Method for combined immunobiological analysis of cells using biochip

FIELD: chemistry; biochemistry.

SUBSTANCE: method for combined immunobiological analysis of cells using a biochip involves incubation of the biochip which contains immobilised antibodies, with suspension of cells, washing the biochip from non-bonded cells, determination of coexpression of antigens on the bonded cells. The obtained result is assessed by determining presence of bonded cells in the region of the stain of the biochip and bonding density of cells and interpretation of the obtained result. Coexpression of antigens on cells bonded to the biochip is determined by carrying out one or more immunocytochemical reactions. When reading out the result, morphological analysis of cells bonded to the biochip is also carried out and presence and character of colouring of cells and their components with the reaction product are determined.

EFFECT: use of the disclosed method provides high reliability and information content of analysis.

9 cl, 6 dwg, 2 ex

 

The invention relates to medicine and can be used for diagnostic purposes in Hematology, Oncology and other fields of medicine and in veterinary medicine and biology.

There is a method of detection of surface antigens of cells with microchips (see Liu, A.Y. Differential Expression of Cell Surface Molecules in Prostate Cancer Cells // Cancer Research. 2000, Vol.60., p.3429-3434), taken as a counterpart, namely, that the biochip (representing a substrate with immobilized in strictly defined areas of antibodies, each specific to a particular cell surface antigen) is incubated with the cell suspension. Cells, having on its surface corresponding antigens bind to the antibodies immobilized in strictly defined areas of the biochip (spots), the remaining cells are not specifically associated with its surface. Then, the chip is rinsed from the non-specifically bound peroxidase cells by rinsing with buffer solution. As a result, the surface of the biochip only those cells that are contacted with the antibody. Carry out reading of the defining any spots of the biochip happened binding cells. The method allows simultaneous determination of different cells a large number of different surface antigens. Maximum panel of antibodies, which may be asesina on a single biochip, limited only by the size of the substrate and areas (spots) of the biochip, in which immobilized antibodies.

The disadvantage of this method is that each individual cell upon binding to a particular spot of the biochip can be defined only one antigen. The inability to identify a larger number of antigens (co-expression) for each individual cell reduces the diagnostic value of analysis and may be the cause of the error. In addition, the method does not allow for carrying out morphological analysis of cells that does not allow to identify them.

The known method of the study of cells using immunological biochips (see Belov L, de la Vega About, dos Remedies C.G., Mulligan SP, R.I. Christopherson Immunophenotyping of leukemias using a cluster of differentiation antibody microarray // Cancer research, 2001. - Vol.61. - P.4483-4489), namely, that the biochip containing immobilized antibody is incubated with the cell suspension. Cells, having on its surface corresponding antigens bind to the antibodies immobilized in strictly defined areas of the substrate. Then biochip washed from the cells, contacting the immobilized antibody. After that incubated the biochip with a solution of antibodies conjugated with fluorochromes, carry out the washing. Determine the density of the spots of the biochip bound peroxidase cells. ODA is dividing the co-expression of antigens explore biochip, for example, using a fluorescent microscope. In the spots of the biochip determine the presence (or absence) of fluorescent cells and estimate their number. Then carry out the interpretation of the result.

The disadvantage of this method, taken as a prototype, is the need to use expensive equipment.

The disadvantage is the necessity of using ultraviolet radiation caused by the use of fluorescent labels. Under the action of ultraviolet radiation is a rapid decrease in fluorescence labels ("fading") due to their destruction. The biochip may not be examined again after a long time.

In addition, when performing the read result it is impossible to conduct morphological analysis of cells (only possible establishment of localization of the label and in some cases, assessment of the size of the cells). It is not possible to identify cells based on morphological characteristics and dramatically reduces informative way.

All of the above limits the use of this method.

Objective of the claimed invention is to enhance reliability, and usefulness of the analysis while reducing its cost and the possibilities run without the use of expensive about is orogovenia.

The problem is solved due to the fact that according to the method of combined immunological studies of cells using a biochip, comprising the incubation of the biochip containing immobilized antibodies, cell suspension, washing the biochip from unbound cells, the determination of the co-expression of antigens on the linked cells, the reading of the result, including the detection of binding cells in the stained area of the biochip and the determination of the density of binding cells, and interpretation of the obtained result, the definition of co-expression of antigens on related biochip cells is accomplished by implementing one or more of immunocytochemical reactions when reading the additional spend morphological study associated with the biochip cells, and determine the presence and the nature of the staining of cells and their components in the reaction product.

Implementing the additional staining of cells which do not prevent the detection of the product (s) of the enzymatic reaction.

To study a single sample of cell suspension use multiple biochips, by immunocytochemical study of cells using antibodies specific for different antigens.

Carry out morphometric study associated with the biochip cells.

Process biochip one or more solutions containing one or more substances that increase the strength of bonding of the cells with the surface of the biochip.

From the biochip prepare long-term preparation for microscopic examination.

On the biochip or made of him a long-term drug caused markup that facilitates finding the right spots.

In the analysis use one or more additional biochips, which perform the staining associated cells for their subsequent morphological studies, with immunocytochemical reaction does not take place.

Using the inventive method allows to determine the presence or absence of co-expression of antigens on each individual contacting biochip cell. When this is determined on a single antigen more than immunocytochemical studies without the use of the biochip. Immunocytochemical (ELISA) research related to the biochip cells can be performed using a direct method (see, for example, Petrov S.V., Raikhlin, ETC Guide immunocytochemical diagnosis of human tumors, edition 3, revised, Kazan: Title, 2004. - p.21). Such a study is the least time-consuming and requires less time and the reaction is ivov, but gives less intense staining of the cells with reaction products. Immunocytochemical study associated with the biochip cells can also be performed using any indirect immunochemical method or by using methods EPOS, En Vision, CSA (see, for example, Petrov S.V., Raikhlin, ETC Guide immunocytochemical diagnosis of human tumors, edition 3, revised, Kazan: Title, 2004. - s-25). In this case, the execution of this phase of the analysis is more time-consuming and laborious, but due to the greater intensity of staining cells with the reaction product increases sensitivity, which allows to identify weakly expressed antigens. In some cases, require the determination of the presence or absence of co-expression of two, for example three antigens on each cell. In this case, using a double cytochemical staining, based on different color visualization of two different antigens of the third antigen is determined by the fact that binding of cells at the different spots of the biochip). This is a two immunocytochemical reactions (possible combination of immunocytochemical methods: direct/direct, direct/indirect direct/indirect method, double EPOS method, a double En-Vision method). (See for example, Petrov S.V., Raikhlin, ETC Guide immunodeficiency is acidogenicity diagnosis of human tumors, edition 3, revised, Kazan: Title, 2004. - s-27). The same principle can be determined and a larger number of antigens.

