Method of simultaneous immunochromatography analysis of psa and cea oncoantigens

FIELD: medicine.

SUBSTANCE: method of simultaneous immunochromatography analysis of PSA and CEA oncoantigens is offered. The analysis involves using a test strip which contains three segments A, B and C; the segment A overlaps 1-2 mm of the segment B. The segment A represents an inert porous carrier made of fibre glass ("ПЭД") with two reaction zones 1 and 2 applied on its surface. Mice monoclonal PSA and CEA antibodies conjugated with colloidal gold are respectively applied on zones 1 and 2. The conjugates are applied in the form of parallel strips in the centre of "ПЭД" perpendicularly to a fluid flow. The segment B represents nitrocellulose immobilised on a lavsan substrate with two test zones applied on its surface (monoclonal PSA and CEA antibodies in each), and a reference area (mice immunoglobulin antibodies).

EFFECT: test enables the simultaneous detection of the patients with the high blood serum PSA and CEA antigen contents in screening assays.

6 ex, 4 dwg

 

The invention relates to biotechnology. A method is proposed for simultaneous lateral flow definition encountering PSA and CEA in serum using test strips.

To implement the method proposed immunochromatographic test strip, consisting of parts a, b and C (figa)mounted on the substrate D in such a way that part And 1-2 mm overlap part of the Century At the same time is an inert porous carrier made of fiberglass (SEM) deposited on the surface of the reaction zone 1 (coated with a conjugate of mouse monoclonal antibodies against PSA with nanocolloidal gold), and reaction zone 2 (coated with a conjugate of mouse monoclonal antibodies against CEA, labeled nanocolloidal gold). Conjugates caused in the reaction zone of parallel strips in the Central region of the pad perpendicular to the flow of the fluid at a distance of 2 mm from each other. Part of the test strips is a nitrocellulose immobilized on a polymer basis, on the surface of which is applied to the test area 6 test area 7 and the control zone 10. Test area 6 is the area coated with a stripe perpendicular to the long side of the test strip 2 mm from the middle of nitrocellulose mouse monoclonal antibodies against PSA epitope different from the epitope to which specific antibodies, the locale is used in the reaction zone 1. The test area 7 represents a zone with applied stripe perpendicular to the long side of the test strip 2 mm from the middle of nitrocellulose mouse monoclonal antibodies against epitope of CEA, is different from the epitope to which specific antibodies, lokalizovana in the reaction zone 2. Control area 10 is an area coated with a parallel strip of 3 mm from the middle closer to the suction filter With monospecific rabbit antibodies against mouse IgG 9. This has In capillary communication with the reaction area of the carrier A. - suction filter to absorb unreacted reagents. The decision on whether PSA and/or CEA take a color test zones 6 and 7. Color zone 6 indicates the presence of an increased number of oceantiger PSA in the sample, the color area 7 in the presence of an increased number of oceantiger CEA, simultaneous color zones 6 and 7 indicates the presence in the sample of both antigens. The lack of color in zones 6 and 7 indicates normal content of these encountering. The color of the control area indicates the health of the test strip. The use of the invention provides simultaneous detection of encountering PSA and CEA for the purposes of screening men at risk for identifying patients with suspected cancer of prestate the Noah cancer and some other diseases.

Description of the INVENTION

The invention relates to immunology, biotechnology and Oncology. The developed method for the simultaneous rapid diagnosis of the presence of tumor markers PSA and CEA in serum based on lateral flow (THEIR) determining the presence of these antigens in the samples. The essence of the invention is to use a sandwich lateral flow analysis using monoclonal antibodies. Monoclonal antibody (MAB) to the PSA and CEA, respectively immobilized in the test zones 6 and 7, strip on nitrocellulose membrane, react with the corresponding antigen (AG) in serum samples in the process of tangential movement of the sample material on THEIR strip. Simultaneous binding of the conjugate (KG) MAT against other epitopes PSA and CEA with nanocolloidal gold (SCP) with the PSA AG and AG CEA, respectively. As a result, when the presence in the sample of AG PSA in the test area 6 immunochromatographic strip is formed complex KG PS-AG PSA-AT PSA. When the presence in the sample of AG CEA in the test area 7 is formed complex KG CEA-AG CEA-at CEA. In the control zone is formed complex AT against mouse IgG with unbound KG PSA KG CEA. The formed complex allows to visualize the presence of PSA AG and AG CEA in the serum, and the complex in the test area visualizes the safety strip. This has allowed the et to conduct screening to diagnose patients with prostate cancer and other cancer risk groups.

