Product and method for multi immunoassay serum

 

(57) Abstract:

The invention relates to an immunochemical methods of analysis of the sera of humans and animals and can be used for simultaneous detection in the samples of specific antibodies (ATA) to a number of markers of infectious, parasitic, allergic and somatic diseases in medicine, veterinary medicine, biotechnology, environmental research and scientific experiments. The invention is a product which is a substrate (strip) in the form of comb teeth 5-12 mm wide, on which is applied to all tested antigens. With the substrate carry out the method immunochemical analysis. Effect: invention allows to reduce the cost, simplify and speed up the procedure serological surveys by simultaneously (in the same analysis) obtain information about the presence in the sample at a number of markers of various diseases, ease of production and application of patented products, as well as reduce energy and labor costs to perform the analysis. 2 N. and 10 C.p. f-crystals, 3 tables.

The invention relates to an immunochemical methods of analysis of the blood of humans and animals and can be used the traditional, parasitic, allergic, and oncogenic diseases in medicine, veterinary medicine, environmental studies and scientific experiments.

The essence of such detection is the formation of specific complexes between AT the sample and known antigens in a specific order discretely recorded on a dense substrate of the analytical device. The resulting complexes are then detected using a secondary immunoreagent specific to AT test sera (individule AT, staphylococcal protein a and G, and others) and associated with easily detectable label.

From the variety of methods of immunological analysis developed to date, the most popular solid-phase systems in which one component is associated with a dense substrate (matrix). This approach allows easy separation of the formed complexes from non-bound peroxidase components by washing the matrix, which significantly reduces the time and simplifies the analysis.

There are many options for the realization of solid-phase immunoassay, however are all created to date, the test system to determine AT have monospecificity, i.e. allow seasonspeech as the primary immunoreagents.

On the origin of the antigens may be natural, recombinant or synthetic. Until recently, in immunochemistry was used only natural antigens derived from natural biological agents. The properties of these antigens (immunological, adsorption, electrochemical and others) significantly varied depending on the species and strain differences in the source of antigen, method and environment for its cultivation, the methods used inaktivirovanie, separation, purification and conservation. A variety of antigens caused the need for careful selection of components of the test system and conditions of analysis in each case.

The success of molecular biology in recent years have made possible large-scale accumulation of the recombinant antigens that every year more and more widely implemented in practice immunoassay. Such popularity of recombinant antigens, along with job security and relatively low material and labor costs, due to their high specificity and purity, ensuring a high quality immunoassay.

On the other hand, recombinant technology allows to standardize the antigens of putanov and analysis in the test systems of different specificity and create diagnostics for simultaneous multi-disciplinary analysis of clinical samples.

Technology for production of natural antigens recently also significantly progressed. The use of high quality components for producing material and effective means of separation from him and purification of antigens in some cases make these products compatible (including recombinant proteins and are suitable for use in medical analysis. But still multidisciplinary analysis was not implemented.

The closest technical solution is the test-system "Orgenics immunocomb HIV 1+2 bi-spot firm "Orgenics, Israel [1, prototype]. This test system is one of the variants of the dot-immunoassay (dot, blot, spot overlay immunoassay) on the surface of non-porous media. Her work item (solid phase) are presented in the form of a flat plastic combs, each prong of which is plotted in the form of separate spots antigens HIV 1 and 2, as well as human immunoglobulins control to verify operation of the conjugate. Another element of the test system is a plastic container that is divided into individual cells by the number of teeth of the comb (horizontal row) and the number used in the system of working solutions (vertical row). Each horizontal row of cells filled is immunoreagent labeled - alkaline phosphatase, a wash solution, a substrate solution for the manifestation of Alp, a wash solution. In the analysis in the first cell in the row are made a certain amount of the investigated sera and then comb consistently moves at certain intervals along the rows of cells. After removal from the last row visually counted result of the analysis by the presence of blue spots in the ground causing antigens and control. The system is fully equipped with everything necessary to analysis components and is suitable for use in unelaborated conditions.

The disadvantage of this prototype is that the system allows to identify only two related strains of human immunodeficiency virus.

An object of the invention is to obtain products for multiparametric immunoassay of serum and use this device for immunoassay.

