WO2012166755A1 - Essai par immunosorbant lié à une enzyme pour la quantification de trypsine résiduelle dans un échantillon biologique - Google Patents

Essai par immunosorbant lié à une enzyme pour la quantification de trypsine résiduelle dans un échantillon biologique Download PDF

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Publication number
WO2012166755A1
WO2012166755A1 PCT/US2012/039915 US2012039915W WO2012166755A1 WO 2012166755 A1 WO2012166755 A1 WO 2012166755A1 US 2012039915 W US2012039915 W US 2012039915W WO 2012166755 A1 WO2012166755 A1 WO 2012166755A1
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trypsin
enzyme
protease
analyte
binding
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PCT/US2012/039915
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English (en)
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James A. BRAAZ
Michael MERGES
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Lonza Walkersville, Inc.
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Publication of WO2012166755A1 publication Critical patent/WO2012166755A1/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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/976—Trypsin; Chymotrypsin

Definitions

  • the present invention relates to a method and kit for performing an enzyme- linked immunosorbent assay (ELISA) to detect a catalytically active analyte.
  • ELISA enzyme- linked immunosorbent assay
  • the present invention relates to an ELISA for determining the amount of residual trypsin in biological and/or biopharmaceutical samples.
  • Degradative enzymes include, for example, trypsin, chymotrypsin, hyaluronidase, subtilisin, pancreatin, elastase, papain, collagenase, and pronase.
  • Trypsin for example, porcine trypsin
  • Trypsin is a proteolytic enzyme that is commonly used for detaching adherent cells from the surface of tissue culture vessels. Trypsin cleaves peptide bonds on the carboxy terminal side of the basic residues lysine and arginine.
  • the U.S. Food and Drug Administration regulates certain products that contain cells and cell-derived products.
  • FDA requires that any reagent used in manufacturing a drug product but not intended to be part of the final product be listed in the Investigational New Drug (IND) application and accounted for in the final product.
  • a proteolytic enzyme such as trypsin
  • trypsin used in harvesting adherent cells from culture vessels is an example of such a reagent.
  • trypsin may be a residual contaminant. To the extent that trypsin may be present in a cell product, its presence must be determined and demonstrated to be below acceptable limits.
  • ELISA is the method generally used to demonstrate levels of residual protein impurities.
  • Commercial kits or reagents for the specific detection of porcine trypsin are not widely available. This unavailability may reflect the nature of the binding antigen, in this case a protease.
  • a protease that is, an enzyme that cleaves peptide bonds, could potentially degrade the capture antibody.
  • a degraded capture antibody, either partially degraded or completely degraded, could interfere with capturing and detecting the antigen the ELISA seeks to determine.
  • Conventional ELISA determines protein in a sample using an immobilized antibody for capturing an antigen to be quantified, and an antibody-enzyme conjugate for detecting the sandwiched antigen.
  • a protease such as trypsin
  • the resulting binding response levels off at relatively low antigen concentrations. As stated, this response may be due to the proteolytic nature of the trypsin antigen. For example, trypsin may have degraded the antibody used for capture. Regardless of the cause, there is therefore a need for an ELISA to detect a catalytically active analyte over a wide range of concentrations.
  • the present invention provides a method and kit for performing an ELISA to detect a catalytically active analyte.
  • a catalytic inhibitor is used as a primary binding or capture agent in the assay under conditions such that the analyte retains immunological activity to bind a secondary binding or capture agent, but its enzymatic activity is inhibited by the catalytic inhibitor.
  • the detection of the analyte is thus not compromised by catalytic activity of the analyte degrading the secondary binding or capture agent.
  • Figure 1 compares results from an ELISA detecting trypsin using alpha-1 - antitrypsin as the capture protein and using an anti-lgG-horseradish peroxidase secondary antibody at a dilution of 1 :500 to results from an ELISA detecting trypsin using alpha-1 -antitrypsin as the capture protein at 5 ⁇ g/well detected with rabbit anti- goat IgG-horseradish peroxidase at a dilution of 1 :5000.