The use of enzyme reactions eliminates the disadvantages associated immunofluorescence analysis: the fading marks, the impossibility of re-examination, and others. In addition, the implementation of the study the proposed method does not require expensive equipment. The study of cells on the chip can be carried out using a conventional light microscope. Microscopic examination can be carried out repeatedly and over a long time after the execution immunocytochemically reaction without distorting the results.

The morphological examination can be defined shape and dimensions of the cells and their nuclei and other structures, as well as other signs. Is determined not only by the presence of staining cells with the reaction product, but his character (intensity of staining, localization of the colored product), which allows to assess the specific structures of the cell is defined antigen and evaluate its number.

For a more complete morphological studies can be carried out painting-related biochip cells which do not prevent the detection of the product (s) of immunos chimicheskoy reaction. For example, when used as Chromogen 3-diaminobenzidine tetrachloride (DAB), you can paint Romanovsky-Giemsa or staining with hematoxylin (figure 2). This significantly increases the value of the examination. It becomes possible to identify different subpopulations of cells, who contacted in any of the spots of the biochip (and, therefore, having a particular surface antigen). This subpopulation of cells can be determined on the basis of results of determination of the co-expression of antigens using immunocytochemical reactions, and based on the results of morphological studies. When processing results can be quantitatively determined the content of the cells belonging to a particular subpopulation. Can be determined as a proportion of the number of cells bound peroxidase in this spot biochip, or the total number of cells.

In some cases, require the definition of several variants of the co-expression of antigens that cannot be performed on a single biochip. In this case, can be used multiple biochips, which are carried out immunocytochemical reaction (one or more) with multiple conjugated with the enzyme antibodies specific to different antigens.

Can be morphometric investigated the e bound peroxidase cells, that significantly increases the information content of the analysis. This approach can be used for research purposes.

To eliminate errors in the analysis of the same sample can be used for more control biochips (one or more). For example, can be used biochips containing antibodies specific to different epitopes of the antigens detected by using the first biochip. In this case, the comparison of the results obtained using the main and control biochips (one or more)allow to eliminate errors associated with cross-reactivity of antibodies, as well as errors associated with loss of activity of the used antibodies. To avoid errors associated with immunocytochemical staining of the drug, can be used to control the biochips are associated cells fixed in any other way (for example, using the fixation of absolute ethanol instead of acetone or Vice versa), or use a different cytochemical reaction or use another Chromogen. It is also possible for various positive and negative controls to determine the specificity of binding to cellular antigens antibodies used in cytochemical reactions (reactions).

After washing from not contacting antibodies cells in some cases, the mod is no take to process the biochip substances, increase the strength of binding to the surface of the biochip of the remaining cells. This increases the accuracy of the analysis, because it helps prevent separation of the cells when performing the following manipulations and to exclude false negative results. This approach also increases the sensitivity analysis, because it allows you to explore the cells, loosely bound to the immobilized antibody (e.g., due to the low expression of the relevant antigen).

Preparation of the biochip long-term drug allows you to repeatedly carry out a morphological study of linked cells using immersion lens without pollution and without damaging the surface of the biochip and related to her cell. This approach is very useful, for example, in cases where for correct interpretation of the result may need to view the drug by several specialists. In addition, it allows for a comparative study of cells of the same patient, subjected to analysis using different biochips at different times. This can be very useful, for example, to assess the dynamics of disease or to assess treatment outcomes. Preparation of long-term drug allows you to store biochip unlimited time and makes us the CSOs more resistant to various injuries. It is possible to make long-term medication in different ways. Note that using this approach is possible if used for this substance does not lead to the dissolution of the product (s) of the enzymatic reaction.

On the biochip or made of him a long-term drug may be the markings (6)facilitating the searching and identification of spots and to avoid possible errors, for example, when manually reading the result.

In some cases, additional coloring biochip most preferred for the implementation of morphological analysis of cells by the method prevents the detection of the reaction product. For example, when using no DUB, and some other Chromogens implementation of colouring Romanovsky-Giemsa prevents the detection of the reaction product. In these cases, the analysis can be used one or more additional biochips, which perform only one colouring related cells, necessary for carrying out morphological studies. Immunocytochemical reactions if this is not carried out. The results of the study of cells obtained through additional biochip (biochips), compared with the results of the study of cells obtained using the bio is IPA (biochips), which carried out the conduct cytochemical reactions. This increases the reliability of the analysis and allows to avoid possible contradictions between the results of immunophenotyping and results of morphological studies.

The proposed method can be used in various modifications, suitable for clinical studies and for various research and other tasks.

The claimed method is illustrated by drawings (Fig.1-6).

Figure 1 is a micrograph of a fragment of the biochip, including spots with immobilized antibodies. In the area of spots associated cells isolated from the blood of a patient with chronic B-lymphocytic leukemia (b-CLL). The density of binding cells in the stained area with a variety of different antibodies. The increase of 17.5 times.

Fig.- 2 micrograph of cells of the patient In chronic-lymphocytic leukemia (b-CLL), who contacted the biochip in the area of the stain with antibodies anti-CD23. Immunocytochemical determined the expression of the CD19 antigen. The nature of the staining of the cells with the reaction product indicates the local surface antigen CD19. Thus, it coexpress CD19/CD23 characteristic of cells of chronic b-lymphocytic leukemia. Cell nuclei zakrasheny with hematoxylin. The magnification of 400 times.

Figure 3 is a diagram showing the result of determining the content in the investigated su is pensii cells, expressing different surface antigens. Using the biochip was investigated cells isolated from the peripheral blood of a patient with chronic B-lymphocytic leukemia (b-CLL).

