WO2011093745A1 - Procédé de détermination de niveau d'autoanticorps par essai immunoenzymatique - Google Patents
Procédé de détermination de niveau d'autoanticorps par essai immunoenzymatique Download PDFInfo
- Publication number
- WO2011093745A1 WO2011093745A1 PCT/RU2011/000034 RU2011000034W WO2011093745A1 WO 2011093745 A1 WO2011093745 A1 WO 2011093745A1 RU 2011000034 W RU2011000034 W RU 2011000034W WO 2011093745 A1 WO2011093745 A1 WO 2011093745A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- solid phase
- physical sorption
- natural autoantibodies
- antibodies
- antigen
- Prior art date
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
Definitions
- the invention is related to the field of medicine, in particular to laboratory diagnostics, and it can be used to improve efficiency and reliability of quantitative determination of natural autoantibody concentration in human biological fluids.
- a method of enzyme immunoassay includes adsorption of antigens at a solid phase of physical sorption, incubation of tested biological specimens, incubation of a conjugate-containing solution, and spectrophotometric analysis of the reaction of extinction of a chromatic agent (RU 2014610 C1 , G01 N33/535, 1994).
- the invention is intended to develop a technologically simple, sensitive and specific method for quantitative determination of natural autoantibody level in human biological fluids using the EIA method.
- the set task is solved due to the fact that in the method of quantitative determination of natural autoantibody level in human biological fluids with the aid of the enzyme immunoassay, including the antigen treatment of physical sorption solid phase, addition of tested biological specimens, solid phase treatment with a conjugate-containing solution, separation of solid and liquid phases, and spectrophotometric analysis of a reaction for extinction of a chromatic agent solution, according to the invention, the physical sorption solid phase, coated with streptavidin, is used as the physical sorption solid phase, and the physical sorption solid phase is treated with the pre-biotinylated antigen and blocking agent for closing the sites of nonspecific binding at the physical sorption solid phase, for which purpose are used proteins biotinylated according to the standard procedure.
- conjugate-containing solution monoclonal or polyclonal enzyme- labeled antibodies which react with one or all isotypes of human immunoglobulins.
- the tested biological fluid is preliminary diluted in a buffer, containing proteins which are used to close the sites of nonspecific binding at the physical sorption solid phase, and also substances which protect natural autoantibodies from destruction during heat treatment, and subject it to heat treatment.
- a corresponding control physical sorption solid phase is used, at which the biotinylated antigen is not immobilized (i.e.
- a biotinylated control protein is used which is similar to protein used for closing the nonspecific binding sites at physical sorption solid phase, which makes it possible to measure spectrophotometric signal, specific and nonspecific for natural autoantibodies), and the number of natural autoantibodies is determined with the aid of a calibration curve which is standardized over monoclonal or polyclonal antibodies to antigen.
- the tested biological fluid diluted in a buffer containing proteins which are used for closing the nonspecific binding sites at the physical sorption solid phase, and substances protecting natural autoantibodies from destruction during thermal treatment, are additionally treated with an iron- containing oxidizer.
- Preliminary dilution of the investigated specimen in a buffer, containing proteins which are present in the blocking buffer and are used for closing the sites of nonspecific binding at physical sorption solid phase minimizes the possibility of nonspecific binding the antibodies of the investigated specimen with the solid phase, at which proteins are immobilized, which also increases specificity and sensitivity of the claimed method.
- Addition of iron-containing oxidizer and heat treatment of the investigated specimen makes it possible to destroy the complex of natural autoantibodies with antigens and anti-idiotypic antibodies, or to ensure availability of antigen paratopes to antigen epitopes due to various demasking effects, and preliminary dilution of the investigated specimen in a buffer protects natural autoantibodies from destruction during heat treatment, which allows to determine the total level of natural autoantibodies (i.e. as free and antigen- bounded forms of natural autoantibodies or natural antibodies with closed paratopes), and thus reduces the possibility of obtaining the false negative results, increases sensitivity, efficiency and functional capabilities of the claimed method.
- Figure 1 shows the calibration curve for example 1 ;
- Figure 2 shows the calibration curve for example 2;
- Figure 3 shows the calibration curve for example 3.
- Solid phase of physical sorption (wells) of a standard well plate is coated with streptavidin or its analogs (for example, avidin, etc), the physical sorption solid phase is blocked with a blocking agent and incubated with a biotinylated agent to which it is needed to determine the level of natural autoantibodies, or (control wells) with biotinylated blocking protein (for example, bovine serum albumin, ovalbumin, human serum albumin, rabbit serum albumin, gelatin, etc), which is the control of nonspecific binding.
