ES2727261B2 - ARTIFICIAL IMMUNOCOMPLEXES AND THEIR USE AS CALIBERS IN CIRCULATING IMMUNOCOMPLEX DETECTION SYSTEMS B2GP1-aB2GP1 - Google Patents

Description

ARTIFICIAL IMMUNOCOMPLEXES AND THEIR USE AS CALIBERS IN CIRCULATING IMMUNOCOMPLEX DETECTION SYSTEMS B2GP1-aB2GP1

TECHNICAL SECTOR

The present invention falls within the technical field of medicine, more specifically in the field of immunopathology.

BACKGROUND OF THE INVENTION

Antiphospholipid syndrome (APS) or Hughes syndrome is a multisystem autoimmune disorder characterized by the appearance of thrombosis and / or gestational morbidity in patients who have antiphospholipid antibodies (aPL) in the blood.

APLs are a heterogeneous group of autoantibodies directed against phospholipids, phospholipid complexes associated with proteins, or phospholipid binding proteins in isolation.

Some of the associated APS manifestations are: blood clots (thrombosis), miscarriages, fetal death, premature delivery, stroke or transient ischemic accident.

Currently, there are no specific diagnostic criteria for PHC, although the patient classification criteria for clinical trials are used to diagnose the disease. These classification criteria, currently in force, were established in Sydney in 2004 (Miyakis S et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006; 4: 295-306) . These criteria mark the practice in all clinical laboratories and are summarized in Table 1.

Table 1: APS classification criteria.

One or more abortions before week 10 of documented anti-beta2 glycoprotein antibodies (aB2GPI) gestation.

One or more premature babies (<34 weeks)

pre-eclampsia or eclampsia Three or more miscarriages

Many patients with APS symptoms go undiagnosed as no consensus aPL is found that meets the laboratory criteria.

Currently, the diagnosis of the disease in clinical laboratories is carried out by detecting aPL using solid phase techniques such as ELISA, Fluoroenzyme Immunoassay or Multiplex technology, which detect anti-cardiolipin (aCL) antibodies or anti-beta-2 glycoprotein 1 (aB2GP1 or anti -B2GP1) in serum or plasma. To meet the criteria for laboratory classification as a subject with APS, antibodies must be maintained in the serum for at least 12 weeks.

Despite the relationship between the presence of aPL and disease, the value of the presence of aPL to predict events related to APS (for example, thrombosis or complications during pregnancy) is low. For this reason, new methods with a better predictive value are necessary for the identification of subjects carrying aPL antibodies and, therefore, are at risk of suffering, for example, a thrombotic event. An effective system to identify these patients will allow them to be treated more quickly, minimizing harm to the subject and its cost derived from the corresponding national health system.

Detection of circulating B2GP1-aB2GP1 immunocomplexes is one of the identification systems for patients at increased risk of developing thrombotic events among those who are carriers of aPL. These immunocomplexes have been described as risk biomarkers in the study of the pathogenesis of APS (Serrano M et al. Beta2-Glycoprotein I / IgA Immune Complexes: A Marker to Predict Thrombosis After Renal Transplantation in Patients With Antiphospholipid Antibodies. Circulation 2017, 135 (20): 1922-34).

These circulating B2GP1-aB2GP1 immunocomplex detection systems detect their concentration in the plasma or serum previously obtained from a subject. In order to determine its concentration, it is necessary to previously prepare a calibration (or calibration) curve based on known concentrations of the immunocomplexes (which function as calibers or calibrators), to determine, from the calibration curve equation, the concentration of the B2GP1-aB2GP1 immunocomplexes in the subject's sample and conclude if said subject has an amount of immunocomplexes above the risk point that will determine if he is at risk of suffering any pathology related to APS.

Current methods of determining circulating immunocomplexes in patient serum samples use known concentrations of native B2GP1-aB2GP1 immunocomplexes as calibers (Martinez-Flores JA et al. Detection of circulating immune complexes of human IgA and beta 2 glycoprotein I in patients with antiphospholipid syndrome symptomatology. J Immunol Methods 2015, 422: 51-8).

The closest state of the art is the document Martinez-Flores JA et al. 2015, which detects circulating B2GP1-aB2GP1 (IgA) immunocomplexes in patients with APS-associated symptoms. To determine the concentration of the immunocomplexes, a previous calibration curve is performed with native B2GP1-aB2GP1 immunocomplexes.

Native immunocomplexes or natural immunocomplexes are those obtained from human subjects. The native immunocomplexes have the drawback of being labile and unstable, which makes their detection difficult, generates great variability between detection assays, and their use is also limited to a few days or weeks. To increase the shelf life of these calibers, it is necessary to freeze native immunocomplexes, but these molecules are very sensitive to freeze-thaw cycles. In addition, because the number of analyzes for which the gauges can be used is limited and it is necessary to replace new gauges periodically in order to continue the analyzes on the patient samples, with the consequent element of uncertainty that generates the variability of the batches of sample gauges. Therefore, calibration curves for the detection of circulating B2GP1-aB2GP1 immunocomplexes prepared with known concentrations of native immunocomplexes are not adequate to establish the actual and reliable concentration of circulating immunocomplexes in the patient sample.

The objective technical problem is the need for stable gauges and to prepare a calibration curve in a B2GP1-aB2GP1 circulating immunocomplex detection system.

