RU2373540C1 - Method of simultaneous detection of two analytes through bioluminescent molecular microanalysis - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biochemistry, more specifically to methods of carrying out immunoenzymometric and DNA-hybridisation analysis. A conjugate is proposed for bioluminescent molecular microanalysis, obtained from an enzyme, which is bonded to a biospecific reagent, where the enzyme used is Ca²⁺ regulated photoprotein obelin with an altered bioluminescent spectrum: recombinant obelin W92F; H22E or recombinant obelin Y138F. The method for simultaneous detection of two analytes through bioluminescent molecular microanalysis involves treating a solid phase with biospecific reagents, separation of unreacted liquid phase, treatment of the solid phase with an enzyme containing conjugate, separation of the liquid and solid phases and analysis of the solid phase. The biospecific reagent used for the first treatment of the solid phase is two immumoglobulins or one immunoglobulin with double specificity or two different oligonucleotides. Final treatment of the solid phase is done simultaneously with two conjugates, which respectively consist of recombinant obelins with different bioluminescent spectra and molecules with different biospecificity, where the recombinant obelins used are W92F;H22E and recombinant obelin Y138F with subsequent analysis on a double-channel bioluminometre with broad-band light filters.

EFFECT: use of this invention allows for accurate, fast, universal and easy realisation of the method of bioluminescent molecular microanalysis.

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Description

The invention relates to biochemistry, and in particular to methods for conducting an enzyme-linked immunosorbent assay and DNA hybridization analysis.

Known conjugate and method for the simultaneous detection of both components of a binding pair [application No. 2002104927/15, IPC G01N 33/48, publ. 2005.04.27], which describes a solid-phase immunological microanalysis using fluorescent dyes as labels and recording signals by flow cytometry.

The disadvantage of this method is that it uses a fluorescence analysis to simultaneously detect 2 components. The disadvantages of which, in turn, are the toxicity of the dyes used, a high background fluorescent signal, which reduces its sensitivity, and a limited scope (only for immune analysis).

Known conjugate for enzyme-linked immunosorbent assay and enzyme-linked immunosorbent assay [RF patent No. 20144610, IPC G01N 33/535, publ. 1994.06.15, (prototype)], where the conjugate is obtained by chemical crosslinking of a biospecific molecule with enzymes, followed by chromatographic purification. For analysis, the method uses two phases: a solid phase for immobilization of the formed biospecific complex and a liquid phase for introducing solutions of reacting substances and intermediate washes, and it uses conjugates of a biospecific molecule with a repetitive enzyme to detect the presence of analyte and its amount.

The disadvantage of the conjugate is that natural laccose is used as an enzyme, the preparation of which is quite a costly and time-consuming process.

The disadvantages of the method are that it is suitable only for enzyme-linked immunosorbent assay and when it is carried out on the solid phase only one biospecific complex is immobilized and, accordingly, one procedure gives an answer about the content of only one analyte. The measurement is carried out by the colorimetric method, which has insufficient sensitivity and a narrow linear range.

The technical result of the invention is the development of an express, more accurate, universal and technologically advanced bioluminescent molecular microanalysis method.

The technical result is achieved in that in the conjugate for bioluminescent molecular microanalysis obtained from an enzyme connected to a biospecific reagent, followed by purification of the conjugate on a chromatographic column, it is new that the recombinant Ca ²⁺ dependent photoprotein obelin with an altered bioluminescence spectrum is used as an enzyme. : recombinant obelin W92F; H22E or recombinant obelin Y138F.

The technical result is also achieved by the fact that in the method for the simultaneous determination of two analytes by a bioluminescent molecular microanalysis, which includes treating the solid phase with biospecific reagents, separating the unreacted liquid phase, treating the solid phase with an enzyme-containing conjugate, separating the liquid and solid phases and analyzing the solid phase, is new that as a biospecific reagent for the first treatment of the solid phase, two different immunoglobulins, or one immunoglobulin with double special use specificity, or two different oligonucleotides, and the final processing of the solid phase is carried out simultaneously by two conjugates consisting respectively of recombinant Ca ²⁺ dependent obelin photoproteins with different bioluminescence spectra and molecules with different biospecificity, where W92F is used as recombinant obelins; H22E and recombinant obelin Y138F, followed by analysis on a two-channel bioluminometer with wide-band light filters.

