Method for simultaneous detection of several markers of congenital diseases in dry blood stains

FIELD: medicine.

SUBSTANCE: invention refers to immunological methods of analysis and can be used for mass screening of congenital diseases in newborn children. That is ensured by immobilising in a microplate well first immune-specific components to thyrotropin, immunoreactive trypsin, thyroxin and 17α-OH-progesterone in the form of discrete microregions, placing in the well a paper disk of a dry blood sample, extracting the detectable markers from dry blood stains by adding into the well a buffer containing danazol and 8-anilinonaphthalene-1-sulfonic acid and 17α-OH-progesteron antibodies, which are bound to extracted 17α-OH-progesterone to form an immune complex with anti-species antibodies in the respective microregion simultaneously, then adding into the microplate well a reaction solution of second immune-specific components, containing a mixture of biotin-conjugated antibodies to thyrotropin, immunoreactive trypsin and thyroxin, and a 17α-OH-progesteron-protein-biotin conjugate for preparing the immune biotin-labelled complexes in the discrete microregions, removing the paper disk of the dry blood sample, adding a streptavidin and Pt-coproporphyrin conjugate to prepare the phosphorescent biotin-streptavidin complexes in the microregions on the bottom of the microplate well and detecting the label phosphoresce emission, by scanning the discrete microregions by a focused laser beam sequentially.

EFFECT: using the given method enables detecting biomarkers specifically and qualitatively in the newborn's capillary blood stain sample that enables diagnosing three newborn's congenital conditions.

10 cl, 7 dwg, 10 tbl

 

The invention relates to immunological methods of analysis and can be used for mass screening of congenital diseases of the newborn.

Early diagnosis of congenital anomalies in newborns that are subject to therapeutic correction in timely detection, allows for proper physical and mental development of the child. Diagnosis is based on quantitative determination of the concentration of markers of congenital diseases when used as an investigational clinical material dried capillary blood spots of newborns. A limited number of tested material necessitates the simultaneous quantitative detection of multiple markers from a single blood sample. The analysis in dry spots of whole blood put additional requirements on accuracy, sensitivity and specificity of the methods used. This method of detection has to be adapted for mass surveys.

Known diagnostic test to determine thyroid diseases (application U.S. No. 2007/0178604, class NCI 436/500). The invention presents a method of immunological studies of biological markers in the biological sample used for the detection of thyroid disorders by using a combination of "sen is HIV-analysis, sequential competitive analysis and serological studies. As a solid phase using different groups of particles coated with various immunological binding agents, immunological reaction is carried out on the surface of the particles. For detection of biological markers associated with labeled particles using flow cytometry. The method allows to identify in a biological sample thyrotropin, thyroxine, trijodthyronin and thyroid-peroxidase.

The method is not intended to identify 17α-Oh-progesterone, and immunoreactive trypsin simultaneously with thyroid hormones.

The known method mnogoukladnogo immunoassay (patent of Russia №2184970, IPC class GOIN 33/53). The method is designed for mnogoukladnogo analysis of samples of dry blood stains and can be used for mass screening of congenital anomalies in newborns. The method consists in the fact that at the bottom of the wells of microtiter Board immobilized first biospecific components in the form of discrete microplasma, extracted with detectable analytes from a dried blood spots by adding to the well a mixture of buffer and conjugate with the second biospecific components defined analytes. The reaction biospecific binding with labeled Pt-scat - or uroporphyrin biospecific component of the mi is carried out in discrete areas of the first immobilized biospecific components, and the emission of fluorescence detected by sequential scanning of microplasma focused laser beam.

The disadvantage of this method is the low sensitivity for quantitative detection of analytes.

A known method of detecting biomarkers (application U.S. No. 2011/01599530, class NCI 435/28). In the invention described method of detecting two or more of the biomarkers used in the screening of newborns for congenital diseases. The method uses a sample of dried blood spots with a diameter of 3.2 mm, which is placed in the hole microtiter Board (microplate), extracted with blood by adding universal buffer, which allows you to extract all desired biomarkers, in particular thyrotropin, thyroxine, immunoreactive trypsin and 17α-Oh-progesterone. As a solid phase using a set of microparticles labeled with two fluorescent labels for detection of particle type and the corresponding biomarker, while the concentration of biomarkers clarify by defining value from the measured hematocrit hemoglobin concentrations. Detection is carried out by use of flow cytometry.

The disadvantage of this method is the large number of operations and the use of flow cytometry, which complicates and increases the cost method.

The closest I have is a way to detect multiple analytes in the sample dry blood (Jong-Juan, at all. Simaltaneous Quadruple-Label Fluorometric Immunoassay of Thiroid-Stimulating Hormone, 17α-hidroxyprogesterone, Immunoreactive Tripsin, and Creative Kinase MM Isoenzyme in Dried Blood Spots. B: CLIN.CHEM., vol.38, No. 10, 1992, p.2038-2043). The method allows to simultaneously detect four of the analyte in the sample dry blood: thyrotropin (TSH), 17α-Oh-progesterone, and immunoreactive trypsin (IRT) and creatine kinase MM. The analysis carried out in the wells microturbulence strip, the surface of which is coated with a mixture of monoclonal antibodies against β-TSH IRT and ck-MM and polyclonal antibodies against 17α-Oh-progesterone, for the detection of desired analytes as labels use ions of lanthanides: Eu3+Sm3+, Tb3+and Dy3+. The fluorescence intensity of each label is measured fluorimeter with a temporary permit. To achieve the required sensitivity in the way are precompensation ions of lanthanides from one chelate complex to another, and adding the solution to enhance the luminescence label, which results in a complicated way.