DESCRIPTION OF THE INVENTION

The present invention relates to an artificial immunocomplex comprising a fragment of human beta-2 glycoprotein 1 protein (B2GP1) linked to a fragment of the constant region of the heavy chain of an immunoglobulin (Fc).

Immune complexes or immunocomplexes (CICs) are macromolecules made up of subunits consisting of antigen (s) and antibody (s) linked together by low energy chemical bonds (Van der Waals forces, hydrogen bonds, or hydrophobic interactions).

For the purposes of the present invention, various types of immunocomplexes are distinguished:

1) circulating immunocomplexes: they are immunocomplexes produced naturally by the body of a subject, they circulate in the blood in a soluble way and they can be deposited on the inner wall of the blood vessels;

2) native (or natural) immunocomplexes: they are circulating immunocomplexes that have been isolated from blood or serum of a subject; and

3) artificial immunocomplexes (or artificial immune complexes): they have been produced in the laboratory. The sequence of these immunocomplexes does not necessarily correspond to that of circulating or native immunocomplexes. Antigen-antibody binding does not take place through low energy chemical bonds but through high energy peptide bonds or covalent bonds between the two subunits of the immunocomplex. Artificial immunocomplexes are stable for several months at 4 ° C.

Apolipoprotein H (Apo H), also called beta-2 glycoprotein 1 (B2GP1, reference UniProtKB P02749, Reference NCBI No.: NP_000033.2), is a 345 amino acid plasma glycoprotein that binds negatively charged substances such as heparin , phospholipids and dextran sulfate. This protein can bind phosphatidylserine and prevent activation of blood clotting by binding to phospholipids on the surface of damaged cells. Its sequence is encoded in the APOH gene.

The artificial immunocomplexes of the invention are made up of two subunits: one subunit corresponds to the B2GP1 protein described above (antigen) and the other subunit corresponds to a fragment of the constant region of the heavy chain of an immunoglobulin (antibody). The artificial immunocomplex immunoglobulin heavy chain constant region fragment is linked via a peptide bond, a covalent bond, to the amino or carboxyl terminus of the B2GP1 protein. In a preferred embodiment, the immunoglobulin heavy chain constant region fragment binds to the carboxyl terminus of the B2GP1 protein. The union of the subunits of the immunocomplexes by means of peptide bonds or covalent bonds has the advantage that, being a high energy chemical bond, it is very stable to changes in ionic strength, pH or temperature. In contrast, antigen-antibody binding of natural immunocomplexes is accomplished through low-energy chemical bonds, such as hydrogen bonds, hydrophobic interactions, and Van der Waals forces. These bonds are weak and the bonds mediated by them are unstable.

In one embodiment, artificial immunocomplexes can have at least one amino acid linker between the B2GP1 protein sequence and the immunoglobulin heavy chain constant region fragment.

In a preferred embodiment. The immunoglobulin heavy chain constant region fragment is selected from the IgM, IgG or IgA isotypes.

The first artificial immunocomplex of the invention is characterized in that it comprises the sequence SEQ ID NO: 1. The sequence SEQ ID NO: 1 corresponds to the B2PG1-IgM chimeric protein, consisting of the human B2PG1 sequence defined above in which the stop codon has been replaced by the amino acid lysine (K) and by means of a peptide bond the amino acids 240 to 590 in the chain weighing of the constant region of human immunoglobulin M (NCBI Accession No. AAS01769.1).

The first artificial immunocomplex of the invention is considered to have at least 98%, 99% or 100% identity with the sequence SEQ ID NO: 1.

The first artificial immunocomplex of the invention can be encoded in any DNA sequence whose translation takes place in SEQ ID NO: 1, or in place of a protein whose amino acid sequence has at least 98%, 99% or 100% of identity with the sequence SEQ ID NO: 1. As an example, and without thereby limiting the present invention, the nucleotide sequence of the first artificial immunocomplex comprises the sequence SEQ ID NO: 2.

The second artificial immunocomplex of the invention is characterized in that it comprises the sequence SEQ ID NO: 3. The sequence SEQ ID NO: 3 corresponds to the B2PG1-IgG chimeric protein, consisting of the human B2PG1 sequence defined above, in which the stop codon has been replaced by a linker peptide composed of the amino acids lysine (K), asparagine (D) and proline (P), linked by peptide bond to amino acids 31 to 246 of the constant region of the heavy chain of human immunoglobulin G (Accession No. NCBI AAW65947.1).

The second artificial immunocomplex of the invention is considered to have at least 98%, 99% or 100% identity with the sequence SEQ ID NO: 3.

The second artificial immunocomplex of the invention can be encoded in any DNA sequence whose translation of place to the sequence SEQ ID NO: 3, or of place to a protein whose amino acid sequence has at least 98%, 99% or 100 % identity with the sequence SEQ ID NO: 3. As an example, and without thereby limiting the present invention, the nucleotide sequence of the second artificial immunocomplex comprises SEQ ID NO: 4.

The third artificial immunocomplex of the invention is characterized in that it comprises the sequence SEQ ID NO: 5. The sequence SEQ ID NO: 5 corresponds to the B2PG1-IgA chimeric protein, consisting of the human B2PG1 sequence defined above in which the stop codon has been replaced by a linker peptide consisting of the amino acids lysine (K) and asparagine. (D), linked by peptide bond to amino acids 117 to 352 of the constant region of the heavy chain of immunoglobulin A (NCBI Accession No. AAC82528.1).