4 is a diagram showing the result of determining the content in the studied suspensions of cells expressing different surface antigens. Using the biochip was investigated cells isolated from the peripheral blood of a healthy person.

5 is a micrograph of cells of the patient In chronic-lymphocytic leukemia (b-CLL)connected on the secondary chip in the area of the stain with antibodies anti-CD19. Color Romanovsky-Giemsa. The increase in 900 times.

6 is a photograph of a plot is made of the biochip long-term medication, which prior to conducting manual reading of the marking, to facilitate finding the right spots. The markup in the form of a grid divides the spots of the biochip group in the form of "rows" and "columns". This avoids errors in the sequential photographing spots of the biochip through the microscope. In this case, the markup applied by hand. The increase in 3 times.

The claimed method is performed as follows. Before analysis biochip (representing a transparent plate, which in strictly defined areas (spots) of immobilized antibodies specific the major surface antigens of cells) fix in any capacity, for example, in a Petri dish, container or cuvette. Instead, the biochip can be mounted in a flow chamber (see A.V. Shishkin, Shmyrev A.I., Kuznetsov S.A., Ovchinina N.G., Butylin A.A., Ataullakhanov FI, Vorob'ev A.I. Immunological biochips for the study of human erythrocytes, Biological membranes, 2008, volume 25, No. 4, S. 267-276). Carry out incubation biochip with a given cell suspension. The cells are deposited on the surface of the biochip and come into contact with the immobilized antibodies. If the cells have the appropriate surface antigens, they are binding in these areas (spots) of the surface of the biochip. Then carry out washing of the biochip to eliminate cells that are not contacting antibodies. If the chip is in a flow chamber, the washing is carried out by passing over the surface of the biochip flow of the washing solution at a given speed. If the chip is in a Petri dish, a dish (container) or other containers, washing may be carried out by rinsing wash solution or by multiple premises (immersion) in this solution. After washing the stained area of the biochip remain bound only those cells that have the appropriate surface antigens. The quality control of washing is carried out using microscopic examination. Mark the WHC is completed, if outside spots of the biochip (background areas) associated cells are absent.

After completion of the washing-off is not in keeping with antibodies to the cells in some cases (for example, when using the flow-through chambers with narrow capillary to prevent separation of the cells at the time of filling of the capillary air before removing the biochip can be processed biochip solutions of substances that increase the strength of binding of the remaining cells to the surface. When working with leukocytes using biochip fabricated on a plastic substrate can be used, for example, incubation (10-20 minutes) with a solution containing salts of calcium and magnesium ions which participate in the processes of cell adhesion to solid surfaces. When selecting the substances it is necessary to take into account that carrying out such processing should not interfere with the subsequent stages of the study, primarily conducting immunocytochemical reactions (reactions). After such processing, the biochip was washed with isotonic buffer solution.

Then the chip is extracted from the tank (or flow cells), where he performed the previous stages of analysis. If you do not specify reuse capacity, the biochip can not be removed and to carry out all subsequent manipulations associated with treatments is coy biochip used solutions.

Then contacting biochip cells is fixed. For example, the biochip dried (optimum drying time in air - 1 hour) and within 1-3 minutes is treated in absolute acetone at 4°C. we can also carry out the fixing of the biochip with related cells absolute ethanol (10-15 minutes) with subsequent drying. When using the selected method of fixation should not happen dissolution of the substrate of the biochip or loss of its transparency under the action of the locking substance.

Next, perform immunocytochemical reactions (reactions) by any known method. It is possible as holding one immunocytochemical reactions and double immunocytochemical staining (see, for example, Petrov S.V., Raikhlin, ETC Guide immunocytochemical diagnosis of human tumors, edition 3, revised, Kazan: Title, 2004. - p.21-27). Possible to execute a larger number of immunocytochemical reactions in the cells associated with a single biochip, but this approach is too time-consuming.

In the case of using immunocytochemical method EnVision for immunocytochemical reaction sequence is as follows :

Spend processing a 3% solution of hydrogen peroxide for 10 minutes, then washed twice biochip Boo is Arnim solution (TBS) for 5 minutes. Then within 20 minutes, incubated biochip with a solution of non-immune serum to block nonspecific binding of antibodies. Then remove the excess whey and within 40 minutes, incubated with a solution of primary antibody after incubation biochip rinsed with buffer and twice washed his TBS (5 minutes). Then remove the remainder of the liquid and add the reagent EnVision representing macromolecule polymer, conjugated with molecules of the secondary antibody and molecules of the enzyme, in this case of horseradish peroxidase. Within 40 minutes spend incubation. Then biochip rinsed with buffer and twice washed his TBS (5 minutes). Followed by incubation of the biochip with a solution DUB, which is carried out in the dark for 5-10 minutes (when the determination of some antigens optimal incubation time may vary up or down). Washed biochip distilled water and optionally dried. You can spend additional staining of cells, for example, hematoxylin or Romanovsky-Giemsa (in the latter case, the preferred fixation with acetone). This staining cells in this case does not prevent the detection of the product of immunocytochemical reactions (when using DAB Chromogen), if there are "repaints of the drug". Should note the e l e C when conducting additional staining Romanovsky-Giemsa must be carefully installed to withstand the processing time of the biochip dye, because the "recolor" begins to interfere with the detection of the reaction product. The optimal time of incubation of the biochip with the dye (dye) is determined experimentally, as it may be different for different batches of dye, and also depends on the concentration of the dye solution. After additional staining biochip rinsed with distilled water.

In some cases you may need to define the co-expression of multiple pairs (or groups) antigens, which cannot be performed on a single biochip. In this case, can be used multiple biochips, which immunocytochemical determine the different antigens. It also can be used any immunocytochemical method. If necessary, can also be accomplished painting.

