WO2022005408A1 - Procédé et kit immunochromatographique pour détecter un anticorps anti-interféron gamma - Google Patents

Procédé et kit immunochromatographique pour détecter un anticorps anti-interféron gamma Download PDF

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Publication number
WO2022005408A1
WO2022005408A1 PCT/TH2021/000029 TH2021000029W WO2022005408A1 WO 2022005408 A1 WO2022005408 A1 WO 2022005408A1 TH 2021000029 W TH2021000029 W TH 2021000029W WO 2022005408 A1 WO2022005408 A1 WO 2022005408A1
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Prior art keywords
complex
region
antibody
interferon gamma
ifn
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PCT/TH2021/000029
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English (en)
Inventor
Chatchai Tayapiwatana
Umpa YASAMUT
Wachira KOCHAKARN
Weeraya THONGKUM
Supachai SAKKHACHORNPHOP
Nattapong POLPONG
Rawiwan PORNPRASIT
Wanwisa THONGKAMWITOON
Jirapan CHAICHANAN
Jaruwan KHAOPLAB
Chonnikarn CHANPRADAB
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Mahidol University
Chiang Mai University
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Application filed by Mahidol University, Chiang Mai University filed Critical Mahidol University
Priority to JP2022555985A priority Critical patent/JP7417231B2/ja
Priority to CN202180017176.6A priority patent/CN115176157A/zh
Publication of WO2022005408A1 publication Critical patent/WO2022005408A1/fr

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    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow

Definitions

  • the present disclosure relates to an immunochromatographic-based testing apparatus for detecting presence of any anti-interferon gamma (IFN-g) antibody in a biological sample, preferably a bodily fluid sample, acquired from a subject. Further, the present disclosure also offers method for detecting presence of any anti-IFN-g antibody in a biological sample of a subject based upon an immunochromatographic approach. The disclosed apparatus and method can be employed for detect presence of anti-IFN-g antibody Background
  • Interferon gamma is a cytokine required for activation of innate and specific immunity by creating CD4+ T helper cell type 1 (Till) lymphocytes, CD8+ cytotoxic lymphocytes, and NK cells.
  • Cell such as B cells, NKT, and professional antigen-present cells (APCs) can secrete IFN-g.
  • Interferons protect cells against invasion of foreign microorganism and regulate various mechanisms of human immune system, as well as inhibiting the activity of viruses, bacteria, and carcinogenesis (Miller CH, Maher SG, Young HA. Clinical use of interferon-gamma. Ann N Y Acad Sci.
  • IFN-g has antiviral activities. Hence, it has been thoroughly research as an idea candidate for development of antiviral treatment. Moreover, IFN-g has also been found to have anti-cancer effects. Although the mechanisms are not fully understood, XFN-g has shown inhibitory effects towards both normal and cancer cells during cell division. Considering that the cancer cells divide more rapidly than normal cells, the inhibitory effect of XFN-g is expected to have a greater impact on cancer cells wTdie directly affecting the natural killer cells in further attacking the cancer cells.
  • IFN-g has also been used in animal stimulation to produce monoclonal antibodies against it that these monoclonal antibodies were subsequently used in enzyme-linked immunosorbent assay (ELISA) systems or interferon gamma release assays (IGRAs) for the diagnosis of tuberculosis infection (Alfa MI, Dembinski JJ, Jay FT.
  • ELISA enzyme-linked immunosorbent assay
  • IGRAs interferon gamma release assays
  • IFN-g has been used, both, in studies of fundamental research and development of medicinal purposes, including production of diagnostic kits.
  • diagnostic kit manufactured using monoclonal antibody of anti-IFN-g antibody can be expensive due to high production costs.
  • recombinant interferon gamma can be produced in systems using at least one of Escherichia coii (E. coli) cells, mammalian cells, yeasts, protozoan, or plant cells. Nevertheless, the production systems that employ bacteria are known to give highest yield generally vet with a relatively low production cost.