The third artificial immunocomplex of the invention is considered to have at least 98%, 99% or 100% identity with the sequence SEQ ID NO: 5.

The third artificial immunocomplex of the invention can be encoded in any DNA sequence whose translation to SEQ ID NO: 5, or to a protein whose amino acid sequence has at least 98%, 99% or 100% of identity with the sequence SEQ ID NO: 5. As an example, and without thereby limiting the present invention, the nucleotide sequence of the third artificial immunocomplex comprises SEQ ID NO: 6.

In a preferred embodiment, the artificial immunocomplex comprising the human beta-2 glycoprotein 1 protein (B2GP1) linked to a fragment of the constant region of the heavy chain of an immunoglobulin, is characterized in that the sequence of said immunocomplex comprises a sequence identified by SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5.

The object of the present invention is a method for calibrating a B2GP1-aB2GP1 immunocomplex detection analytical system comprising:

a) diluting an artificial immunocomplex to at least three different known concentrations;

b) determine the optical density of each concentration of the previous stage; c) carry out a calibration curve that relates each concentration in step a) with its respective optical density obtained in step b); d) obtain a mathematical function that defines the calibration curve of step c).

The calibration curve is performed with at least three known concentrations of the same type of artificial immunocomplexes (B2GP1-IgG, B2GP1-IgM or B2GP1-IgA) in step a). The curve is performed with at least 3, at least 4, at least 5, at least 6 or at least 7 known concentrations of the same type of artificial immunocomplexes.

Gauges or calibrators are known concentrations of certain molecules that serve to establish a standard curve (or calibration curve) that relates a quantifiable physical property (eg concentration) and a response or physical property of the same (eg optical density). In a preferred embodiment, the calibration curve it relates the optical density (OD) of the average of several measurements of the same caliber against the known concentration of said caliber.

The optical density value is proportional to the concentration of the substance of interest in a sample (artificial immunocomplexes), which allows the calibration curve to be made that relates the optical density and the concentration of the immunocomplexes. In a preferred embodiment, the optical density of a sample is absorbed at 450 nm.

The detection of the optical density can be carried out by any method known in the state of the art, such as colorimetry or turbidity. Analytical immunocomplex detection systems refer to any detection system and to quantify proteins, such as spectrometry, immunoassays, or immunoblotting. An analytical detection system is ELISA ( Enzyme-Linked ImmunoSorbent Assay) which, for example, may be based on alkaline phosphatase or peroxidase.

The mathematical function, curve function or curve equation is a quadratic equation of type y = ax bx2 c, in which the variable "y" is the known concentration of artificial immunocomplexes in a sample caliber, the variable "x" is the optical density value for said sample and the values a, b and c are the coefficients of the calibration curve. Values a, b and c are specific to each calibration curve and can be determined manually or automatically by software installed on the analytical detection system.

For the purposes of the present invention, the concentration of immunocomplexes in a sample is represented in units per milliliter (U / mL).

Another embodiment of the present invention is a method of determining the concentration of a circulating B2PG1-aB2GP1 immunocomplex in a sample obtained from a subject comprising:

a) dilute an artificial immunocomplex to at least three different concentrations; b) determining the optical density of each concentration of immunocomplexes from the previous step;

c) carry out a calibration curve that relates each concentration in step a) with its respective optical density obtained in step b); d) obtain a mathematical function that defines the calibration curve of the step c);

e) measuring the optical density of a biological sample obtained from a subject; and f) determining the concentration of circulating B2GP1-aB2GP1 immunocomplexes in the sample from the function obtained in step d).

In a preferred embodiment, the subject's biological sample is serum or plasma. In a more preferred embodiment, the subject's biological sample is serum. Serum is a cell-free liquid that is obtained from the blood after the clotting process. Serum differs from plasma fundamentally by lacking fibrinogen and platelets.

To determine the concentration of a circulating immunocomplex in a sample from a subject, a calibration curve must be previously performed, as previously indicated, that relates the known concentration of immunocomplexes with respect to a quantifiable physical characteristic, such as optical density.

The determination of the concentration of the circulating immunocomplexes in step d) is carried out by substituting in the equation obtained in step d) the variable "x" by the value of the optical density obtained in step e).

Another embodiment of the present invention is directed to a method of determining whether a subject is at risk for manifestations associated with antiphospholipid syndrome (APS). This method comprises determining the concentration of circulating B2GP1-aB2GP1 immunocomplexes in a sample from a subject according to any of the methods defined above, in which a concentration value of circulating B2GP1-aB2GP1 immunocomplexes is equal to or greater than 20.5 U / mL is indicative that the subject is at high risk of suffering pathological manifestations associated with APS. In another embodiment, a value of less than 20.5 U / mL of circulating immunocomplexes is considered to be indicative that the subject has a low risk of suffering manifestations associated with APS, a risk that is equal to that of the healthy population, a population carrying aPL antibodies or aB2GP1 antibody-bearing population that do not form circulating immunocomplexes.

Determining the concentration of immunocomplexes in patient samples allows the identification of subjects with antiphospholipid antibodies (aPL) who are at high or low risk of suffering a thrombotic event or other manifestations. pathologies associated with APS. The risk is determined by the presence of immunocomplexes above a numerical value corresponding to a cut-off point that is determined by analysis of ROC curves.

