RU2848031C1 - Method for simultaneous semi-quantitative determination of autoantibodies to neuroantigens for diagnosis of brain lesions - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: invention relates to the field of biotechnology and medicine, in particular immunology, and is intended for the simultaneous determination of the level of autoantibodies to markers of brain lesions of various aetiologies for the purpose of early diagnosis, monitoring and personalisation of therapy in patients. The essence of the invention lies in the use of commercial recombinant protein preparations (NMDAR: NR2A, NR2B; D, DR1, DR2 and protein S100B) manufactured by Cloud-Clone Corporation (USA) (producer - Escherichia coli, cell localisation - membrane), which were immobilised in the wells of a removable polystyrene plate at a concentration of 3 μg/ml and incubated for at least 14 hours at 4^oC. To block free binding sites on the plate, a 1% solution of Tween 20 (pH 7.2) was used and incubated for 2 hours at room temperature (18-25°C). The conjugate was a conjugate of monoclonal antibodies against light and heavy polypeptide chains of human immunoglobulin G labelled with horseradish peroxidase (epitope C2 domain of IgG, no cross-reactions with IgE, IgA, IgD and IgM). A positive control sample (K+) (a mixture of recombinant IgG MCTAs to the studied neuroantigens produced by Cloud-Clone Corporation (USA)) and a negative control sample (K-) based on cow serum were used in the analysis. The samples analysed were blood serum.

EFFECT: enzyme immunoassay test system has been developed.

1 cl, 5 dwg

Description

The invention relates to the field of biotechnology and medicine, namely, it is used in immunology and is intended for the simultaneous determination of the level of autoantibodies to antigen markers of brain damage of various etiologies for the purpose of early diagnosis, monitoring and personalization of therapy in patients.

Currently, the incidence of central nervous system (CNS) diseases is steadily increasing, with most of them causing long-term disability and even severe incapacitation. One of the main pathological processes occurring in various types of acute and chronic CNS diseases is the detection of elevated levels of autoantibodies to various neurotransmitters and neuroreceptors. This has led to growing interest in both the clinical characteristics of such disorders with elevated autoantibody levels and the prevalence of these antibodies among psychiatric patients, which is important for timely diagnosis and the identification of personalized therapy options. A close relationship has been identified between dysfunction of neurotransmitter systems and the induction of autoantibodies to them in various forms of CNS pathology, where a number of neuroantigens play a key role, including N-methyl-D-aspartate receptors (NMDARs), in particular the NR2A and NR2B subunits; dopamine (D); dopamine receptors (DR1 and DR2); and the neuron-specific protein S100B.

NMDARs are important pathogenetic targets in many CNS diseases [4, 8], including stroke, hypoxia, ischemia, head injury, Huntington's, Parkinson's, and Alzheimer's diseases, epilepsy, alcoholism, neuropathic pain, schizophrenia, and mood disorders. Any homeostatic dysfunction of NMDAR activity can lead to various pathologies.

NMDAR hyperreactivity, which causes excitotoxicity, contributes to an increase in the level of autoantibodies and is of significant importance in the development of epilepsy, dementia, encephalitis of various etiologies, in the pathogenesis of stroke and other conditions [1, 2, 3, 4].

Elevated serum levels of autoantibodies to the S100B protein are found in patients with stroke [3]. Changes in dopamine autoantibody levels have been found in patients with epilepsy and severe alcoholism [2]. Elevated levels of autoantibodies to both NMDAR and dopamine receptors are found in autoimmune encephalitis [1]. Thus, the detection of a significant increase in autoantibody levels to a number of neuroreceptors and neurotransmitters during simultaneous analysis can be used both for the early diagnosis of an emerging pathological process and for the selection and correction of personalized therapy.