- biotinylated blocking protein for example, bovine serum albumin, ovalbumin, human serum albumin, rabbit serum albumin, gelatin, etc
- Antigens are biotinylated by the minimum quantity of biotin (for example, D-biotinoyl-e-aminocaproic acid-N-hydroxysuccinimide ester) (1 :2 antigen to biotin molar ratio) to minimize epitope destruction.
- biotin for example, D-biotinoyl-e-aminocaproic acid-N-hydroxysuccinimide ester
- Biological fluid is prepared for the assay (for example, blood serum, blood plasma, etc) by dilution (in 50 to 200,000 time range) in a buffer solution which contains proteins in the composition of the blocking buffer (for example, bovine serum albumin, ovalbumin, human serum albumin, rabbit serum albumin, gelatin, etc), preservatives (for example, timerosal, etc), surface active substances (for example, Triton-X100, etc), which protect natural autoantibodies from destruction during heat treatment , and the obtained solution of biological fluid is heat treated within the temperature range of 50 S C to 80 2 C.
- a buffer solution which contains proteins in the composition of the blocking buffer (for example, bovine serum albumin, ovalbumin, human serum albumin, rabbit serum albumin, gelatin, etc), preservatives (for example, timerosal, etc), surface active substances (for example, Triton-X100, etc), which protect natural autoantibodies from destruction during heat treatment , and the obtained solution of biological fluid is heat treated within the temperature range of 50 S C to
- the tested biological fluid shall be treated additionally with an iron-containing oxidizer (for example, ferric chloride (III) [FeCI 3 ]).
- an iron-containing oxidizer for example, ferric chloride (III) [FeCI 3 ]
- polyclonal or monoclonal antibodies for example, goat polyclonal antibodies, sheep polyclonal antibodies, mice monoclonal antibodies, etc
- an enzyme for example, alkaline phosphatase, horseradish peroxidase, etc
- the level of natural autoantibodies is determined with respect to the reaction of extinction of chromatic agent solution which changes its color depending on the quantity of chromatic agent isolated from the substrate upon its decomposition by an enzyme (form example, alkaline phosphatase, etc); the substrate produces a soluble product whose color characteristics can be measured spectrophotometrically at a certain wavelength.
- Blood serum specimens were diluted by 20 times (100 ⁇ of serum were diluted in 1900 B iild IF N tt ⁇ onae yy - ⁇ of phosphate buffer containing 1 % Triton-X100 and 0.002% timerosal), and were incubated for 20 minutes at 75°C.
- B iild IF N tt onae yy - mice monoclonal antibodies to human gamma interferon, clone MD-2 within the concentration range from 16 units/ml to B ii B Sld A tt on y ae 0.5 units/ml. 100 picogram/ml of murine monoclonal antibodies was arbitrarily defined as 1 Unit/ml.
- the prepared standard antibodies, the s B iild IF N tt on y ae y o- lution used as negative control phosphate buffer containing 1 % BSA, 1 % Triton-X100 and 0.002% timerosal
- negative control phosphate buffer containing 1 % BSA, 1 % Triton-X100 and 0.002% timerosal
- Table 1 shows the chart of specimen inoculation into plate wells.
- Negative control - phosphate buffer containing 1 % BSA, 1 % Triton-X100 and 0.002% timerosal
- a chromatic agent solution was prepared by dissolving 1 tablet of a substrate buffer and 1 tablet of substrate (para-nitrophenylphosphate) from Sigma Company (Catalog N Q . N-2770) in 20 ml of distilled water.
- the mean OD for negative control (the mean arithmetic value of OD in wells 1G, 1 H, 2G, 2H) was calculated.
- the mean OD was calculated for the investigated specimens in plate wells in which biotinylated IFN- ⁇ (rows 3,5,7,9,11 in Table 1) is immobilized, and in which biotinylated BSA (rows 4,6,8,10,12 in Table 1) is immobilized.
- the mean OD value was calculated in wells A3, B3 and A4. B4, respectively
- OD true value was calculated for the investigated specimens as the difference of the OD mean value for the investigated specimens measured in plate wells, where biotinylated IFN- ⁇ is immobilized, and OD mean value for the investigated specimens measured in plate wells, where biotinylated BSA is immobilized.
- the investigated specimen S1 from the OD mean value, measured in plate wells A3, B3, the OD mean value, measured in plate wells A4, B4, is deducted.