A person is at risk of developing a manifestation associated with APS when the probability of suffering the associated pathology is at least twice the risk of the general population. For example, people with a concentration of circulating B2GP1-aB2GP1 immunocomplexes (IgA or IgG isotypes) equal to or greater than 20.5 U / mL have a risk of developing an APS-associated manifestation of at least 3%, compared to the general population that has a risk of 0.6% (five times higher). Studies have been described in which the risk of immunocomplex carriers is greater than 30% (Serrano et al., 2017).

In a preferred embodiment, the subject who is at risk for manifestations associated with antiphospholipid syndrome (APS) is a carrier of antiphospholipid antibodies (aPL). A subject carrying aPL is a person who has been detected at least one aPL of any target: lupus anticoagulant (aAL), anticardiolipin (aCL) or anti-beta2 glycoprotein (aB2GP1), of any isotype (IgG, IgM or IgA). A subject carrying aPL antibodies may manifest signs and symptoms of any of the pathological manifestations associated with APS or be an asymptomatic carrier.

In a preferred embodiment, the method determines whether the subject is at risk for antiphospholipid syndrome or any of its associated pathological manifestations, such as blood clots (thrombosis), miscarriages, fetal death, premature delivery, stroke, or transient ischemic accident.

Thrombosis or a thrombotic event is any incident in which a thrombus (clot) develops in an artery. It can manifest as angina pectoris, ischemic stroke, or peripheral arterial disease. Minor thromboses such as thrombophlebitis and hemorrhoids are excluded from the definition of thrombotic events.

Another embodiment of the invention relates to the use of any of the artificial immunocomplexes of the invention B2GP1-IgG, B2GP1-IgM or B2GP1-IgGA as a gauge in a detection system of circulating immunocomplexes comprising at least a fragment of the bound B2GP1 protein to an aB2GP1 antibody fragment (B2GP1-aB2GP1).

A stable and reliable calibration system allows the detection of circulating immunocomplexes of a subject allows to determine more precisely the concentration of these immunocomplexes and favors that a patient at risk of suffering antiphospholipid syndrome or any of its associated manifestations, is treated more quickly. minimizing harm to the patient.

Artificial immunocomplexes have the same anti-B2GP1 antigenic recognition properties as native aBGP1 antibodies or anti-B2GP1 monoclonal antibodies. In addition, they are more than 100 times more stable than natural immunocomplexes, allowing them to be used as gauges in circulating immunocomplex detection systems for weeks thanks to their stability and reproducibility between assays.

The advantages of artificial immunocomplexes and their use as gauges in detection systems of circulating B2GP1-aB2GP1 immunocomplexes are:

- Stable for several months at 4 ° C;

- Reproducibility between assays (little inter-assay and intra-assay variability); - Ease of obtaining using standard molecular biology techniques;

- Antigenic recognition by monoclonal anti-B2GP1 antibodies identical to the recognition by native immunocomplexes.

DESCRIPTION OF THE FIGURES

Figure 1: shows the detection of artificial complexes by ELISA developed with peroxidase (A, B) or alkaline phosphatase (C). The dilutions used were: 1: 1 (dilution -); 1: 2; 1: 4; 1: 8; 1:16; 1:32; 1:64 (Dilution); white (C-). A : B2GP1-IgM, B : B2GP1-IgG, C : B2GP1-IgA.

Figure 2: Inter-assay variability of 10 trials with 8 dilutions of the B2GP1-IgM construct. The X-axis represents the concentration of artificial immunocomplex and the Y-axis represents the optical density value measured at 450 nm. A : medium, B : medium.

Figure 3: Inter-assay variability of 10 trials with 8 dilutions of the B2GP1-IgG construct. The X-axis represents the concentration of artificial immunocomplex and the Y-axis represents the optical density value measured at 450 nm. A : medium, B : medium.

Figure 4: Inter-assay variability of 10 trials with 8 dilutions of the B2GP1-IgA construct. The X-axis represents the concentration of artificial immunocomplex and the Y-axis represents the optical density value measured at 450 nm. A : medium, B : medium.

Figure 5: Calibration curve and corresponding equation for the calibers B2GP1-IgM ( A ), B2GP1-IgG ( B ) and B2GP1-IgA ( C ), calculated with the mean values for each point of the 10 experiments referred to in Figures 2 to 4 respectively. To the right of each curve, the equation of the curve, the coefficient of determination (R2) and the P value of the study for each gauge are represented.

Figure 6 . Comparison between B2GP1 immunocomplex calibration systems. A (blank), BH serial dilutions 1, 1: 2, 1: 4, 1: 8, 1:16, 1:32 and 1:64 in PBS respectively of concentrations of artificial B2GP1-IgA immunocomplexes (in duplicate, samples a and b ) and from three samples of native B2GP1-IgA immunocomplexes (aB2GP1) obtained from three patients (samples # 1, # 2 and # 3).

EXAMPLES

With the intention of showing the present invention in an illustrative way, although in no way limiting, the following examples are provided.

Example 1: Obtaining artificial immunocomplexes

Fusion proteins, chimeric proteins, or artificial immunocomplexes were generated by fusing the B2GP1 protein sequence to a fragment of the heavy chain constant region of IgM, IgG, or IgA immunoglobulins.