The disadvantage of this method is the complexity of the analysis due to the use of a large number of operations. In addition, the accuracy of the analysis is reduced due to the cross-impact of emission of luminescence labels when determining quantitative values of analytes.

The objective is to provide a method for detecting markers of congenital diseases novaro the military.

The technical result that is achievable with the use of the invention is to improve the accuracy and specificity of the quantitative detection of biomarkers in the sample dried blood spots collected from newborns.

The technical result is achieved by the fact that in the known method for the simultaneous detection of several markers of congenital diseases in dry blood stains, including immobilization in the microplate well first immunospecificity components to thyrotropin, immunoreactive trypsin and 17α-OH-progesterone, accommodation in well paper disk sample dry blood, the extraction of detected markers from dried blood spots by adding in the hole buffer containing danazol, holding immune response with specific monoclonal antibodies and 17α-OH-progesterone, labeled with a fluorescent label, and the detection mode time resolution of the emission of luminescence labels associated with complexes, in the microplate well advanced immobilized first immunospecificity components for detection of thyroxine, all immunospecificity components for each token immobilized at the bottom of a microplate well as discrete areas in the buffer for the extraction of markers add 8-anilinonaphthalene-1-sulfonic acid and antibodies to 17α-HE-progestero is, which is associated with the extracted 17α-OH-progesterone for the simultaneous formation of an immune complex with antivirovym antibodies in the appropriate microregions, then in a microplate well add the reaction solution second immunospecificity component containing a mixture of three biotinylated antibodies to thyrotropin, immunoreactive trypsin and thyroxine (T4), and conjugate 17α-Oh-progesterone-protein-Biotin for obtaining immune labeled biotype complexes in discrete microblasted, remove the paper disk of dry blood sample, add conjugate of streptavidin with Pt-coproporphyrin for education phosphorescent Biotin-streptavidin complexes on microblasted at the bottom of the wells of microtiter fees, and emission of phosphorescence tag detects sequentially scanning discrete microregions focused laser beam.

The technical result is achieved by the fact that the extraction of danazol is used to displace 17α-Oh-progesterone from complexes with proteins.

The technical result is achieved by the fact that the extraction of 8-anilinonaphthalene-1-sulphonic acid is used to destroy complexes thyroxine with proteins.

The technical result is also achieved by the fact that discrete microblast put on the bottom of a microplate well as on the edge is her least three micro-watersheds for each token.

The technical result is achieved by the fact that as immunopurification component to identify thyrotropin use of monoclonal antibodies to thyrotropin.

The technical result is also achieved by the fact that as immunospecificity component to identify thyroxine use heterogeneous conjugate triiodthyronine with protein carrier.

The technical result is also achieved by the fact that as immunospecificity component to identify 17α-Oh-progesterone use individule antibodies to rabbit immunoglobulins.

The technical result is also achieved by the fact that as immunospecificity component for detection of immunoreactive trypsin using monoclonal antibodies to trypsin.

The technical result is also achieved by the fact that as the second immunospecificity component to identify thyroxine using mouse monoclonal antibodies.

The technical result is also achieved by the fact that as the second immunospecificity component to identify 17A-Oh-progesterone using polyclonal rabbit antibodies.

Not known to the authors of the technical solution with a set of features similar to the declare, therefore, the present invention meets the criterion of novelty.

Known technical solutions is tion, used to identify--markers for congenital diseases of newborns in a dry blood stain (Jong-Juan, at all. Simaltaneous Quadruple-Label Fluorometric Immunoassay of Thiroid-Stimulating Hormone, 17α-hidroxyprogesterone, Immunoreactive Tripsin, and Creative Kinase MM Isoenzyme in Dried Blood Spots, application U.S. No. 2011/01599530, class NCI 435/28, patent of Russia №2184970, IPC class GOIN 33/53 and others). In the known technical solutions detects two or more markers of congenital diseases of the newborn, or more thyroid hormones, using biospecific immobilization components to detektivami markers in the hole or at the bottom of a microplate well, extracting markers from a sample of dried blood spots, holding biospecific reactions with the formation of biospecific complexes labeled with fluorescent or luminescent label, and detecting the emission of luminescence labels or other known method. The proposed technical solution, by introducing into the well of the reaction solution containing biotinylated antibodies to thyrotropin, immunoreactive trypsin and thyroxine and conjugate 17α-Oh-progesterone-protein-Biotin, 8-anilinonaphthalene-1-sulfonic acid and danazol together with other signs creates the possibility of more accurately be extracted from the blood samples and to detect these markers, which increases the accuracy and specificity of the quantitative analysis of the sample with the steamboat blood stains, taken from newborns. Therefore, the present invention meets the criterion level of technology.

The invention can be used in health care, particularly in Pediatrics and neonatology for screening, early detection of congenital anomalies of child development and implementation of appropriate corrective therapy. Therefore, the present invention meets the criterion of industrial applicability.