- the calibration curve ( Figure 1) was used to determine the concentration of diluted natural autobodies to human gamma interferon in the investigated specimens. In order to obtain the true value of concentration of natural autoantibodies to human gamma interferon in the investigated specimens, the obtained result was multiplied by the degree of specimen dilution (by 100). The results are presented in Table 3.
- Serum specimens were taken from twenty patients with infectious mononucleosis, at which the level of natural autoantibodies to human gamma interferon increases.
- Blood serum specimens were diluted by 10 times (10 ⁇ of serum were diluted in 90 ⁇ of a phosphate buffer containing 1 % Triton-X100 and 0.002% timerosal), treated with 2 mM FeCU and incubated for 40 minutes at 56°C.
- the incubated solution was diluted additionally by 5 times (100 ⁇ of incubated solution were diluted in 400 ⁇ of a phosphate buffer containing 1 % bovine serum albumin (BSA),1 % Triton-X100 and 0.002% timerosal), and it was further used for inoculating into the plate wells ( 00 ⁇ per well).
- BSA bovine serum albumin
- Triton-X100 0.002% timerosal
- solutions of standard antibodies (mice monoclonal antibodies to human gamma interferon, clone MD-2) were prepared within the range of concentrations from 16 Units/ml to 0.5 Units/ml. 100 picogram/ml of murine monoclonal antibodies was arbitrarily defined as 1 Unit/ml.
- the prepared standard antibodies, a solution used as negative control (a phosphate buffer containing 1 % BSA, 1 % Triton-X100 and 0.002% timerosal), and solutions of the investigated specimens were inoculated into plate wells as shown in Table 5, and they were incubated at 37 S C for 2 hours. After incubation, fluid was removed from the plate by decanting, and the plate was rinsed 5 times with a standard washing solution (a phosphate buffer containing 0.05% Twin-20 and 0.01 % timerosal).
- Table 5 shows the chart for placing specimens into the plate wells.
- Negative control - a phosphate buffer containing 1 % BSA, 1 % Triton-X100 and 0.002% timerosal
- a chromatic agent solution was prepared by dissolving 1 tablet of a substrate buffer and 1 tablet of a substrate (para-nitrophenylphosphate) from Sigma Company (Catalog Ne. N-2770) in 20 ml of distilled water.
- the obtained measurement results were used to calculate the level of natural autoantibodies to gamma interferon.
- the true value of optical density (OD) of standard antibodies and the true value of OD of the investigated specimens were determined as follows:
- the mean OD was calculated for negative control (the mean arithmetic OD value in wells 1 G, 1 H, 2G, 2H)
- the mean OD was calculated for standard antibodies (the mean arithmetic OD value in wells of the respective standard P1-P6).
- the true value of OD of standard antibodies was calculated as the difference of the OD mean value of standard antibodies and the mean OD value of negative control.
- the mean OD for the investigated specimens was calculated in plate wells in which biotinylated IFN- ⁇ (rows 3,5,7,9,11 in Table 5) is immobilized, and in which biotinylated BSA (rows 4,6,8,10,12 in Table 5) is immobilized.
- the true OD value for the investigated specimens was calculated as the difference of the mean OD value for the investigated specimens, measured in plate wells, where biotinylated IFN - ⁇ is immobilized, and the mean OD value in which biotinylated SA is immobilized.
- the mean OD value measured in plate wells A4, B4
- concentration of diluted natural autoantibodies to human gamma interferon was determined in the investigated specimens.
- concentration of diluted natural autoantibodies to human gamma interferon was multiplied by the degree of dilution of the specimens (by 100). The results are presented in Table 7.