The sequences coding for the B2GP1-IgM (SEQ ID NO: 1), B2GP1-IgG (SEQ ID NO: 3) and B2GP1-IgA (SEQ ID NO: 5) constructs were amplified by PCR (30 cycles with a temperature of priming at 60 ° C 1 min and an expansion time of one minute at 72 ° C). The amplified sequences are then introduced into the plasmid pLVX-IRES-tdTomato (Clonteck, Takara Bio USA, Inc., Mountain View, CA 94043, USA). 3 plasmids were obtained, each containing respectively the nucleotide sequences B2GP1-Mu-C2C3C4 (B2GP1 fused to constant domains 2, 3 and 4 of the IgM heavy chain, SEQ ID NO: 2), B2GP1- FcIgG (SEQ ID NO: 4) and B2GP1-FcIgA (SEQ ID NO: 6). Each of the plasmids was introduced into the Stellar competent E. Coli cells bacteria (Clontech, Takara Bio USA, Inc.) by heat shock according to the manufacturer's protocol. Positive colonies were selected by antibiotic selection and cultured for 24-48h at 37 ° C. Plasmid DNA from the transforming colonies was extracted and purified using a purification kit, as directed by the manufacturer (Clontech, Takara Bio USA, Inc.).

Human HEK-293 cells (embryonic kidney) were then transfected with Lipofectamine 2000 according to manufacturer specifications (Thermofisher Scientific).

During cultivation the encoded fusion protein is expressed in the respective gene construct. The expressed proteins are secreted into the cell culture supernatant. After 500 ml of supernatant accumulated, the fusion protein was purified by affinity columns.

This procedure was performed for each of the three constructions. Once purified, fusion proteins (or artificial chimeric proteins or immunocomplexes B2GP1-IgM (SEQ ID NO: 1), B2GP1-IgG (SEQ ID NO: 3) and B2GP1-IgA (SEQ ID NO: 5) were obtained.

Figure 1 shows the detection of the constructs by ELISA developed with the peroxidase substrate (A, B) or alkaline phosphatase (C). The experiment was performed in quadruplicate using various dilutions of the purified fusion protein. The dilutions used were: 1: 1 (dilution -); 1: 2; 1: 4; 1: 8; 1:16; 1:32; 1:64 (Dilution); white (C-). A: B2GP1-IgM, B: B2GP1-IgG, C: B2GP1-IgA.

The fusion zone between the B2GP1 protein and the corresponding immunoglobulin heavy chain fragment is essential for the fusion protein to be stable and therefore to be used as a gauge in circulating immunocomplex detection systems.

The amino acids selected for the fusion of the fragment of the immunoglobulin heavy chains in each isotype, have the objective that the generated chimeric molecule conserves the physiological folding of the two fused subunits. Structure maintenance is key in the design of artificial immunocomplexes. The selected area of the heavy chain of each immunoglobulin that binds to the B2GP1 sequence is critical so that in the fusion product it maintains the three-dimensional structure of both subunits (B2GP1 and corresponding immunoglobulin heavy chain fragment), and therefore , its antigenic characteristics.

It has been found that if the binding between B2GP1 and the IgG heavy chain occurs, for example, at the beginning of the hinge region of the heavy chain, the resulting protein is of adequate size, but this construct is not recognized by anti-antibodies -B2GP1 H219 (Mabtech, Stockholm, Sweden) or by polyclonal anti-human IgG 1 antibodies (Sigma, Jackson and Inova).

In contrast, these anti-B2GP1 H219 antibodies (Mabtech, Stockholm, Sweden) and the three polyclonal anti-human IgG 1 antibodies (Sigma, Jackson and Inova) perfectly recognize their target molecule in the native form (B2GP1 or IgG1, respectively). ).

In the case where the fusion between B2GP1 and IgG takes place by excluding 21 amino acids from the hinge region of IgG1 from the heavy chain, it has been found that the resulting fusion product can be recognized by the anti-B2GP1 H219 monoclonal antibody and by the three aforementioned polyclonal anti IgG 1 antibodies.

These experiments demonstrate that the fusions between B2GP1 and fragments of the immunoglobulin heavy chain must be performed at certain positions in the sequence. From the data obtained with B2GP1-IgG, homologous regions in the IgM and IgA heavy chain sequences were identified.

Example 2: Calibration of circulating immunocomplex detection systems with artificial immunocomplexes.

Next, it was determined whether the constructs can be used in the calibration of circulating immunocomplex detection systems.

Detection of immunocomplexes was carried out as described in following the protocols written in Martínez-Flores et al. 2015.

A stock solution of each fusion protein (gauge) was prepared at 400 U / ml by diluting in PBS. Then 0.1% Proclin was added to each stock solution (preservative for antibody solutions). From the stock solution of each construct, dilutions were prepared in PBS with known concentration values to be used as calibres: 1.5 U / mL, 3.1 U / mL, 6.3 U / mL, 12.5 U / mL, 25 U / mL, 25 U / mL, 50 U / mL, 100 U / mL, and 200 U / mL.

10 independent assays (calibration curves) were performed for each concentration (gauge) of each of the artificial immunocomplexes in triplicate. The coefficients of variation were always less than 5%, for all points of all the experiments, indicating that the intra-assay variability of triplicates for each dilution is very low.

The inter-assay variability for the 10 8-point tests (dilutions of each construct) was also very low. Figures 2-4 show the variability of the measurements of each artificial immunocomplex ( Figure 2 : B2GP1-IgM, Figure 3 : B2GP1-IgG, Figure 4 : B2GP1-IgA) in which graph A shows the mean and the Graph B shows the median of the optical density (OD) values at 450 nm for each concentration. The determinations were carried out in an automated way in a Triturus® autoanalyzer (Grifols, Barcelona, Spain).