Then from the biochip can be cooked long-term medication. To do this, on the surface of the glass (or transparent plates of other material) put a drop of Canada balsam (or another substance with similar properties, for example, a liquid for the preparation of histological preparations "Shandon-Mount"; the manufacturer, Thermo-electron corporation, USA, Pitsburg). SV is the Rhu village, it imposes a biochip (a party with bound peroxidase cells up) and press so that so between him and the glass did not leave air bubbles. Then, on the surface of the biochip put another portion of Canada balsam. Top impose and thus press the cover glass. The drug is placed under load or press to solidification of Canada balsam. Preparation of long-term drug may, if used for this substance does not lead to the dissolution of the product (s) of the enzymatic reaction. For example, the above-described variant of the method of preparation of a long-term remedy can be applied when using DAB as Chromogen. It is possible to make long-term drug and other methods.

Then, if necessary, on a biochip or made of him a long-term drug may be the markings (6), which facilitates finding the right spots with manual reading of the result. For example, in the simplest case can be held line between the spots of the biochip group (for example, rows and columns)that allows to avoid errors in the sequential photographing spots of the biochip through the microscope.

Then carry out the reading of the result, for receiving the image of the spots and the background areas of the biochip (1), (for example, in the simplest case by means of photography through the microscope) and define p is otnesti binding of cells (number of cells, bound peroxidase on the surface of the biochip given area) in the area of the spots of the biochip and the background areas. Depending on the purpose of the analysis may be qualitative, semi-quantitative or quantitative determination of the concentration of the investigated cell suspension having a data surface antigens. For the quantitative determination of the density of binding cells in the simplest case, can be used direct counting of cells in the specified area of the surface of the biochip. For example, the photos of the spots of the biochip can be selected sections of a given area, by counting cells. This process can be automated.

Next, determine the content in the studied suspensions of cells with defined (using biochip) surface antigens (3, 4). For this purpose, the density of binding of cells in the different spots is expressed in percentage relative to 100% of the maximum possible under these conditions the density of binding (defined, for example, by calculation) or relative density of binding cells in the area of the stain with antibodies able to contact all the analyzed cells (e.g., with antibodies anti-CD45 in the study of suspensions of leukocytes). The obtained values correspond well to the actual content in suspense the cells, having defined surface antigens. This approach is possible if the incubation biochip with cell suspension was carried out without stirring or flow of liquid, and the sizes of the cells belonging to different subpopulations, close enough.

Next, perform morphological examination of the cells, bound peroxidase in the region of each of the spots of the biochip. This determine morphological features necessary for the identification of cells, determine their staining product immunocytochemical reactions (reactions) and evaluate its characteristics (intensity, localization). Morphological study can be done without automation (for example, by viewing the drug under a microscope or by examining micrographs of cells bound peroxidase in the stained area of the biochip, and the use of automation (for example, when using computer programs that can recognize the image and performing its analysis). If necessary, can also be carried out morphometric study of bound peroxidase cells. In case of the biochip was cooked long-term preparation, holding his microscopic research as possible with the use of lenses, not coming in contact with the surface preparation and application of the immersion lens.

P and conducting a microscopic examination of each spot on the biochip sections of a given area can be determined the number of cells, colored reaction product and, therefore, expressing the corresponding antigen together with the antigen specific binding of the cells in this spot biochip. Similarly, it can be determined the number of cells with any other distinctive (morphological, immunocytochemical or morphometric) sign. Can be determined, the proportion of such cells from the number of cells bound peroxidase in this spot on the plot of a given area, or determined by calculation of their percentage in the sample cell suspension.

In some cases, the research requires the use of additional biochips.

1) In some cases require the use of additional control biochips (one or more), which is only the color of cells needed to perform their morphological research. Immunocytochemical reactions if this is not carried out. This approach can be used, for example, in the case where staining is most acceptable for morphological research method prevents the detection of the reaction product formed by using the selected Chromogen, or when required the use of several types of additional color. Processing of map data is received using primary and secondary biochips. Carrying out morphological studies on additional biochip (biochips) improves the reliability of the analysis and allows to avoid possible contradictions.

2) Additional biochips can be used for control. The number of used control biochips and conditions of conducting follow-up research depend on a specific task (assess the binding specificity of cells immobilized on the biochip antibodies, reproducibility studies, monitoring the implementation of the immunocytochemical reactions, positive and negative control at the immunocytochemical reactions and others) and can be individualized in each case.

The claimed invention is directed to improving the reliability of the method, cost reduction analysis, achieving the possibility to perform research in laboratories that do not have expensive equipment, raising awareness and diagnostic value of analysis results, and to achieve the possibility of long-term preservation of biochips, which was completed analysis for re-microscopic studies.

Examples of use of the method.

Example 1

Were investigated cells A. patient, 61 years of age, with seleciton lymphoproliferative disease, soprovojdalos is MSA bone marrow failure. When the blood smear was marked by an increased content of Mature lymphocytes. It was necessary to determine the T - or b-cell nature of the tumor and determine its nosological form. For this purpose it was necessary to determine immunophenotype tumor cells.

Used set of 3 biochips containing immobilized antibodies (IgG)specific to antigens: CD2, CD3, CD4, CD5, CD7, CD8, CD9, CD10, CD16, CD19, CD20, CD21, CD22, CD23, CD27, CD29, CD31, CD36, CD38, CD41, CD44, CD45, CD45RA, CD56, CD71, CD72, CD95, CD98, HLA-DR, IgM.

Leukocyte fraction of cells was isolated by centrifugation on a density gradient. Selected cells were resuspension in the working solution. The biochips were fixed in cuvettes. Fill the cuvette cell suspension and incubated for 40 minutes without stirring.

Then biochips who remained in cells) were rinsed wash solution to resolve not contacting antibodies cells. The quality control of washing was carried out using microscopy. Biochips was treated with absolute ethanol for 10 minutes and dried.

Next conducted immunocytochemical reaction method EnVision (the course of the reaction described above) by the following procedure: 1) treatment with 3% hydrogen peroxide solution for 10 minutes; 2) rinsing buffer (TBS), followed by double washing TBS for 5 minutes; 3) incubation with a solution Pervin the x antibody, specific to a defined antigen, for 40 minutes; 4) rinse buffer (TBS), followed by double washing TBS for 5 minutes; 5) removal of residual liquid; 6) incubation of the biochip with EnVision reagent ("Daco", USA) for 40 minutes; 7) incubation of the biochip with a solution of Chromogen DAB in the dark for 10 minutes; 8) washing biochip with distilled water and drying.

