2, Marcus Kehrel1, Alexander Buhl1, Karl J. Lackner2 and Peter B. Luppa1,a

1 Institute of Clinical Chemistry and Pathobiochemistry, Klinikum Rechts der Isar der TU München, München, Germany.
2 Institute of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg-Universität Mainz, Mainz, Germany.

aAddress correspondence to this author at: Institute of Clinical Chemistry and Pathobiochemistry, Ismaninger Str. 22, D-81675 Munich, Germany. Fax 49-89-4140-4875; e-mail luppa{at}klinchem.med.tum.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: For the laboratory diagnosis of the antiphospholipid syndrome (APS) we developed a biosensor with the ability to distinguish between disease-relevant anti-ß2-glycoprotein I (ß2GPI) autoantibodies (anti-ß2GPI) and pathogen-specific ß2GPI cross-reactive antibodies that occur transiently during infections.

Methods: We used a surface plasmon resonance (SPR) biosensor device. For the detection of anti-ß2GPI in serum samples, affinity-purified human ß2GPI was covalently attached to a functionalized n-alkanethiol self-assembling monolayer on the biosensor chip. After verifying the specificity of the biosensor system with a panel of monoclonal antibodies to ß2GPI, we analyzed sera from healthy donors and patients suffering from APS, systemic lupus erythematosus (SLE), syphilis, or parvovirus B19 infections. The SPR results were compared with ß2GPI-specific ELISA.

Results: Using the SPR biosensor, we recorded antigen binding curves with response levels in the range of 50–500, resonance units (RU) for anti-ß2GPI ELISA-positive APS patient sera. The amplitudes of the antiphospholipid antibody (APL) responses in the biosensor correlated with the overall IgG and IgM anti-ß2GPI ELISA titers with a correlation coefficient of 0.87. Moreover, we observed immunoglobulin isotype-specific association and dissociation profiles for APL binding of different APS patient sera to the biosensor-immobilized ß2GPI. In contrast to APS patient samples, no significant anti-ß2GPI binding (response levels <35 RU) was observed in samples from healthy individuals or from patients suffering from SLE, syphilis, or parvovirus B19 infection.

Conclusions: The SPR biosensor system enables specific detection of APS-associated ß2GPI-reactive APL and differentiation from ß2GPI cross-reactive antibodies that occur frequently during acute infections. The established association/dissociation plot for anti-ß2GPI responses in APS patient sera gives additional information regarding the influence of anti-ß2GPI IgG and IgM isotype distribution.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The antiphospholipid syndrome (APS)1 is an autoimmune disorder characterized by arterial and venous thromboses, recurrent fetal loss, and increased serum titers of antiphospholipid antibodies (APLs), such as lupus anticoagulants and antibodies against cardiolipin (CL) (1).

According to the Sapporo classification criteria for APS, identification by ELISA of APLs in moderate to high serum titers is a significant finding only when it persists for more than 6 weeks and is closely associated with clinical features (2). The poor positive predictive value of APL detection is mainly attributed to the frequent presence of these autoantibodies in other autoimmune diseases, particularly systemic lupus erythematosus (SLE), and in response to various infectious agents such as Treponema pallidum or parvovirus B19. In the latter, occurrence of serum APLs is usually not correlated with thrombotic events.

Although APL was initially thought to bind directly to the phospholipid CL, results of 3 independent studies revealed that ß2-glycoprotein I (ß2GPI), a highly glycosylated plasma protein, is involved in immune complex formation (3)(4)(5). Since then, evidence has suggested that ß2GPI reactivity is related to the pathogenic potential of APLs, leading to the classification of ß2GPI-dependent autoimmune- and ß2GPI-independent infection-associated types of APLs (6)(7).

