RU2568242C2 - Method of treatment of blood sample at immunological analysis of allergen-specific and total immunoglobulins e - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: before the immunoanalysis the immunoglobulins G are removed from the sample, in particular, by interaction of the sample with the sorbent which is specifically binds immunoglobulins G then the immunological analysis of allergen-specific and total immunoglobulins E is conducted using the allergens or mixes of allergens and antibodies against the immunoglobulins E immobilised in the elements of the biological microchip which is the array of three-dimensional hydrogel elements containing elements with covalently immobilised allergens and antibodies against the immunoglobulin E, while the array elements are microdrops obtained by the polymerisation immobilisation method.

EFFECT: decrease of non-specific signals, increase of the ratio signal-background, increase of reproducibility of the measured specific signals and improvement of sensitivity and reproducibility at analysing for determination of total and allergen - specific immunoglobulins.

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Description

FIELD OF THE INVENTION

The invention relates to biochemistry and medicine, in particular to analytical biochemistry and immunochemical analysis using biologic microchips, and relates to a method for processing an analyzed blood sample when quantifying general and allergen-specific immunoglobulins E. A biological microchip is an array of elements (cells) containing immobilized allergens (mixtures of allergens) and antibodies against immunoglobulins E. The present invention may find application in analytical Joachim, immunology and medicine, in particular, during the diagnosis and monitoring of treatment of allergies.

State of the art

An allergy is a hypersensitivity reaction initiated by a specific immune mechanism. The term “allergy” is most often used for a type 1 hypersensitivity reaction: an immediate type reaction, the clinical symptoms of which appear 30-60 minutes after exposure to an allergen. The reaction is based on the release of histamine, which occurs with the participation of allergen-specific immunoglobulins (antibodies) of class E (sIgE). The most common symptoms are hay fever (rhinitis), conjunctivitis, urticaria, asthma; the development of symptoms can lead to anaphylactic shock.

Recently, there has been an increase in the number of allergic diseases all over the world, which is associated, in particular, with the deterioration of the environmental situation and an increase in the number of products of economic activity in the environment. According to various estimates, allergic diseases occur in 20-40% of the population.

A classic and widespread method for diagnosing type 1 allergies is by staging skin samples. The disadvantages of this method are discomfort for the patient, the risk of provoking increased sensitization and anaphylactic shock (Liccardi G., D'Amato G. et al. Systemic reactions from skin testing: literature review. J. Investig. Allergol. Clin. Immunol., 2006, v.16, p. 75-78). A safe alternative to the skin test method is the immunological analysis of allergen-specific IgE in the patient’s blood, including the radio allergosorbent assay (RAST), enzyme-linked immunosorbent assay (ELISA), etc. (Hamilton RG and Adkinson NF, In vitro Assays for the Diagnosis of IgE-Mediated Disorders, J. Allergy Clin. Immunol., 2004, v. 114 (2), p. 213-225).

Test systems for semi-quantitative and quantitative analysis of sIgE are produced by several companies in Europe and America, for example, Dr. Fooke Laboratorien (Germany), Phadia (Sweden), Cypress Diagnostics (Belgium), Hycor Biomedical (USA ), and the immunological method of analysis in recent years has become increasingly popular. In most cases, an analysis option is used in which allergens or allergen mixtures are immobilized on a solid phase, for example, in the wells of an immunological tablet, inside capillary tubes (IVT Allergy Screen test system from Arlington Scientific) (USA) (www.arlingtonscientific. com), on nitrocellulose strips (test systems from Heska, Switzerland (www.heska.com), etc. Cellulose discs are also used, which are placed in the wells of an immunological plate during analysis (allergy discs R-Biopharm AG, Germany) and Dr. Fooke Laboratorien, Germany). as well as test systems for IgE analysis by reverse immunoassay, in which anti-IgE antibodies are adsorbed in the plate wells, for example, a test system manufactured by Dr. Fooke Laboratorien (Germany).