To determine the strength and reproducibility of the assays, the intraclass correlation coefficients of the data from the 10 calibration curves for each artificial immunocomplex were analyzed (Figures 2, 3 and 4). The intraclass correlation coefficient for the B2GP1-IgM fusion protein is 0.985 (95% CI: 0.964-0.997; p <0.0001), for the B2GP1-IgG fusion protein it is 0.991 (95% CI: 0.978- 0.998; p <0.0001) and for the IgA fusion protein 0.988 (95% Confidence interval 0.970 0.997; p <0.0001). These data indicate that the calibration curves performed using the artificial immunocomplexes of the invention they have a very low variability. Statistical analysis was performed using MedCalc V. 14.12 (MedCalc Software Bvba, Oostende, Belgium).

Figure 5 shows the calibration curves of the mean values of the 10 tests for each of the artificial immunocomplexes (AC). To the right of each curve is indicated the function (or equation) of the corresponding curve, its coefficient of determination (R2) and the P value of each test, which expresses the probability that the results obtained are due to chance. Values of p <0.01 are indicative that the results are not due to chance. The formulas are automatically obtained from the autoanalyzer software. The functions of the calibration curves correspond to a type equation y = ax bx2 c (U / mL = aOD bOD2 c). Substituting in the calibration curve the optical density values of a patient's sample, the concentration of circulating immunocomplexes is obtained.

Example 3. Comparison between calibration systems.

In order to show the differences between the native B2GP1-aB2GP1 gauges and the artificial B2GP1-Ig gauges, an experiment was conducted in which the two calibration systems were compared.

For this, serial dilutions 1, 1: 2, 1: 4, 1: 8, 1:16, 1:32 and 1:64 were performed in PBS of known concentrations of three samples of native B2GP1-IgA immunocomplexes (aB2GP1) obtained from three patients (samples # 1, # 2 and # 3) and another from a sample of artificial B2GP1-IgA immune complexes. The artificial immunocomplex assay was performed in duplicate (a, b).

The samples of the patients were obtained in the Immunology Service of the Hospital 12 de Octubre from samples sent for a health examination. Only the surplus serum from the samples was used anonymously as samples for testing.

The concentrations of immunocomplexes used as calibres were those indicated in Table 2 .

Table 2: Immunocomplex gauges

^B2GP1-IgA Patient # 1 Patient # 2 Patient # 3 Dilution _{(U / mL)} (U / mL) (U / mL) / mL) A Blank 0 0 0 0

B 1 800 204 156 50

C 1: 2 400 102 78 26

D 1: 4 200 56 39 13

E 1: 8 100 28 20 3.5

F 1:16 50 14 10 3.8

G 1:32 25 7 5 2

12,5

3,5

2,5

1 H 1:64

12.5

3.5

2.5

one

The determination of the levels of immunocomplexes was carried out following the protocol described in Martínez-Flores et al. (2015), in which calibers were added instead of sera: native immunocomplexes (patients # 1, # 2 and # 3) and artificial immunocomplexes (B2GP1-IgA).

Figure 6 shows the result of the experiment in which the differences in the concentration of immunocomplexes in relation to the intensity of color in each well are observed. Lanes AH correspond to the concentration of immunocomplexes indicated in Table 1. It is observed that artificial B2GP1-IgA immunocomplexes (in duplicate, samples a and b) can be used as gauges in the determination of circulating immunocomplexes in the same way as up to Date native B2GP1-IgA immunocomplexes (aB2GP1) were used. This experiment was repeated with the artificial immunocomplexes B2GP1-IgG and B2GP1-IgM, obtaining the same result as with B2GP1-IgA.

The function to calculate the units of each patient from the optical density of the assay based on the reference values of the calibration curve is performed automatically by the autoanalyzer software. The curve function is calculated by the autoanalyzer for each calibration test independently.

Example 4: Detection of circulating immunocomplexes in serum

In order to determine whether artificial immunocomplexes can be used as gauges in B2GP1-aB2GP1 circulating immunocomplex detection systems, A capture ELISA was performed in which the presence of the circulating immunocomplexes in the serum of the patients was detected.

86 consecutive samples were obtained from the seroteca of the Hospital 12 de Octubre (Spain) and the concentration of circulating immunocomplexes was determined as described in Martínez-Flores et al. (2015), using as concentrations the concentrations 200, 100, 50, 25, 12.5, 6.3, 3.1 and 1.5 of the artificial immunocomplexes B2GP1-IgM, B2GP1-IgG and B2GP1-IgA.

Nunc maxisorp ™ 96-well plates (A / S Nunc, Kamstrup, Roskilde, Denmark) were covered with 100 µl per well of 2 pg / ml solution of monoclonal anti-human B2GP1 antibody H219 (Mabtech AB, Nacka Strand, Sweden) in PBS at pH 7.4. The plates were incubated for 16 hours at 4 ° C and then washed three times with PBS-Tween-200.05% (PBS-T). The wells were then blocked for 1 hour at room temperature with 0.1% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) in PBS.

The plates were washed three times with PBS-T and then 100 pL of the test sera diluted in PBS 1: 100 in PBS were added. They were incubated 2 hours at room temperature, washed with PBS-T three times and the presence of each aB2GP1 antibody isotype was detected with 100 pL of goat anti-IgG, anti-IgM or anti-human IgA antibody (Jackson ImmunoResearch Laboratories , Inc., West Grove, PA, USA) conjugated to horseradish peroxidase, diluted 1: 1000 in PBS. The plates were incubated 30 minutes with the anti-human Ig antibody at room temperature and washed 3 times with PBS-T.