The cells associated with the biochip No. 1, conducted immunocytochemical determination of antigen CD19.

The cells associated with the biochip No. 2, performed immunocytochemical determination of antigens CD2.

For staining of nuclei from related cell biochips No. 1 and No. 2 were additionally treated with hematoxylin.

Biochip No. 3 no reagents required for the implementation of immunocytochemical studies were not processed. Instead, after washing from not contacting the antibody of his cell were fixed with methanol (15 minutes) and were stained with Romanovsky-Giemsa, which for 40 minutes, treated with a solution containing Azur and eosin.

Then from biochips made long preparations and performed microscopic examination.

Was determined the density of binding cells in various spots of the biochip, based on what determined the percentage of cells. The density of binding cells was determined by p is the direct counting. To do this, each spot biochip counted the number of cells bound peroxidase in areas specified area. Counting was performed at least 3 areas of 100×100 μm, the obtained values were averaged. The density of binding cells in each spot was expressed in percentage. 100% took the density of binding cells in the area of the stain with antibodies anti - CD45 (11500 cells/mm2). The results obtained are given in figure 3.

In addition to the control spots with antibodies anti-CD44 and anti-CD45 (where you can contact all leukocytes) the highest density of binding cells was observed in the stained area of the biochip with antibodies specific to antigens: CD5, CD19, CD20, CD21, CD22, CD23, CD27, CD29, CD29, CD31, CD72, CD98, HLA-DR. The morphological examination of the cells, bound peroxidase in the area of these spots, it was found that almost all of them are Mature lymphocytes.

On the biochip No. 1 the accumulation of cells (all or the greater part) of the colored product of the enzymatic reaction was noted in the area of the stain with antibodies specific to antigens: CD5, CD19, CD20, CD21, CD22, CD23, CD27, CD29, CD31, CD44, CD45, CD45RA, CD72, CD98, HLA-DR. This indicates a joint expression in the same cells (co-expression) antigen CD19 with antigens CD5, CD20, CD21, CD22, CD23, CD27, CD29, CD31, CD44, CD45, CD45RA, CD72, CD98, HLA-DR. Figure 2 is a micrograph illustrating an example of defining a joint expression of antigens CD19 and CD23 (maxpri what these CD19/CD23).

On the biochip No. 2 the accumulation of cells colored product of the enzymatic reaction was noted in the area of the stain with antibodies specific to antigens CD2, CD3, CD4, CD8 (all related in these stained cells) and CD7 (cell parts), in addition, it was painted a small portion of the cells, bound peroxidase in the area of the stain with antibodies anti-CD5, anti-CD44, anti-CD45, anti-CD45RA. Thus, it was noted typical of Mature T-cells coexpressed CD2/CD3, CD2/CD4, CD2/CD5, CD2/CD7, CD2/CD8 etc. Thus, the reaction product was reacted only cells located in the area of the stain with antibodies specific for T-cell antigens and the antigens that are found on different types of cells. The density of binding cells in the area with antibodies specific for T-cell antigens was low, indicating a low content of T-lymphocytes in the sample (figure 3). These cells are normal T-lymphocytes, which are present in small numbers on the background of a much larger number of tumor cells. The density of binding cells in the area of the stain with antibodies specific to the antigen CD5 was high, but the accumulation of a colored product had only a small proportion of these cells. Therefore, most of the cells, bound peroxidase in this spot biochip was not T-lymphocytes. This testifies to their abberant immunogenet the PE. The reaction product was reacted also some of the cells, bound peroxidase in the area of the stain with antibodies anti-CD31 (CD31 antigen is present on part of T-lymphocytes), and isolated cells in the stained area with antibodies anti-CD16 and anti-CD56 (CD2 antigen is present on the part of NK-cells).

Thus, this tumor is In the cell. The presence of the co-expression of CD5/CD19/CD23 in the absence of expression of the lymphocyte antigen CD10 typical cell chronic b-lymphocytic leukemia. The results were subsequently confirmed clinically.

Example 2

Were investigated cells of a healthy donor b, 29 years. The aim of the study was to verify the binding specificity of different subpopulations of lymphocytes (T-, b - and NK-cells). Used set of 3 biochips, such the same as in example 1. The selection of leukocytes from the peripheral blood of their incubation with the biochip, washing biochip and fixation was carried out in the same manner as described in example 1.

Then on each of the biochips were carried out immunocytochemical reaction.

The cells related to the biochip No. 1, was determined by immunocytochemical expression of CD19 antigen.

The cells related to the biochip No. 2, was determined by immunocytochemical expression of CD16 antigen.

The cells related to the biochip No. 3, was determined by immunocytochemical expression of CD2 antigen.

Methodology for immunocytochemically reactions by the method of EnVision was the same as described in example 1.

In the same manner as in example 1,was determined density of binding cells in various spots of biochips based on what was determined by the percentage in the sample of cells expressing different antigens. The results obtained are given in figure 4. They are typical indicators of a healthy person.

By microscopic examination determined the accumulation of the product of the enzymatic reaction in the cells that are in different spots of the investigated biochips.

On the biochip No. 1 the accumulation of cells colored product of the enzymatic reaction was noted in the area of the stain with antibodies specific to antigens CD19, CD20, CD21, CD22, CD38, CD45RA, CD72, CD98, HLA-DR, which is typical for b-lymphocytes, as well as in the area of the stain with antibodies specific to antigens CD44, CD45, present on all leukocytes. In the area of the stain with antibodies specific to antigens CD19, CD20, CD21, CD22, CD72, the reaction product was reacted almost all cells, and in the other above mentioned spots of the biochip was painted only part of the bound peroxidase cells.

On the biochip No. 2 the accumulation of cells colored product of the enzymatic reaction was noted in the area of the stain with antibodies specific to antigens CD16, CD56, which is typical of NK cell. It was painted all bound peroxidase cells. In the field of stains and what citelli, specific to antigens CD44, CD45, CD7, the reaction product was reacted fraction of the cells. This is because these antigens Express not only NK cells, but other cell types. Stained a small fraction of the cells, bound peroxidase in the area of the stain with antibodies specific to antigens CD2 and CD7. This is the norm. Thus was discovered coexpressed CD16/CD56, CD16/CD44, CD16/CD45, CD16/CD2, CD16/CD7 that is the norm.