The problem is solved using the proposed device, the teeth of the comb or separate strips which discrete applied several antigens (different infectious and parasitic diseases, different somatic markers, allergic or carcinogenic pathology), can effectively suvislosti from immunodiagnostics. The immunoassay involves simultaneous (one analysis) identify the presence of specific antibodies to multiple (up to several tens) antigens, corresponding to different infectious, parasitic, autoimmune or cancer.

Set of antigens may include a compilation of several antigens of the same type (for example, antigens of pathogens of sexually transmitted infections, or antigens of infectious agents that require monitoring during transfusion, or antigens of infectious and parasitic diseases in animals) or be relatively universal, including dozens of antigens medical or veterinary profile.

The selection of antigens for such a system can be implemented in the tablet version of the ELISA with the same conditions sorption of antigen and production analysis. In the same way can be chosen concentration of antigen for sorption on a solid substrate. The application of the antigen may be in the form of a measured aliquot of not more than 1 mm, or in the form of electrospray. In the latter case, the antigen can be applied in the form of spots or lines of certain shapes, which further increases the reliability of the results. For weakly sorbero is his connection with the substrate by means of bifunctional agents.

After fixation, antigen-free areas of the substrate (strip) are blocked by one or more natural or synthetic polymers, such as bovine serum albumin, casein, polyethylene glycol, etc.

The analysis includes sequential incubation articles:

1) in a dilution of test sera or plasma;

2) in a wash solution;

3) in solution secondary immunoreagents (antivitamin antibody or Staphylococcus protein a or G), associated with easily allocated label (enzyme or Sol of inorganic catalyst);

4) in a wash solution;

5) in the substrate for a display of labels;

6) in a wash solution.

Analysis is carried out visually or by using a scanner and subsequent computer analysis of the image. In the latter case possible densitometric analysis of the results with obtaining approximate quantitative characteristics of the content analyzed AT in the sample.

The present invention involves the use as the label of the secondary immunoreagent as enzymes (peroxidase, Alp and others) and visokodispersnie and others). Sols can be associated with secondary immunoreagents (Immunocal) sorption or covalently. When using porous substrates sols may be associated with larger (particle diameter in the range of 0.1-10 μm) a carrier having a low density (latex, coal), and immunoreagents [2]. In the latter case, we have a stable immunoresponse, particles which, due to its dimensions do not penetrate into the pores of the dense substrate, which greatly facilitates the procedure of cleaning (Patent RF №2133469). The manifestation of the label is held in one of known substrates, providing a sensitivity of detection.

Conducted by the applicant's analysis of the prior art, including searching by the patent and scientific and technical information sources, and identify sources that contain information about the equivalents of the claimed invention, has allowed to establish that the applicant had not discovered similar, characterized by signs, identical to all the essential features of the claimed invention.

Therefore, the claimed invention meets the criterion of "novelty" and "inventive step".

This is followed by examples of implementation of the proposed technical solution.

In the experimental trial is Spirochaetaceae);

2) recombinant antigen cytomegalovirus (CMV) (family Herpesvirdae);

3) recombinant antigen of the virus Crimean-Congo hemorrhagic fever (WCCP) (family Bunuaviridae);

4) recombinant antigen of Toxoplasma gondii (class Sporozoa);

5) the particulate antigen Chlamidia psitacci (family Chlamudiaceae);

6) corpuscular antigen of herpes simplex virus (HSV) (family Herpesvirdae);

7) particulate antigen vaccinia virus (BOB) (from the poxviridae family);

8) corpuscular antigen Brucella suis.

Solutions:

1) FSB - 0.1 M matrifocality buffer pH of 7.2 to 7.4, with 0.15 M NaCl;

2) FSB-T with 0.05% tween-20;

3) of the CBD concentrate blocking solution containing E. coli lysate, a commercial product manufactured by “Drops”, Moscow;

4) KB - 0.025 M Na-carbonate-bicarbonate buffer, pH 9,5;

5) RBR-To - diluent conjugates and immunosera - FSB-T 6 with 0.05% casein;

6) RBR-To - solution for dilution of serum - FSB, the pH of 9.6, 0.1% tween-20 and 5% CBD;

7) the substrate for a display of peroxidase - commercial preparation “Buffer substrate Kit DAB" (Cat. SK-41100), production Vector lab. Inc.", Burllingem, CA, United States;

8) PH-a/IgG - peroxidase conjugate with MA proimer particles 15 nm), sorption is associated with staphylococcal protein A [3], the original Immunocal had OP520=2,0 TH;

10) Ag-a/IgG - colloidal silver (average particle diameter 10 nm), sorption is associated with antibodies against human IgG [4];

11) physical developer” for signal amplification of immunosera gold and silver - aqueous solution containing 0.5% citric acid, 0.2% metol and 0.2% silver nitrate [4, 5].