Although a complex of CL and ß2GPI was identified as the main antigenic determinant, Arvieux et al. (8) reported that most APLs also recognize ß2GPI linked to a solid support in the absence of any phospholipid. Other studies have revealed significant correlations between APL reactivities against ß2GPI and those against CL, providing additional evidence that ß2GPI alone may be used as a specific marker for APS diagnosis (9)(10). Even in the case of anti-ß2GPI antibodies (anti-ß2GPI), however, patients exhibit heterogeneous polyclonal antibody responses. The identification of heterogeneous responses strongly associated with clinical symptoms subsequently leads to the detection of infection-induced APLs not involved in the development of an APS. This process makes testing at 2 time points necessary for differentiation of APS-associated from infection-associated APLs. Anti-ß2GPI may occur as IgM, IgG, or IgA. The significance of IgM and IgA isotypes, however, is questionable (11)(12)(13). The finding that particularly high affinity anti-ß2GPI IgGs are associated with clinical signs (14)(15) suggests that antigen-induced affinity maturation of polyreactive natural IgM antibodies and IgG isotype switching both contribute to the development of APS (16). Thus, avidity testing was considered an appropriate method for differentiating between pathogenic and nonpathogenic anti-ß2GPI (17).

ELISA does not allow reliable differentiation of affinity and avidity of antibody binding. Therefore, we applied a surface plasmon resonance (SPR) biosensor device that allows resolution of association and dissociation phases to monitor the reactivity of APLs in APS patient sera to surface-immobilized human ß2GPI.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The principles of SPR technology are described in the Data Supplement Text that accompanies the online version of this article at http://www.clinchem.org/content/vol53/issue6 . In a typical biosensor run, one of the interaction partners is coupled to the sensor surface, whereas the dissolved opponent passes over the surface under continuous flow conditions. The SPR signal is generated by a change in mass concentration over the biosensor chip as the soluble analyte binds to or dissociates from the immobilized ligand. Binding is expressed in an increase of arbitrary resonance units (RU) and plotted in the form of a sensorgram. The interaction curves were evaluated for total amounts of bound antibody and apparent percentage of dissociation, the latter being a measure of immune complex stability. Our goal in analyzing both variables was to gain information on the overall avidity of the autoantibody response.

patients
We studied 30 patients with APS, 9 with SLE, 10 with positive results for the Venereal Disease Research Laboratory (VDRL) test for syphilis, and 20 with parvovirus B19 infection. All serum samples were leftover human specimens that were not individually identifiable, in accordance with Food and Drug Administration guidelines (18). The serum samples were accompanied by clinical information, but the sample source was not identifiable to the investigator. All APS patients whose sera were used in this study satisfied the Sapporo classification criteria for APS (19), and the SLE patients fulfilled >4 of the revised American College of Rheumatology criteria for the classification of SLE (20). We also obtained 20 samples from apparently healthy controls recruited from the laboratory staff. The controls were age matched and were assumed on the basis of a medical and clinical chemistry examination to be free of any acute or chronic disease. Written informed consent was obtained from these participants.

Each blood sample was collected in 10-mL tubes without anticoagulant, and after clotting was centrifuged at 1500g for 15 min. Sera were collected and stored in aliquots at –70 °C.

elisa
Concentrations of ß2GPI-specific IgG and IgM APLs in sera were determined with ELISA reagent sets manufactured by Orgentec and Phadia, subsequently referred to as ELISA-A and -B, respectively. Results were interpreted according to the following cutoff values: ELISA-A, 8 kU/L; ELISA-B, 15 kU/L.

apparatus
ß2GPI immobilization and APL interaction analyses were carried out in a BiaCoreX® SPR biosensor (Biacore AB) at 25 °C. For all measurements, SIA Kit Au biosensor chips from Biacore AB were used.

removal of IGG and IGM
We used agarose beads coated with goat antibodies (Sigma) against the IgG Fc-fragment or the IgM µ-chain. We added 20 µL of a 50% suspension of antibody agarose in HEPES-buffered saline to 180 µL of 1:90 prediluted serum. After a 30-min incubation, the suspensions were centrifuged at 10 000g for 10 min. Supernatants were collected and assayed immediately for ß2GPI binding activity in the SPR biosensor.