The LEVEL of TECHNOLOGY

Prostatespecific AG (PSA) is a glycoprotein produced by the secretory epithelia of the prostate and providing the liquefaction of semen. The concentration of PSA in serum of blood donors - not more than 4 ng/ml is Known that the increase of PSA levels in tumors is a mechanical pressure hyperplastic tissue on unmodified fabric that is including prostate cancer. Increasing the concentration of PSA greater than 4 ng/ml at age 60 and up to 8 ng/ml after the age of 60 years for men consider a diagnostic symptom of disorders of the prostate, including its zlokacestvennoe. In Russia, mortality in the first year of life after diagnosis is less than 30%, which is associated with the diagnosis of prostate cancer on the 3rd and 4th stage in most cases. Early detection of patients with rising PSA in the blood of older men is one of the most effective ways to reduce mortality from prostate cancer, which is one of the most common malignant pathologies of older men. Immunochromatographic tests is one of the most suitable for diagnostic screening method that can be widely implemented as medical test, and as a home test. These tests do not require qualified personnel and specialloyalty for their production.

Known test cassette for rapid detection of specific AG prostate cancer (PSA) firm AFC Biotech Co, Ltd, China. The disadvantage of this rapid test is the lack of sensitivity and specificity in the definition of the same antigen, increased levels of which are negligible in the early stages of prostate cancer. Besides its diagnostic value reduces elevated PSA in benign tumors and inflammatory diseases of the prostate. Similar disadvantages inherent in similar tests firm "Syntron" US, "Nnimap France and LLC "xema-Medica Russia ("Bioorganic chemistry 2007, 33(5): 550-554).

Recombinantly AG (UBA) - oncofetal protein formed during embryonic (fetal) development. Adults CEA is produced in very limited quantities and its concentration does not exceed 2.5-5 u/ml in non-smokers and 7-10 u/ml in smokers. Increasing the concentration of CEA above this norm is usually evidenced by the presence of malignant diseases of different etiology (prostate cancer, cancer of the gastrointestinal tract, breast cancer, lung cancer and some other). The detection sensitivity increase of CEA in the serum for a different cancer is 20-60%. The quick tests on this AG is produced by the firm "Syntron" US. The disadvantage of the test is low pacificnet and predictive value for prostate cancer detection.

We have proposed a method of simultaneous detection of PSA and CEA, which increases its sensitivity to patients with prostate cancer and simultaneous significant increase screening values due to the discovery of other malignant diseases by detecting the increase of concentration of CEA.

Accordingly, required when implementing device technical result consists in the elimination of the disadvantages inherent counterparts.

Drawings

Fig. 1. Schematic diagram of the device immunochromatographic test strip that implements the proposed method immunochromatographic identify antigens PSA and CEA.

a) a Test strip prior to application of the sample.

b) the Test strip after the reaction in the absence of antigens.

Fig. 2. Scheme immunochromatographic reaction in the presence of antigen PSA in the sample.

a) a Test strip prior to application of the sample.

b) the Test strip after the reaction.

Fig. 3. Scheme immunochromatographic reaction in the presence of antigen CEA in the sample.

a) a Test strip prior to application of the sample.

b) the Test strip after the reaction.