Currently, several hundred different types of allergens are known, and in order to maximize the effectiveness of allergy diagnosis, it is necessary to determine the many types of sIgE, and the cost of the analysis should be low. The use of biological microarrays - arrays of elements containing immobilized biological probes, in particular protein microarrays, makes it possible to conduct simultaneous parallel analysis of many compounds in a minimal amount of a biological sample. The biological microarrays described in the patent literature and open sources for sIgE analysis in most cases are substrates (glass, plastic, membrane) onto which allergen solutions are applied (Kim E., Park SW, Cho NY, et al, Quantitative Measurement of Serum Allergen -Specific IgE on Protein Chip, Exp. Mol. Med., 2002, v. 34 (2), p. 152-158; Jahn-Schmid B., Harwanegg C, Hiller R, et al, Allergen microarray: comparison of microarray using recombinant allergens with conventional diagnostic methods to detect allergen-specific serum immunoglobulin E. Clin. Exp.Allergy, 2003, v. 33, p. 1443-1449; Fall BI, Eberlein-Konig B., Behrendt H., et al. , Microarrays for the Screening of Allergen-Specific IgE in Human Serum, Anal. Chem, 2003, v. 75, p. 566-562; U.S. Patent Application No. 20090068233; patent application WO 2002/029415 (VBC Genomics Bioscience Research GmbH); US patent No. 7846713 ("Randox Laboratories Ltd.").

The method of analysis of sIgE on microchips with immobilized allergens involves the interaction of the analyzed sample with allergens (in this case, specific sIgE antibodies are captured by immobilized allergens) and further development of microarrays with labeled species-specific antibodies (Harwanegg C.and Hiller R., Protein Microarrays for the Diagnosis of State Disease of State Allergic -of-the-Art and Future Development, Clin. Chem. Lab. Med. 2005, v. 43, p. 321-1326).

As a rule, blood samples of patients are used for determination: usually undiluted serum or blood plasma. However, it should be noted that 75-80% of serum antibodies are immunoglobulins G (IgG), and IgE is only about 0.002%. Thus, the concentration of IgG in the blood is several orders of magnitude higher than the concentration of IgE (10-20 mg / ml for IgG and ~ 50 ng / ml for IgE). The presence of IgG at such high concentrations makes it difficult to determine sIgE, since (1) there is a competition between IgG and IgE for binding to immobilized allergens, (2) anti-IgE autoantibodies can form an IgG-IgE complex, preventing the further binding of IgE to species-specific antibodies, containing a label for detection (developing antibodies) (Johansson SG, Anti-IgE antibodies in human serum, J. Allergy Clin. Immunol., 1986, v.77 (4), p.555-557; Gleich GJ, Zimmermann EM, Gleich MC, Yunginger JW, Measurement of IgG blocking antibodies by interference in the radioallergosorbent test, J. Immunol., 1981, v. 126 (2), p. 575-579).

Thus, the presence of IgG in the blood sample prevents the determination of sIgE during immunoassay, leading to a deterioration in the interaction of sIgE with allergens immobilized in the elements (cells) of the biological microchip and an increase in background signals due to non-specific sorption. Nonspecific interactions and background signals are especially pronounced in the case of analysis using biochips, since a biochip for simultaneous parallel analysis of a number of compounds in a sample contains many elements with different immobilized probes, and non-specific background signals can be observed for each of the many elements, which leads to to a significant decrease in the sensitivity and reproducibility of the analysis. In addition, competition between IgE and IgG for binding to immobilized allergens often leads to underestimated results of the determination of allergen-specific IgE.

These disadvantages are overcome by the present invention, which provides a method for processing a blood sample during the analysis of allergen-specific and general immunoglobulins E on biological microarrays, including the removal of IgG from the analyzed sample.

Disclosure of invention

The invention provides a method for processing a blood sample during the analysis of allergen-specific and general immunoglobulins E using biological microarrays. A biological microchip is an array of elements containing immobilized allergens (mixtures of allergens) and antibodies against immunoglobulins E. The term "immobilized" includes allergens and antibodies bound to the carrier in the elements of the microchip in any way, that is, by adsorption, through hydrophobic interactions, covalent binding, complexation, etc.