Details on the preparation of the self-assembling monolayer (SAM), biosensor measurements, kinetic analyses, and statistics, as well as data on reagents and antibodies, are given in the online Data Supplement Text.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
immobilization of ß2gpi
Preliminary experiments with ß2GPI, linked to a carboxymethyl dextran (CMD) hydrogel matrix of CM5 chips from Biacore AB, revealed that binding of ß2GPI-reactive APLs on the SPR biosensor strongly depends on the surface density of ß2GPI (data not shown). To gain surfaces with high ligand densities, we established planar carboxyl-terminated SAM surfaces, allowing ß2GPI to be coupled in the range of 2600–3000 RU. Because 1 RU resembles a surface mass change of 1 pg protein/mm2, these response levels correspond to 2.6–3.0 ng/mm2 of immobilized protein. With the SAM-based immobilization strategy, we obtained significantly higher sensor signals for ß2GPI binding of the APLs than with the commonly used CMD matrix. Moreover, the SAM-covered gold surface was characterized by extraordinarily high stability attributable to reorganization of alkyl chains to a semicrystalline structure (21). Reproducibility was assessed on 3 separate biosensor chips by making multiple injections of 1 APS serum with ß2GPI IgG ELISA titers of >100 kU/L. After 50 serum injections and rigid regenerations to remove the surface-bound antibodies, the mean loss of ß2GPI-specific activity obtained with the reference serum was at most 8%.

binding of control antibodies to the ß2gpi-coated surface
We first tested the functionality of the antigen-coated surface with the mouse IgG1 monoclonal antibody (mAb) 5F10.F3 raised against purified human ß2GPI, and the mAbs HL5B and JGG9 originally derived from APS patients with ELISA-defined specificities against CL and ß2GPI (16)(22). 5F10.F3 bound specifically to ß2GPI (Fig. 1A ), with no reactivity against transferrin on the reference cell. In contrast, the human IgG1{kappa} control antibody, injected at the same concentration as 5F10.F3, showed no reactivity on either the transferrin- or the ß2GPI-coated biosensor surface. We determined the value of the apparent first dissociation constant (KD1) by fitting 4 binding curves of 5F10.F3, representing concentrations of 34, 17, 11, and 9 nmol/L, to the bivalent binding model of the BIA evaluation program. This KD1 value, 9.52 nmol/L, was taken as a measure for the affinity of 1 antibody binding site. The quality of the fitting analysis was indicated by a {chi}2 value of 7.8. For a rough approximation of avidity, we calculated the overall KD by applying the 1:1 Langmuir model to these sensorgrams. We obtained a value of 2.27 nmol/L, considered reasonable because the calculated {chi}2 value of 8.04 was in the same range as the value for the bivalent binding mode. From the 2 human mAbs, the IgG HL5B depicted no ß2GPI-specific reactivity, whereas the IgM JGG9 showed a strong binding signal (Fig. 1B ). By kinetic analyses of 4 JGG9 sensorgrams, representing concentrations of 1.0, 0.4, 0.2, and 0.1 nmol/L, the apparent KD1 and KD values were calculated as 0.71 nmol/L ({chi}2 = 3.5) and 0.13 nmol/L ({chi}2 = 4.4), respectively.


Figure 1
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  		Figure 1. Specificity analysis of the ß2GPI-coated SPR biosensor surface with a panel of mAbs.

(A), binding profiles of the mouse antihuman ß2GPI IgG1 F510.F3 (black) and a human myeloma-derived IgG{kappa} (gray) as irrelevant control. (B), binding profiles of the anti-ß2GPI ELISA-positive human APL JGG9 (black) and the anti-ß2GPI ELISA-negative human APL HL5B (gray). Each antibody was applied at a concentration of 25 mg/L.

biosensor analyses of patient sera
Whereas sera of healthy donors showed no ß2GPI-specific activity, for sera of syphilis patients under low-salt conditions a low-to-moderate degree of binding was observed. This binding was completely blocked by the addition of 300 mmol/L NaCl to the HEPES-based running and dilution buffers.