Fig. 4. Scheme immunochromatographic reaction in the presence of antigen PSA and CEA in the sample.

a) a Test strip prior to application of the sample.

b) the Test strip after the reaction,

where the following notation is used a compound of the elements of the specified device and outlines the key stages of its operation:

A. SEM.

B. Nitrocellulose membrane.

C. Suction filter.

D. Assembly substrate.

1. The application zone conjugate (KG) mouse monoclonal antibodies against PS.

2. The application zone conjugate (KG) mouse monoclonal antibodies against CEA.

3. Conjugate of mouse monoclonal antibodies against PSA.

4. Conjugate of mouse monoclonal antibodies against CEA.

5. Mouse monoclonal antibodies against PSA.

6. The test area S (test zone).

7. The test area CEA (test zone).

8. Mouse monoclonal antibodies against CEA.

9. Rabbit antibodies against mouse IgG.

10. The control area.

11. Sample.

12. Unbound products of the reaction.

13. Complex antibodies against mouse IgG with unbound by the conjugates.

14. Antigen PSA.

15. The complex KG PSA-PSA.

16. Complex AT PSA-PSA-KG PSA.

17. Antigen CEA.

18. The complex KG CEA-CEA.

19. Complex AT CEA-CEA-KG CEA.

1 schematically shows the sequence of the reaction zone, consisting of regions 1 and 2, as well as test zones, control zones and the suction filter on the surface of the mounted substrate D (implied adjacency zones a, b, C to D). Also shown is the distribution of reagents 3, 4, 5, 8 and 9 in regions 1, 2, 6, 7, 10. On figa presents the General scheme of the proposed test strip prior to the reaction. Adding to the area And sample 11 containing the tested antigens (figb), the reaction occurs, ending with the formation of the complex 13 and the imaging area 10, which corresponds to a negative reaction (the formation of one colored band in the control zone).

Figure 2 shows the formation of complex 15 (figa) adding a sample containing antigen PSA, the leaching of the complex and unbound conjugates 3 and 4 and the formation of complexes 16 and 13 (figb) in zones 6 and 10, respectively. Education conjugate 16 leads to the visualization of the bands in zone 6, which indicates the presence in the sample of AG PSA, and the formation of the complex in the zone 10 renders the control band, which indicates the suitability of the test and the full completion of the reaction.

Figure 3 shows the formation of the complex 18 (figa) adding a sample containing the antigen CEA, leaching of the complex and unbound conjugates 3 and 4 and the formation of complexes 19 and 13 (figb) in zones 7 and 10, respectively. Education conjugate 19 leads to the visualization of the bands in zone 7, which indicates the presence in the sample of AG CEA, and the formation of the complex in the zone 10 renders the control band, which indicates the suitability of the test and the full completion of the reaction.

<> Figure 4 shows the formation of complexes 15 and 18 (figa) adding a sample containing the antigen CEA and antigen PSA, the leaching of the complex and unbound conjugates 3 and 4 and the formation of complexes 16, 19 and 13 (figb) in zones 6, 7 and 10, respectively. Education conjugate 19 leads to the visualization of the bands in zone 7, which indicates the presence in the sample of AG CEA, which indicates the suitability of the test and the full completion of the reaction. Education conjugate 16 leads to the visualization of the bands in zone 6, which indicates the presence in the sample of AG PSA, which indicates the suitability of the test and the full completion of the reaction.

Unreacted reagents 12 (3 and 4) are absorbed in the zone C.

The INVENTION

To eliminate the disadvantages of the known technical solutions, due to the fact that the simultaneous identification of two markers of cancer increases the value of such definitions for the purposes of screening due to a higher probability of detecting prostate cancer and simultaneous detection of cancers other nosology, developed immunochromatographic method of simultaneous determination of antigens CEA and PSA in serum, consisting in the fact that the sample under test serum is applied to the input end of the test strip, move it to the front of the liquid medium due to capillary forces through the reaction C is well, consisting of regions 1, containing conjugate of a monoclonal antibody to a specific epitope of PSA with nanocolloidal gold, and 2 containing conjugate of a monoclonal antibody to a specific epitope of CEA with nanocolloidal gold, then move the front of the fluid through the test zone 6 containing immobilized monoclonal antibody to a different epitope of PSA, then to zone 7, containing immobilized monoclonal antibody to a different epitope of CEA, and further to the control zone 10 with the dissolution of floating liquid reagents that are responsible for binding antigens PSA and CEA, and their marker properties, complex 16 consisting of antigen PSA the affine corresponding reagent 5 and marker 15, immobilized in the test area 6 and 19, containing the reagent, affine corresponding antigen CEA, and the marker reagent 4, immobilized in the test area 7, unbound marker Regency 3 and 4 immobilized in the control zone 13, and the excess of reagents for the control area absorb suction filter, and as a marker reagent 3 use the conjugate particles of colloidal gold with a monoclonal antibody against a specific epitope of PSA, and as a marker reagent 4 use the conjugate particles of colloidal gold with a monoclonal antibody against a specific epitope CEA, the color of the test zones 6 and 7 prin is thought the decision about the presence or absence of antigens PSA and CEA in the test sample, and the color of the control area 10 make a decision on the performance of the test strips.