On the biochip No. 3 accumulation colored product of the enzymatic reaction by virtually all cells were noted in the area of the stain with antibodies specific to antigens CD2, CD3, CD4, CD5, CD8. In the area of the stain with antibodies specific to antigens CD7, CD27, CD29, CD31, CD44, CD45, CD45RA, CD95, stained portion of the cells. This is because these antigens Express not only T cells, but other cell types. Thus was discovered coexpressed CD2/CD3, CD2/CD4, CD2/CD5, CD2/CD7, CD2/CD8, CD2/CD27, CD2/CD29, CD2/CD31, CD2/CD44, CD2/CD45, etc. that is the norm. The presence of the co-expression of CD2/CD95 in some cells may be indicative of their activation. Stained also part of the cells, bound peroxidase in the area of the stain with antibodies anti-CD16. This is due to the expression of the CD2 antigen NK cells. Thus, in the experiment data, indicating cross-reactivity of antibodies, have been received, which indicates the prigoda the spine of the party of biochips for use.

1. The method combined immunological studies of cells using a biochip, comprising the incubation of the biochip containing immobilized antibodies, cell suspension, washing the biochip from unbound cells, the determination of the co-expression of antigens on the linked cells, the reading of the result, including the detection of binding cells in the stained area of the biochip and the determination of the density of binding cells, and interpretation of the received result, wherein determining the co-expression of antigens on related biochip cells is accomplished by implementing one or more of immunocytochemical reactions when reading the additional spend morphological study associated with the biochip cells and determine the presence and nature of the staining of cells and their components the reaction product.

2. The method according to claim 1, wherein implementing the additional staining of cells which do not prevent the detection of the product (s) of the enzymatic reaction.

3. The method according to claim 1, characterized in that, for the study of a single sample of cell suspension use multiple biochips, by immunocytochemical study of cells using antibodies specific for different antigens.

4. The method according to claim 1, characterized in that the wasp is estlat morphometric study associated with the biochip cells.

5. The method according to claim 1, characterized in that in the analysis use one or more additional control biochips.

6. The method according to claim 1, characterized in that the process biochip one or more solutions containing one or more substances that increase the strength of bonding of the cells with the surface of the biochip.

7. The method according to claim 1, characterized in that the biochip prepare long-term preparation for microscopic examination.

8. The method according to claim 1, characterized in that the biochip or made of him a long-term drug caused markup that facilitates finding the right spots.

9. The method according to claim 1, characterized in that in the analysis use one or more additional biochips, which perform the staining associated cells for their subsequent morphological studies, with immunocytochemical reaction is not carried out.



 

Same patents:

FIELD: medicine.

SUBSTANCE: after antigen-antibody complex is prepared in a reaction compartment wherein said antigen to be modified and antibody are bound, said reaction compartment is washed with using a sample solution. The invention allows detecting a signal reflecting quantity of said antigen to be modified at precision comparable when using a washing solution to this without using said solution. Herewith, the washing solution is not required to be supplied from the outside of a chip or to be ensured therein beforehand.

EFFECT: immunoassay is easily realised on the chip.

6 cl, 6 dwg, 1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention can be used for evaluating the antibody level in blood serum by enzyme-linked immunosorbent assay (ELISA) in diagnostics of the diseases caused by capsular forms of such agents, as Meningococcus, Pneumococcus, Streptococcus, Haemophilus, Neisseria, Salmonella, Klebsiella, Pseudomonas, and also for evaluating the postvaccinal immunity level.

EFFECT: covalent binding of polysaccharide to the protein pre-immobilised on the tray surface ensures reliable and simple antigen fixation that does not involve effect of foreign chemical groups on the analysis results that leads to simplification of the method for tray sensitisation in the ELISA reliability control.

1 dwg, 1 tbl, 3 ex

FIELD: instrument making.

SUBSTANCE: set of invention relates to devices to determine the level of analysed substances in biological fluids. Proposed system comprises (a) measuring device with housing, logical circuit arranged in said housing, visual display arranged on said housing and measuring system arranged therein. It comprises also (b) cartridge incorporating at least one test strip to analyse lateral flow. Note here that said test strip incorporates (i) lateral flow transport matrix, (ii) zone of analysing specific binding on transport matrix to take up fluid specimen to produce required reaction, and (iii) zone of general chemical analysis on transport matrix to take up fluid specimen to produce required reaction. Note also that sizes of cartridges are selected to allow arranging analysed biological fluid substances in measuring device so that measuring system in above described zones on test strip. Mind that transport matrix first and second segments are made from different materials and bound so that said segments does not allow the product formed on first segment to contribute into reaction on second segment. Proposed invention covers other versions of aforesaid device.

EFFECT: higher efficiency, accuracy and reliability.

162 cl, 27 dwg

FIELD: veterinary.

SUBSTANCE: claimed is test-system of immuno-enzyme analysis, which allows to determine antibodies to viruses of infectious rhinotracheitis (IRT), viral diarrhea-disease of mucous membranes (VD-DMM), parainfluenza viruses -3 (PIV-3), respiratory syncytial (RS) and adenoviral (AVI) infections of livestock. Serological examination of animals allows to detect zones of infection spreading and estimate post-vaccination immunity.

EFFECT: application of claimed test-system IEA will allow to carry out simultaneously epizootological monitoring of five important infections of livestock, retrospective diagnostics of respiratory infections, and estimation of immunity stress in animals resulting from application of vaccines, determination of level of colostral antibodies in young animals in the first weeks or days of life, estimation of therapeutic medicine quality.

10 tbl

FIELD: medicine.

