Method for carrying out immunochromatographic assay with dissociating fluorescent tag
SUBSTANCE: invention refers to medicine and concerns a method for carrying out an immunochromatographic assay with a dissociating fluorescent tag, wherein complexes comprising molecules of an antigen or antigens, specific antibodies and tag molecules are formed on a membrane test strip. After the assay is completed, the tag is extracted from the working membrane of the test strip, and a fluorescent signal is measured in a liquid volume.
EFFECT: method according to the invention provides creating the optimum conditions for tag fluorescence, as well as eliminating the harmful influence of the membrane which is impermeable to decrease an intensity of both exciting and emitted light.
1 dwg, 1 ex
The invention relates to immunology and biotechnology and is a way immunochromatographic analysis dissociable fluorescent label.
Immunochromatographic method for content evaluation of various compounds widely used in the practice of medical diagnosis [Raphael C. Wong l Harley Y. Tse (Editors) (2009) Lateral Flow Immunoassay. Springer, USA].
Lateral flow tests can be implemented in several different ways. Let us consider the General form of the scheme of "sandwich"format chromatography as one of the most frequently used. Sample potentially containing the designated connection, under the action of capillary forces is moved along the test strip. However, it first interacts with the labeled component (usually antibodies against defined connection), then the front of the liquid overcomes area (analysis area) to the immobilized component, linking the designated connection. The degree of binding of the label in the analytical zone and, accordingly, the intensity of staining of the membrane is determined by the concentration of the analyte in the sample. The main advantages of immunochromatographic analysis is the rapidity of results (5-15 min) and the ability besprovodnoy detection. As the label in the vast majority of cases are particles of colloidal gold. [Babaar Ngom & Yancheng Guo & Xiliang Wang & Dingren Bi. (2010) Development and application of lateral flow test strip technology for detection of infectious agents and chemical contaminants: a review // Anal Bioanal Chem 397:1113-1135]. However, if you want to increase the sensitivity analysis using other markers, such as fluorescent dyes [Yoo, Jisun, Jung, Young), Hahn, Jong Hoon, Pyo, Dongjin (2010) 'QUANTITATIVE ANALYSIS OF A PROSTATE-SPECIFIC ANTIGEN IN SERUM USING FLUORESCENCE IMMUNOCHROMATOGRAPHY', Journal of Immunoassay and Immunochemistry, 31:4, 259-265]. Although in this case, the signal requires additional equipment, this approach allows to combine the rapidity of results (due to the use of the principle of chromatography and highly sensitive detection (due to the low detection limit of fluorescent dye).
Compared with organic fluorescent dyes fluorescent lanthanide (Eu3+Sm3+, Tb3+, Dy3+, ...) complexes have the advantage of a long lifetime fluorescence (up to 1000 microseconds), a large Stokowski shift (>250 nm), narrow profile issue, allowing their use in a method microsecond spectroscopy with a time resolution [J. Yuan, G. Wang (2005) Lanthanide complex-based fluorescence label for time-resolved fluorescence bioassay. J. Fluorescence, 15, 559-568].
Fluorescent labels based on lanthanide complexes are successfully used in vysokochuvstvitel resolved in time fluorescent immunologicals the x methods. Due to the fact that the technology of reception of the signal with a time resolution allows easy separation of specific long-lived fluorescent signal marks from the short-lived background noise in the bioassay and get rid of the effects of light scattering (scattering Tindale, Rayleigh and Raman), the limit of detection of the method is significantly increased [J. Yuan, G. Wang (2005) Lanthanide complex-based fluorescence label for time-resolved fluorescence bioassay. J. Fluorescence, 15, 559-568.], [Hemmila I. (1985) Fluoroimmunoassays and immunofluorometric assays. Clin. Chem., 31, 359-370].