  • Chinese patent application no. CN103869084A discloses an antibody against IFN-g, which can be used in ELISA system based on doubleantibody sandwich ELISA., in which two antibodies employed will bind to different locations on the molecule of IFN-g, leading to the development of IFN-g detection.
  • the platform employed is far from being considered convenient and user friendly.
  • the present disclosure aims to provide an apparatus or test kit for detecting presence of anti-IFN-g antibody in a bodily fluid sample such as blood sample obtain from a subject.
  • the apparatus or test kit disclosed is operating on an immunochromatographic approach to provide the result needed.
  • test kit for screening anti-IFN-g antibody being capable of delivering a test result free from using any laboratory tool and outside of a laboratory environment.
  • the disclosed test kit is relatively user friendly compared to general ELISA platform.
  • another object of the present disclosure is directed to offer a test kit, which can be manufactured at relatively low cost, for screening anti-IFN-g antibody.
  • the IFN-g employed in the assembly of the disclosed test kit is a recombinant peptide expressed by and harvested from a compatible cell line.
  • Another object of the present disclosure associates to a method for screening presence of anti-IFN-g antibody in a bodily fluid sample using an immunochromatographic platform equipped with recombinant IFN-g.
  • the apparatus comprises a sampling region for receiving deposition of a solvent phase comprising the bodily fluid sample; a reaction region impregnated with a plurality of a first complex comprising a first recombinant IFN-g tagged with a biotin and a first antibody attached with a signaling moiety that the first antibody coupled to the first recombinant IFN-g to form the first complex by binding onto the tagged biotin; a testing region having a plurality of second recombinant IFN-g immobilized thereto.
  • the sampling region, the reaction region, and the testing region are arranged consecutively from a first end towards a second end of a chromatographic solid phase.
  • the solvent phase migrates from the first end towards the second end of the solid phase along with the anti-IFN-g antibody present in the bodily fluid sample such that the first complex attaches onto the anti-IFN-g antibody forming a second complex at the reaction region, and the first complex and the second complex migrate further into the testing region which the second complex binds onto the immobilized second recombinant IFN-g to provide a first visual signal thereto.
  • the first visual signal established at the testing region indicates presence of the anti-IFN-g antibody in the bodily fluid sample.
  • the disclosed apparatus further comprises a control region comprising a substrate being anchored thereto and configured to bind onto the first antibody.
  • the control region is arranged after and adjacent to the testing region on the chromatographic solid phase.
  • the first complex and the second complex unbounded at the testing region further migrate into the control region which the first complex and the second complex bind onto the substrate through the first antibody to provide a second visual signal thereto.
  • the disclosed apparatus further comprises an absorbent element connecting to one end of the control region forming at least part of the second end.
  • the absorbent is any one or combination of cellulose fiber sheet, filter paper, and pulp.
  • the second recombinant IFN-g and/or the substrate is immobilized on a synthetic membrane.
  • the signaling moiety of the disclosed apparatus is gold nanoparticles (AuNPs), fluorescence, or quantum dots.
  • the method essentially comprises the steps of receiving deposition of a solvent phase comprising the bodily fluid sample at a sampling region; directing the solvent phase into a reaction region along with anti-IFN-g antibody present in the bodily fluid sample, the reaction region being impregnated with a plurality of a first complex comprising a first recombinant IFN-g tagged with a biotin and a first antibody attached with a signaling moiety that the first antibody coupled to the first recombinant IFN-g to form the first complex by binding onto the tagged biotin, the first complex being configured to attach onto the anti-IFN-g antibody forming a second complex at the reaction region; and directing the solvent phase to move further into a testing region having a plurality of second recombinant IFN-g immobilized thereto along with the formed second complex and the first complex, the second complex being
  • the first visual signal established at the testing region indicates presence of the anti-IFN-g antibody in the bodily fluid sample.