Cell-based assays (CBA) on cell cultures can be used to detect antibodies to various neuroantigens. However, CBA results provide a more qualitative assessment. Furthermore, the method involves the use of both primary neuronal cultures functionally expressing these receptors and transformed cell lines presenting various parts of the receptor domains [7]. However, CBA analysis is extremely expensive, requiring qualified personnel and expensive equipment. One of the most accurate, rapid, and accessible methods in laboratory diagnostics is enzyme-linked immunosorbent assay (ELISA). The closest technical approach to the claimed method is the enzyme-linked immunosorbent assay for determining autoantibodies to NMDAR in serum [5]. The essence of the described method is as follows: recombinant protein preparations (GRIN Ed1 and GRIN Ed2) are diluted with bicarbonate buffer (0.1 M NaHCO3, pH 9.2) to a final concentration of 10 μg/ml and immobilized in the wells of a 96-well plate (Greiner) at 100 μl per well. Incubate for at least 12 hours at 4° C in a humidified chamber. Next, the wells are washed twice with wash buffer (PBS, 0.05% Tween-20 pH 7.4). In order to block free binding sites on the plate, 100 μl of blocking buffer (PBS, 3% BSA pH 7.4) are added to the wells and incubated for 1 hour at 20° C on a rotary shaker (500 rpm). After incubation, the wells are washed three times with wash buffer. Before adding the biomaterial to the wells, patient serum is preincubated with lysates dialyzed against PBS, non-transformed strain DE3 BL21 E. coli , and the samples are pre-diluted 1:50, followed by incubation for 2 hours at 20°C. After washing, 100 μl of goat biotinylated antibodies to human IgG, IgM, IgA (H+L) (ThermoFisher Sci) are added to each well of the plate and incubated for 1 hour on a rotary shaker (500 rpm). After washing three times, 100 μl of streptavidin solution conjugated with peroxidase are added (according to the manufacturer's instructions, ThermoFisher Sci). After incubation and washing, the reaction is developed with tetramethylbenzidine (TMB) solution for 15-30 minutes, followed by the addition of 100 µl of 1M H2SO4. The results are detected using a photometer at 450 nm. The method for recording and interpreting the results is described as a ROC analysis for ELISA using GRIN1_ED2_TrxA as the antigen. To obtain diagnostic thresholds for the ELISA, its results are compared with verified cases of anti-NMDA encephalitis confirmed by the CBA method (INVITRO LLC, Russia). Based on the comparison results, an ROC curve analysis was performed to determine the ELISA results that offer the optimal balance between specificity and sensitivity.

Disadvantages of this method include the relatively high antigen concentration (10 μg/ml) immobilized in the wells, which reduces the ability of the immunoglobulin Fc fragments to unfold for complete capture. Since autoantibodies are classified as class G, the use of a polyclonal conjugate may distort sensitivity and affect the final test result, and the use of two conjugates in the assay increases the time required. Furthermore, the additional preincubation of patient serum with lysates dialyzed against PBS of the non-transformed DE3 BL21 E. coli strain, followed by incubation for 2 hours at 20°C, complicates the reaction process and increases the time required, which may impact both the sensitivity and specificity of the assay.

The aim of the invention is to develop a sensitive, specific, and easy-to-implement method using commercial recombinant antigens in an enzyme-linked immunosorbent assay system for the simultaneous testing of patients' serum for the presence of autoantibodies to neurotransmitters and neuroreceptors that are causative in CNS damage. This integrated approach can be recommended as a highly sensitive marker for the development of a system for early diagnosis, outcome prognosis, and therapy monitoring, enabling timely treatment adjustments.