The formation of complexes with some organic ligands significantly enhances the fluorescence of lanthanide ions. After light absorption ligand is efficient energy transfer from the excited singlet energy level through the triplet level at the resonant energy level of the metal [Ferrari M, Ozkan M. & Heller, M. J. (2007) BioMEMS and Biomedical Nanotechnology. Volume II: Micro/Nano Technologies for Genomics and Proteomics. - Springer US].
For dissociative analysis method use the primary complexes polyaminopolycarboxylate of chelators with lanthanides, demonstrating the high stability and solubility in water. Most chelating agents form a lanthanide chelates with a binding constant of order 1015M-1. For conjugation of the label with antibodies in the structure complexone there is a corresponding chemical group, such as, for example, the R, diisopentyl-EDTA or AMINOPHENYL-EDTA. These chelating agents are well suited for dissociative analysis with subsequent increase in fluorescence by adding β-diketonates. Based on the same principle analysis system kits are used (Dissociative-Enhansed Lanthanide Fluorescence Immunoassay). In this system, europium dissociates from complexone (ITC-DTTA-Eu) in acidic medium, and then measured the fluorescence of the chelate Eu with β-diketone-tri-n-octylphosphine in micellar aqueous solution [J. Yuan, G. Wang (2005) Lanthanide complex-based fluorescence label for time-resolved fluorescence bioassay. J. Fluorescence, 15, 559-68].
To register fluorescence of lanthanide complexes using the method of fluorimetry with a temporary permit. After short-term exposure of the sample luminescence of all molecules and the cell (tablet) begins to grow exponentially. Short-lived background fluorescence decreases rapidly and does not interfere with the measurement of the fluorescence of the label. This allows the measurement of extremely long-lived fluorescence of lanthanide labels. Usually spend many cycles of measurements for one sample (within 1 s), which improves the signal-to-noise calculations [Hemmila I. (1985) Fluoroimmunoassays and immunofluorometric assays. Clin. Chem., 31, 359-70].
Despite the above advantages dissociable lanthanide labels, previously, this approach was not used for signal amplification in the lateral flow systems.
If the detection with the persecuted label is not with the membrane surface, and out of solution, volume of solution, you can create optimal conditions for fluorescence labels, as well as to fix the light shielding signal membrane test strips. To do this, applicants are encouraged after conducting a lateral flow analysis to extract related in the analytical zone of the label molecules and hold the instrument activity registration fluorescent label in the volume of extracting solution.
The advantages set forth above is confirmed experimentally and are shown in the following example.
The concentration of monoclonal antibodies HTM81 against recombinant 38 kDa antigen of Mycobacterium tuberculosis.
1. Getting conjugate antibodies with ITC-DTTA-Eu
For conjugation used a set of "kits are used® Eu-Labelling kit 1244-302" manufactured by PerkinElmer™ (Wallac Oy, Finland) containing:
1) Eu-labelling reagent" is 0.2 mg (300 nm) N1-(p-isothiocyanatobenzene)-Diethylenetriamine-N1N2N3N4-leads to compounds, which Eu3+(ITC-DTTA-Eu), freeze-dried.
2) Reinforcing solution "kits are used® Enhancement Solution)) - Triton-X100, acetic acid and chelators.
2 mg of monoclonal antibody (MAB) T C1.81 in 1 ml of 0.1 M carbonate buffer, pH 8.5 (antibodies were transferred into dialysis buffer for 35 min.) was mixed with 0.2 ml of 7.5 mm aqueous solution of Eu-labelling reagent (R-DTTA-Eu, 500-fold excess is for). The mixture was left overnight at +4°C. Purification of the conjugate Mat-ITC-DTTA-Eu carried out by the method of gel filtration using a column of 1.5×28 cm with medium Sephadex G-50, equilibrated with 50 mm Tris-HCl and 0.15 M NaCl and 0.05% NaN3, pH 7.75.
The optical density of the obtained fractions was measured on a spectrophotometer Shimadzu 1202 at a wavelength of 280 nm. To determine the amount of incorporated label aliquots of antibodies were diluted in the amplifying solution in the ratio of 1:10,000 to a final volume of 200 μl in the wells of the microplate, incubated for 2 min, and then recorded the fluorescence on a tablet reader EnSpire 2300 production company PerkinElmer™.