In Fig.1, 2 and 3 presents a schematic of the various stages of conducting the detection of markers from a sample of dried blood spots from 1 - well 96-well microplate, 2 - the bottom of the wells, 3 - microregions for detection thyrotropin (TSH), 4 - microregions for detection of immunoreactive trypsin (IRT), 5 - microregions for detection of total thyroxine (T4), 6 - microregions for detection of 17α-Oh-progesterone (OP), 7 - studied TSH blood, 8 - researched RTI, 9 - studied T4the 10 - researched OP, 11 - antibodies to OP, 12 - labeled Biotin anti-TSH antibodies, 13 - labeled Biotin anti-T4antibodies, 14 - labeled Biotin anti-IRT antibodies, 15 - conjugate OP-Biotin and 16 - conjugate streptavidin-Pt-coproporphyrin; Fig.4 shows a calibration curve for determining the concentration of TSH in Fig.5 shows a calibration curve to determine the concentration of RTIs; f is g 6 presents the calibration curve to determine the concentration of T 4Fig.7 shows a calibration curve to determine the concentration OF;

The method is as follows.

In samples dried capillary blood spots of newborns at the same time quantitatively detects four of the analyte, which serve as markers of congenital diseases of the newborn. Low blood levels of newborn thyroid hormone thyroxine in combination with high levels of the hormone thyrotropin are markers of possible development in congenital hypothyroidism. Simultaneous determination of the concentrations of the two hormones allows you to more accurately diagnose different forms of this disease. For the diagnosis of congenital adrenal hyperplasia, adrenogenital syndrome, use the detection in the blood of newborns increased levels of corticosteroid hormone 17α-Oh-progesterone and to diagnose other congenital diseases, cystic fibrosis, using the detection of elevated levels of immunoreactive trypsin.

A scheme for detecting markers shown in figures 1, 2, 3.

At the bottom of each hole 1 microplate form discrete microregions for the quantitative detection of these four markers. For this purpose, the bottom 2 holes 1 immobilized in the form of discrete microplasma first immunospecificity components: microapl is here 3 for the detection of TSH - mouse monoclonal antibodies to TSH, microblast 4 to identify IRT - mouse monoclonal antibodies immunoreactive trypsin, microblast 5 for detection of T4- conjugate triiodthyronine with bovine serum albumin (BSA), microblast 6 to identify OP - individule antibodies to rabbit immunoglobulins. At the bottom of each of the wells to identify each of the four markers immobilized at least three identical microregions, which allows to reduce the variability and increase the accuracy of the results of the quantitative analysis of markers in the blood.

The diameter of each of the microregions is about 0.5 mm At this size microregions on a solid phase immobilized small number immunospecificity components. In this connection, when the immobilization microregions to identify OP use individule of 6 antibodies to rabbit immunoglobulins, because the direct immobilization of specific rabbit antibodies to OP does not provide the necessary level and range of detected signal. When immobilization microregions 5 to identify thyroxine used heterologous conjugate with triiodthyronine, because you know that for some of monoclonal antibodies to T4this can lead to inhibition of antibody binding in the course of competitive reactions in more low is x levels of the investigated analyte in the blood, accordingly, increases the sensitivity analysis. In the solution for immobilization add glycerine to reduce speed drying drops and, consequently, increase the completeness and uniformity of biospecific sorption components.

For analysis of dried on filter paper spots analyzed blood cut out a disk with a diameter of 3.2 mm and placed in the sensitized wells of the microplate. The analysis is performed in three stages: two stages immunoanalytical (Fig.1 and Fig.2) and one detector (Fig.3). In the first stage (see Fig.1) in a microplate well add extracting buffer containing danazol, 8-anilinonaphthalene-1-sulfonic acid (8-ANS) and highly specific rabbit polyclonal antibodies to OP. In the process of extraction from the blood-soaked paper disk in the solution is extracted with four of the studied marker. Two of the extracted token TTG and ART associated with the respective monoclonal antibodies, immobilized in the form of discrete regions 3 and 4 on the bottom of the wells (see Fig.1). The other two marker OF 9 and T410 is extracted in the form complexes with proteins of the blood, which prevents them from binding to antibodies and, therefore, the correct measurement. To displace UP of complexes with proteins use steroid compound danazol, and for destruction of the complex is impressive thyroxine - acid 8-ANS. The destruction of the complexes in the presence of data of chemically active reagents on stage extraction can reduce known for their negative impact on the affinity of monoclonal antibodies specific to other markers. Released EP 9 associate in solution with specific rabbit antibodies 11 with simultaneous formation of the immune complex with antivirovym antibodies immobilized in microregions 6 to identify OP (see Fig.1).

For the second stage of the analysis (see Fig.2) in a microplate well add the reaction solution second immunospecificity component containing a mixture of three biotinylated monoclonal antibodies: TSH, T4and RTIs, and conjugate 17α-Oh-progesterone-BSA-Biotin, respectively, Ref.12, 13, 14 and 15 in Fig.2. At the same time spend four immunoreactive (Fig.2): marked by biotype second monoclonal antibody TG and ART form a "sandwich"complexes PA respective microblasted 3 and 4; T4from the sample 9 competes for binding centers labeled with Biotin monoclonal antibodies to thyroxine 13 with immobilized triiodthyronine 5; and conjugate 17α-Oh-progesterone-BSA-Biotin 15 displaces OP 10 of the immune complex on the microregions 6 to identify OP. Biotinylation second immunospecificity components used for growing the Oia signal of immune complexes, formed on the specific microblaster.