  • the disclosed method may further comprises the step of directing the solvent phase to move further into a control region comprising a substrate being anchored thereto and configured to bind onto the first antibody, the control region being arranged after and adjacent to the testing region on the chromatographic solid phase, the first complex and the second complex unbounded at the testing region migrated into the control region which the first complex and the second complex bind onto the substrate through the first antibody to provide a second visual signal thereto.
  • Fig. 1 is a schematic illustration for the general principle employed in the disclosed apparatus and method for detection of anti-IFN-g antibody in a biological sample acquired from a subject based on double antigen sandwich ELISA;
  • Fig. 2 is a schematic illustration of the present disclosure (a) general arrangement of one embodiment of the disclosed apparatus and respective configurations of the apparatus in providing; (b) positive result; and (c) negative result;
  • Fig. 3 is a graph showing results of recombinant IFN-g analysis by indirect ELISA using anti- his tag mAh;
  • Fig.4 is a graph results of recombinant IFN-g analysis by indirect ELISA using anti- IFN-g mAh
  • Fig. 5 is a graph showing the results of dimer analysis of recombinant interferon gamma by double-antibody sandwich ELISA
  • Fig. 6 is a graph showing results of anti-IFN-g detection in AOID patient serum of 20 patients by indirect ELISA using a group of active AOID (A) and a group of inactive AOID (I));
  • Fig. 7 is a graph showing results of anti-IFN-g detection in AOID patient serum of 20 patients by double- antigen sandwich ELISA of a cut-off value of 0.17 using a group of active AOID (A) and a group of inactive AOID (I));
  • Fig. 8 is a graph showing a comparison of anti-IFN-g detection in AOID patient serum of 20 patients between indirect ELISA and double-antigen sandwich ELISA by O.D. normalization using a group of active AOID (A) and a group of inactive AOID (I));
  • Fig. 9 shows a signaling establishment of a complex of the goat anti-biotin tagged with colloidal gold (CGC) and the biotinylated-IFN-g by performing direct dot blotting assay;
  • Fig. 10 shows results of one embodiment of the disclosed apparatus with recombinant IFN-g at concentrations of 1.0 and 0.5 mg/mL coated on test line at the testing region by dipping tests respectively performed towards phosphate buffered saline (control), pooled positive serum (No.1-5), and pooled negative serum (No.6- 10), respectively.
  • the terms “approximately” or “about”, in the context of concentrations of components, conditions, other measurement values, etc., means +/- 5% of the stated value, or +/- 4% of the stated value, or +/- 3% of the stated value, or +/- 2% of the stated value, or +/- 1% of the stated value, or +/- 0.5% of the stated value, or +/- 0% of the stated value.
  • detectable moiety refers to compounds which capable of releasing signal that is detectable either by a device or naked eye to reveal the presence of the formed antigen-antibody complexes to ascertain presence of a target compound, molecule, the diseased state of a test subject.
  • the signal can be in the form of the low frequency wave or color changes due to substrate-enzyme activity as in the ELISA or any other modified methods derive thereof.
  • recombinant interferon gamma and “recombinant IFN-g” are used interchangeably throughout the specification referring to a man-made IFN-g harvested from one or more engineered cell lines conferred with the ability to secrete or produce IFN-g.
  • one aspect of the present disclosure is directed to an apparatus for detecting presence of anti-IFN-g antibody (30) in a bodily fluid sample acquired from a subject.
  • the anti-IFN-g antibody (30) can be of IgG, IgM or IgA.
  • the apparatus comprises a sampling region (1) for receiving deposition of a solvent phase comprising the bodily fluid sample; a reaction region (2) or a conjugate region impregnated with a plurality of a first complex, which comprises a first recombinant IFN-g (10) tagged with a biotin and a first antibody (20) attached with a signaling moiety that the first antibody (20) coupled to the first recombinant IFN-g (10) to form the first complex by binding onto the tagged biotin; and a testing region (3) having a plurality of second recombinant IFN-g (40) immobilized thereto.