SUBSTANCE: group of inventions refer to medical diagnostics and covers diagnostics of early myocardial infarction by direct determination of myoglobin from human myocardium. Said biosensor represents a disposable graphite electrode modified by colloidal gold and monoclonal antibodies to myoglobin from human myocardium. It is produced the way as follows. A three-contact graphite main electrode is covered with colloidal gold solution stabilised with didodecyl methylammonium bromide in chloroform. It is dried at room temperature. The electrode structure is modified by antibodies to myoglobin from human myocardium by covering the main graphite electrode to myoglobin from human myocardium modified with didodecyl methylammonium bromide and colloidal gold. Then it is dried, settled at +2°÷+6°C, rinsed with water, processed with a blocking buffer, dried, settled at room temperature and rinsed with water.

EFFECT: possibility to determine myoglobin in aqueous solutions.

3 cl, 2 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, immunology and biotechnology. Substance of the invention consists in developing the method to lower the content of thyroid hormone antibodies by blood passage through prepared original granulated magnetic agent with immobilised forms of thyroid hormones on the basis of polyacrylamide granules with magnetic properties.

EFFECT: improved effectiveness and reduced consumption of the agent as compared to a prototype, owing to higher specific activity and effective multiple regenerability, simplified manipulations with the agent and maintained suspension of granules thereof in sorption process ensured by constant magnetic field that improves the active sorption area, and reduced destruction of perfused blood corpuscles.

5 ex, 2 tbl

FIELD: chemistry.

SUBSTANCE: invention refers to the biochemistry and molecular biology, in particular to the immunochemical analysis. The claimed microchip for multiple parallel immunological analysis of the compounds represents the array of three-dimensional hydrogel elements with specified volume formed on the support by the photo- or chemically induced polymerisation and containing the biological molecules (ligands) of identical or different nature. The method of quantitive immunoassay implies microchip incubation to the reaction media including the analysed sample for the purpose of complex generation. The biological microchips for quantitive immunological analysis and method for implementation of multiple parallel quantitive analysis of the compounds can be used in the analysis of the wide range of low- and high-molecular compounds, in medicine, pharmacology, food industry, environmental protection, scientific researches including proteomics.

EFFECT: possibility of multiple parallel immunological analysis of the compounds.

33 cl, 12 ex, 2 tbl, 11 dwg

FIELD: medicine.

SUBSTANCE: invention concerns medicine, in particular to medical diagnostics. A nano-biochip for registration of proteins and albuminous complexes and a way of its reception is offered. As a biochip substrate a non-stratified material, for example polycarbonate or polystyrene with a relief grid, is used and then the surface is leveled by means of a die providing equal surface with the size of roughnesses not more than 1 mm; after that updating and immobilisation of the molecules-probes capable to cooperate with investigated proteins and albuminous complexes on a surface of the chip specifically is carried out.

EFFECT: way allows to raise reliability of the received biochip at the expense of exception of its stratification and to raise reliability of diagnostics at the expense of rising of registration sensitivity of specific proteins and albuminous complexes.

12 cl, 2 dwg, 1 ex

FIELD: veterinary science.

SUBSTANCE: invention refers to veterinary microbiology and biotechnology can be used for development of specific diagnostic aids. According to the invention the method covers producing antigen erythrocytic colibacillosis diagnosticum by extracting an adhesive antigen from Escherichia cultures, centrifuging the extract, and separating the supernatant. Thereafter formalinised tanninised animal erythrocytes are sensitised with the produced antigen. Extraction is performed in culture fluid containing Escherichia cell culture with phosphate-carbamide buffer 1.8-2.0 M of pH 7.2-7.4 at temperature 40-45°C during 25-30 min. The culture fluid containing Escherichia cell culture and phosphate-carbamide buffer are taken in mass ratio 1:0.4-0.6 respectively. After extraction the supernatant is heated up at 65-68°C within 25-30 min.

EFFECT: method allows for high-quality end product due to improved sensitivity and specificity.

3 ex

FIELD: veterinary science.

SUBSTANCE: invention refers to veterinary microbiology and biotechnology can be used for development of specific diagnostic aids. According to the invention the method covers producing antigen erythrocytic pasteurellosis diagnosticum by extracting pasteurellosis capsular antigen from Pasteurella cultures with sodium chloride brine, with centrifuging the extract and separating the supernatant exposed to sterilisation filtration and used for sensitisation of formalinised anionised animal erythrocytes. Pasteurellosis capsular antigen is extracted with 2.0-2.5% sodium chloride brine at 40-42°C during 30-40 min. Prior to sterilisation filtration, the supernatant is heated up at 65-70°C within 15-30 minutes.

EFFECT: application of the method allows for high-quality end product due to improved sensitivity and specificity.

3 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to gastroenterology and clinic immunology, and can be used to predict risk of development of destructive changes of gastrodoudental zone mucous. Method includes estimation of total quantity of positive by CD80-receptors neutrophil granulocytes of peripheral blood, and if value of said index is higher than cell/ml, presence of risk of developing destructive changes of gastroduodental zone mucous tunic is determined.

EFFECT: claimed method can be applied for screening assessment of evidence and character of inflammation in mucous tunic in case of Helicobacter pylori-associated inflammatory diseases of gastroduodental zone, allowing quickly, without large material and technological costs, to detect among patients with H pylori-associated inflammatory diseases of gastroduodental zone risk group as to development of destructive inflammation (erosion, ulcer) in mucous tunic with high probability (75%) .

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: method lies in determining caspase-3 and titre of antibodies to herpes virus by EIA method in placenta. If titre of antibodies to herpes virus is 1:12800 and content of caspase-3 is 103.7±0.8 ng/ml conclusion about increased risk of damaging action of caspase-3 on syncytiotrophoblast of placenta is made.

EFFECT: method has high sensitivity and allows to predict morphofunctional damage of syncytiotrophoblast of placenta in case of exacerbation of herpes-viral infection during pregnancy.

FIELD: medicine.

SUBSTANCE: clinic, endoscopic, electrophysiological examination, morphologic examination of mucous membrane of large intestine, as well as immunologic examination of blood serum are carried out. If frequency of slow waves of electromotive activity reduces by 35-50%, detection of hyperplasia of goblet cells, large macrophages with dense inclusions, activated plasmocytes, which form rosettes; as well as detection of antibodies to bactericidal protein, which increases permeability of cells (BPI) from 4 to 18 U/ml, antibodies to Saccharomyces cerevidiae (ASCA) from 7 to 22 U/ml early stage of diverticular disease of colon is diagnosed.