2. Determining the amount of antibodies labeled ITC-DTTA-Eu, with the use of amplification fluorescence
In the method used chelate Eu3+with N1(p-isocyanatobenzyl)dietilen triamine-N1N2N3N3-tetraoxane acid (ITC-DTTA-Eu) as aiming reagent. To the drug labeled antibody or conjugate antibodies with KZ added a solution of β-diketone (2-afterdepolarization β-NTA) and slightly amplifying the fluorescence of the solution (pH 3,2) containing trioctylphosphine (TORO) and Triton X-100. Ion Eu3+of the labeled agent is passed into the solution and contacted with the ligand. The solution was turned into a highly fluorescent micellar solution containing Eu(β-ITA)3(TORO)2chelate, f is uorescence which was measured by the method of fluorometry with a temporal resolution on the device Wallac 1234 Fluorimetr (Perkin Elmer, Finland) [Ferrari M., Ozkan M., Heller, M. J. (2007) BioMEMS and Biomedical Nanotechnology. Volume II: Micro/Nano Technologies for Genomics and Proteomics. - Springer US]. Registration settings fluorescence of Eu3+: delay time 0.1 MS, frequency flash 1 MS, the time of signal reading 0.4 MS, excitation at 340 nm and registration of fluorescence at 615 nm.
Preparation of calibration graph to determine the amount of bound peroxidase label on the antibody molecule. Labeled antibodies (2-20 ál, the concentration of 1-100 μg/ml) were diluted in the amplifying solution to a final volume of 200 μl in the wells collapsible transparent microplate from the set of "kits are used® Eu-Labelling kit 1244-302", incubated 10 min at room temperature, then measured the fluorescence. The content of antibodies in the fractions and the amount of incorporated label in the received drugs labeled antibodies was calculated by the following formulas:
where A=10000 - dilution factor;
B=1000000 - signal standard preparation 1 nm Eu3+;
where 1,34 - absorption of 1 mg/ml antibody at 280 nm, the 160,000 - molecular weight IgG, 0,008 - absorption of 1 μm chelate complex of europium at 280 nm.
The ratio of the number of bound peroxidase label to the antibody was 0.57.
3. Construction of calibration curve for determining the concentration of labeled antibodies immobilized on the lateral flow membrane, fluorescence Eu3+
Antibodies were applied to the working membrane CNPC-SN12 L2-P25 (pore size 15 μm) "mdi Easypack" company "Advanced Microdevices" (India). Then cut off the portion of the membrane to the immobilized antibodies and were placed in Eppendorf, was filled with 100 ál Eppendorf amplifying solution "kits are used® Enhancement Solution, incubated for 2 min, were collected aliquots of 50 µl and registered fluo is ascencio on a tablet reader EnSpire 2300 production company PerkinElmer™.
The calibration curve for the detection of antibodies shown in the drawing.
The limit of detection according to current recommendations [Burdon D., Markov, B. N. (1975) fundamentals of Metrology, 2nd ed., M], [Charykov A. K. (1984) Mathematical processing of results of chemical analysis, L.] was defined as the concentration giving a signal value equal to the background signal (640 units) plus 3 standard deviations of the background signal (3·197 units). The limit of detection antibody was 0.9 ng/ml, which corresponds to the limit of detection labels 3,1·10-12M with regard to the ratio of the amount of bound peroxidase label to the antibody.
4. Determination of limit of detection of colloidal gold as a label in the lateral flow assay (for comparison of limits of detection of two labels)
On the lateral flow membrane CNPC-SN12 L2-P25 inflicted solutions, representing a series of dilutions of particles of colloidal gold size 25 nm. Then scanned the image of the test strip and aziraphale, quantitatively assessing the intensity of the color of colloidal gold on the membrane with the help of the program "Nonlinear Dynamics TotalLab TL120 v2009". As in the case of a fluorescent label, the limit of detection was defined as the concentration of the label, giving the intensity of staining at three standard deviations above background signal. The limit of detection of colloidal gold amounted to 1.3·10-10M, h is about (at a ratio of 1 antibody at a 1-particle colloidal gold) corresponds to the limit of detection antibodies, equal to 20 ng/ml.