The problem is solved by the described method. In the work, the indirect ELISA method was used [6]. Recombinant protein preparations (NMDAR: subunits NR2A, NR2B; D, DR1, DR2 and protein S100B) manufactured by Cloud-Clone Corporation (USA) (producer - Escherichia coli , cellular localization - membrane) are diluted with bicarbonate buffer (0.1 M NaHCO3, pH 9.2) to a final concentration of 3 μg / ml and added to the wells of a 96-well plate (Greiner) at 100 μl per well, incubated for at least 14 hours at 4 ° C. Then the wells are washed twice with wash buffer (PBS, 0.05% Tween-20 pH 7.4). To block free binding sites on the plate, 100 μl of blocking buffer (1% HSA, pH 7.2) is added to each well and incubated for 2 hours at room temperature (18-25°C). After incubation, the wells are washed three times with wash buffer. The conjugate is a conjugate of monoclonal antibodies against the light and heavy polypeptide chains of human immunoglobulin G labeled with horseradish peroxidase (MAB-anti-IgG-HRP) (the C2 epitope of the IgG domain does not cross-react with IgE, IgA, IgD, and IgM). The assay uses a positive control sample (K+), which is a substance containing a mix of recombinant IgG MABs to the neuroantigens under study, manufactured by Cloud-Clone Corporation (USA) HYPERLINK. Fetal bovine serum, which does not contain IgG antibodies to the neuroantigens being studied, serves as a negative control sample ( ^K- ). During incubation with the biomaterial being studied, the antigens interact with the antibodies present in the sample. In the first stage of the analysis, the autoantibodies form an antigen-antibody immune complex, which (stage 2) is detected by the conjugate. When the substrate (TMB) is added to the wells of the plate, the solution turns blue. The reaction is stopped by adding the stop reagent to the wells. The blue color of the solution changes to yellow. The optical density (OD) of samples containing specific autoantibodies (immunoglobulins class G) exceeds the OD of the negative control samples and is proportional to the content of the autoantibodies being studied. The analysis results are recorded using a vertical scanning photometer at a wavelength of 450 nm. Due to the lack of international reference samples of specific IgG to NMDAR: NR2A, NR2B; D; DR1; DR2 and S100B protein, the content of autoantibodies is expressed in units per milliliter (U/ml) and calculated using the formula given in the instructions for use.

The technical solution for implementing the proposed method is a test system that includes:

1. Immunosorbent - polystyrene tablet 1 pc., ready for use and consisting of 6 blocks of 2 strips for each neuroantigen.

2. Conjugate (MCAT-anti-IgG-HRP), 100-fold concentrate;

3. Washing buffer solution, 30x concentrate;

4. TMB (tetramethylbenzidine), ready to use;

5. Stop reagent, ready to use;

6. Positive control sample (K ⁺ ), ready for use;

7. Negative control sample (K- ⁾ , ready for use.

The test system is designed to test 6 patients for all studied antigens in duplicate, or 12 patients in single samples (96 determinations in total).

The analyzed samples are blood serum. The required volume of serum sample for analysis is 10 µl.

The method is implemented as follows: according to the scheme shown in Figure 4, 90 µl of the working buffer solution, 10 µl of the controls, and 10 µl of the test samples are added to each well of the plate. The plate is covered with a protective film and its contents are mixed by shaking on a shaker or gently tapping the frame for 20-30 seconds, after which it is incubated in a thermostat at 37°C for 1 hour.

At the end of the incubation, the liquid is removed from the wells of the plate by shaking, and the washing procedure is performed. 250 µl of the working buffer solution is added to each well of the plate. The washing procedure is repeated twice more. Residual moisture is carefully removed from the wells by tapping the inverted plate on filter paper. 100 µl of the working conjugate solution is added to each well of the plate, the plate is covered with a protective film, and incubated at 37°C for 1 hour. At the end of the incubation, the liquid is removed from the wells by shaking, and the washing procedure is repeated, after which the wells of the plate are additionally washed twice with purified or deionized water (250 µl each). Traces of liquid are removed from the wells by tapping the inverted plate on filter paper. 100 µl of TMB is added to each well. Incubate for 7-15 minutes (depending on the degree of blue coloration) in a dark place at room temperature (18-25°C). The reaction is stopped by adding 50 µl of stopping solution to each well; the well contents turn bright yellow. The optical density (OD) of the solutions in the wells is measured using a vertical scanning photometer at a wavelength of 450 nm. The time between stopping the reaction and measuring the optical density should not exceed 5 minutes.