  • Figure 2 shows antisera reactivity titer from a porcine trypsin immunization to a first goat.
  • Figure 3 shows antisera reactivity titer from a porcine trypsin immunization to a second goat.
  • Figure 4 shows antisera reactivity titer from a porcine trypsin immunization to a third goat.
  • Figure 5 shows an antisera reactivity titer for a first goat against bovine and porcine trypsin with antisera dilution expressed fractionally (FIG. 5A) and
  • Figure 6 shows an antisera reactivity titer for a second goat against bovine and porcine trypsin with antisera dilution expressed fractionally (FIG. 6A) and logarithmically (FIG. 6B).
  • Figure 7 shows an antisera reactivity titer for a third goat against bovine and porcine trypsin with antisera dilution expressed fractionally (FIG. 7A) and
  • Figure 8 shows an antisera reactivity titer for a first goat of the first two test bleeds from an immunization against immobilized porcine trypsin.
  • Figure 9 shows an antisera reactivity titer for a second goat of the first two test bleeds from an immunization against immobilized porcine trypsin.
  • Figure 10 shows antisera reactivity titer for a third goat of the first two test bleeds from an immunization against immobilized porcine trypsin.
  • Figure 11 shows isolation of IgG from antisera of a second goat, on a Protein G column.
  • Figure 12 shows isolation of IgG from antisera of a third goat, on a Protein G column.
  • Figure 13 shows titers of IgG fractions from immunizations to a second goat and a third goat.
  • Figure 14 shows a standard curve for protein detection of bovine gamma globulin.
  • Figure 15 shows electrophoretic separation of an IgG fraction obtained from Protein G chromatography.
  • Figure 16 shows enzymatic (horseradish peroxidase) activity of fractions eluted from a desalting column.
  • Figure 17 shows results from an ELISA detecting trypsin using IgG as the capture protein using an IgG-horseradish peroxidase secondary antibody at a dilution of 1 :500.
  • Figure 18 shows results from an ELISA detecting trypsin using alpha-1 - antitrypsin as the capture protein using an IgG-horseradish peroxidase secondary antibody at a dilution of 1 :500.
  • Figure 19 shows results from an ELISA detecting trypsin using alpha-1 - antitrypsin as the capture protein at 5 g/well detected with rabbit anti-goat IgG- horseradish peroxidase at a dilution of 1 :5000.
  • the disclosed method and kit determine levels of a protease, such as trypsin, in cell-based products using a non-immunoglobulin capture protein that inhibits the protein degradation activity of, for example, trypsin in an ELISA.
  • the method quantifies residual process biochemicals used in producing cell-based therapeutics.
  • the method is useful to meet FDA requirements that any reagent used in manufacturing a cell-based product, but not intended to be part of the final product, be listed in the IND application submission and accounted for in the final product.
  • the method of the present invention uses a non- immunoglobulin capture protein in place of an immunoglobulin capture protein in an ELISA.
  • the disclosed method uses a trypsin inhibitor, such as alpha-1 -antitrypsin, as the capture protein in place of an antibody.
  • a trypsin inhibitor such as alpha-1 -antitrypsin
  • Alternative trypsin inhibitors and/or capture proteins are contemplated including, but not limited to, trypsin inhibitor from chicken egg white, trypsin inhibitor from Phaseolus limensis, and serum proteins such as alpha-2-macroglobulin and inter-alpha trypsin inhibitor.
  • ELISA for determining levels of chymotrypsin alpha-1 -antichymotrypsin is contemplated.
  • a protease inhibitor is immobilized, by passive absorption, to a vessel.
  • a well of a microtiter plate can serve as a vessel.
  • a sample that contains a protease, such as trypsin, is added to the vessel.
  • the protease inhibitor binds protease that is present in the sample.
