The method of simultaneous determination of several analytes

 

(57) Abstract:

The invention relates to medicine, in particular for immunochemical analysis, and can be used to improve analytical and technological characteristics of immunochemical analysis. The essence of the method is the simultaneous determination of multiple analytes antigens or antibodies by contacting the sample with an immobilized on the carrier antibody or antigen capable of contacting the analyte, followed by the introduction of the hybrid molecules, constructed for each of the designated analyte, comprising the ligand portion capable of contacting the analyte, polynucleotide fragments having an area of binding with a complementary primers and matrix zones with a unique nucleotide sequence, given, respectively, each defined analyte. This is followed by polymerase chain reaction (PCR), using hybridization analysis to determine the received amplificatoare products PCR, detection of unique nucleotide sequences are used to indicate the presence in the sample of certain analytes. The technical result is the creation of highly sensitive sposor> The invention relates to medicine, in particular for immunochemical analysis, and is intended to improve analytical and technological characteristics of immunochemical analysis.

There is a method of detection of immunoglobulin antibodies in a sample, including immunoassay, and mixed ligand antigen, antibody or hapten associated with Biotin or its functional derivatives, antibody directed against detected antibodies associated with a paramagnetic particle, chemiluminescent acridine compound associated with Avidya, streptavidin or a functional derivative, with a break for the formation of solid-phase bound complex, conduct magnetic separation of the solid phase from the liquid phase, initiating the chemiluminescent reaction and analysis of the separated solid phase in the presence of the chemiluminescent complex, and the presence of chemiluminescence is an indication of the presence of the aforementioned antibodies in the sample (RU, 2132070, CL G 01 N 33/533, 1999).

The disadvantage of this method is the inability to simultaneously detect a large number of different complexes of antigen-antibody with a parallel analysis of different pedestalize multiple analytes using a hybrid molecule.

To achieve the technical result in the method of simultaneous determination of several analytes, the sample in contact with an immobilized on the carrier antibody or antigen capable of contacting the analyte, after incubation and washing contribute constructed for each of the designated analyte hybrid molecule containing ligand portion capable of contacting the analyte, and polynucleotide portion having an area linking with complementary primers, and matrix area with a unique nucleotide sequence, given, respectively, each defined analyte, the resulting complex, consisting of a hybrid molecule, analyte and the immobilized antibody or antigen is washed and is carried out after adding the necessary components polymerase chain reaction (PCR) received amplificatoare the PCR products was determined by hybridization analysis, the discovery of a unique nucleotide sequence is judged on the presence in the sample of certain analytes.

Moreover, the primer has a length of from 10 to 30 nucleotides.

The ratio of a-T and G-C nucleotides in the primer is 1:1.

Ligand polynucleotide and the parts are connected using the avidin-Biotin.

Hybridization analysis is performed using the technology of DNA chips.

Hybridization analysis is performed using oligonucleotide probes, enzyme labeled or radioactively.

The method is designed to determine antigens and/or antibodies.

The determination is performed in the serum or any other biological fluid.

For positive control ligand portion of the hybrid molecule is approaching the analyte, obviously in the sample and the negative control ligand selected part to the analyte, obviously missing in the sample.

The method is illustrated by the following example.

The principle of the method immunochemical determination of multiple analytes with possibility of simultaneous production of positive and negative internal controls using a "hybrid molecule".

The media immobilizerturning antibodies and/or antigens, the ability to communicate with different analytes, add the analyzed solution. After the first incubation and washing make "hybrid molecule" of various types. Each type of gcoi of analytes. For positive control ligand part of the "hybrid molecule" is selected for this analyte, which obviously must be in the sample. For the negative control is selected ligand such part of the "hybrid molecule", which obviously cannot communicate with any of the analytes in the sample. Each type of ligand part must comply with its own unique sequence of nucleotides in the matrix zone polynucleotide part of the "hybrid molecule". After a second incubation and washing contribute primers and other components for PCR. The unique nucleotide sequence of the matrix area of each type of "hybrid molecule", which became part of the "sandwich", repeatedly copied. At the final stage is the identification of unique nucleotide sequences using any of the varieties hybridization analysis (DNA-chip) or other suitable method.