The maximum binding signals after injection of diluted sera of healthy donors under appropriate experimental conditions (300 mmol/L NaCl) was as small as ~35 RU, whereas sera of anti-ß2GPI ELISA-positive APS patients showed values of 50–550 RU (Fig. 2A ).


Figure 2
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  		Figure 2. SPR biosensor detection of anti-ß2GPI in serum.

(A), APS patients (black lines) and healthy donors (gray lines). (B), non-APS patient groups tested positive in the VDRL test for syphilis (top), suffering from SLE without secondary APS (middle), or affected by parvovirus B19 infection (bottom).

By calculating the mean RU value plus 3 SDs for the negative control sera, we obtained a cutoff value of 51 RU. By use of this cutoff, positive and negative results for specimen anti-ß2GPI activity were the same as those measured with ELISA (see Table in the online Data Supplement). On the basis of the biosensor and ELISA data presented in the Table in the online Data Supplement, the Spearman rank coefficients between the amplitudes of the APL biosensor responses and the sum of the respective IgG and IgM anti-ß2GPI titers assessed with the 2 ELISA were calculated. In both cases the statistical test gave a coefficient r of 0.87 with P <0.0001. All sera of SLE patients without secondary APS and of patients with positive VDRL test or parvovirus B19 infection were negative for ß2GPI-specific APLs (Fig. 2BUp ).

kinetic analyses
Because of the polyclonal nature of the APL response, sensorgrams recorded for diluted patient serum samples are the sums of multiple antibody-ß2GPI interactions. As a consequence, equilibrium and kinetic rate constants cannot be determined by nonlinear regression analysis. Therefore, we calculated the relative binding affinity of APLs to ß2GPI by determining the amplitude of the SPR signal after 270 s of sample injection and the percentage decrease of the maximum binding signal after 300 s of dissociation. Whereas the first value serves as an indicator of the amount of antibody-antigen complex formation, the latter reflects mainly complex stability. A representative sensorgram, demonstrating the selection criteria of these 2 SPR-derived binding variables, is presented in Fig. 3A .


Figure 3
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  		Figure 3. Representative sensorgram depicting the SPR binding variables.

(A), association levels are defined by the SPR response after 270 s of serum injection (response at t270s), whereas the amount of dissociation is expressed as percentage decrease of the SPR response at t270s after 300 s of buffer flow. (B), list of classification criteria for the division of APS sera into subgroups on the basis of anti-ß2GPI IgG and IgM antibody titers as determined by ELISA-B. Each group is marked with a unique symbol for better identification in the subsequent comparison of anti-ß2GPI IgG and IgM distribution and SPR binding variables. (C), association/dissociation plot with values derived from sensorgrams. The SPR binding variables are plotted as described in A, with the response at t270s on the y axis and the percentage of dissociation on the x axis. Data points are labeled according to the classification scheme presented in B and represent the mean of serum injections over 3 biosensor chips on which the same amount of ß2GPI was immobilized. SDs are displayed as gray lines. Because the percentage of dissociation (reflecting the stability of the antibody-ß2GPI complex) is plotted in reverse order, serum samples with a fast dissociation from the ß2GPI biosensor surface are located on the left, and those with a slow dissociation are located on the right. Sera with negative ELISA results are clustered together in the bottom right corner of the plot. As indicated by the different symbols, APS sera with dissociation values of 40%–80% are characterized by high IgG and low to moderate IgM titers, whereas those with 10%–30% dissociation contain high titers of ß2GPI-specific IgM.