In addition, as a biochemical reagent control zone 7 using rabbit antibodies against mouse IgG.

In addition, as a porous medium reagent zone using SEM And (inert porous carrier made of fiberglass), and as porous media test zones 6 and 7 and the control area 10 using the layer is a microporous nitrocellulose deposited on a Mylar base and having a capillary communication with the porous carrier reagent zone (SEM). All elements a, b, C are mounted on the substrate D overlap, as shown in figa.

The work of the proposed method is based on the binding of conjugated and unconjugated monoclonal antibodies with different epitopes of antigens PSA and CEA and immobilization of the resulting complexes in the test zone on the nitrocellulose. And then the complexes are visualized by the local accumulation of complexes containing colored particles nanocolloidal gold.

In the reaction detect the presence of antigens PSA and CEA analyzed blood serum sample of the patient is applied in the area of the Pad A. It dissolves conjugates 3 and 4, and in the process of dissolution is formed a complex of these conjugates with present in the samples antigens 14 and 17, respectively. Under the action of capillary force is tangential migration of the complex in the test zones 6 and 7, where the pre-immobilizing antibodies to PSA and CEA, respectively. In the case of sample antigen PSA in the test area 6 is formed a complex of 16, and in the case of the presence in the sample of the antigen CEA in the test area 7 is formed complex 19. The presence of complexes nanocolloidal gold allows you to visualize the test zone in the presence in the sample of relevant antigens that allows bespribornoj detection of the investigated antigens PSA and CEA in the sample water. Complexes that do not contain the investigated antigens, but containing conjugates, migrate into the control zone 10, staining it and thus confirming the operability of the test strip. Excess unbound conjugate and antigen migrate to the suction filter C.

Thus, the appearance of a colored band in zone 6 after the reaction indicates the presence in the sample antigen PSA, the appearance of a colored band in the zone 7 - the presence in the sample antigen CEA, simultaneous staining zones 6 and 7 indicates the presence in the sample simultaneously antigens CEA and PSA, and the absence of staining in both zones in the color control area - about the absence of the studied antigens in the sample.

EXAMPLE 1. Conjugation of monoclonal antibodies to PSA and CEA with nanocolloidal gold with a particle diameter of 30 nm (BioCell) was performed, the AK is described in the patent Fuchs et. all (US patent 5968758, Oct. 19, 1999). The conjugates were applied parallel stripes in the Central region of the Pad perpendicular to the flow of the fluid at a distance of 2 mm from each other (KG PSA in zone 1, KG CEA in zone 2 and after drying was mounted on one of the adhesive areas-block CNPF-PD31-L2-P25 (MDI, India), including nitrocellulose with a pore diameter of 20 μm, so that the SEM 1-2 mm overlap nitrocellulose. On the opposite adhesive area of the block overlap mounted filter paper. 2 mm from the middle of nitrocellulose parallel to the long side of the block was applied strips of monoclonal antibodies to other epitopes PSA (zone 6) and CEA (zone 7), and zone 10 was applied monospecific antibodies to mouse IgG (control area). After drying over Pad and suction filter mounted protective film, cut the block on the test strip width of 5 mm and sealed in waterproof bags with desiccant. The resulting test strip used for analysis.