Thus, the limit of detection of antibodies using dissociable fluorescent label more than 20 times lower than when using traditional label is colloidal gold (0,9 ng/ml and 20 ng/ml, respectively).
BRIEF DESCRIPTION of DRAWINGS
A drawing. The calibration graph to determine the concentration of labeled antibodies immobilized on the lateral flow membrane, fluorescence Eu3+.
The method of conducting lateral flow analysis with dissociative fluorescent label, in which the membrane test strip to form complexes, which are composed of the molecules of the antigen or antigens that are specific to them antibodies, and molecules of the label, characterized in that after analysis of the label is extracted from the working membrane test strip and a fluorescent signal is measured in the liquid volume.
SUBSTANCE: invention relates to medicine and deals with a method of quantitative determination of D-antigen of polioviruses of 1-3 types by enzyme-linked immunoassay (ELISA). As an immunosorbent used is polyvalent (to three types of poliovirus) affinity-purified immunoglobulin of a class Y (IgY), obtained from egg yolks of hens, immunised with polioviruses, and as detector antibodies - an affinity-purified preparation IgG of the rabbit against each of three polioviruses.
EFFECT: invention provides a lower non-specific background and, respectively, higher reliability of the analysis result.
1 cl, 3 ex, 1 tbl
SUBSTANCE: invention refers to medicine, particularly to laboratory diagnostics. Substance of the method: cytomegalovirus antibody titre is measured; disturbed haemoglobin oxygenation in erythrocytes is detected; a histochemical method is used to determine the activity of glucoso-6-phosphate dehydrogenase and the content of erythrocyte methaemoglobin; if the cytomegalovirus antibody titre is 1:1,600, the activity of glucoso-6-phosphate dehydrogenase diseases to 15.0±0.8 standard units, and the content of erythrocyte methaemoglobin increases up to 1.7±0.09% and peripheral blood oxyhaemoglobin decreases up to 90.0±1.3%, threatening hemic hypoxia is stated.
EFFECT: invention can be used to state threatening hemic hypoxia in the pregnant women suffering aggravated cytomegalovirak infection in the third trimester of gestation.
SUBSTANCE: analysed blood sample is taken that is combined with a dynamic cytofluorometrical analysis of the taken test sample for endothelial dysfunction markers - soluble adhesion molecules sICAM-1 and sVCAM-1. If the expression level of the adhesion molecules sICAM-1 is 591.9 ng/ml and more, and the expression level of sVCAM-1 is 632 ng/ml and more on the first days of a peracute period of the ischemic stroke to be persistent in dynamics or increasing in relation to the reference, the unfavourable course of ischemic stroke in the given category of patients is predicted.
EFFECT: using the method provides high sensitivity, specificity and accuracy of the prediction on the first days following the peracute period of the ischemic stroke in diabetic patients.
5 dwg, 1 tbl, 3 ex
SUBSTANCE: invention refers to medicine, namely to pathologic anatomy, and enables providing the morphological differential diagnosis of trophoblastic, endometrial and mixed forms of primary placental insufficiency in spontaneous miscarriage in the first trimester. The technique consists in performing an immunohistological analysis of the villous chorion and decidual cells of the gravidary endometrium to evaluate a vascular endothelial expression index (VEEI) and the transforming growth factor (TGF-β2). If the VEEI villous chorion expression index is 1.5 standard units or less, while the TGF-β2 expression index is 1.4 standard units or less, the trophoblastic form of primary placental insufficiency is diagnosed. If the VEEI decidual cell expression index is 1.6 standard units or less, while TGF-β2 is 2.7 standard units or more, the endometrial form of primary placental insufficiency is diagnosed; if observing the above values simultaneously, the mixed form of primary placental insufficiency is diagnosed.