For the competitive analysis OF use conjugate 17α-Oh-progesterone-BSA-Biotin obtained by attaching Biotin to the 17α-OH-progesterone, pre anywherefrom with BSA, which allows, firstly, to increase the load-labeled conjugate, the ratio of Biotin/conjugate, and, secondly, the spatial spread UP and Biotin in the conjugate, and thereby to simplify the interaction with antibodies and the subsequent binding of Biotin with streptavidin. Using conjugate with such properties leads to an increase in the intensity of the recorded signal and the expansion of the dynamic range of the measurements and, accordingly, to improve the accuracy of measuring the concentration of the marker.

Holding immunoanalytical reactions in two stages: first, the binding of redundant markers with antibodies (see Fig.1), and then the competition-added, conjugated 17α-Oh-progesterone-BSA-Biotin for 17α-Oh-progesterone, or linking with the second labeled with Biotin monoclonal antibodies for TSH and RTIs (see Fig.2), allows to increase the specificity of the analysis, to reduce the influence of other structurally similar compounds in the blood, which is especially important when determining the level OF newborns with low gestation in the blood which may contain a very high level intermediate Stero the underwater hormone 17α-Oh-pregnenolone. This method of holding immunoreactive allows to reduce the number of false-positive results, especially in premature newborns and thereby significantly improve the specificity and accuracy of the detection of the markers.

After incubation the disk with the blood sample is removed and the microtiter plate is washed.

For the third stage of the analysis (see Fig.3) into the wells of the microplate add the detecting solution containing a conjugate of streptavidin with Pt-coproporphyrin 16, which forms a phosphorescent Biotin/streptavidin complexes at all specific microblasted at the bottom of the wells of the microplate. Then, the microplate is washed and dried. The detection of emission of phosphorescence hold mode time resolution luminescence by sequential scanning of the focused laser beam of each of the microregions at the bottom of a microplate well.

The results of the analysis multianalyte calibration samples are building four calibration curves to determine the concentration values of the markers in the sample of blood is shown in Fig.4-7. Multianalyte calibration samples are a collection of dried blood spots, each of which contains a mixture of four of the studied markers in different ratios.

The recorded signal in the analysis of TSH and RTI is directly proportional to the concentratie markers in the blood (see Fig.4 and 5), and the signal recorded in the analysis of T4and OP, is inversely proportional to the concentration of these markers in the blood (see Fig.6 and 7).

Using this method, carry out the simultaneous quantitative determination of four markers, extracted in a microplate well from one impregnated analyzed the blood of a paper disk with a diameter of 3.2 mm, that is, the analysis is performed in the same dilution of the sample of blood in a single reaction solution for all markers. For each of the markers has a clinically significant concentration range for diagnosis, in particular, it is necessary to measure low concentrations of TSH (from 2 mkme/ml) and very high levels of RTIs (up to 300 ng/ml). Improving the accuracy and specificity of the simultaneous measurement of the concentration of the four markers in clinically relevant ranges are due to the entire set of features including immobilization of several micro-watersheds biospecific reagents in each of the microregions, using immunospecificity components (heterologous conjugate T3-BSA, specific and high-affinity antivitamin antibody conjugate OP-BSA-Biotin), and Biotin-streptavidin complex, vysokofosforistye label and method of detecting the phosphorescence on the surface of micro-watersheds. Special value which has the sequence of operations when adding reagents in the sensitized wells of the tablet when conducting multianalyte: in the first stage, the extraction is carried out markers from samples of dried blood spot the release of complexes with proteins in the blood and bind with specific antibodies, and the second is the binding of the labeled second specific antibodies and conducting competitive immune responses. This method of analysis allows to avoid cross-influence present in the reaction mixture reagents specific for different markers, and to increase the sensitivity and specificity of simultaneous detection of markers in the blood that is necessary for effective identification of neonates with suspected congenital hypothyroidism, adrenogenital syndrome and cystic fibrosis during neopallium screening.

Example 1. Detection of thyrotropin in dried blood spot.

On the surface of the bottom of the wells of polystyrene 96-well microtiter plate reader (Labsystems, cat. No. 95029140) immobilized specific discrete microregions to identify markers TSH IRT, T4and OP. For detection of each marker immobilized 4 identical microregions with a diameter of 0.5 mm, 16 microplasma at the bottom of each well. For this purpose on the bottom of each well of the microplate using the device for printing chips put in the appropriate point of the droplets with a volume of 25 nl solutions corresponding immunospecificity components in a concentration of 0.1 mg/ml To create microplasma specific to TSH, COI is lsout mouse monoclonal antibodies to TSH, for microplasma for the detection of RTI using mouse monoclonal antibodies immunoreactive trypsin, for microplasma specific to T4use heterologous conjugate triiodthyronine with BSA, and microblasted to identify OP use individule antibodies to rabbit immunoglobulins. Conjugate triiodthyronine with BSA synthesize carbodiimide method (Beckman N. And. and the other Wedge.lab.diagnosis of 2004, No. 8). To reduce the speed of drying of the droplets in the solution for sorption add 5% glycerol (Sigma). The microplate is incubated for 18 hours at 4°C, washed and treated with a blocking solution of 15 mm phosphate buffer containing 1% BSA (bovine serum albumin) for 1 hour at room temperature, remove the blocking solution and dried microplate.

