Method for the diagnosis of systemic lupus erythematosus (SLE)

Abstract

A method for the diagnosis of systemic lupus erythematosus (SLE) based on an interfacial process of antigen-antibody molecular recognition, specifically between anti-Ro52 and Ro52 protein, in a piezoelectric resonator, for application in the diagnosis of autoimmune diseases such as SLE.

Claims

1. A method for real time quantification of circulating autoantibodies in a human suffering from systemic lupus erythematosus (SLE), comprising: (a) incubating a blood serum sample obtained from said human in a piezoelectric resonator, wherein the piezoelectric resonator comprises a protein antigen Ro52 immobilized on a surface of the resonator forming a self-assembled monolayer and dispensing Ro52 protein on the surface such that a selective interaction of autoantigens with specific autoantibodies are obtained; (b) measuring, in the piezoelectric resonator, a variation of a surface concentration dΔΓ and a variation of a quality factor dΔQ during the selective interaction of autoantigens with specific autoantibodies; (c) determining a maximum value of dΔΓ/dΔQ with respect to time through formula $\begin{array}{cc}\frac{d\Delta \Gamma }{d\Delta Q}\approx -\frac{A\sqrt{{\rho }_0{\mu }_0}}{2f}\frac{Q_0{\delta }_3^2}{D_{0}2{\eta }_3^2}\frac{d{\rho }_1}{d\left(\frac{1}{{\mu }_1}\right)}& \end{array}$ wherein ΔΓ is a surface concentration shift and ΔQ is a quality factor shift, A is a surface area of the resonator, ρ.sub.0 is a density, D.sub.0 is a dissipation factor, μ.sub.0 is an elastic shear modulus, and Q.sub.0 is a quality factor of the piezoelectric sensor, f is a resonance frequency, ρ.sub.1 is a density and μ.sub.1 is an elastic shear modulus of a viscoelastic film coating the sensor, and η.sub.3 is a shear viscosity and δ.sub.3 is a viscous penetration depth of a Newtonian fluid in which a film is immersed, wherein the maximum value of dΔΓ/dΔQ is greater than 600 ng/cm.sup.2.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1. Representation of the geometry of a piezoelectric crystal coated with a viscoelastic film (general depiction of a concept that is known in the state of the art).

(2) FIG. 2. Graphic representation of function dΔΓ/dΔQ over time during the interaction of Ro52 protein with autoantibodies coming from anti-Ro+ autoimmune patients (a) and healthy individuals (b).

DESCRIPTION OF EMBODIMENTS

Example 1. Study of the Interaction Antibody Anti-Ro52/Ro52 Protein

(3) The chemical immobilization of specific autoantigens in the piezoelectric resonator and the determination of circulating autoantibodies were performed.

(4) Ro52 protein was immobilized by covalent anchoring, forming a self-assembled monolayer. The monolayer was created by submerging the piezoelectric surface for 16 h in a 10 mM mercaptopropionic acid solution and subsequent activation by 10 mM EDC/NHS for 60 min. Next, the surface was treated with 5 mM carbohydrazide for 60 min. Then 100 μL of Ro52 protein (33 mg/L) were dispensed on the sensor surface, being incubated for 60 min. Finally, the free active residues were blocked by means of treating the sensor surface with an aqueous solution containing EDTA, BSA and Polysorbate (TWEEN 20) at 0.05%, pH 8.5.

(5) The antibodies were purified from the serum of anti-Ro+ patients.

(6) Next, the variation of the surface concentration and the quality factor during the selective interaction of autoantigens with specific autoantibodies present in a PBS1x solution was recorded using a piezoelectric sensor.

(7) Subsequently, function ∂ΔΓ/∂ΔQ, wherein “Γ” is the surface concentration and “Q” is the quality factor, during the studied molecular recognition process was calculated (FIG. 2a). FIG. 2a shows the fingerprint characteristic of the molecular recognition process, presenting a maximum value at 3,000 ng/cm.sup.2 at 1,000 seconds. Therefore, as described above, this function shows that the monitored process corresponds to cooperative binding. The antibodies bind the antigen by means of a specific epitope-paratope interaction, for the Fc fragment of these antibodies to subsequently be recognized by the PRY-SPRY domain of Ro52 protein. This experiment demonstrates the use of the function to monitor transition states and molecular recognition patterns, establishing reaction pathways of the studied process.

Example 2. Study of the Application of Function dΔΓ/dΔQ in Healthy Patients and Anti-Ro+ Autoimmune Patients

(8) To demonstrate the goodness of use of function dΔΓ/dΔQ for the discrimination of patients with autoimmune diseases, the molecular recognition of anti-Ro52 antibodies from anti-Ro+ autoimmune patients and healthy individuals was studied. As shown in FIG. 2, the antibodies of both populations show a similar recognition behavior when represented over time.

(9) The pattern shown is a function with a peak shape having a maximum value around 1,000 seconds. Therefore, both processes have one and the same cooperative reaction mechanism. However, the antibodies from anti-Ro+ patients have a maximum value close to 3,000 ng/cm.sup.2.

(10) This characteristic pattern remains unchanged when solutions containing anti-Ro52 antibodies of a population of 130 anti-Ro+ autoimmune patients are analyzed. In contrast, when the serum of healthy patients is analyzed, the antibody recognition pattern presents a maximum value at 500 ng/cm.sup.2.

(11) Therefore, function ∂ΔΓ/∂ΓQ allows unequivocally discriminating anti-Ro+ patients by establishing a cutoff value at 600 ng/cm.sup.2, identifying characteristic recognition patterns.