EFFECT: presented method is high-informative, increases the quality of a pathology report, promotes an individual approach to the further diagnosing, treatment and rehabilitation of the female reproductive health depending on the pathogenetic form of placental insufficiency.
SUBSTANCE: invention relates to the field of medicine, in particular to neurosurgery, and deals with a method of diagnosing the brain tumour in patients. The essence of the method: the percent quantity of CD25-lymphocytes is determined in sampled blood. If its value is 13.7 the absence of the brain tumour is stated, in case if its value is lower than 13.7 the presence of the brain tumour in the patient is diagnosed. The method is simple for the practical application.
EFFECT: invention ensures a zero level of errors, which testifies to a high probability of a correct distribution of the tested people for groups of healthy people and patients with the brain tumour, which makes it possible to practically apply the claimed method in the additional diagnostics of a cerebral pathology.
2 tbl, 1 dwg
SUBSTANCE: pathogenetic treatment of chronic tonsillitis and/or hypertrophy of palatine tonsils in preschool children suffering from lymphoproliferative syndrome is ensured by the palatine tonsils debridement. An interleukin-1β(IL-1β) level is measured in the palatine tonsils washing. If the measured value is less than 5.8 pg/ml, recombinant interleukin-1β (IL-1β) is to be administered orally by phonophoresis with the use of the Tonsillor MM apparatus. Two courses of 10 procedures every 14 days are performed. The clinical effectiveness is assessed if observing a positive dynamics of IL-1β measured in the palatine tonsils washing 17 and 41 days after the beginning of the immunomodulatory therapy.
EFFECT: higher clinical effectiveness ensured by the differentiated selection of children for carrying out the immunomodulatory therapy, reducing a rate of infectious involvements of the palatine tonsils in the declared group of patients by the pathogenetically reasoned application of recombinant IL-1β.
3 cl, 2 tbl, 1 ex
SUBSTANCE: implementing the method involves determining the content of biomarkers - matrix metalloproteinase 2 (MMP 2) and tissue inhibitor of metalloproteinase 1 (TIMP 1) in the patient's oral fluid sampled before or not earlier than 30 minutes following meals. Using the derived values enables diagnosing papilloma or cancer of patient's oral floor mucosa. Using the invention provides achieving a considerably higher accuracy of the differential diagnosis of benign and malignant new growths of the patient's oral floor mucosa on the day of vising, a considerably higher sensitivity of the differential instant diagnosis, a higher probability of detecting the processes of benign and malignant new growths in the patient's body both at the early stages of the disease, and at the later ones, as well as a considerably simpler preparation of the patient within certain time frames for sampling and storing the diagnostic material.
EFFECT: lower loss of sensitivity of the detection channel in the presence of multiple non-synchronous pulse noise and mutual interference.
3 cl, 5 ex
SUBSTANCE: invention represents a method for the Mycoplasma hominis DNA detection from a blood sample by dividing it into a supernatant and precipitate by centrifugation followed by enriching the analysed material, recovering DNA by affine sorption with a sorbent and a lysis buffer added, incubating, washing the sorbent precipitate three times by centrifugation and removing the supernatant, drying the sorbent precipitate, adding a DNA elution solution and detecting by the PCR method. The method is characterised by the fact that the material to be analysed is a whole blood precipitate and to be enriched while recovering DNA by doubling up a number of test tubes per one sample with the whole blood precipitate 100 mcl and the sorbent 20 mcl in each; before the washing procedure, each test tube is added with the washing solution 375 mcl, while the content of the test tubes is combined in one test tube with adding the DNA elution solution 40 mcl to the sorbent precipitate.
EFFECT: higher diagnostic accuracy.