For analysis of the dried filter paper blood spots with known content of TSH (the production company "IMMUNOGEN", Russia) cut out a disk with a diameter of 3.2 mm and is placed in the hole sensitised microplate. Then in a microplate well add 25 ál reaction buffer for universal detection of all four markers, containing 15 mm Tris buffer at pH of 7.75, 0.05% tween-20, 5% BSA (all reagents SIGMA, USA), 0.15 mg/ml 8-anilinonaphthalene-1-sulfonic acid (Biomedicals, USA) and 0.3 mg/l of danazol (SIGMA, USA). Mick is olyset incubated with shaking at a temperature of 18-25°C for 5 minutes, add 25 ál of a solution of biotinylated mouse monoclonal antibodies to TSH in a concentration of 2.5 μg/ml in reaction buffer. To attach bitenova labels to antibodies using the reaction with Biotin-N-hydroxysuccinimide ester Sigma, USA, cat. No. H1759). The microplate is incubated with shaking for 1 minute at a temperature of 18-25°C and 18 hours at a temperature of 2-8°C without shaking. Remove the disks from the wells and washed tablet. After incubation in a microplate well add 25 ál of a solution of a conjugate of streptavidin with Pt-coproporphyrin and incubated for 15 minutes at room temperature, washed and dried the microplate. Conjugate synthesized via N-hydroxysuccinimide Pt-coproporphyrin obtained carbodiimide method (De Haas R. R. et.al., J. Histochem.&Cytochem. 1997. - 45. - 1279-1292).

The detection of emission of phosphorescence is carried out by sequential scanning of the bottom of the wells dry microplate laser beam with a wavelength of excitation 365 nm and the signal in the measurement mode long-lasting phosphorescence Pt-coproporphyrin at a wavelength of 645 nm. The scan results for each of the studied marker count the average number of photoemulsion all specific for the marker to microblaster.

Output values of the intensities of the signal from microblasted privedennyu table 1. As can be seen from the presented results, the rise of a signal specific to the TSH microblasted by increasing the concentration of TSH in the sample is linear, with a significant, more than 6 times, the excess signal in the analysis sample TSH 10 mkme/ml above the signal from the sample with zero content of TSH, indicating a high sensitivity of the method and the ability to accurately measure low concentrations of the marker. The intensity of the specific signal persists in the presence of supplements (8-ANS and danazol) in the composition of the reaction buffer. However, the signal strength value from microblasted specific to the other three markers vary in the range not exceeding the values obtained for the reference sample TSH (see tab.1), which proves the absence of cross-interactions with this method of conducting multianalyte analysis and, accordingly, the high specificity of the method.

Example 2. Detection of total thyroxine in dried blood spot.

In a microplate well, sensitized as described in example 1 is placed blood-soaked disc with a diameter of 3.2 mm, cut from the dried filter paper blood spots with a known content of T4(production of CJSC "Immunogen", Russia) add 25 ál universal reaction buffer described in the example 1, the microplate and incubated with shaking at a temperature of 18-25°C for 5 minutes. In a microplate well add 25 ál of a solution of biotinylated mouse monoclonal antibodies to T4in the reaction buffer at a concentration of 1 μg/ml For compounds bitenova label with antibodies using the reaction with Biotin-aminohexyl-N-hydroxysuccinimide ester (Sigma, USA, cat. No. V). The microplate is incubated with shaking for 1 minute at a temperature of 18-25°C and 18 hours without shaking at a temperature of 2-8°C, remove the disks from the wells and wash the microtiter plate.

The detection carried out as described in example 1.

The detection results are shown in table 2. As in example 1, the dependence of the signal specific to T4microblasted on the concentration of T4in the sample has a linear character. As can be seen from table 2, this example shows that the levels of total thyroxine in the blood may be measured under the condition of separation immunoassay on two stages and holding it in a universal reaction buffer. The signal strength value from microblasted specific to the other three markers, do not exceed background values, which confirms the absence of cross-interactions and high specificity of the method.

Example 3. Detection of immunoreactive trypsin in dry five the fuck blood.

In a microplate well, sensitized as described in example 1 is placed blood-soaked disc with a diameter of 3.2 mm, cut from the dried filter paper blood spots with known content of RTIs (production of CJSC "IMMUNOGEN", Russia) add 25 ál universal reaction buffer composition described in example 1, and incubated with shaking at a temperature of 18-25°C for 5 minutes. Then in a microplate well add 25 ál of a solution of biotinylated monoclonal antibodies to RTI in the reaction buffer at a concentration of 2.5 µg/ml For compounds bitenova label with antibodies using the reaction with Biotin-N-hydroxysuccinimide ester (Sigma, USA, cat. No. n). The microplate is incubated with shaking for 1 minute at a temperature of 18-25°C and 18 hours at a temperature of 2-8°C without shaking. Remove the disc from the wells and wash the microtiter plate.