  • the first antibody (20) (anti-biotin antibody) and the signaling moiety are in a ratio of 0.1-0.5 mg: 0.5-5.0 mL.
  • the sampling region (1), the reaction region (2), and the testing region (3) are arranged consecutively from a first end towards a second end of a chromatographic solid phase as shown in Fig. 2.
  • the chromatographic solid phase in the present disclosure can be a composite assembled using different materials such that the solvent phase carrying the bodily fluid sample can move from the sampling region (1) towards the testing region (3) passing though the reaction region (2) for progressively and intrinsically performing antigen ELISA thereby based on chromatography capillary action.
  • the solvent phase and the target anti-IFN-g antibody are configured to come into contact and react with various reagents predisposed on each region demarcated on the solid phase.
  • the solid phase preferably comprises a cellulosic or plastic strip forming a base layer having the first end and the second end, a synthetic membrane overlaying portion of the surface of the base layer for establishment of the testing region (3) and an optional control region (4) next to the testing region (3), and an absorbent material incorporated around the second end to absorb excessive solvent phase reaching the second end by the completion of the reaction time using the disclosed apparatus.
  • the recombinant IFN-g is immobilized on the synthetic membrane in an amount of 0.1-1.0 pg on a test line within the testing region (3).
  • the synthetic membrane is made of glass fiber.
  • the first complex is impregnated or disposed thereto for readily binding on the anti-IFN-g antibody (30) found in any of the biological sample tested using the disclosed apparatus.
  • the sampling region (1) it forms the first end or part of the first end of the disclosed apparatus.
  • the solvent phase which can be the bodily fluid of the subject or a mixture of the bodily fluid with one or more buffer, to be deposited thereto for the initiation of the testing and/or detection of the anti-IFN-g antibody.
  • the bodily fluid can be a blood sample or serum sample of the subject.
  • the bodily fluid may be subjected to dilution or mixed with other reagents to effectuate the double- antibody sandwich ELISA upon deposition of the solvent phase onto the sampling region (1).
  • Filtering element may be equipped at the sampling region (1) in some embodiments of the disclosed apparatus to trap some large cellular components found in the bodily fluid from moving towards the second end. These cellular components may affect the results produced at the end of the reaction or testing.
  • the solvent phase preferably migrates from the first end towards the second end of the solid phase along with the anti-IFN-g antibody (30) present in the bodily fluid sample such that the first complex attaches onto the anti-IFN-g antibody (30) forming a second complex at the reaction region (2).
  • the solvent phase migrates from the first end towards the second end under the influence of the chromatographic capillary action.
  • the first complex not binding to any anti-IFN-g antibody (30) and the second complex formed migrate further into the testing region (3), where the second complex binds onto the immobilized second recombinant IFN-g (40) readily for providing a first visual signal thereto.
  • the anti-IFN-g antibody, if any, present in the bodily fluid sample has a pair of binding site each can immunologically couple with the first recombinant IFN-g (10) and the second recombinant IFN-g.
  • the anti-IFN-g antibody (30) being displaced into the reaction region (2) will have one of its binding sites reacted onto the first recombinant IFN-g (10) incorporated into the first complex predisposed at the reaction region (2) giving rise to the second complex.
  • the unreacted or unbound first complex and the second complex further migrate into the testing region (3) under the chromatographic capillary action along with the solvent phase.
  • another binding site of the anti-IFN-g antibody which already has one biding site attached to the first recombinant IFN-g (10) in the formed second complex, immunologically attaches onto the second recombinant IFN-g (40) anchored or immobilized on the test line of the testing region (3).
  • the signaling moiety tagged to the first antibody (20) becomes concentrated at the testing line hence accumulatively giving out a signal sufficiently strong to be seen or observed by preferably naked eye or a compatible a signal reader vouching for detection of anti-IFN-g antibody (30) in the testing bodily fluid sample.