As an example to illustrate the method for the simultaneous detection of multiple analytes proposed test for detection of antibodies to core, NS3, NS4, NS5 antigens of the hepatitis C virus (HCV) in serum with positive and negative internal controls.

- NS4, The NS5) regions of the HCV genome, as well as antibodies to human immunoglobulins (positive internal control) are sorbed on the surface of 96-well polystyrene plates. To do this, each well make a 0.2 ml solution of recombinant HCV antigens and immunoglobulins of human rights in optimal concentration in 0.01 M K-phosphate buffer (pH 7.4) with 0.1 M NaCl. Cover with a lid and incubated at a temperature of +4oC. Then the wells are washed 4 times with phosphate buffer solution containing 0.5% tween-20 or Triton-100.

- Getting "hybrid molecule". In this test involved six different types of "hybrid molecule". As the ligand part of the used core, NS3, NS4,NS5 recombinant HCV antigens, antibodies to human immunoglobulins (positive internal control). For negative internal control as the ligand part may be used any inert protein, such as bovine serum albumin (BSA). Polynucleotide parts are odnozadachnoy DNA length 130 nucleotides and have two zones linking with PCR primers with a length of 20 nucleotides that is identical for all types of hybrid molecules" that limit a matrix area. The sequence of the nucleus polynucleotide. For covalent crosslinking ligand (recombinant HCV antigens, antibodies to human immunoglobulins) and polynucleotide parts "hybrid molecule", to the 5'-end of the polynucleotide chain attached to the amino group, which then in the course of a chemical reaction binds the ligand. This method is described in detail in the work of Chu C. C. F., Orgel L., (1985), DNA, 4, 327.

For non-covalent binding of the parts "hybrid molecule" using the avidin-Biotin produce biotinylation polynucleotide chains of DNA and avidin kongugiruut with ligands. Mixing the resulting reagents, receive a "hybrid molecule".

For biotinidase polynucleotide chains of DNA can be used 3'-terminal attaching Biotin using terminal deoxynucleotidyltransferase (TdT), the technique described in the book "Molecular clinical diagnostics. Methods.", Herrington, McGee, M., Mir, 1999

Materials

5 x d-buffer: 5 mm l2, 700 mm cacodylate sodium, 0.5 mm DTT, 150 mm Tris-Hcl, pH 6.8

SIV-11dU: 0.4 mmol

Polynucleotide (up to mkg)

TdT: 50 U/ál

Technique

In placed in ice microcentrifuge test tube add the following reagents:

5 × TdT buffer 10 ál

Polynucleotide 10 ál (mcg)

centrifuge at 12000 g. Incubated at 37 g for 2 h

The volume was adjusted with polynucleotides to 100 ál and clean the probe by centrifugation on a column. The resulting polynucleotide contains the 3'end, up to three Biotin residues.

Methodology immunohistochemistry analysis:

- In wells make test sera. Incubated at temperature 37oC for 30 minutes In case analyzed in the sera are desired antibody complex is formed: solid phase - recombinant HCV antigens-antibodies.

The first washing. Five-washed wells phosphate buffer with tween-20 or Triton-100.