The detection carried out as described in example 1.

From the detection results shown in table 3, it is seen that in the analysis of samples containing IRT, the method allows to register a high level signal from the respective microplate significant, more than 50 times, the separation of the study sample with the minimum level of RTIs from the study of the zero sample, indicating a high sensitivity SP is soba. As can be seen from table 3, the specificity of the method is confirmed by the absence of significant signals microblasted specific to other tokens.

Example 4. Detection of 17α-Oh-progesterone in dried blood spot.

In a microplate well, sensitized as described in example 1 is placed blood-soaked disc with a diameter of 3.2 mm, cut from the dried filter paper blood spots with known content OF (production of CJSC "IMMUNOGEN", Russia) add 25 ál of a solution of rabbit polyclonal anti-AP antibodies in the reaction buffer and incubated with shaking for 5 minutes at a temperature of 18-25°C. Then, in a microplate well add 25 ál of conjugate solution OP-BSA-Biotin at a concentration of 1 μg/ml in reaction buffer, which are synthesized using Biotin-aminohexyl-N-hydroxysuccinimide ether (Sigma, USA, cat. No. V) of the conjugate OM-BSA obtained by joining carbodiimide method 17α-Oh-progesterone-3-oxime (Sigma, USA) to BSA (Sigma, USA). The microplate is incubated with shaking for 1 minute at a temperature of 18-25°C and 18 hours at a temperature of 2-8°C without shaking.

The detection carried out as described in example 1.

The detection results shown in table 4, indicate that the carrying out immunoassay in universal buffer of the research complexes antivitamin antibodies with specific antibodies and conjugate OP-BSA-Biotin allows you to achieve value for specific signals, significantly different from background values, and the signals recorded on the specific other markers microblasted, do not exceed the level of background values, which can be seen from table 4.

As can be seen from examples 1-4, the method allows to obtain high specificity and sensitivity simultaneous detection of each of the four investigated in dry blood samples tokens.

Example 5. Construction of calibration curves for quantitative determination of markers TSH IRT, T4and OP in dried blood spot.

Multicomponent calibration sample containing TSH IRT, T4and OP, prepared from refined markers of whole blood donors by adding appropriate quantities of thyrotropin (ICN, USA), trypsin (USBiological, USA), thyroxine (ICN, USA) and 17α-Oh-progesterone (ICN, USA), and coating the obtained solution of the blood in the form of drops 100 ál of special forms of filter paper (Schleicher &Schuell, Germany, cat. No. 903) with subsequent drying forming spots of blood on the air. Prepared sample certificate level markers using commercial lanthanide mono tests TSH-NEOSKIN", "RTI-NEOSKIN", "T4screen" and "17α-Oh-PROGESTERONE-NEOSKIN" (CJSC "IMMUNOGEN", Russia). The results of the evaluation are presented in table 5.

For analysis of dry mult is a component of the calibration samples cut out blood-soaked disks with a diameter of 3.2 mm and placed one in each of the sensitized wells of the microplate, as described in example 1. The analysis of each calibrator carried out in duplicates. To each well of the microplate was added 25 μl of a solution of rabbit polyclonal anti-AP antibodies in the universal reaction buffer containing 15 mm Tris buffer at pH of 7.75, 0.05% tween-20, 5% BSA (all reagents SIGMA, USA), 0.15 mg/ml 8-anilinonaphthalene-1-sulfonic acid (Biomedicals, USA) and 0.3 mg/l of danazol (SIGMA, USA). The microplate is incubated with shaking for 5 minutes at a temperature of 18-25°C. Then, to each well of the microplate was added 25 μl of a mixture of three biotinylated mouse monoclonal antibodies to TSH in a concentration of 2.5 μg/ml, to RTIs in concentration of 2.5 μg/ml and T4at a concentration of 1 μg/ml and 1 μg/ml conjugate OP-BSA-Biotin in the reaction buffer. The microplate is incubated with shaking for 1 minute at a temperature of 18-25°C and 18 hours at a temperature of 2-8°C without shaking.

The detection carried out as described in example 1.

The final assessment of the results of determination of each hormone use average values of the scan data of the wells of the microplate, which was a re-analysis of the calibration samples. The results of the analysis multianalyte calibration samples are building four calibration curves of average intensity values measured at the respective microblasted signal from which concentratie markers in multianalyte samples. The obtained curves for the determination of TSH is shown in Fig.4, for IRT in Fig.5, for T4 in Fig.6 and OP - Fig.7. The resulting signal characterizing TSH or RTI, is directly proportional, and the signal characterizing the T4or OP, is inversely proportional to the concentration of the respective markers in the sample of blood, as simultaneously in the same reaction mixture are two "sandwich" immune response to TSH and RTIs, and two competitive immune reaction with low-molecular markers of T4and OP. As follows from the calibration curves shown in Fig.4-7, the proposed method allows the simultaneous quantitative determination of markers in dried blood samples in the concentration ranges from 0 to 250 mkme/ml TSH, from 0 to 300 ng/ml IRT, from 0 to 200 nmol/l T4and from 0 to 300 nmol/l OP, which is consistent with the ranges of concentrations of markers that are required for the diagnosis of congenital hypothyroidism, congenital adrenal hyperplasia and cystic fibrosis in newborns. Thus the sensitivity of the method of detection is 2 km mkme/ml for TSH, 4 ng/ml for RTIs, 10 nmol/l for T4and 2 nmol/l for OP that also meets established performance criteria of method of blood analysis for the purposes of the neonatal screening of diseases.