  • the immobilized second recombinant IFN-g (40) and the first complex are in a mole ratio of 1:1 to 1:10.
  • the signaling moiety is gold nanoparticle (AuNP) that the first antibody (20) equipped with the signaling moiety is an AuNPs-tagged anti-biotin antibody (20).
  • AuNP gold nanoparticle
  • the signaling moiety maybe selected from metallic particles (i.e. AuNPs or colloidal gold, gold/silver, platinum, etc.), fluorescence or quantum dots.
  • the disclosed apparatus may further comprise a control region (4) succeeding to the testing region (3) on the solid phase.
  • the control region (4) is located on the synthetic membrane as well like the testing region (3).
  • the planar surface of the synthetic membrane permits a substrate (50) possessing affinity towards the first antibody (20) or the anti-biotin antibody, as illustrated in Fig. 2, to be permanently anchored on a control line within the control region (4) similar to permanent placement of the second recombinant IFN-g (40) on the test line.
  • the disclosed apparatus further comprises a control region (4) comprising a substrate (50) being anchored thereto and configured to bind onto the first antibody (20) and the control region (4) being arranged after and adjacent to the testing region (3) on the chromatographic solid phase.
  • the substrate (50) can be rabbit anti-goat IgG immobilized in an amount of 0.5- 1.0 pg on the control line (4) while the first antibody (20) is of goat origin such that the substrate (50) has the needed immunological affinity towards the first antibody.
  • some embodiments of the disclosed apparatus have the AuNPs-tagged goat anti-biotin or first antibody (20) in an amount of 0.1-0.5 mg to react with the biotinylated-IFN-g (10) of a concentration of 1-500 pg/mL for forming the first complex prior to impregnating the first complex onto the reaction region or conjugate region (2).
  • the first complex and the second complex unbounded at the testing region (3) further migrate into the control region (4) which the first complex and the second complex bind onto the substrate (50) through the first antibody (20) to provide a second visual signal thereto.
  • the signaling moiety become concentrated at the control line that the concentrated signaling moieties accumulatively provide a sufficiently strong signal being detectable by naked eye or a compatible signal reader.
  • the first complex from the reaction region (2) inevitably flows through the testing region (3), without binding onto the anchored second recombinant IFN-g (40) thus giving out no first visual signal, along with the solvent and reaches the control region (4) to be captured by the substrate (50) anchored thereby.
  • control line is designated to give out the second visual signal regardless the presence of the anti- IFN-g in the testing bodily fluid sample or not to ascertain that the absence of the first visual signal or the negative result acquired with respect to the detection of anti-IFN-g is not caused by defect of the disclosed apparatus.
  • the control line serves as a negative control for the disclosed apparatus.
  • the substrate (50) can be a second antibody immunologically effective against the first antibody (20) for effectively capturing the first complex and/or second complex comprising the first antibody (20) coming into contact at the control line or control region (4). More preferably, the immobilized second recombinant IFN-g (40) and the substrate (50) are in a mole ratio of 1:2 to 1:10.
  • the disclosed apparatus has the synthetic membrane having the test line of the testing region (3) and the control line of the control region (4) overlaid onto a plastic or paper strip with one end of the synthetic membrane overlapping with the sampling region (1) and the reaction region (2) interposing between sampling region (1) and the testing region (3).
  • the free space on the planar surface which the solvent phase passing through is further overlaid with a layer of coating (60), which create chemically inert environment towards the reagent and/or cellular compounds passing along with the solvent phase.
  • the layer of coating (60) is preferably fabricated from non-specific signal blocking protein.
  • the coating can be composed of bovine serum albumin and/or phosphoprotein of skimmed milk, which are dissolved in PBS before coating onto the surface of the synthetic membrane.
  • some embodiments of the disclosed apparatus may further comprise an absorbent element connecting to one end of the control region (4) forming at least part of the second end.
  • the existence of the absorbent at the second end may enhance the capillary effect to drive the solvent phase and the other reagents to flow towards the second end in addition to absorbing excessive solvent phase arrived at the second end.