At the first stage of the analysis, the sample interacts with immobilized compounds. Blood samples contain a high concentration of IgG, several orders of magnitude higher than the concentration of measured IgE. Because of this, incomplete binding of IgE with immobilized probes and high background signals are observed, which vary from sample to sample, which leads to insufficient sensitivity and reproducibility of the analysis, as well as to underestimated results in the analysis of allergen-specific sIgE.

The present invention provides a method of treating a serum or blood plasma sample before immunoassay on biological microchips, characterized in that immunoglobulins G are removed from the sample.

Processing a sample of serum or plasma is carried out in any way that allows you to remove immunoglobulins G, without reducing the number of defined compounds: immunoglobulins E, both general and allergen-specific. In one aspect of the invention, the removal of immunoglobulins G is carried out by the interaction of a blood sample with a sorbent specifically binding immunoglobulins G.

When analyzing using biological microarrays with immobilized allergens after processing the sample to remove IgG, there is a decrease in background (non-specific) signals, an increase in the signal-to-background ratio, an increase in the reproducibility of the measured specific signals and, as a result, an increase in the sensitivity and reproducibility of the definition of common and allergen -specific immunoglobulins compared with immunoassay of the untreated sample.

For a number of allergens, an increase in specific signals from microarray elements is also observed in comparison with the analysis of the untreated sample.

Further, the invention will be disclosed in more detail with reference to the figures and examples, which are given solely to illustrate and explain the essence of the claimed invention, but which are not intended to limit the scope of claims.

Brief Description of the Figures

Figure 1 represents the fluorescence images of biological microarrays during simultaneous analysis of specific and total IgE in a human serum sample before (A) and after processing the sample to remove IgG (B). A microchip is an array of hydrogel elements in the form of microdrops. Each element contains an immobilized allergen or antibody: 1) pollen of trees and shrubs, 2) weed herbs and flowers, 3) meadow herbs and cereals, 4) epidermal, 5) household, 6) mold and yeast, 7) food, 8) elements that do not contain biomaterial, and elements with immobilized antibodies 9) IgE in different concentrations (to form an “internal calibration curve”), 10) anti-IgE.

Microchips were incubated with the sample and developed with anti-IgE antibodies labeled with fluorescent dye Cy5.

Figure 2 presents the signal profiles (fluorescence intensity, rel. Units) of the elements of the biological microchip during simultaneous analysis of specific and total IgE in a human serum sample before and after processing the sample to remove IgG. Microchips were incubated with the sample and developed with anti-IgE antibodies labeled with fluorescent dye Cy5. Analysis of blood serum # 5 (A) and # 6 (B) (Table 1).

The implementation of the invention

The present invention provides a method for processing a blood sample during quantitative immunological analysis of allergen-specific and general immunoglobulins E, using biological microchips containing elements with immobilized allergens or mixtures of allergens, as well as immobilized antibodies against immunoglobulins E. Allergens and antibodies can be immobilized in elements of the microchip in any way: sorbed, linked through hyphrophobic interactions, linked through complexation (complexes like avidin (streptavidin) -biotin, antigen-antibody, metal - chelator, etc.), by the formation of covalent bonds, etc.

The present invention provides a method for processing a blood sample during analysis using biological microarrays, characterized in that immunoglobulins G are removed from the sample. Diluted or undiluted serum and plasma samples are used as analysis samples. Preliminary processing of the sample is carried out by any method that allows you to remove immunoglobulins G, without reducing the number of defined compounds: immunoglobulins E, both general and allergen-specific. In one embodiment of the invention, the removal of immunoglobulins G is carried out by the interaction of a blood sample with a sorbent specifically binding immunoglobulins G, for example, with a sorbent containing immobilized protein G, a sorbent containing immobilized antibodies against human IgG, but not limited to. An example of an sorbent specifically binding IgG is a resin with immobilized protein G (Protein G Sepharose).

Sample processing can be carried out using a chromatographic column or spin column with a specific sorbent, add the sorbent in the form of a suspension to the sample, or in any other way. The processing of blood samples to remove IgG using sepharose containing immobilized protein G is described in Example 1. As can be seen from Table 1 (Example 1), the processing of blood samples does not change the concentration of total and allergen-specific IgE in serum and plasma samples (within the measurement error), i.e. does not affect the measurement of the concentration of the analyzed compounds.