Results assessment (validation criteria). Research results are considered reliable if the average OD value in the wells of the negative control sample (K-) is in the range of 0.01–0.15, and the OD value in the well of the positive control sample (K+) is at least 0.6.

The average OD of positive and negative controls is calculated.

The level of autoantibodies to the studied neuroantigens is estimated in U/ml using the formula: U/ml = (OD _sample - OD K ^- _avg ) : (OD K ⁺ - OD K ^- _avg ) × 100.

Recommended interpretation of analysis results:

if the antibody level is <10 U/ml - negative sample;

if the antibody count is > 20 U/ml – the sample is positive;

in the range of 10-20 U/ml - borderline sample (gray zone) and in this case it is recommended to re-examine samples from these patients after 7-10 days.

The obtained results are recommended for evaluation by a physician in conjunction with clinical and anamnestic data, taking into account clinical indicators and other diagnostic procedures.

A distinctive feature of the proposed method is that commercial standardized recombinant proteins NMDAR: NR2A, NR2B; D: DR1; DR2 and protein S100B ( E. coli , membrane localization) are used as antigens, immobilization of which in wells is carried out from an aqueous buffer solution with a pH of 7.2 ± 0.2 at + 4 ° C for 18 ± 2 hours at a concentration of 3 μg / ml; using a conjugate of monoclonal antibodies against light and heavy polypeptide chains of human immunoglobulins G labeled with horseradish peroxidase (epitope C2 domain of IgG, which does not have cross-reactions with IgE, IgA, IgD and IgM); using positive and negative control samples for recording and evaluating the results.

To confirm the feasibility of achieving a technical result using the proposed method, 69 patients with paranoid schizophrenia were also examined. The average age of the patients was 35.3±1.2 years. The duration of illness ranged from 1 to 38 years. Inclusion criteria for the study included a history of exacerbation of mental illness leading to hospitalization in a psychiatric hospital, treatment with antipsychotics, and adherence to biomedical ethics standards during the examination. Exclusion criteria included clinical and laboratory signs of inflammatory, infectious, or autoimmune pathology identified within the 2 months preceding the examination, and a history of substance abuse.

The examination and treatment of patients included in the study were conducted in accordance with approved clinical guidelines and with the informed consent of the patients or their legal representatives. The study was approved by the Ethics Committee of the Stavropol State Medical University (Protocol No. 114 dated December 15, 2022). The examination was conducted using a clinical psychopathological method and an assessment of negative and positive symptoms using the PANSS scale. Two groups of patients were identified: the first - with an attack-like progressive course of the disease without a pronounced emotional-volitional defect (n - 25); the second - with a continuously progressive course of the disease and a pronounced emotional-volitional defect (n - 44).

All patients received antipsychotic therapy. Venous blood was collected during routine patient examination. Serum was isolated and analyzed for specific anti-inflammatory antibodies (IgG) to neuroantigens. Anti-inflammatory antibodies to the S100B protein were measured, as were anti-inflammatory antibodies to neuroreceptors (NMDA receptors (NR1, NR2A, NR2B subunits) and dopamine receptors (DR1 and DR2). Statistical analysis of the obtained measurement results was performed using STATISTICA 10 software (StatSoft Inc., USA).

Patient examination revealed elevated levels of autoantibodies to neuroreceptors. In some patients, blood autoantibody levels were as low as 20 U/ml. However, the vast majority of patients examined had elevated autoantibody levels (above 20 U/ml). In some cases, low autoantibody levels (below 10 U/ml) were detected.

It should be noted that elevated levels of AAT to NR1 and NR2A were found in more than 70% of patients, and to DR1 and DR2 were detected in more than 80% of patients (Fig. 1). Elevated levels of AAT to the S100B protein were detected in 63.4% of patients.

Correlation analysis revealed a relationship between autoantibody levels and psychometric test scores on the PANSS scale. A moderate relationship was established between the level of autoantibodies to NR2A and the indicators of positive and negative symptoms (r= -0.48, p<0.05; r=0.58, p<0.05, respectively). A moderate negative relationship was found with the composite index (r= -0.59, p<0.05), and a moderate positive relationship was found with the anergy cluster indicator (r=0.4, p<0.05).