  • the absorbent is any one or combination of cellulose fiber sheet, filter paper, and pulp.
  • the method comprises the step of receiving deposition of a solvent phase comprising the bodily fluid sample at a sampling region (1); directing the solvent phase into a reaction region (2) along with anti-IFN-g antibody present in the bodily fluid sample, the reaction region (2) being impregnated with a plurality of a first complex comprising a first recombinant IFN-g (10) tagged with a biotin and a first antibody (20) attached with a signaling moiety that the first antibody (20) coupled to the first recombinant IFN-g (10) to form the first complex by binding onto the tagged biotin, the first complex being configured to attach onto the anti-IFN-g antibody forming a second complex at the reaction region (2); directing the solvent phase to move further into a testing region (3) having a plurality of second recombinant IFN-g (40) immobilized there
  • the sampling region (1), the reaction region (2), and the testing region (3) are arranged consecutively from a first end towards a second end of a chromatographic solid phase. It is important to note that the directing steps are performed intrinsically under the influence of the chromatographic capillary action of the chromatographic solid phase upon depositing the solvent phase at the sampling region (1). Also, the first visual signal established at the testing region (3) indicates presence of the anti-IFN-g antibody (30) in the bodily fluid sample.
  • the solvent phase and the target anti-IFN-g antibody are configured to come into contact and react with various reagents predisposed on each region demarcated on the solid phase.
  • the solid phase preferably comprises a cellulosic or plastic strip forming a base layer having the first end and the second end, a synthetic membrane overlaying portion of the surface of the base layer for establishment of the testing region (3) and an optional control region (4) next to the testing region (3), and an absorbent material incorporated around the second end to absorb excessive solvent phase reaching the second end by the completion of the disclosed method.
  • the recombinant IFN-g is immobilized on the synthetic membrane in an amount of 0.1- 1.0 pg on a test line within the testing region (3).
  • the first complex is impregnated or disposed thereto for readily binding on the anti- IFN-g antibody (30) found in any of the biological sample tested.
  • the solvent phase which can be the bodily fluid of the subject or a mixture of the bodily fluid with one or more buffer, to be deposited thereto for the initiation of the testing and/or detection of the anti-IFN-g antibody.
  • the bodily fluid can be a blood sample or serum sample of the subject.
  • the disclosed method may further comprise a pre-treatment step to subject the bodily fluid to dilution or to mix with other reagents to effectuate the double-antibody sandwich ELISA upon deposition of the solvent phase onto the sampling region (1).
  • the disclosed method further comprising the step of filtering the bodily fluid sample using a filtering element, which may be equipped at the sampling region (1) for trapping some large cellular components found in the bodily fluid.
  • the disclosed method further comprises the step of directing the solvent phase to move further into a control region (4) comprising a substrate (50) being anchored thereto and configured to bind onto the first antibody.
  • the control region (4) is arranged after and adjacent to the testing region (3) on the chromatographic solid phase that the first complex and the second complex unbounded at the testing region (3) inevitably migrate into the control region (4) along with the solvent phase. Owing to the immunological affinity towards the substrate, the first complex and the second complex bind onto the substrate (50) through the first antibody (20) to provide a second visual signal thereto.
  • the first complex and the second complex unbounded at the testing region (3) further migrate into the control region (4) which the first complex and the second complex bind onto the substrate (50) through the first antibody (20) to provide a second visual signal thereto.
  • the signaling moiety becomes concentrated at the control line that the concentrated signaling moieties accumulatively provide a sufficiently strong signal being detectable by naked eye or a compatible signal reader.
  • the first complex from the reaction region (2) inevitably flows through the testing region (3), without binding onto the anchored second recombinant IFN-g (40) thus giving out no first visual signal, along with the solvent and reaches the control region (4) to be captured by the substrate (50) anchored thereby.