- In wells contribute 100 μl of phosphate-saline buffer containing six different types of "hybrid molecule". Incubated at temperature 37oC for 30 minutes Produces complex: solid phase - recombinant HCV antigens or antibodies against human immunoglobulin (positive internal control) antibodies contained in the analyzed sample to the appropriate type of "hybrid molecule"

- Second rinse. Five-washed wells phosphate buffer with tween-20 or Triton-100 to remove hybrid molecules, not contacting pourny solution containing 20 mm Tris-HCl, pH of 8.4, 50 mm KCl, 3.5 mm MgCl2, 0.01% gelatin solution, primers 0.5 pmol/l, DNA polymerase Taq 0.025 units/μl, 0.2 mm dATP, dGTP, dCTP, dTTP (for all components of the reaction mixture indicated final concentration), 50 ál. mineral oils. The tablet is placed in thermal cycler and spend 20-25 cycles of amplification. Each cycle consists of a denaturation - 95oC 1 min., annealing - 55oC 30 s, elongation - 72oC 30 s

At the final stage of hybridization analysis of PCR products. The presence in the reaction cell a nucleotide sequence corresponding "hybrid molecule" with antibodies to human immunoglobulins as a ligand portion (positive control) indicates the correct analysis (the presence of sample in the well, the absence of substances inhibiting the immune reaction, PCR, and so on). The lack of reaction cell a nucleotide sequence corresponding to the "hybrid molecule" with BSA as a ligand part, indicates the absence of non-specific adsorption hybrid molecules on the solid phase. Detection of polynucleotide sequences corresponding to the hybrid molecules with core, NS3, NS4, NS5 antigen as the ligand part, Stateline hybridization analysis using the technology of DNA chips, that allows you to analyze large number of samples with a minimum expenditure of time and reagents. However, it is possible to use traditional options hybridization analysis. As an example, the methodology hybridization analysis in 96-well polystyrene tablet.

On the surface of the wells of 96-hole tablet separately are sorbed odnozadachnaya DNA with a nucleotide sequence that is complementary to one of the six matrix zones "hybrid molecule".

- Material with PCR products from the reaction cell is transferred to each of six wells.

All wells are oligonucleotide probes complementary zone of binding to a polynucleotide primers part of the "hybrid molecule", enzyme labeled.

Tablet incubated. In the case of the analyzed material complementary DNA sequences is the hybridization matrix zone polynucleotide part of a hybrid molecule with the DNA adsorbed on the surface of the hole. In turn, the area of binding to a polynucleotide primers part of the "hybrid molecule" 's hybrid with oligonucleotide probes labeled with an enzyme.

the rata. In fact, in any holes has been the development of painting, judge of the matrix area what types of "hybrid molecule" amplificadores during PCR.

As an example illustrating the method for simultaneous detection of antibodies and antigens, we offer a test for the detection of antibodies to human immunodeficiency virus (HIV) type 1 and 2, the antigen of HIV-1, HBsAg and antibodies to HCV.

- Getting media immobilizerturning antibodies and antigens. Viral protein gp160 of HIV-1, a synthetic peptide ANT70 HIV-1 group 0, a synthetic peptide gp36 env HIV-2, mouse monoclonal antibodies to the antigen P24 HIV-1 polyclonal antibody to HBsAg recombinant HCV antigens corresponding parts of the proteins encoded by the structural (core) and nonstructural (NS3, NS4, NS5) areas of genomewise HCV, and antibody to human immunoglobulins (positive internal control) are sorbed on the surface of 96-well polystyrene plate.

- Getting "hybrid molecule". As ligand parts are used viral protein gp160 of HIV-1, a synthetic peptide ANT70 HIV-1 group 0, a synthetic peptide gp36 env HIV-2, mouse monoclonal antibodies to the antigen P24 HIV-1 polyclonal antibodies to HBsAg, recom areas of genomewise hepatitis C, antibodies to human immunoglobulins (positive internal control), bovine serum albumin (negative internal control). Polynucleotide parts are odnozadachnoy DNA length 130 nucleotides and have two zones binding primarliy PCR with a length of 20 nucleotides that is identical for all types of hybrid molecules" that limit a matrix area. The nucleotide sequence of the matrix area is unique for each type of "hybrid molecule". Conjugation of ligand and polynucleotide parts is the same as it was described in the previous example.

Methodology immunohistochemistry analysis:

- In wells contribute 100 μm analyzed serum and 100 μl of phosphate-saline buffer containing all types of hybrid molecules". Incubated for 1 h at a temperature of 37oC. In the presence in the sample of the analyte forms a complex: solid - phase adsorbed antigens or antibody - analyte - appropriate type of "hybrid molecule".