A positive moderate relationship was found between the assessment of negative symptoms and the level of autoantibodies to the S-100 protein (r=0.4, p<0.05) and the serum concentration of autoantibodies to DR2 receptors (r=0.48, p<0.05).

To confirm the feasibility of obtaining a technical result using the proposed method, we studied the levels of autoantibodies to neurospecific proteins in children with type 1 diabetes mellitus (T1DM). Diabetic encephalopathy (DE) is a serious and long-term complication of diabetes. Neuropsychological tests for diagnosing DE are far from perfect, so an active search for laboratory-specific markers for this complication is underway. Neurospecific proteins are markers of brain damage in many neurological and mental disorders and can be considered as indicators of DE in diabetes. The study was approved by the local ethics committee of Stavropol State Medical University (Protocol No. 100 dated June 17, 2021). All studies were conducted in accordance with international guidelines for working with human biomaterials. Statistical analysis of the measurement results was performed using STATISTICA 10.0 software (StatSoftInc, USA). Quantitative variables were described using the arithmetic mean and standard error of the mean, median, and quartiles. The Shapiro-Wilk and Kolmogorov-Smirnov tests were used to assess normal distribution. If the variables did not conform to a normal distribution, the Mann-Whitney U test was used. Differences between groups were considered significant at p < 0.05.

The open comparative study included 42 children aged 4 to 17 years, the average age was 11.7±0.96 years, including 20 boys (47.6%) and 22 girls (42.4%). Two groups were formed. Group 1 - patients with type 1 diabetes mellitus (T1DM), 21 children: 10 boys and 11 girls, the average age was 13.01±0.72 years. Group 2 - 21 children, 10 boys and 11 girls, the average age was 10.01±0.32 years, who did not suffer from T1DM. The average duration of the disease in children with T1DM was 9.6±0.36 years. Parents (or their legal representatives) of all children gave informed consent for their children to participate in the study.

The conducted analysis shows that the average values of autoantibodies to NMDA receptors, DR2 and S-100B protein were statistically significantly higher in patients with type 1 diabetes compared to “conditionally healthy children” (Fig. 2). No statistically significant changes were found in the average values of dopamine (D) and DR1 levels. The minimum value of autoantibodies to NMDA (NR1 subunit) in group 1 was 7.08 U/ml, the maximum was 98.48 U/ml, in group 2 it was 3.05 U/ml and 43.14 U/ml, respectively (p=0.0021). The diagnosed high levels of autoantibodies to NMDA receptors suggest the presence of excitotoxicity. Mean levels of DR2 autoantibodies were also significantly higher (p=0.0082) in children with diabetes compared to "conditionally healthy children." The minimum value was 1.42 U/ml, the maximum was 119.46 U/ml in the study group, and 1.03 U/ml and 86.3 U/ml, respectively, in the comparison group. High levels of DR2 autoantibodies suggest changes in the activity of the dopaminergic system. The average values of autoantibodies to the S-100B protein were also significantly higher (p=0.0045) in children of the 1st group (the minimum value of the level of autoantibodies to the S-100B protein was 42 U/ml and the maximum 76.81 U/ml, in "conditionally healthy children" -1.42 U/ml and 19.34 U/ml, respectively. High levels of autoantibodies to the S-100B protein may indicate damage to brain tissue, possibly due to developing excitotoxicity in type 1 diabetes in children and serve as a marker for the development of DE. Statistically significant differences in the average values of autoantibodies were revealed, depending on the duration of the disease. The level of autoantibodies to NMDA and DR2 receptors in children with a duration of type 1 diabetes over 5 years, as well as the average values of autoantibodies to the S-100B protein were significantly higher, compared with patients with a shorter duration of the disease (Fig. 3).