The reaction was developed with a tetramethylbenzidine (TMB) solution for 30 minutes and finally stopped with H2SO41N. The optical density (OD) of each well was read with a 450nm filter (reference filter, 620nm) and the absorbance of the wells containing the blank subtracted. The OD values obtained for each sample were used to calculate the amount of each type of immunocomplex according to the previously obtained calibration values. The entire procedure was performed in Triturus Analyzer (Diagnostics Grifols S.A., Barcelona, Spain).

Table 3 shows the OD values of the calibration curves for each artificial immunocomplex concentration. The values in the table correspond to the value recorded by the autoanalyzer minus the value of the blank in each case.

Table 3. Optical density (OD) of the calibres of each immunocomplex.

Concentration

DO B2GP1-IgM DO B2GP1-IgG DO B2GP1-IgA _{(U / mL)}

0 (white 0.066 0.047 0.043

200 3,150 2,417 2,944

100 2,223 1,425 2,485

50 1,384 0.906 1,599

25 0,752 0,529 0,882

12.5 0.363 0.309 0.490

6.3 0.205 0.226 0.260

3.1 0.103 0.092 0.130

0,054 0,012 0,0641.5

0.054 0.012 0.064

Table 4 shows the concentration for the samples as a function of their OD at 450 nm, calculated from the function of the calibration curve obtained from the results of Table 3 for each of the artificial immunocomplexes. For a subject to be considered to be at risk, it is necessary that the immunocomplex concentration value of at least one of the analyzed isotypes (IgM, IgG or IgA) is greater than 20.5 U / mL.

Table 4: Determination of circulating immunocomplexes in patient sera

DO 450 nm ^{Concentration}

_(U/mL)

_{(U / mL)}

Sample ^B2GP1-aB2GP1 B2GP1-aB2GP1

_{IgM IgG IgA IgM IgG IgA}

1 0.097 0.061 0.019 5.1 2.9 <2

2 0.183 0.091 0.063 6.3 4.3 2.5

3 0.378 0.116 0.155 10 5.6 4.9

4 0.043 0.065 0.159 4.5 3.1 5

5 0.085 0.1 0.079 5 4.8 2.9

6 0.056 0.063 0.049 4.6 3 2.1

7 0.144 0.09 0.136 5.8 4.3 4.4

8 0.086 0.427 0.08 5 22.5 2.9

9 0.075 0.074 0.075 4.9 3.5 2.8

10 1,952 0.156 0.467 85.3 7.6 16.1

11 0.105 0.125 0.118 5.2 6 3.9

12 0.071 0.059 0.083 4.8 2.8 3

13 0.135 0.118 0.054 5.6 5.7 2.3

, 218 0.082 0.058 6.9 3.9 2.4 Negative, 121 0.102 0.024 5.4 4.9 <2 Negative, 027 0.076 0.098 4.3 3.6 3.3 Negative, 049 0.065 0.036 4.6 3, 1 <2 Negative, 259 0.082 0.043 7.6 3.9 2 Negative, 095 0.061 0.099 5.1 2.9 3.4 Negative, 142 0.061 0.022 5.7 2.9 <2 Negative, 064 0.143 0.162 4.7 6.9 5.1 Negative, 341 0.094 0.18 9.2 4.5 5.6 Negative, 056 0.088 0.059 4.6 4.2 2.4 Negative, 066 0.145 0.123 4.8 7 4 Negative, 387 0.177 0.177 10.2 8.7 5.5 Negative, 06 0.04 0.021 4.7 <2 <2 Negative, 032 0.123 0.02 4.4 5.9 <2 Negative, 072 0.11 0.037 4.8 5 , 3 <2 Negative, 44 0.057 0.026 11.4 2.7 <2 Negative, 041 0.056 0.072 4.5 2.6 2.7 Negative, 029 0.071 0.017 4.3 3.4 <2 Negative, 225 0.045 0.074 7 2.1 2.7 Negative 0.1 0.063 0.093 5.2 3 3.2 Negative, 245 0.072 0.12 7.4 3.4 3.9 Negative, 24 0.057 0.027 7.3 2.7 <2 Negative, 125 0.064 0.099 5.5 3 3.4 Negative, 114 0.123 0.15 5.4 5.9 4.7 Negative, 051 0.11 0.116 4.6 5.3 3.8 Negative, 072 0.107 0.131 4.8 5.1 4.2 Negative, 031 0.137 0.046 4.4 6.6 2.1 Negative, 043 0.015 0.017 4.5 <2 <2 Negative, 073 0.055 0.057 4.8 2.6 2.3 Negative, 047 0.036 0.237 4.5 <2 7.4 Negative, 035 0.024 0.027 4.4 <2 <2 Negative, 145 0.048 0.582 5.8 2.2 21.4 IgA positive , 052 0.065 0.051 4.6 3.1 2.2 Negative, 64 0.094 0.069 16.9 4.5 2.6 Negative, 08 0.067 0.048 4.9 3 , 2 2.1 Negative, 568 0.053 0.099 14.8 2.5 3.4 Negative, 256 0.043 0.036 7.6 2 <2 Negative, 076 0.281 0.05 4.9 14.2 2.2 Negative, 044 0.058 0.035 4.5 2.7 <2 Negative, 035 0.077 0.022 4.4 3.6 <2 Negative 3.7 0.124 0.872 264.6 6 37.6 Positive IgM, IgA , 049 0.036 0.167 4.6 <2 5, 2 Negative, 57 0.076 0.082 14.8 3.6 2.9 Negative, 227 0.105 0.062 7.1 5 2.5 Negative, 217 0.024 0.062 6.9 <2 2.5 Negative, 05 0.055 0.024 4.6 2, 6 <2 Negative, 043 0.062 0.029 4.5 2.9 <2 Negative, 059 0.041 0.024 4.7 <2 <2 Negative 62 0.22 0.046 0.022 6.9 2.2 <2 Negative