Example 1. THE MANUFACTURE AND USE OF DEVICES FOR MULTIDISCIPLINARY ANALYSIS OF SERA USING AS THE SUBSTRATE MATERIAL OF POLYSTYRENE

The manufacturer of the device.

Option A.

White polystyrene (packaging from food) cut into strips (stripe) size 8×60 mm, the top layer of plastic is scraped with a scalpel. On a fresh surface of the plastic in two rows along the strip at intervals of 5-7 mm put antigens at a concentration of 50-100 μg/ml for KB, aliquot 2 μl, and dried at room temperature. The length of the working part of the strip (the area coated with antigens) is about 20-25 mm, free part of the strip is used as the handle. Strips 2/3 the length of the working part is immersed in 0.2% solution of casein on the FSB and incubated 1 h at 37°C. Polaski The same as in option a, polystyrene strips incubated for 2 h at room temperature in a 2% solution of glutaraldehyde in 0.01 M califorina buffer, pH 7.0 and dried in air. A further operation on the application of antigens and the blocking strip is conducted similar to that described in option A.

The use of the device

Strips coated with antigens in a vertical position incubated for 30 min at 37°With the 1/40 dilution of the investigated sera on RBR, washed three times by immersion for 1 min in the FSB-T, incubated for 30 min at 37°With immunocore Ag-a/IgG (dilution 1:20 for RDB-K) or the conjugate HRP/IgG (working dilution on RBR). Washed three times by immersion for 1 min in the FSB-T and rinse the glass with distilled water. Showing related Sol dipping strips 10 to 15 minutes in the physical developer, and associated conjugate - 20 min a substrate for peroxidase. Manifested strips pasted on white paper and passed through the scanner. Test results are transmitted to the computer and using a special program, process and print.

The results obtained in comparison with immunoperoxidase test monospecific test systems are given in table 1.

The receiving device.

Fat-free cover glass for microscopy boiled for 4 hours in 6N hydrochloric acid, washed with water and dried. Boil glass 12 h in 10% solution of 3-amoxicilpin-1-amine in anhydrous toluene, washed with acetone and dried. Put in certain points 2 μl of a 10% aqueous solution of glutaraldehyde, incubated for 12 h at 20°C and 100% relative humidity (wet cell), washed with water, with acetone and dried. In activated with glutaraldehyde point put 2 μl of solutions of different antigens with a concentration of 0.5-1 mg/ml in phosphate buffer, pH 7.5, and incubated for 12 h at 20°C in a humid chamber. Scrub the glass with water, dried. Incubated glass in 0.2% solution of casein in phosphate buffer, pH 7.5, 1 h at 20°C, rinsed with water, dried.

The use of the device.

Glass coated antigens is incubated for 30 min at 37°C in the tested sera (dilution 1/20 at RBR, washed twice by immersion for 1 min in the FSB-T, incubated for 30 min at 37°C in a solution of immunosera Au-SpA (dilution 1/50 to RDB). Washed twice by immersion for 1 min in the FSB-T and rinse the glass with distilled water. Are associated gold will escaut through the scanner. Test results are transmitted to the computer and using special programs to process and print.

Results multivariate dot-analysis compared with monospecific tablet option ELISA are shown in table 2.

Example 3. PREPARATION AND APPLICATION DEVICE FOR A MULTIDISCIPLINARY ANALYSIS OF SERA USING AS THE SUBSTRATE MATERIAL OF THE POROUS MEMBRANE

The receiving device.

Nitrocellulose membranes firm Millipore with a pore diameter of 0.45 μm (type) cut in strips of a width of 7-8 mm and a length of 25 mm, antigens at a concentration of 50-100 μg/ml for KB put in two rows along the strip at intervals of 5-7 mm aliquot 5 ál and dried at room temperature. Strips dipped in 0.2% solution of casein on the FSB and incubated 1 h at 37°C. Rinse strips with distilled water and dried at room temperature.

The use of the device.