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Abstract
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
UAA201210068A UA109893C2 (uk) | 2010-01-26 | 2011-01-24 | Спосіб кількісного визначення рівня природних аутоантитіл в біологічних рідинах людини за допомогою імуноферментного аналізу |
US13/575,320 US20130189707A1 (en) | 2010-01-26 | 2011-01-24 | Method of determination of autoantibody level by means of enzyme immunoassay |
JP2012551116A JP5785959B2 (ja) | 2010-01-26 | 2011-01-24 | 酵素イムノアッセイを用いて自己抗体の濃度を測定する方法 |
EP11737346.4A EP2529228A4 (fr) | 2010-01-26 | 2011-01-24 | Procédé de détermination de niveau d'autoanticorps par essai immunoenzymatique |
EA201200936A EA020484B1 (ru) | 2010-01-26 | 2011-01-24 | Способ количественного определения уровня естественных аутоантител в биологических жидкостях человека путем иммуноферментного анализа |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
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RU2010102114 | 2010-01-26 | ||
RU2010102114/15A RU2465600C2 (ru) | 2010-01-26 | 2010-01-26 | Способ количественного определения уровня естественных аутоантител в биологических жидкостях человека путем иммуноферментного анализа |
RU2010117620 | 2010-05-05 | ||
RU2010117620/15A RU2465601C2 (ru) | 2010-05-05 | 2010-05-05 | Способ количественного определения уровня естественных аутоантител в биологических жидкостях человека |
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WO2011093745A1 true WO2011093745A1 (fr) | 2011-08-04 |
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PCT/RU2011/000034 WO2011093745A1 (fr) | 2010-01-26 | 2011-01-24 | Procédé de détermination de niveau d'autoanticorps par essai immunoenzymatique |
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EP (1) | EP2529228A4 (fr) |
JP (1) | JP5785959B2 (fr) |
EA (1) | EA020484B1 (fr) |
WO (1) | WO2011093745A1 (fr) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN117405877A (zh) * | 2023-11-27 | 2024-01-16 | 山东帝俊生物技术有限公司 | 一种elisa试剂盒抗原间接包被酶标板的包被方法 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2004088315A1 (fr) * | 2003-03-31 | 2004-10-14 | Council Of Scientific And Industrial Research | Procede rapide utilisant la chaleur pour procedure de dosage immunoenzymatique |
RU2240561C1 (ru) * | 2003-04-14 | 2004-11-20 | Азимов Александр Гитальевич | Способ определения уровня циркулирующих аутоантител в биологических жидкостях |
Family Cites Families (4)
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US4650772A (en) * | 1984-08-09 | 1987-03-17 | Abbott Laboratories | Monoclonal antibody stabilization |
US6706486B1 (en) * | 1998-06-19 | 2004-03-16 | Amersham Biosciences Ab | Method for the quantitative release of natural or recombinant proteins, polypeptides or peptides |
DE60234435D1 (de) * | 2002-07-23 | 2009-12-31 | Guy S & St Thomas Hospital Nhs | Methode zum Entfernen von MHC Antikörpern |
US8574848B2 (en) * | 2006-09-13 | 2013-11-05 | Oncimmune Ltd. | Immunoassay methods |
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2011
- 2011-01-24 EA EA201200936A patent/EA020484B1/ru not_active IP Right Cessation
- 2011-01-24 WO PCT/RU2011/000034 patent/WO2011093745A1/fr active Application Filing
- 2011-01-24 EP EP11737346.4A patent/EP2529228A4/fr not_active Ceased
- 2011-01-24 JP JP2012551116A patent/JP5785959B2/ja not_active Expired - Fee Related
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004088315A1 (fr) * | 2003-03-31 | 2004-10-14 | Council Of Scientific And Industrial Research | Procede rapide utilisant la chaleur pour procedure de dosage immunoenzymatique |
RU2240561C1 (ru) * | 2003-04-14 | 2004-11-20 | Азимов Александр Гитальевич | Способ определения уровня циркулирующих аутоантител в биологических жидкостях |
Non-Patent Citations (3)
Title |
---|
MASYAGO A.V.: "Nekotorye oshibki pri postanovke IFA, Informatsionno-metodicheskoe posobie", NOVOSIBIRSK, 2006, pages 10, 15 * |
METODY IMMUNOANALIZA., 12 October 2007 (2007-10-12), pages 1 - 51, Retrieved from the Internet <URL:http://il.ks.ua/tem_razdel_files> [retrieved on 20110520] * |
See also references of EP2529228A4 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117405877A (zh) * | 2023-11-27 | 2024-01-16 | 山东帝俊生物技术有限公司 | 一种elisa试剂盒抗原间接包被酶标板的包被方法 |
CN117405877B (zh) * | 2023-11-27 | 2024-04-12 | 山东帝俊生物技术有限公司 | 一种elisa试剂盒抗原间接包被酶标板的包被方法 |
Also Published As
Publication number | Publication date |
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EA201200936A1 (ru) | 2013-02-28 |
EP2529228A1 (fr) | 2012-12-05 |
EP2529228A4 (fr) | 2013-09-04 |
EA020484B1 (ru) | 2014-11-28 |
JP5785959B2 (ja) | 2015-09-30 |
JP2013518277A (ja) | 2013-05-20 |
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