The technical result. Using the obtained test strips explored the patient's serum K. with the verified diagnosis of prostate cancer. Schematically the study are presented in figure 2. Reaction time from introduction of the sample into the zone And before taking account of the reaction was 10 minutes. The presence of antigen PSA concentration greater than 10 ng per ml in the sample was detected by the immune method is enzymatic analysis using the test system of the company "DRG" (Germany). Pad was immersed 2 mm in the sample. The liquid sample was dissolved conjugates, dried pad, and the formed complex 15 migrated to the test area, where he formed a complex of 16, which visualized the presence of AG PSA in the sample. Nesvetaevskaya conjugate migrated in the control zone, where he formed a complex 13. This complex is visualized suitability test strips to work. All other unbound components of the reaction was absorbed suction filter. Five test serum by using the received test strip gave a consistent result - staining zones 6 and 10.

EXAMPLE 2. Produced strip as in example 1. Investigated the serum of a patient with P. verified prostate cancer (schematically research presented on figure 4), containing antigen PSA concentration greater than 10 ng per ml, and antigen CEA in concentrations greater than 15 units per ml of the Reaction was carried out as in example 1, resulting in the formation of complexes 16 in zone 6, 19 in zone 7 and 13 in the zone 10. These complexes were visualized zones 6, 7 and 10, testified to the presence of antigens PSA and CEA in the sample and the suitability of the test strip for analysis. Five test serum by using the received test strip gave a consistent result - staining zones 6, 7, and 10.

EXAMPLE 3. Made the strip so W is, as in example 1. Explored the patient's serum F. with verified colorectal cancer, containing the antigen CEA (schematically research presented on figure 3) at a concentration of greater than 15 units per ml of the Reaction was carried out as in example 1, resulting in the formation of complexes 19 in zone 7 and 13 in the zone 10. These complexes were visualized zone 7 and 10, indicating that the presence of antigen CEA in the sample and the suitability of the test strip for analysis. Five test serum by using the received test strip gave a consistent result - staining zones 7 and 10.

EXAMPLE 4. Produced strip as in example 1. Investigated the serum of patient Days with verified prostate cancer (schematically research presented on figure 3), containing the antigen CEA in concentrations greater than 15 units per ml, but not containing antigen PSA. The reaction was carried out as in example 1, resulting in the formation of complexes 19 in zone 7 and 13 in the zone 10. These complexes were visualized zone 7 and 10, indicating that the presence of antigen CEA in the sample and the suitability of the test strip for analysis. Five test serum by using the received test strip gave a consistent result - staining zones 7 and 10.

EXAMPLE 5. Produced strip as in example 1. Investigated the serum of the patient A. verify the new cancer of the pancreas (schematically research presented on figure 3), containing the antigen CEA in concentrations greater than 15 units per ml of the Reaction was carried out as in example 1, resulting in the formation of complexes 19 in zone 7 and 13 in the zone 10. These complexes were visualized zone 7 and 10, indicating that the presence of antigen CEA in the sample and the suitability of the test strip for analysis. Five test serum by using the received test strip gave a consistent result - staining zones 7 and 10.

EXAMPLE 6. Produced strip as in example 1. Explored the patient's serum HP without clinical pathology and containing antigens PSA less than 1 ng/ml and CEA less than 5 IU/ml (schematically research are presented in figure 1). The reaction was carried out as in example 1, resulting in the formation of complex 13 in the zone 10 and zone 6 and 7 remained unpainted. It showed the absence of a pathological number of antigens PSA and CEA in the sample and the suitability of the test strip for analysis. Five test serum by using the received test strip gave a consistent result - staining zone 10.

Next we show that due to the considerable differences of the proposed method provided the required technical result.

What test is used to determine various oncological disease increases sensitivity when screening studies is that it is seen from examples 2-5.

What the test reveals the presence of cancer when prostate cancer in the absence of pathological concentrations of antigen PSA (example 4) also increases its sensitivity and predictive value.