SUBSTANCE: invention refers to medicine, and represents a method for the prediction of a risk of congenital infections by measuring specific Ig M and Ig G antibodies in a biological material, differing by the fact that the biological material is presented by the first-screening cervical smear at the 12th week of gestation; the smears are tested for the IgG antibodies to the rubella virus, cytomegalovirus, B19V parvovirus, toxoplasm viruses, type 1 and 2 herpes simplex viruses and an avidity of the specific Ig G to this agents; additionally, the same smear is tested for secretory non-specific Ig A by IFA to cytomegalovirus, Chlamydia, Mycoplasm antigens, and a genetic material of this microorganisms by PCR, and depending on the findings, groups of a high, moderate and low risk of congenital infections are predicted.
EFFECT: invention provides the more accurate prediction of the risk of the most actual congenital infections by the integral assessment of a collection of clinical anamnestic data, and the qualitative parameters of the laboratory findings at the first pregnancy screening.
SUBSTANCE: invention refers to medicine. The method involves a sequence of the qualitative measurement of an HLDF cell differentiation factor in the patient suffering from a cardiovascular disease.
EFFECT: sequence provides preparing the valid specific and reproducible immunoenzyme test system enabling detecting a vascular system biomarker in blood serum both of healthy individuals, and of the patients suffering from vascular diseases.
2 ex, 2 dwg
SUBSTANCE: method involves: peripheral blood is examined for cytomegalovirus titre, blood pH and oxyhaemoglobin on a biochemical analyser; if observing an increase of the cytomegalovirus titre to 1:1600, a decrease of blood pH to 7.25±0.03, and oxyhaemoglobin - to 90.15±0.35% with the reference of 95.20%, a threatening formation of hemic hypoxia is stated.
EFFECT: higher assessment accuracy.
SUBSTANCE: carried out are: determination of the serum concentration of a factor of a tumour necrosis alpha (TNF-α), evaluation of a relative content of Th17-lymphocytes (%CD3+CD4+CD161+ ot CD3+CD4+) and an activity of succinatedehydrogenase in a population of regulatory T-cells (CD3+CD4+CD127low) (SDH-Treg) before and on the following day after infliximab introduction. A prognostic factor of the medicine efficiency K is calculated by formula. If the factor value is K<0.5, a minimal therapeutic effect of infliximab is predicted. When K=0.5-1.5, a moderate therapeutic effect, which demands correction of the medicine introduction plan, is predicted. If K is from over 1.5 to 2.5, a positive effect from introduction of infliximab is predicted.
EFFECT: possibility to predict long-term, not less than 1 year, effect from the infliximab therapy at any stage of therapy.
4 dwg, 2 tbl, 3 ex
SUBSTANCE: invention relates to analytical chemistry, in particular to aspects, related to methods of the nonenzymatic determination of the presence or a quantity of carbohydrates in a sample, and describes a method, device and set for the sample analysis to determine the presence or a quantity of an analysed substance, in particular a carbohydrate, in particular, sugar, in the sample with the application of fabric. The method includes the application of the sample on a synthetic fabric; chemical modification of the said carbohydrate, present in the sample, with an oxidising agent with a sufficient oxidising potential in order to cleave the carbohydrate between two hydroxyl groups; inactivation of the said oxidising agent, which would prevent the identification of the chemically modified carbohydrate; identification of the presence or the quantity of the said chemically modified carbohydrate by means of a copper-containing compound to obtain the visible change of colour.
EFFECT: invention can be used for the determination of the presence of a carbohydrate on the surface to indicate the surface contamination, in particular contamination with a substance, promoting the microbial growth.
20 cl, 2 dwg, 7 tbl, 4 ex
SUBSTANCE: invention relates to medicine, namely to method of determining expression of inflammatory process in case of osteoarthrosis. Essence of method consists in the following: carried out is luminol-dependent iron-induced chemi-luminescence of model system, which has the following composition: 2.72 g of KH2PO4, 7.82 g of KCl, 1.5 g of sodium citrate C6H8O7Na3*5,5H2O per 1 liter of distilled water, pH 7.45 with 0.2 ml of 10-5 M luminol solution, after that, intensity of model system luminescence is determined in presence of synovial fluid before and after addition of synovial fluid. Degree of suppression of intensity of model system chemi-luminescence is calculated by formula. If its value is from 1.71 to 6.48%, high activity of inflammatory process is determined, if its value is from 6.49 to 21.55%, medium activity is determined, from 21.56 to 55.46 - small activity.