We analyzed a series of 30 patient sera on 3 separate ß2GPI biosensor chips and calculated the mean (SD) value of both SPR-derived binding variables for each serum sample. APS sera were divided into subgroups on the basis of their anti-ß2GPI IgG and IgM ELISA titer (Fig. 3BUp ). As shown in the association/dissociation plots in Fig. 3CUp , APS sera with dissociation values of 40%–80% are characterized by high IgG and low to moderate IgM titers, whereas those with 10%–30% of dissociation contain high titers of ß2GPI-specific IgM. Overall, it is evident that the position of a ß2GPI-positive serum along the x axis in the scatter diagram depends on the ratio of IgG and IgM isotype distribution.

Next, we investigated whether these differences in affinity and avidity between the 2 antibody isotypes are also detectable in APS sera depicting increased titers of both anti-ß2GPI IgG and IgM. For separate examination, the 2 antibody isotypes were selectively removed from 3 APS sera by preincubation with antihuman IgG- or IgM-specific agarose beads. As shown in Table 1 , the original dissociation profile of all 3 sera could be resolved into an IgG-mediated fast and IgM-mediated slow component. Values for the SPR response and percentage of dissociation in Table 1 were derived from the SPR sensorgrams presented in the Figure in the online Data Supplement. No correlation was found between the amount of IgG and IgM binding to biosensor-immobilized ß2GPI and the corresponding ELISA titers, which can be explained by the fact that the amplitude of the SPR signal depends not only on the concentration of the analyte but also on its molecular mass and the kinetic rate constants of the ligand interaction.


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  		Table 1. Differentiation of the overall anti-ß2GPI dissociation profile into its IgG-mediated fast and IgM-mediated slow component as determined for 3 APS patients with both increased anti-ß2GPI IgG and IgM titers.1


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ELISA detection of anti-ß2GPI is considered to be more specific for the diagnosis of APS than that of antiphospholipid autoantibodies, including CL (23)(24). Some studies, however, have indicated that antibodies against ß2GPI can also be found in patients with parvovirus B19, leptospirosis, syphilis, and other infections (25)(26)(27). Because these patients did not show clinical signs of APS, the specificity of anti-ß2GPI detection by ELISA is questionable. Therefore, our aim was to evaluate ß2GPI binding of APS- and infection-associated anti-ß2GPI in an SPR biosensor system. As demonstrated, our biosensor assay allows reliable detection of ß2GPI-reactive APLs in sera of APS patients. No antibody purification steps are required before analysis. The excellent specificity of the biosensor assay for APS-associated anti-ß2GPI was demonstrated by the complete absence of ß2GPI-specific responses in the control serum samples from patients with SLE without secondary APS, syphilis, or parvovirus B19 infection.

In accordance with ELISA, we observed that high ß2GPI surface densities are required for the sensitive SPR detection of anti-ß2GPI in serum of APS patients. With this finding it became clear that the analytical quality of the biosensor strongly depended on the composition of the functionalized biolayer attached to the gold surface. By application of an SAM rather than a CMD hydrogel matrix, we achieved high ß2GPI immobilization rates. The assembly of ß2GPI onto the planar and densely packed rigid monolayer may also promote increased access by APS-associated anti-ß2GPI to their antigenic sites.

We tested the functionality of the biosensor with antibody controls of known specificities, including the monoclonal APLs JGG9 and HL5B. As expected from the ELISA measurements, JGG9 demonstrated strong ß2GPI binding, whereas HL5B showed no significant binding. A kinetic evaluation of the sensorgrams resulted in the determination of KD1 and KD values for the JGG9-ß2GPI interaction of 0.71 and 0.13 nmol/L. By comparison with the mouse antihuman mAb 5F10.F3, it became clear that JGG9 can bind to ß2GPI as potently as high-affinity anti-ß2GPI IgG antibodies. Although the high affinity of the JGG9 mAb can be in part explained by the occurrence of somatic hypermutations (16), the unexpectedly low influence of avidity on the overall dissociation rate (indicated by a KD1:KD ratio of 5.46 between the values) may arise from the inability of the biosensor to differentiate between mono- and multivalent binding if kinetics are very fast. Additional SPR measurements with Fab-fragments derived from the mAbs by papain digestion will be required to elucidate this process.