- Washing. Wash wells phosphate buffer with tween-20 or Triton-100 eight times.

- Carrying out PCR. In wells make the components necessary for rimary 0.5 pmol/l, DNA polymerase Taq 0.025 units /μl, 0.2 mm dATP, dGTP, dCTP, dTTP (for all components of the reaction mixture indicated final concentration), 50 μl of mineral oil. The tablet is placed in thermal cycler and spend 20-25 cycles of amplification. Each cycle consists of a denaturation - 95oC 1 min, annealing at 55oC 30 s, elongation - 72oC 30 s

At the final stage of hybridization analysis of PCR products. The fact of detection of the nucleotide sequence inherent in a particular type of "hybrid molecule" is evidence of the presence of this analyte in the sample.

As an example illustrating the method for simultaneous detection of analytes in biological fluids, it is proposed test to determine the following drug and narcotic substances in urine: opiates, amphetamine, barbiturate, methadone, benzodiazepine metabolites, propoksifen, metabolites of cocaine, phencyclidine, kannabia-IDA, methaqualone.

- Antibodies to defined in the test drug and narcotic compounds are sorbed on the surface of 96-well polystyrene plate.

- Getting "hybrid molecule". As the ligand part also used antibodies to defined lekarstv and have two zones binding primers and PCR length of 20 nucleotides, identical for all types of hybrid molecules" that limit a matrix area. The nucleotide sequence of the matrix area is unique for each type of "hybrid molecule". Conjugation of ligand and polynucleotide parts is the same as it was described in the previous examples.

Methodology immunohistochemistry analysis:

- In wells make the sample. Incubated at room temperature for 10 minutes if the sample has the desired substance is formed complex: solid phase - adsorbed antibody - analyte - "hybrid molecule".

- The wells washed with phosphate-saline buffer five times.

- After adding all the necessary components to carry out PCR, as described in the previous examples.

- Analyze hybridization of PCR products. Identification of a unique nucleotide sequence of the matrix part of the "hybrid molecule" will indicate the presence of analyte in the sample.

In General, the definition of PCR products can be performed not only by using hybridization analysis. For example, if the uniqueness of the matrix zone set as a sequence of nucleotides, and their number is on the determination of several analytes antigens or antibodies in a single sample, characterized in that the test sample is contacted with an immobilized on the carrier antibody or antigen capable of contacting the analyte, after incubation and washing make a hybrid molecule constructed for each of the designated analyte consisting of ligand part, can specifically bind with the analyte, and polynucleotide fragments having an area linking with complementary primers and matrix zones with a unique nucleotide sequence, given, respectively, each ligand part of the formed complex, consisting of hybrid molecules of the analyte and the immobilized antibody or antigen, washed and performed polymerase chain reaction (PCR), received amplificatoare the PCR products was determined by hybridization analysis, detection of a unique nucleotide sequence of the matrix zone hybrid molecules determine the presence in the sample of certain analytes.

2. The method according to p. 1, characterized in that the primer has a length of from 10 to 30 nucleotides.

3. The method according to PP.1 and 2, characterized in that the ratio of a-T and G-C nucleotides in the primer is 1:1.

4. The method according to p. 1, otlichalis the traveler and the nucleotide part linked covalently.

6. The method according to p. 1, characterized in that the ligand polynucleotide and the parts are connected using the avidin-Biotin.

7. The method according to p. 1, wherein hybridization analysis is performed using the technology of DNA chips.

8. The method according to p. 1, wherein hybridization analysis is performed using oligonucleotide probes, enzyme labeled or radioactively.

9. The method according to PP.1, 6, 7, characterized in that for the positive control ligand portion of the hybrid molecule is approaching the analyte, obviously in the sample and the negative control ligada part is matched to the analyte, obviously missing in the sample.

 

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