EFFECT: application of the method makes it possible to reduce time of determination and increases accuracy of assessment of inflammatory process degree in case of osteoarthrosis.
1 tbl, 1 dwg, 3 ex
SUBSTANCE: diagnostic test element (110) for the detection of an analyte in a sample (126) of a body fluid, in particular whole blood with the volume not less than 2 microlitres, contains a test field (116) with a reagent-indicator, where the reagent-indicator is capable of feeling a detected change, in particular an optic change, in case of the analyte presence. The test field (116) includes a detector layer (118), containing the reagent-indicator, where the detector layer contains particles (137). 90% of all particles (137) of the indicator layer (118) have the actual size smaller than 10 microns. The diagnostic test element (110) contains a bearing element (112), which has a transparent region (114), where the test field (116) with its detection side (120) is partially applied on the transparent region (114).
EFFECT: detected change is an optically detectable change, where an optic detector with spatial resolution is applied for the detection of the detected change.
9 cl, 8 dwg, 1 tbl, 2 ex
SUBSTANCE: invention relates to immunology and medical diagnostics and represents method of carrying out immunochromatographic analysis for serodiagnostics. Claimed invention is intended for immunochromatographic determination of antibodies to causative agents of infectious diseases or other antigens, for instance, allergens, in liquid samples. Claimed method of identifying antibodies is characterised by the following: solution for sample dilution contains specific antibodies against test strip of antigen (or antigens) immobilised in analytic zone. Used concentration of antibodies in diluting solution is lower than lower limit of determination of antibodies by said method with application of diluting solution, which does not contain specific antibodies, which results coloration of analytic zone of test strip is not observed if specific antibodies are absent in sample. If sample contains specific antibodies, presence of additional quantity of specific antibodies in diluting solution results in enhancement of intensity of coloration of test strip analytic zone.
EFFECT: application of invention makes it possible to register total concentration of specific antibodies in sample and in diluting solution, which results in reduction of limit of analysis identification due to displacement of working range of concentrations, determined by test, into the area of lower values.
3 dwg, 1 ex
SUBSTANCE: invention refers to medicine and represents a method for accelerated staining of histological specimen for identifying tuberculosis mycobacteria involving the preparation of the histological specimen according to a common technique, the Ziehl-Nelsen staining differing by the fact that the standard preparation of the specimen for staining is followed by a de-waxing procedure by placing them successfully into xylol and alcohols of the decreasing concentration for 5 minutes in a thermostat at 54°C and then placing into water, then in a 1% periodic acid for 2 minutes and washing in flowing water for 10 seconds; thereafter, Ziehl carbol-fuxine is poured on a section coated with a filter paper and heated on an alcohol burner lamp until evaporating for 2 minutes; the stain is left on the section after the heating procedure is completed, for 3-5 minutes; the filter paper is removed, and the sections are washed in water, differentiated in a 1% acid buffer, washed in main water for 1 minute; thereafter Leffler methylene blue is placed on the section and heated on the alcohol burner lamp until evaporating, washed in water, differentiated in a 1% acid buffer and washed with a plenty of water, de-watered in alcohols of the increasing concentration for 1 minute, placed in xylol and balm.
EFFECT: developing the method for the accelerated stain of the histological specimen.
SUBSTANCE: invention refers to medicine and aims at the prediction of postoperative suppurative complications in patients suffering from colorectal cancer by a dynamic blood test. On the 2nd and 5th postoperative day, blood plasma of the patient suffering from colorectal cancer is examined for C-reactive protein (CRP). If the 5th-day CRP level is less than 10.0 mg/dl, a relatively mild postoperative course is predicted. If the CRP level decreases in relation to the second postoperative day, a risk of the complications is considered to be low. If the CRP level increases in relation to the second postoperative day or remains unchanged, a risk of the complications is moderate. If the 5th-day CRP level is more than 10.0 mg/dl, a risk of the postoperative suppurative complications is considered to be high.