EFFECT: increase of accuracy of diverticular disease of colon diagnostics at early stage.

3 ex

FIELD: medicine.

SUBSTANCE: diagnostics of vagina infections is carried out by detection of clinical indications, determining amount of leukocytes, epithelium, lacto bacteria, carrying out of pH-metrics of vaginal secretion and aminotest. Additionally secretory immunoglobulin A (slgA) is examined and if it equals 200.5±26.3 aerobic vaginitis is diagnosed. If sIgA equals 168±29.9, pH is over 5.6-6.0 and aminotest is positive bacterial vaginosis is diagnosed. If slgA equals 172.1±53.2, pH does not exceed 6.2 and leukocytes are present in presence of trichomonads trichomoniasis is diagnosed. If slgA equals 375.61 ±23.1, pH is 4.0-4.5 and aminotest is negative candidiasis is diagnosed, slgA value of 248.4±18.8 corresponds to absence of infection.

EFFECT: invention allows unifying of existing methods of slgA detection in vaginal secretion.

1 tbl

FIELD: medicine.

SUBSTANCE: invention refers to a method of evaluating the damaging action of protein p53 on placenta in acute herpes virus infection. The method consists in evaluating protein p53 and a herpes virus antibody titre by EIA method in placenta homogenate. The herpes virus antibody titre 1:12800 and protein p53 4.52±0.16 E/ml ensure to observe higher risk of damaging morphofunctional condition of syncytiotrophoblast.

EFFECT: method exhibits high sensitivity and allows predicting damaging placenta syncytiotrophoblast in acute herpes virus infection in the pregnancy.

FIELD: medicine.

SUBSTANCE: device comprises a measuring cavity for receiving and introducing a fluid sample. The measuring cavity has a set fixed thickness not exceeding 170 micrometres. The measuring cavity has a section fit for acquisition of its image. Within the measuring cavity, there is a dry reagent. The reagent contains as a component, a molecule conjugate with phosphor used for binding with biological components and with all other reacting components. The reacting components are soluble and/or suspended in the fluid sample. The method involves mixing of the reagent with the liquid sample to be introduced in the measuring cavity. A section of the sample in the measuring cavity is exposed to electromagnetic radiation of wavelength corresponding to wavelength of phosphor excitation. Phosphor marked biological components are detected through-thickness of the measuring cavity. Further, numerical analysis of the digital image follows to identify the biological components showing phosphor and to determine amounts of the biological components showing phosphor in the sample. The biological components are discernible on the digital image as fluorescing points emitting electromagnetic radiation of wavelength corresponding wavelength of phosphor emission.

EFFECT: device and method allow for higher effectiveness of numerical volume concentration of fluorescent marked biological components of the fluid sample.

24 cl, 4 dwg

FIELD: medicine.

SUBSTANCE: initially anamnestic data and clinical features of laryngotracheitis are specified, microbiological and virological studies are conducted in a smear from posterior pharyngeal wall, level of cytokines is determined (interleukine-4, interleukine-6, interferon-α, interferon-γ) and general IgE in peripheral blood, and at presence in a child the following symptoms: allergic pathology in case history, effuse of Streptococcus agalacticae, viral-viral and bacterial-bacterial associations, development of croup syndrome up to 2 stage, reducing of red blood cell level <2.9*10 billion/l and hemoglobin <89 g/l in clinical blood analysis, as well as determining the level of cytokines in peripheral blood: interleukine-4 is higher than 14.8 ng/ml, interleukine-6 is higher than 16.1 ng/ml, interferon-α is higher than 147.8 ng/ml, interferon-γ is lower than 16.9 ng/ml; the level of total IgE in the serum is higher than 47.0, relapsing course is predicted. Using the method enables to determine characteristics of the course of laryngotracheitis in children at an early stage, to take adequate programs of anti-recidivating treatment and conduct targeted immunotherapy.

EFFECT: reduction in frequency of laryngotracheitis recuring in children.

3 ex

FIELD: medicine.

SUBSTANCE: method of study of cells by the means of immunological biochip is proposed, which contains capture antibodies, specific to cell membrane antigens. Biochip is incubated with cell suspension, and then it is washed out from cells not linked to the antibodies. Then the biochip is treated with a solution which increases the strength of binding the biochip and cells remaining on its surface due to interaction with immobilised antibodies. Preparation for microscopic studies is prepared of the biochip, research study of related to the biochip cells is conducted, reading and analysing the result is performed. In the course of the study colouring of connected cells can be done or their treatment with labeled antibodies can be conducted.

EFFECT: method enables to prevent cells detachment from the surface of a biochip at conducting of different manipulations, which leads to increased sensitivity of analysis.

13 cl, 6 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: number of peripheral blood lymphocytes expressing Fas-L is used as an indicator. At increase of the number of lymphocytes expressing Fas-L after antigen-specific immunotherapy the treatment is evaluated as of 3 or more times more effective as compared to the level prior to immunotherapy.

EFFECT: improved prediction of treatment of bronchial asthma.

2 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to laboratory analysis and can be used in dermatology. The method aims at higher accuracy of prediction ensured by more reliable profiling of target antigens of autoimmune pemphigus. That is ensured by analysing a clinically intact skin area by the direct immunofluorescence. Fixed immunoglobulin G is localised. When localising IgG in an intercellular substance of all epidermic layers, potential pemphigus deformation is predicted; IgG observed not in all layers shows low probability of variability of pemphigus clinical form. The offered method allows for prediction of potential transition between clinical forms.

EFFECT: it ensures more effective specification of therapeutic approach and management of the patient.

3 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: embryonic tissue is flushed with enzyme solution at 25-37°C temperature. For enzyme neutralisation is used medium, made of Eagle medium with lactalbumine hydrolysate or bovine serum.

EFFECT: due to reducing of trypsin proteolitic activity and higher development temperature in stable cell cultures manufacture, yield of viable diploid cells is increased in 1,5-2 times compared to closest analogue of this invention.

7 cl, 4 tbl

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