Strips coated with antigens is incubated for 30 min at 37°With the 1/40 dilution of the investigated sera on RBR, washed three times for 5 min on a shaker in the FSB-T, incubated for 30 min at 37°C in Konsole Ag-a/IgG (dilution 1:20 for RDB-K) or the conjugate HRP/IgG (working again is knit Sol dipping strips for 10-15 min in a physical developer”, and associated conjugate for 20 min in a substrate for peroxidase. Manifested strips pasted on white paper and passed through the scanner. Test results are transmitted to the computer and using special programs to process and print.

The results obtained in comparison with immunoperoxidase test monospecific test systems are given in table 3.

The proposed product and method of immunoassay assume a complete set of components and can be used for mass screening of sera and for the individual analyses. They can be used not only in laboratories, but also (in medicine) at the bedside, in the doctor's office, for self-diagnosis; (ecology) field experiments; (in veterinary medicine) in animal husbandry and household. The invention allows for the definition in a single analysis right number of parameters significantly reduce the time for examination of the patient. In addition, as one analysis of the proposed method is equivalent to several tests in monospecific systems and the cost of these tests is close, the implementation of the invention in the broad practice can provide significant economic effect work on the analysis.

REFERENCES

1. HIV 1+2 bi-spot Cat: 432102920922.

2. RF patent №2133469 Marker for superficial blot immunological and hybridization analysis in porous media”, G 01 N 33/53.

3. Raska I. Electron microscopic immunocytoshemistry with colloidal gold. - Laboratory manual of the practical course organized by the Institute of Experimental Medicine, Czechoslovak Academy of Sciences in collaboration with the Czechoslovak Biochemical Society of the Czechoslovak Academy of Sciences, Prague, May 29th-Jine 3rd, 1988.

4. Poltavchenko A. G., Karavaev B. C., Tuzikov F. C. Use of silver sols as markers immunoassay in microtiter tablets // JAMI, 1998, No. 2, S. 108-111.

5. Poltavchenko A. G., Lavrinenko, I. A., Kostrovsky Century, and others Use silver immensely to detect HIV antibodies in microtiter tablets // Matters. Virusol., 1997, No. 3, S. 120-123.

1. Product for immunochemical analysis, representing a plastic substrate (strip) in the form of a comb with sorption fixed in a limited area of its surface antigen and blocked natural or synthetic polymers, free of antigen sites, characterized in that the surface of the substrate with a teeth width 5-12 mm on each is applied in the form of separate spots all used the P CLASS="ptx2">2. The product under item 1, characterized in that the substrate is glass or a porous membrane.

3. The product under item 1, characterized in that the coating antigens on the substrate is dosing the product in the form of restricted spots.

4. The product under item 1, characterized in that the coating antigens on the substrate is performed in the state of spray (electrospray) in the form of restricted spots or lines of some form.

5. The product under item 1, characterized in that the fixing antigens on its surface is produced by covalent bonds or by bifunctional agents.

6. The product under item 1, characterized in that each fixed on the substrate antigens or part of them presents of recombinant proteins.

7. The product under item 1, characterized in that each fixed on the substrate antigens or part of them are natural proteins.

8. The product under item 1, characterized in that each fixed on the substrate antigens or part of them are synthetic proteins.

9. The way immunochemical analysis, including the incubation of the strip with a fixed antigen at a dilution of test sera, the shaded strip, incubation stri is bstrate for the manifestation of the label, the shaded strip, visual analysis, wherein the analysis is performed with use of the product described in paras.1-8, and serum simultaneously determine multiple (more than 3) different types of specificity of antibodies.

10. The method according to p. 9, characterized in that as the conjugate using sols of inorganic catalysts, such as gold, or silver, or selenium, or palladium associated with secondary immunoreagents, or antivirovym antibody or Staphylococcus protein a or G.

11. The method according to p. 9, characterized in that as the conjugate used immensely described in paragraph 10, associated with Macronectes (latex, powdered coal, carbon black, Aerosil), having a density of 1.0-1.5 g/cm3and particle sizes in the range of 0.05-10 microns.

12. The method according to p. 9, characterized in that as the substrate for the manifestation of the catalytic labels use one of the known compositions "physical developers, including reducing agents, and salts of silver.

13. The method according to p. 9, wherein the analysis is performed using a scanner with subsequent computer analysis of the image.



 

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