EFFECT: invention provides the effective prediction of the suppurative complications in the patients on the second and fifth day following colorectal cancer surgery.
SUBSTANCE: ovarian tumour tissue is examined for a portion of the synthesis cycling-state cell; if the derived value is less than 15.9%, a favourable outcome is predicted, while at the derived value being more than 15.9%, an unfavourable outcome is predicted. The invention can be used more than once in clinics for women and special oncological hospitals for the purpose of predicting recurrent ovarian carcinoma according to the tumour tissue DNA-cytometry findings.
EFFECT: higher prediction accuracy.
SUBSTANCE: technique involves preparing a patient's blood serum sample by drying blood serum, milling the dry deposition and suspending in Vaseline oil. The prepared sample is analysed by IR-spectroscopy by determining a peak height of absorption bands with maxima 1165; 1160; 1150; 1100; 1070; 1050, and 1025 cm-1. And the peak height relations are calculated: the relation of the peak height with maximum 1160 cm-1 to the peak height with maximum 1165 cm-1; the relation of the peak height with maximum 1165 cm-1 to the peak height with maximum 1070 cm-1; the relation of the peak height with maximum 1165 cm-1 to the peak height with maximum 1150 cm-1, the relation of the peak height with maximum 1165 cm-1 to the peak height with maximum 1050 cm-1, the relation of the peak height with maximum 1100 cm-1 to the peak height with maximum 1050 cm-1, the relation of the peak height with maximum 1025 cm-1 to the peak height with maximum 1165 cm-1. The derived values are used to draw a differential diagnostic profile of the patient's blood serum sample to produce a plane polygon formed by 6 beams corresponding to 6 relations and to compare the above polygon to a polygon that is a reference differential diagnostic profile of pulmonary thromboembolism, wherein 6 relations make: 1 (1.85±0.26), 2 (0.28±0.13), 3 (0.39±0.07), 4 (0.25±0.13), 5 (0.58±0.10), 6 (4.47±1.70). If the derived patient's differential diagnostic profile has a similarity with the reference profile, and all 6 relations of the patient's blood serum sample coincide with the reference relations, pulmonary thromboembolism is diagnosed in the patient.
EFFECT: invention possesses high accuracy, easy to implement, and requires no heavy material and time expenses; it enables diagnosing thromboembolism of all pulmonary arteries both in the presence of clinically significant symptoms of the disease, and in the absence thereof.
2 ex, 3 dwg
FIELD: medicine, analytical biochemistry.
SUBSTANCE: invention relates to laboratory methods of investigations. Method involves sampling specimen from patient to be inspected, extraction of serotonin and histamine from a specimen, chromatography of extract and determination of concentration of serotonin and histamine by the fluorescence intensity value. Saliva is used as biological fluid. Saliva by volume 1 ml is extracted with 4 ml of 1 N hydrochloric acid solution, 2 g of anhydrous potassium carbonate and 5 ml of mixture of butanol and chloroform in the ratio 3:2 are added, extract is shaken up and centrifuged. Organic phase (4 ml) is sucked off from extract and passed through chromatography column (diameter is 3 mm, height is 16 mm) filled with ion-exchange resin KB-4 or KB-4P-2 or Bio Rex-70 in H+-form, size of granules is 0.1 ± 0.02 mm. Histamine is eluted with 4 ml of 0.1 N hydrochloric acid at the rate of eluting solution 0.4 ml/min. Histamine concentration is determined by reaction with ortho-phthalic aldehyde dissolved in ethanol. Serotonin concentration is determined by reaction with ninhydrin in organic passed through column. Method provides assaying the saliva concentration of serotonin and histamine with high precision.
EFFECT: improved assay method.