The above distinguishing features from the prototype signs allow us to conclude that the claimed technical solutions meet the criterion of "novelty."

The features distinguishing the claimed technical solutions from the prototype are not identified in other technical solutions and, therefore, provide the claimed solution with the criterion of "inventive step".

On figa presents the bioluminescence spectra of recombinant obelins W92F; H22E (-) and Y138F (- -) and transmission spectra used to separate the signals of wide-band photo filters and FS6 (1) and ZhS 16 (2). On figb shows the passage of signals from the "color" obelins through photofilters installed in the channels of the bioluminometer 1 and 2. The arrows indicate the moment of injection of a solution of CaCl ₂ . Dotted lines show the transmission of each channel of the corresponding signals without a filter.

Figure 2 (above) shows a diagram of the simultaneous analysis of two different immunoglobulins - rabbit (RIgG) and human (HIgG). In figure 2 (below), the results of the determination of murine anti-RigG (■) and anti-HigG (▲) antibodies obtained by separation of signals using light filters. The common cue signal without separation is shown (●).

Figure 3 (left) shows a diagram of the simultaneous solid-phase immunoassay of gonadotropic hormones (luteinizing - LH and follicle-stimulating - FSH) in standard human sera; on the right are the obtained calibration curves with simultaneous two-color determination of LH (▲) and FSH (●).

When implementing the method, recombinant forms of Ca ²⁺ -activated obelin photoprotein with altered bioluminescence spectra are chemically conjugated with haptens (e.g. biotin or oligonucleotides) or other proteins (e.g. streptavidin or immunoglobulins) and the derivatives obtained are used as specific binding assays for molecules in the sample (for example, blood serum, amplicons after the PCR procedure). On the solid phase (for example, the surface of plastic tubes or microparticles), specific complexes (for example, antibody antigens or nucleotide hybrids) are immobilized, the composition of which depends on the presence of the analyte in the sample. After processing the solid phase with a mixture of specific labels and the necessary washes, the signal from each analyte is recorded immediately after the CaCl ₂ solution is introduced into the test tube using a two-channel bioluminometer. A photo filter is built into each channel, ensuring the passage of a light signal of only one color. Thus, each channel registers the signal of only one label (figb).

Example 1. Obtaining chemical conjugates of mutant forms of obelin with immunoglobulins

Thionylation of mutant obelin forms was performed in 50 mM BICINE, pH 8.5, 250 mM NaCl, 5 mM EDTA, using a 10-fold molar excess of 2-iminothiolan for 30 min at room temperature. Excess reagent was separated by gel filtration on a column equilibrated with 20 mM PIPES, pH 7.5, 250 mM NaCl, 5 mM EDTA. Sulfo-SMCC (4- (N-maleimidomethyl-) cyclohexanoic acid sulfo-succinimide ester) -activation of immunoglobulins was carried out in 20 mM PIPES, pH 7.5, 250 mM NaCl, 5 mM EDTA using a 50-fold molar excess of Sulfo-SMCC for two hours at room temperature. Excess reagent was separated by gel filtration on a column equilibrated with 20 mM PIPES, pH 7.5, 250 mM NaCl, 5 mM EDTA. Conjugation was performed by incubation of thionylated obelin mutants with SMCC-activated immunoglobulins in a 1: 5 molar ratio at 4 ° C overnight. The resulting conjugates of mutant proteins with immunoglobulins were isolated by gel filtration on a column filled with P-150 biogel balanced with 0.1 M phosphate buffer pH 7.0 containing 0.15 M NaCl and 5 mM EDTA, or on a Superose 6 column balanced with a buffer containing 0.02 M Tris-HCl pH 7.0, 0.1 M NaCl and 5 mM EDTA.

Example 2. Obtaining conjugates of mutant forms of obelin with oligonucleotides

Thionylation of mutant obelin forms was carried out in 50 mM phosphate buffer pH 7.5, 5, 0.15 M NaCl (PBS), 5 mM EDTA, using a 10-fold molar excess of 2-iminothiolan for 30 min at room temperature. Excess reagent was separated by gel filtration on a column equilibrated with the same buffer. SMCC activation of the oligonucleotides NH ₂ -dT (30) and NH ₂ -dA (30) was performed in 0.1 M NaHCO ₃ using a 100-fold molar excess of SMCC for 30 min at room temperature. The excess reagent was separated by gel filtration on a column balanced with 50 mmol / L NaH ₂ PO ₄ pH 7.5, 5 mmol / L EDTA. Conjugation was performed by incubation of thionylated obelin mutants with SMCC oligonucleotides in a molar ratio of 3: 1 at 4 ° C. overnight. The resulting conjugates of mutant proteins with oligonucleotides were isolated by anion exchange chromatography on a MonoQ column equilibrated with 20 mM Tris-HCl pH 7.0, 5 mM EDTA in a NaCl solution gradient (0-47%) in the same buffer.