63 0.054 0.057 0.046 4.6 2.7 2.1 Negative

64 0.125 0.063 0.544 5.5 3 19.6 Negative

65 0.086 0.23 0.021 5 11.4 <2 Negative

66 0.125 0.041 0.093 5.5 <2 3.2 Negative

67 0.087 0.067 0.107 5 3.2 3.6 Negative

68 0.042 0.059 0.048 4.5 2.8 2.1 Negative

69 0.164 0.038 0.123 6.1 <2 4 Negative

70 0.073 0.105 0.07 4.8 5 2.6 Negative

71 0.018 0.05 0.027 4.2 2.3 <2 Negative

72 0.089 0.045 0.029 5 2.1 <2 Negative

73 0.207 0.059 0.036 6.7 2.8 <2 Negative

74 0.08 0.058 0.06 4.9 2.7 2.4 Negative

75 0.145 0.033 0.041 5.8 <2 2 Negative

76 0.011 0.054 0.021 4.2 2.5 <2 Negative

77 0.063 0.051 0.076 4.7 2.4 2.8 Negative

78 0.128 0.06 0.025 5.5 2.8 <2 Negative

79 0.12 0.109 0.367 5.4 5.2 12 Negative

80 0.092 0.082 0.07 5.1 3.9 2.6 Negative

81 0.091 0.084 0.097 5.1 4 3.3 Negative

82 0.077 0.048 0.075 4.9 2.2 2.8 Negative

83 0.615 0.214 0.004 16.1 10.6 <2 Negative

84 0.049 0.099 0.069 4.6 4.7 2.6 Negative

85 0.177 0.076 0.036 6.3 3.6 Negative

1,22

0,052

0,018 40,1 2,4

Positivo IgM 86

1.22

0.052

0.018 40.1 2.4

IgM positive

From this experiment it is concluded that the calibration curve prepared with artificial immunocomplexes B2GP1-IgM, B2GP1-IgG and B2GP1-IgA serves to carry out calibration curves with high reproducibility for use in the detection of circulating immunocomplexes from patients.

SEQUENCE LISTING

The following sequences are described as part of the invention:

- SEQ ID NO: 1 Protein sequence of the artificial immunocomplex B2GP1-IgM. - SEQ ID NO: 2 Nucleotide sequence of the artificial immunocomplex B2GP1-IgM. - SEQ ID NO: 3 Protein sequence of the artificial immunocomplex B2GP1-IgG. - SEQ ID NO: 4 Nucleotide sequence of the artificial immunocomplex B2GP1-IgG. - SEQ ID NO: 5 Protein sequence of the artificial immunocomplex B2GP1-IgA. - SEQ ID NO: 6 Nucleotide sequence of the artificial immunocomplex B2GP1-IgA.

Claims (6)

1. An artificial immunocomplex comprising the human beta-2 glycoprotein 1 protein (B2GP1) linked to a fragment of the constant region of the heavy chain of an immunoglobulin, characterized in that the sequence of said immunocomplex comprises a sequence identified by SEQ ID NO. : 1, SEQ ID NO: 3 or SEQ ID NO: 5. 2. Method for calibrating an analytical detection system for circulating immunocomplexes B2GP1-aB2GP1, comprising: a) diluting the artificial immunocomplex of claim 1 to at least two different known concentrations; b) determine the optical density of each concentration of the previous stage; c) carry out a calibration curve that relates each concentration in step a) with its respective optical density obtained in step b); d) obtain a mathematical function that defines the calibration curve of step c). 3. Method for determining the concentration of a circulating B2GP1-aB2GP1 immunocomplex in a sample, comprising: a) diluting the artificial immunocomplex of claim 1 at least at two different concentrations; b) determine the optical density of each concentration of the previous stage; c) carry out a calibration curve that relates each concentration in step a) with its respective optical density obtained in step b); d) obtain a mathematical function that defines the calibration curve of step c); e) measuring the optical density of a biological sample obtained from a subject, f) determine the concentration of circulating B2GP1-aB2GP1 immunocomplexes in the sample from the function obtained in step d). 4. Method according to claim 3, wherein the subject's biological sample is serum or plasma. 5. Method for determining whether a subject is at risk of suffering pathological manifestations associated with antiphospholipid syndrome (APS), comprising determining the concentration of circulating immunocomplexes in a sample from a patient according to the method of claims 3 or 4, in that a concentration value equal to or greater than 20.5 U / mL is indicative that the subject is at high risk of suffering pathological manifestations associated with APS. 6. Use of any of the artificial immunocomplexes of claim 1 as a gauge in a B2GP1-aB2GP1 circulating immunocomplex detection system.