Example 2 shows the results of a quantitative analysis of allergen-specific and total IgE in a blood serum sample on a biological microchip. The biochip is an array of three-dimensional hydrogel elements containing elements covalently linked to immobilized allergens and antibodies against IgE. The array elements are microdroplets of a given volume, obtained by the method of polymerization immobilization according to the technology patented by the IMB RAS (RF patent No. 2216547 "Method for the polymerization immobilization of biological molecules and compositions for its implementation", international application WO 03/033539).

The results of the analysis of samples before and after processing by the method proposed in this invention are shown in Figures 1 and 2 and in Table 2. When analyzing the sample after removal of IgG for a number of allergens, an increase in specific signals from microchip elements with immobilized allergens is compared (Fig. .1 and 2). As can be seen from Table 2, there is also a decrease in background signals. Thus, for all analyzed specific and total IgE, an increase in the signal / background ratio is observed, which leads to a significant increase in the sensitivity of the analysis.

The sensitivity of the analysis was evaluated as follows. The threshold concentration of IgE, below which the level of IgE is defined as undetectable - 0.35 IU / ml. When conducting a sample analysis after IgG removal, the fluorescent signals of microarray elements with immobilized allergens and antibodies after interaction with a sample containing IgE at a concentration of 0.35 IU / ml exceeded background signals by one and a half to two times. Processing the sample (removal of IgG) leads to the fact that the signal-to-background ratio at an IgE concentration of 0.35 IU / ml increases to 3-4 or more times. Particularly noticeable increase in sensitivity for allergens, where there is an increase in specific signals.

The following examples are not intended to limit claims, but solely to illustrate individual embodiments of the present invention.

Example 1. Processing a blood sample to remove immunoglobulins G

a) Option 1

A Tricorn 5/50 chromatography column (GE Healthcare, USA) was filled with 720 μl of sepharose containing immobilized protein G (Protein G Sepharose 4 Fast Flow, GE Healthcare, USA, sorbent capacity> 10 mg / ml). The column was balanced with 5 volumes of 25 mM phosphate-buffered saline, pH 7.2 (working buffer). Human blood serum (or blood plasma, 360 μl, 0.5 volume of sorbent), pre-filtered with a Millex SLHA 025 OS filter (Millipore Corporation, USA), was applied to the column. Elution was carried out with a working buffer until the peak of IgE peak and other serum proteins that did not bind to the carrier was completely released. Detection was carried out using a UV detector "Uvicord S2" (LKB, Sweden) at a wavelength of 280 nm. The resulting solution was used to measure the content of total and specific IgE. If necessary, the sample was concentrated to the initial volume using a Vivaspin 2 MWCO 10000 concentrator (GE Healthcare, USA) or the sample dilution was taken into account when calculating the concentrations of total and specific IgE.

To regenerate the sorbent, the column was washed with 0.1 M sodium citrate phosphate buffer, pH 2.8, until the IgG peak was completely out and then balanced with 5 volumes of working buffer. After this, the sorbent was reused. For long-term storage, the sorbent was balanced with a working buffer with the addition of 0.2% sodium azide.

b) Option 2

Blood serum (or blood plasma) and Sepharose with immobilized Protein G, equilibrated with working buffer, were mixed in a volume ratio of 1: 1, and the mixture was incubated with stirring for 1 hour at room temperature. Sepharose was removed by centrifugation (10,000 rpm, 4 min), the supernatant was collected and used for analysis. If necessary, the sample was concentrated to the initial volume using a Vivaspin 2 MWCO 10000 concentrator (GE Healthcare, USA) or the sample dilution was taken into account when calculating the concentrations of total and specific IgE.

The results of measurements of concentrations of allergen-specific and total IgE in blood samples before and after treatment are shown in Table 1.