Because the concentrations of ß2GPI-reactive APLs in sera needed for the estimation of affinity constants are unknown, we had to use total response levels and the percentage of dissociation after a defined period of time to describe binding affinity and avidity. We found that SPR association levels correlate well with anti-ß2GPI ELISA titers, whereas the degree of dissociation provided additional information concerning the binding strength of the overall ß2GPI-directed APL response. As a consequence, simultaneous acquisition of the amount and stability of immune complexes on a ß2GPI-coated biosensor surface provided information on the binding properties of APLs that is not accessible through conventional ELISAs. In future studies we will evaluate whether the availability of this information will allow a more accurate estimation of the pathogenicity of APLs in serum of APS patients.

The pathogenic potential of autoantibodies has been suggested to depend mainly on high-affinity and -avidity binding to self-antigens. Evidence has recently accumulated that effector functions associated with the Fc regions of the different isotypes are more likely to play the major role (28). Because they function in an early stage of the immune response, IgMs are usually polyreactive and of low affinity. Low antigen affinity, however, is compensated for by the pentameric structure of IgM, which promotes a relatively high avidity. Because the amount of APL binding correlates well with the density of immobilized ß2GPI on the biosensor surface, enhanced avidity seems a likely reason for the low dissociation rates observed in our biosensor system for APS patient sera with high anti-ß2GPI IgM titers. The capacity of IgM to mediate high-avidity binding, however, does not necessarily implicate association with cellular responses that contribute to clinical manifestations of autoimmune diseases. Indeed, some authors consider the prevalence of IgM-specific autoantigen responses to be protective against disease progression (29)(30). On the other hand, fast dissociation, typically found in sera of APS patients with high anti-ß2GPI IgG titers and low or completely absent IgM, strongly suggests the pathogenicity of ß2GPI-reactive IgG not being attributed to a high affinity of the variable region but instead to the effector functions of the Ig heavy chain constant regions. This explanation is in accordance with a study by Fulpius et al. (31), who found that immune complex–mediated vasculitis was induced by low-affinity anti-IgG2a rheumatoid factor mAb.

In summary, the presented SPR biosensor analyses demonstrate that covalent attachment of ß2GPI through a covalent amide linkage to the SAM-coated sensor chip is a suitable method for obtaining highly reproducible APL measurements in patient sera without noteworthy loss of activity after more than 50 measurement cycles. Our novel biosensor-based analytical assay system for ß2GPI-dependent APL detection in sera of APS patients offers relevant diagnostic advantages compared with conventional ELISA formats and readily lends itself to adaptation to the serological evaluation of other autoimmune diseases.


   Acknowledgments
 
Grant/funding support: Stiftung für Pathobiochemie und Molekulare Diagnostik of the Deutsche Vereinte Gesellschaft für Klinische Chemie und Laboratoriumsmedizin.

Financial disclosures: The authors declare that they have no competing financial interests.

Acknowledgements: We thank Anita Schreiegg for excellent technical assistance.


   Footnotes
 
2 These authors contributed equally to this work.

1 Nonstandard abbreviations: APS, antiphospholipid syndrome; APL, antiphospholipid antibody; CL, cardiolipin; SLE, systemic lupus erythematosus; ß2GPI, ß2-glycoprotein I; anti-ß2GPI, anti-ß2GPI antibodies; SPR, surface plasmon resonance; RU, resonance units; VDRL, Venereal Disease Research Laboratory; HBS, HEPES-buffered saline; SAM, self-assembling monolayer; CMD, carboxymethyl dextran; mAb, monoclonal antibody.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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