Example 6. Quantity is a significant concentration of markers TSH, RTI, T4and OP in the control dried blood spot.

The method is carried out as described in example 5. As the samples used control samples dried blood spot with a known content of markers in the blood (Centers for Disease Control and Prevention, US).

For detection of the markers studied blood spots cut out blood-soaked disc with a diameter of 3.2 mm and placed in a microplate well. The analysis of each sample is carried out in ten repetitions. Further analysis is performed as described in example 5. The concentration of the marker in the sample is determined by the calibration curve for this token, based on the results of the analysis of multicomponent calibration samples as described in example 5. In tables 6-8 shows the obtained average concentrations of markers of TSH IRT, T4and OP in the analyzed control samples, indicating the coefficient of variation of the results. The obtained values correspond to the expected values of the concentrations of the markers in all control samples. The variability of the results of the analysis does not exceed 15%, regulated for the quantitative analysis of dried blood spot. The results show that the method can improve the accuracy of detection of markers in dried blood spot.

Example 7. Simultaneous determination of the concentration of markers TT is, RTI, T4and OP in dried capillary blood spots of newborns.

The method is carried out as described in example 5. As the samples used clinical samples of dried blood spot newborn, surveyed in the framework of the neonatal screening. Clinical samples obtained by drying on a special form of filter paper and a drop of blood from the heel of the child to 3-5 birthday. Examples of pre-certify using lanthanide mono-tests TSH-NEOSKIN", "RTI-NEOSKIN", "T4screen" and "17α-Oh-PROGESTERONE-NEOSKIN" (CJSC "IMMUNOGEN", Russia).

The research results of clinical blood samples of newborns are shown in table 6. These results correlate well with the results of the study using a commercial mono-tests. The study sample No. 1-10 blood of healthy newborns (including four of prematurity) showed that the obtained values normal ranges of concentrations for all four markers. In samples No. 11-15, obtained from infants diagnosed with congenital hypothyroidism detected increased levels of TSH and decreased levels of total T4. Pathologically high level of RTI is fixed according to the results of examination of samples No. 16-18 blood of newborns diagnosed with risk of developing cystic fibrosis. In samples No. 19 and the 20, taken in newborns with congenital adrenal hyperplasia detected elevated levels of the marker 17α-Oh-progesterone. For each pathology detectable blood levels of other, non-specific for this disease markers, fluctuated within the normal range (table 6).

Thus, the proposed method to a sample of dried blood spots has provided an adequate assessment of the four markers in the blood of both healthy and sick newborns. It is shown that the proposed method allows on the basis of the study, one sample of dry spots capillary blood to diagnose at the same time three of congenital abnormalities in newborns with a high degree of specificity and accuracy, and its application can significantly improve the effectiveness of neonatal screening programs.

The results of reception signal intensity of the phosphorescence of microplasma that are specific to different markers in the analysis of samples of dried blood spots containing thyrotropin.

Table 1
Concentration TTGV the sample mkme/mlThe intensity of phosphorescence microregions, imp.
TSHRTIT4OP
02711926
1017371124
25430121218
5082761514
1001696132119
2504107101613

The results of reception signal intensity of the phosphorescence microable the stay, specific to the various markers in the analysis of samples of dried blood spots containing immunoreactive trypsin.

Table 2
The concentration of RTIs in the sample, ng/mlThe intensity of phosphorescence microregions, imp.
TSHRTIT4OP
059914
3598731112
651130511219
13013761215 17
25524130191320
65512154061821

The results of reception signal intensity of the phosphorescence of microplasma that are specific to different markers in the analysis of samples of dried blood spots containing thyroxine.

Table 3
The concentration of total T4in the sample, nmol/lThe intensity of phosphorescence microregions, imp.
TSHRTIT4OP
01211287715
1557217012
25912176117
50106118322
10016356629
200131017312

The results of reception signal intensity of the phosphorescence of microplasma that are specific to different markers in the analysis of samples of dried blood spots containing 17α-Oh-progesterone.

Table 4
Concentration OP in the sample, nmol/mlThe intensity of phosphorescence microregions, imp.
TSHRTIT4OP
01011193110
5,2127131246
12,891212553
33,916619237
1058328127
27820102186

The multicomponent composition of the calibration samples.

Table 5
Caliber is CNA sample TSH, mkme/mlRTI ng/mlT4, nmol/lOP, nmol/l
And0000
In7,016,5135
12,529,42413
D2650,74533
E5094,190100
F100198180260

Results detection of thyrotropin from control samples dried blood spot.

Table 6
Reference material The expected range of concentrations of TSH, mkme/mlThe concentration of TSH measured by the proposed method, mkme/mlThe coefficient of variation of measurement results concentration %
21119,2-29,526,810,2
21231,0-47,334,28,9
21356,6-81,960,87,8

Results detection of immunoreactive trypsin from control samples dried blood spot.