Measurements of total IgE were performed using a kit for determining total IgE by ELISA, allergen-specific IgE - a kit for determining specific IgE by REAST reverse immunoassay (both kits manufactured by Dr. Fooke Laboratorien Gmbh, Germany) according to the procedures described by the manufacturer.

Example 2. Simultaneous quantitative determination of allergen-specific and total IgE in a blood sample on a biological microchip

For the analysis used microchips, the structure of which is shown in Fig.1. Microchips were made using the polymerization immobilization technology developed at the IMB RAS (RF patent No. 2216547, international application WO 03/033539; Rubina A.Yu, Dementieva EL, et al., Hydrogel-based protein microchips: manufacturing, properties, and applications, Biotechniques 2003, v. 34 (5), p. 1008-1022).

A polymerization mixture containing gelling monomers based on methacrylamide and allergens and antibodies to be immobilized was applied to the surface of a glass slide using a QArray robot (Genetix, UK) in the form of 0.1 nl droplets. Polymerization of elements with simultaneous covalent immobilization of proteins was carried out by irradiation with an ultraviolet lamp with a maximum radiation of 350 nm (Sylvania GTE lamp, F15T 8/350 Bl, UK) for 50 min at 20 ° C in a stream of nitrogen. To increase the reliability of the data obtained, all immobilized allergens and antibodies in the microchip are presented in three identical gel elements. After polymerization, microarrays were washed from unreacted components and incubated in 0.01 M phosphate buffer, pH 7.2, containing 0.15 M NaCl, 1% polyvinyl alcohol (“blocking solution”), 40 min to reduce non-specific interactions. at room temperature.

A blood serum sample was processed to remove IgG as described in Example 1 (option 1). For comparison, an analysis was performed with the initial sample of blood serum (without treatment). Microchips were incubated with samples for 20 h at 37 ° C and then with developing anti-IgE labeled with Cy5 fluorescent dye (1 h, 37 ° C). After incubation and washing from non-specifically bound components, fluorescent signals were recorded. For registration, we used a fluorescence microscope equipped with a CCD camera and software for image visualization and processing of the obtained data, developed at the IMB RAS (Barsky V., Perov A. Tokalov S., et al., Fluorescence data analysis on gel-based biochips, J. Biomol. Screening, 2002, v. 7, p. 247-257). The fluorescence intensity was calculated as the median signal from three identical gel elements. The fluorescence images of the microarrays after analysis are presented in FIG. 1.

The signal profiles (fluorescence intensity, rel. Units) of the elements of the biological microchip for samples before and after removal of IgG are shown in Figure 2. The values of the background fluorescent signals in the analysis of samples before and after purification (removal of IgG) are shown in Table 2.

Table 2. Background fluorescent signals in the analysis of blood samples on biological microarrays before and after processing (removal of IgG) Sample Number (Table 1) Fluorescent signals, rel. units Before processing After processing #5 1.6 ± 0.3 0.9 ± 0.1 # 6 1.4 ± 0.2 0.7 ± 0.1 #8 2.6 ± 0.4 0.44 ± 0.06 #10 2.4 ± 0.4 0.50 ± 0.07

After the interaction of the microchip with developing antibodies on the elements of the microchip containing immobilized IgE in different concentrations, fluorescent signals were obtained that formed an internal calibration curve. The calibration dependence “fluorescence signal intensity - IgE concentration” was constructed, which was used to determine the concentrations of specific and total IgE in the test sample.

Claims (3)

1. A method for processing a serum or blood plasma sample during immunological analysis of allergen-specific and general immunoglobulins E using allergens or mixtures of allergens and antibodies against immunoglobulins E immobilized in elements of a biological microchip, which is an array of three-dimensional hydrogel elements containing elements with covalently immobilized allergens and antibodies against immunoglobulin E, while the elements of the array are microdrops obtained by the floor method merizatsionnoy immobilization, wherein before analysis produce removal of immunoglobulin G sample. 2. The method according to claim 1, characterized in that the removal of immunoglobulins G is carried out by the interaction of a sample of serum or plasma with a sorbent that specifically binds immunoglobulins G. 3. The method according to claim 2, characterized in that as the sorbent use a resin with immobilized protein G.

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