  • control line is designated to give out the second visual signal regardless the presence of the anti-IFN-g in the testing bodily fluid sample or not to ascertain that the absence of the first visual signal or the negative result acquired with respect to the detection of anti-IFN- g is not caused by defect of the chromatographic solid phase or reagents or inappropriate administering of the disclosed method.
  • the second recombinant IFN-g (40) and/or the substrate (50) is immobilized on a synthetic membrane for the disclosed method.
  • the signaling moiety is AuNPs, fluorescence or quantum dots.
  • microtiter plates were coated with 50 pF of recombinant IFN-g per well and incubate for overnight at 4 °C in a moisture chamber. The following steps were performed at room temperature. The coated wells were washed 3 times with 0.05% Tween 20 in PBS and blocked with 200 pF of blocking solution (PBS with 2% skimmed milk) for 1 hour. After five times washing, 50 pF of mouse anti-His tag mAh was added and incubated for 1 hour. After washing for five times, 50 pF of HRP-conjugated goat anti-mouse immunoglobulin was added and incubated for 1 h.
  • FIG. 3 shows the results of recombinant IFN-g analysis by indirect EFISA using anti- his tag mAh.
  • the recombinant IFN-g was prepared and confirmed using EFISA.
  • the recombinant IFN-g coated wells displayed a positive signal upon probing with mouse anti-His tag mAh. This result suggested that IFN-g protein including soluble, inclusion bodies, refolded formed reacted with mouse anti-his tag antibody.
  • IFN-g preparation in some conditions showed different binding activity.
  • Figure 4 shows results of recombinant IFN-g analysis by indirect ELISA using anti- IFN- g mAh and tagged with HRP-goat anti-mouse Igs. This result suggested that IFN-g protein including soluble, inclusion bodies, refolded formed reacted with mouse anti-IFN-g mAh antibody.
  • microtiter plates were coated with 50 pL of mouse anti-IFN-g clone B27 (0.5 pg/mL in bicarbonate buffer, pH 9.6) per well and incubate for overnight at 4 °C in a moisture chamber. The following steps were performed at room temperature. The coated wells were washed 3 times with 0.05% Tween 20 in PBS and blocked with 200 pL of blocking solution (PBS with 1% skimmed milk) for 1 h.
  • mouse anti-IFN-g clone B27 0.5 pg/mL in bicarbonate buffer, pH 9.6
  • the following steps were performed at room temperature.
  • the coated wells were washed 3 times with 0.05% Tween 20 in PBS and blocked with 200 pL of blocking solution (PBS with 1% skimmed milk) for 1 h.
  • Figure 5 shows Dimer analysis of recombinant interferon gamma prepared in a soluble form and inclusion body (IB) solution by double- antibody sandwich ELISA was performed from the produced protein samples. High optical density (OD) was observed except for IFN-g solubilized protein in the PBS buffer, which has the lowest OD.
  • IB soluble form and inclusion body
  • microplate wells were coated with purified interferon gamma at the concentration of 10 ⁇ g/mL, 50 pL per well and leave in the humidifying box at 4 °C for 16-18 h. After washed the plate with 0.05% Tween 20 in phosphate-buffered saline (PBS) for 5 times and patted dry, added a blocking solution using 2% bovine serum albumin (BSA) in PBS at 200 pL per well and kept the plate in the humidifying box at room temperature for 1 h.
  • PBS phosphate-buffered saline
  • Biotinylated IFN-g at concentration of 1 pg/mL was added into the microplate 50 pL per well, and kept the plate in the humidifying box at room temperature for 1 h. After washing the plate with a high stringency washing buffer for 5 times and patting dry, the HRP-anti biotin mAh at 1:5000 dilution 50 pL was added per well, and kept the plate in the humidifying box at room temperature for 1 h. Then, washed the plate with 0.05 % Tween 20 in PBS for 5 times. After drying the plate, the TMB substrate (3,3',5,5'-Tetramethylbenzidine) 50 pL was added per well for the HRP reaction. The reaction between horseradish peroxidase (HRP) and TMB was stopped at minute 15 by adding 6 N HCL 50 pL per well. The optical density at 450 nm (OD450) was measured by ELISA microplate reader.