Thus, it can be concluded that with increasing duration of type 1 diabetes, there is a parallel increase in the levels of autoantibodies to NMDA and dopamine receptors, as well as to the S100B protein. The observed increase in autoantibodies to NMDA and dopamine receptors may indicate that, in childhood diabetes, there is an increase in the activity of the dopaminergic and glutamatergic systems. This is believed to cause damage to brain neurons and lead to cognitive deficits [9].

The obtained results allow us to conclude that the proposed method for the simultaneous determination of the level of autoantibodies to NMDAR: NR2A, NR2B; D; DR1; DR2 and S100B protein can be recommended as part of a complex of methods for early diagnosis, selection and monitoring of personalized therapy for the purpose of possible correction in various CNS pathologies:

- allows for standardized, highly accurate determination of the level of autoantibodies to neuroantigens;

- allows its use in all clinical diagnostic laboratories that have a plate or strip spectrophotometer;

- highly sensitive and reproducible;

- the technical solution is optimal in terms of the ratio of specificity - sensitivity - price - ease of use.

The main quality indicators of the proposed technical solution of the method in the form of a test system are presented in Fig. 5.

Bibliography:

1. Azizova U.M., Bembeeva R.Ts., Kozyreva A.A., Zavadenko N.N. Anti-NMDA receptor encephalitis. Neurological Journal named after L.O. Badalyan. 2021;2(3):137-145. https://doi.org/10.46563/2686-8997-2021-2-3-137-145.

2. Kuznetsova L.V., Vetrile L.A., Karpova M.N. NEURIOIMMUNOLOGICAL ASPECTS OF EPILEPSY PATHOGENESIS. SUCCESS OF PHYSIOLOGICAL SCIENCES. 2014; 45(3): 3-22.

3. Ermakov S.V., Karpov S.M., Baturin V.A., et al. Neuroimmune predictors of the outcome of aneurysmal subarachnoid hemorrhage. Kazan Med. J. 2020; 101 (5): 754–759. DOI: 10.17816/KMJ2020-754.

4. Kemp JA, McKernan RM. NMDAR pathways as drug targets. Nature Neurosci. 2002; 5:1039.

5. Pavlova O.V., Murashko A.A., Morozova A.Yu., Pavlov K.A., Gurina O.I., Shmukler A.B. Enzyme immunoassay method for the quantitative determination of autoantibodies to NMDA receptors in blood serum. SOCIAL AND CLINICAL PSYCHIATRY. 2022; 32 (4): 3-7.

6. Voller A., Bartlett A., Bidwell D.E. Enzyme immunoassays with special reference to ELISA technologies. J. Clin. Pathology. 1978; 31 (6): 507-520.

7. Murashko A.A., Pavlov K.A., Pavlova O.V., Gurina O.I. et al. Antibodies against N-Methyl D-Aspartate Receptor in Psychotic Disorders: A Systematic Review. Neuropsychobiology. 2022; 81(1): 1-18.

8. Jansen M, Dannhardt G. Antagonists and agonists at the glycine site of NMDARs for therapeutic interventions. Eur J Med hem. 2003; 38: 661.

9. Robinson R., Krishnakumar A., Paulose C.S. Enhanced dopamine D1 and D2 receptor gene expression in the hippocampus of hypoglycaemic and diabetic rats. Cell Mol. Neurobiol. 2009; 29(3): 365-72.

Claims (1)

A method for determining the level of autoantibodies to neuroantigens, which includes immobilization of proteins in plate wells, blocking of free sites, introduction of analyzed samples, incubation, introduction of a conjugate, characterized by immobilization in plate wells of standardized commercial preparations of recombinant proteins NMDAR: NR2A, NR2B; D; DR1; DR2 and protein S100B at a concentration of 3 μg/ml, using a 1% HSA solution to block free binding sites, using a conjugate of monoclonal antibodies against the light and heavy polypeptide chains of human immunoglobulins G labeled with horseradish peroxidase, using positive and negative control samples to record and evaluate the results calculated in U/ml.