Table 7
Reference materialThe expected range of concentrations RTI ng/mlThe concentration of RTIs, measured by the proposed method, ng/mlThe coefficient of variation of measurement results concentration %
139113,6-20,118a 12.7
139248-60 5410,6
139392-1221209,9
1394164-213204the 10.1

Results detection of total thyroxine from control samples dried blood spot.

Table 8
Reference materialThe expected range of concentrations of total T4, nmol/lThe concentration of total T4measured by the proposed method, nmol/lThe coefficient of variation of measurement results concentration %
10114,0-33,5to 19.911,3
10271-11281,410,2
103112-152141the 9.7

Results of detection of 17α-Oh-progesterone from control samples dried blood spot.

Table 9
Reference materialThe expected range of concentrations UP, nmol/lConcentration OF measured by the proposed method, nmol/lThe coefficient of variation of measurement results concentration %
25117,0-33,220,19,9
25235,1 of 62.338,910,2
25379,9-138,587,712,2

The results of determination of thyrotropin, total thyroxine, immunoreactive trypsin and 17α-Oh-progesterone samples from dried blood spots of newborns.

Table 10
Sample numberThe results of determination of markers of known methodsThe results of the op is adelene markers proposed method
TSH, mkme/mlTotal T4, nmol/lRTI ng/mlOP, nmol/lTSH, mkme/mlTotal T4, nmol/lRTI ng/mlOP, nmol/l
11,693,024,19,41,8to 83.526,27,8
20,2 104,046,33,60,482,5to 49.34,1
38,347,844,76,58,946,947,65,8
40,4of 37.8of 21.212,40,642,025,411,2
513,2100,027,06,412,570,032,8the 5.7
60,669,339,54,10,8of 60.544,74,5
71,159, 50,77,72,9of 60.556,17,1
83,264,529,23,10,262,030,22,8
90,339,718,94,50,339,121,45,2
/td>
10of 21.248,522,12,120,950,0to 25.32,8
1177,513,517,610,9126,611,818,99,9
1222,09,0 43,79,818,3a 12.740,19,1
1365,28,536,51177,6the 10.134,210,2
1427,034,139,86,927,332,842,67,4
1538,726,0to 49.97,133,925,455,36,3
161,51153297,41,91023407,9
175,6111 1428,14,3951508,3
184,31043139,4a 3.9893298,7
197,88726,83698,57621,6341
209,17439,61798,28245,6188

1. The method of simultaneous detection of several markers of congenital diseases in dry blood stains, including immobilization in the microplate well first immunospecificity components to thyrotropin, immunoreactive trypsin and 17α-OH-progesterone, accommodation in well paper disk sample dry blood, the extraction of detected markers from dried blood spots by adding in the hole buffer containing danazol, holding immune response with specific monoclonal antibodies and 17α-OH-progesterone, labeled with a fluorescent label, and the detection mode time resolution of the emission of luminescence labels associated with the formed complexes, characterized in that in the microplate well advanced immobilized first immunospecificity components for detection of thyroxine, the first immunospecificity components for each token immobilized at the bottom of l the NCI microtiter Board in the form of discrete microplasma, in the buffer for the extraction of markers add 8-anilinonaphthalene-1-sulfonic acid and antibodies to 17α-OH-progesterone, which is associated with the extracted 17α-OH-progesterone for the simultaneous formation of an immune complex with antivirovym antibodies in the appropriate microregions, then in a microplate well add the reaction solution second immunospecificity component containing a mixture of three biotinylated antibodies to thyrotropin, immunoreactive trypsin and thyroxine, and conjugate 17α-Oh-progesterone-protein-Biotin for obtaining immune labeled with Biotin complexes in discrete microblasted, remove the paper disk of dry blood sample, add conjugate of streptavidin with Pt-coproporphyrin for education phosphorescent Biotin-streptavidin complexes on microblasted at the bottom of a microplate well, and the emission of phosphorescence tag detects sequentially scanning discrete microregions focused laser beam.

2. The method according to p. 1, characterized in that the extraction of danazol is used to displace 17α-Oh-progesterone from complexes with proteins.

3. The method according to p. 1, characterized in that the extraction of 8-anilinonaphthalene-1-sulphonic acid is used to destroy complexes thyroxine with proteins.

4. The method according to p. 1, characterized in that thesis is specific microregions put on the bottom of the wells in the form of at least three micro-watersheds for each token.

5. The method according to p. 1, characterized in that as immunospecificity component to identify thyrotropin using mouse monoclonal antibodies to thyrotropin.

6. The method according to p. 1, characterized in that the first immunospecificity component to identify thyroxine use heterogeneous conjugate triiodthyronine with protein carrier.

7. The method according to p. 1, characterized in that the first immunospecificity component to identify 17α-Oh-progesterone use individule antibodies to rabbit immunoglobulins.

8. The method according to p. 1, characterized in that as immunospecificity component to identify immunoreactive trypsin using mouse monoclonal antibodies to trypsin.

9. The method according to p. 1, characterized in that as the second immunospecificity component to identify thyroxine using mouse monoclonal antibodies.

10. The method according to p. 1, characterized in that as the second immunospecificity component to identify 17α-Oh-progesterone using polyclonal rabbit antibodies.



 

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