  • the serum dilution was selected to be 1:100.
  • the threshold for determining a positive result were considered from the cut-off value obtained from the background value of individual serum, the mean of which was 0.17, resulting in all positive results of the 20 patients, as shown in figure 7.
  • Impregnating a plurality of first complex comprising gold nanoparticle-labeled anti-biotin (or first antibody) (20) coupled to the biotinylated IFN-g (10) at the reaction region (2), where the biotinylated IFN-g (10) will intrinsically bind to any anti-IFN-g antibody present in the serum sample to produce a positive result.
  • first complex comprising gold nanoparticle-labeled anti-biotin (or first antibody) (20) coupled to the biotinylated IFN-g (10) at the reaction region (2), where the biotinylated IFN-g (10) will intrinsically bind to any anti-IFN-g antibody present in the serum sample to produce a positive result.
  • test kit by placing the synthetic membrane comprising the testing region (3) and control region (4) obtained from process A in the middle of a plastic strip or a chromatographic solid phase.
  • An end of the synthetic membrane adjacent to the reaction region is join at one end of the binding region (2) in a way that the ends of each region are physically contacting one another.
  • the strip may further include a contact region (5) incorporated with an absorbent material connected to one end of the control region (4) in an overlapping fashion.
  • the direct dot blotting assay was conducted by applying the goat anti-biotin tagged with colloidal gold to the biotinylated IFN-g at three different concentrations of 100, 10, and 1 mg/mL, while the rabbit anti-goat IgG 10 mg/mL being a positive control and PBS being a negative control. As shown in figure 9a, red-purple or dark purple dots appeared only at the area or region coated/immobilized with the biotinylated-IFN-g.
  • the CGC-tagged anti-biotin is specifically served for reporting or signaling on the disclosed embodiments.
  • the recombinant IFN-g is applied to the biotinylated IFN-g, there is no appearance of the red- purple or dark purple dot, as shown in figure 9b.
  • FigurelO is a picture showing the accuracy of the disclosed embodiments for detecting a presence or absence of anti-interferon gamma antibody in a sample, where the two visual signals of the testing and the control regions were observed in all 5 positive serums (No.1-5) while control region was solely visualized in the negative serums (No.6- 10).

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  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un appareil permettant de détecter la présence d'un anticorps anti-IFN-γ dans un échantillon de fluide corporel acquis sur un sujet. L'appareil peut comprendre une région d'échantillonnage servant à recevoir le dépôt d'une phase de solvant comprenant l'échantillon de fluide corporel ; une région de réaction imprégnée d'une pluralité d'un premier complexe comprenant un premier IFN-γ recombinant marqué avec une biotine et un premier anticorps fixé à un groupement de signalisation, le premier anticorps étant couplé au premier IFN-γ recombinant pour former le premier complexe par liaison sur la biotine marquée ; et une région de test ayant une pluralité de deuxième IFN-γ recombinant immobilisée dessus. La région d'échantillonnage, la région de réaction et la région de test sont agencées consécutivement entre une première extrémité et une deuxième extrémité d'une phase solide chromatographique.
PCT/TH2021/000029 2020-06-29 2021-06-04 Procédé et kit immunochromatographique pour détecter un anticorps anti-interféron gamma WO2022005408A1 (fr)

Priority Applications (2)

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JP2022555985A JP7417231B2 (ja) 2020-06-29 2021-06-04 抗インターフェロンγ抗体を検出するための免疫クロマトグラフィーによる方法及びキット
CN202180017176.6A CN115176157A (zh) 2020-06-29 2021-06-04 用于检测抗干扰素γ抗体的免疫层析方法及试剂盒

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CN115176157A (zh) 2022-10-11
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