That as a porous medium reagent zone using SEM (inert porous carrier made of fiberglass), and as porous media control area using layer nitrocellulose deposited on a Mylar base and having a capillary communication with the porous carrier reagent zone (SEM), also allows you to provide the required technical result.

Achievable technical result consists in the reduction of expensive reagents and time-saving compared with traditional methods for a similar purpose in improving its economic parameters.

Thus, it is shown that the required technical result, indeed, is achieved due to the significant differences of the proposed method. The experiments showed the feasibility of the invention.

The immunochromatographic method of simultaneous determination of encountering PSA and CEA, characterized in that to determine the use of one test strip, which consists of three parts a, b and C, which are mounted sequentially on the substrate, in this part As is and 1-2 mm overlap portion, where a - is an inert porous carrier made of fiberglass (SEM) deposited on the surface of the reaction zones 1 and 2, where the reaction zone 1 is the area coated with a conjugate of mouse monoclonal antibodies against specific epitopes of PSA, with nanocolloidal gold, and zone 2 is the area coated with a conjugate of mouse monoclonal antibodies against CEA labeled with colloidal gold, and conjugates applied parallel stripes in the Central region of the pad at a distance of not less than 2 mm from each other; nitrocellulose immobilized on a Mylar base, coated on the surface of the test zones designated 6 and 7 and the control area indicated, where the test area 6 is an area coated with mouse monoclonal antibodies against PSA, test area 7 represents the area coated with mouse monoclonal antibodies against CEA, and the control area 10 is an area coated with a monospecific rabbit antibodies against mouse IgG, and monoclonal antibodies against PSA caused by the strip perpendicular to the long side of the test strip 2 mm from the middle of nitrocellulose, monoclonal antibodies against CEA caused by the strip parallel to the runway antibodies against PSA in the middle of the nitrocellulose, and monospecific antibodies against immunol Bulanov mouse applied parallel strip of 3 mm from the middle closer to the suction filter, where C is the capillary communication with the reaction area of the carrier of A; C - suction filter to absorb unreacted reagents, the decision about the presence of PSA and/or CEA accept the presence of color in the test zones 6 and 7 respectively, while the color of the control area indicates the health of the test strip.



 

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3 ex, 1 tbl

FIELD: medicine, gynecology.

SUBSTANCE: invention relates to a method for diagnosis of internal endometriosis in peripheral venous blood of women wherein the relative content of lymphocytes CD25+ is determined. Internal endometriosis is diagnosed at values of this index 6% or above. Proposed method provides carrying out diagnosis of internal endometriosis in women with high precision, sensitivity and specificity that allows carrying out the correct and well-timed necessary complex of curative-prophylactic treatment.

EFFECT: improved method for diagnosis.

1 tbl, 3 ex

FIELD: medicine, immunological laboratory diagnostics.

SUBSTANCE: at terms from 6 to 12 wk of gestation one should study relative content of CD3+CD16+ lymphocytes in peripheral venous blood and at its values being either equal or above 5.4% one should predict the development of light-degree gestosis to carry out the complex of curative-prophylactic means.

EFFECT: higher efficiency of prediction.

3 ex, 1 tbl

FIELD: medicine, laboratory diagnostics.

SUBSTANCE: during the 1st trimester of pregnancy (6-13 wk) in peripheral venous blood in women at risk of failed pregnancy one should detect relative content of CD16+CD56- lymphocytes and at its value being either equal or below 11% it is possible to predict the development of infectious diseases in full-term neonatals during the first 7-10 d of their lives. The innovation enables to predict the development of local form of infectious-inflammatory diseases in full-term neonatals.

EFFECT: higher accuracy of prediction.

3 ex, 1 tbl

FIELD: medicine, clinical laboratory diagnostics.

SUBSTANCE: in the sample of peripheral venous blood one should determine relative content of CD45RO+ lymphocytes and at its value being equal to 31% or lower it is possible to diagnose external genital endometriosis. The method is atraumatic and enables to diagnose external genital endometriosis at high accuracy.

EFFECT: higher efficiency of diagnostics.

3 ex, 1 tbl

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