Example 3. Simultaneous bioluminescent analysis of anti-human and anti-rabbit mouse immunoglobulins

200 μl of a mixture of monoclonal mouse anti-human and anti-rabbit IgG with a final titer of 5,000, 10,000, 25,000, 50,000 and 100,000 were added to the tubes, sorption was performed overnight at 4 ° C. After washing (thrice, PBS, 0.1% Tween 20, 5 mM EDTA), unoccupied surface sites were blocked with 1% bovine serum albumin in PBS buffer (1 hour at 37 ° C). After washing, 200 μl of RIgG ~ W92F conjugate solution were added to the tubes; H22E and HIgG ~ Y138F. The reaction was carried out for 1 h at room temperature. After that, the contents of the wells were discarded, the wells were washed. The bioluminescent obelin signal was measured immediately after injection with 200 μl of a 0.1 M solution of CaCl ₂ in 0.1 M TrisHCl pH 8.8. The signals were separated using wide-band optical filters mounted in the channels of the bioluminometer.

Example 4. Simultaneous bioluminescent analysis of LH and FSH

Anti-αFSH activated tubes (10 μg / ml) were mixed with standard sera at concentrations of 1.25; 5; 10; 40; 80 mIU / ml and a mixture of control sera. After incubation (with stirring for 1 hour at 37 ° C) and washing (three times, PBS, 0.1% Tween20, 5 mM EDTA), a mixture of labels was added in a 1: 1 ratio (anti-βFSH ~ W92F; H22E (7.8 μg / ml) and anti-βLH ~ Y138F (1 μg / ml)). The reaction was carried out for 40 min at room temperature with stirring. Washed again. The bioluminescent signal of the sorbed labels was measured immediately after injection of a 0.1 M CaCl ₂ solution in 0.1 M TrisHCl pH 8.8. The signals were separated using wide-band optical filters mounted in the channels of the bioluminometer.

In the claimed method, enzymes are used recombinant photoproteins isolated from recombinant strains of bacteria E coli with high productivity, which greatly simplifies and reduces the cost of their production.

Bioluminescence of photoproteins occurs with a high quantum yield and is characterized by the absence of a background signal, which ensures high sensitivity of the analysis.

The reaction of photoproteins is extremely simple - bioluminescence is initiated by the addition of a CaCl ₂ solution and has the character of a bright flash, in our case, violet and green (the bioluminescence of wild-type obelin is blue).

The bioluminescent signal of photoproteins has an almost unlimited linear range.

The inventive method provides the ability to simultaneously determine two analytes.

The measurements are carried out in a bioluminescent way, which provides high sensitivity, comparable only with the radioisotope (up to 1 attol).

The method is intended for both immunoassay and DNA hybridization analysis.

Claims (2)

1. The conjugate for bioluminescent molecular microanalysis obtained from an enzyme connected to a biospecific reagent, followed by purification of the conjugate on a chromatographic column, characterized in that the recombinant Ca ²⁺ dependent photoprotein obelin with an altered bioluminescence spectrum is used as an enzyme: recombinant W obelin92; H22E or recombinant obelin Y138F. 2. A method for the simultaneous determination of two analytes by a bioluminescent molecular microanalysis, including treating the solid phase with biospecific reagents, separating the unreacted liquid phase, treating the solid phase with an enzyme-containing conjugate, separating the liquid and solid phases and analyzing the solid phase, characterized in that as a biospecific reagent for the first treatment solid phase use two different immunoglobulins or one immunoglobulin with double specificity or two different oligonucleotides, and the final treating the solid phase is carried out simultaneously by two conjugates composed, respectively, from recombinant Ca ²⁺ dependent obelin photoproteins with different spectra bioluminescence and molecules with different biospetsifichnostyu wherein as recombinant Obelin use W92F; H22E and recombinant obelin - Y138F, followed by analysis on a two-channel bioluminometer with broadband light filters.

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