  • the protease inhibitor inactivates the catalytic activity of the protease.
  • the protein structure remains intact and thus is able to bind to an antibody against the protein which in turn can then bind to a secondary antibody in a detection antibody-enzyme conjugate.
  • a detection antibody-enzyme conjugate is rabbit anti-goat IgG-horseradish peroxidase.
  • an alternative example is anti-lgG-alkaline phosphatase. Binding of the complex and the activity of the secondary enzyme can be measured with an appropriate substrate.
  • contemplated substrates include for example TMB (tetramethylbenzidine), ABTS (2,2'-Azino-di(3-ethyl-benzthiazoline sulfonic acid), o-phenylenediamine, 3-amino-9-ethyl carbazole, 4-chloro-1 -naphthol, and 3,3'-diaminobenzidine tetrahydrochloride.
  • TMB tetramethylbenzidine
  • ABTS 2,2'-Azino-di(3-ethyl-benzthiazoline sulfonic acid
  • o-phenylenediamine 3-amino-9-ethyl carbazole
  • 4-chloro-1 -naphthol 4-chloro-1 -naphthol
  • the level of activity of the secondary antibody reflects the concentration of the protease in the sample.
  • the level of activity of the secondary antibody reflects the concentration of the trypsin in the sample.
  • the IgG is used as a capture protein, in this case anti-trypsin IgG. It is attached to wells of a microtiter plate.
  • the sample containing the protein of interest in this case trypsin
  • Trypsin in the sample binds to the anti-trypsin IgG antibody, and is subsequently detected using a secondary detection antibody.
  • the secondary detection antibody which has an enzyme conjugated to it, binds to the captured protein (in this case trypsin), (by binding the IgG of the primary antibody) and provides a colored signal when a substrate for the secondary detection enzyme, is added.
  • the substrate is TMB; when TMB is oxidized it produces a yellow color after acidification.
  • the yellow color is measured spectrophotometncally with absorbance determining concentration compared to a calibration curve with a series of standards at known concentrations.
  • the above scheme is modified to replace the anti-trypsin IgG-HRP with anti-trypsin IgG then followed by a rabbit-ant-goat IgG-HRP.
  • trypsin when trypsin is the substance to be measured, a potential difficulty arises because trypsin is a proteolytic enzyme. As such, trypsin can potentially degrade the immobilized antibody and interfere with spectrophotometric detection.
  • Scheme 2 illustrates the use of a protease inhibitor in place of the anti-trypsin IgG capture antibody to bind to and inactivate trypsin.
  • the protease inhibitor can be, for example, alpha-1 -antitrypsin.
  • the protease inhibitor is attached to the vessel and captures the trypsin, rendering it proteolytically inactive and thus incapable of degrading capture proteins such as the immobilized antibody or secondary antibody.
  • a goat anti-trypsin IgG antibody raised against porcine trypsin and labeled as G2032 IgG, binds trypsin and is detected with a commercially available antibody-enzyme conjugate after adding the enzyme substrate.
  • Trypsi Trypsi (Trypsin is inactivated on binding to the
  • ELISA plates are Immulon ® 4HBX, Flat Bottom Microtiter Plates, Part # 3855 from Thermo, Milford MA. Costar UV Plates with UV
  • Transparent Flat Bottom, product #3635 are from Corning, Inc., Corning NY may also be used.
  • Rabbit polyclonal antibody to goat IgG - heavy and light chains (HRP conjugated) product # ab6741 is from Abeam, Cambridge MA.
  • SureBlueTM TMB Microwell Peroxidase Substrate (l-Component) is from KPL Labs, Gaithersburg MD.
  • Alpha-1 -antitrypsin is from human plasma, product #A6150, TBST (tris-buffered saline with Tween, product #T9039-IOPAK), porcine pancreatic trypsin, product # T0303-1 G, and gamma globulin from bovine blood, product #G5009-1 G are from Sigma-Aldrich, St. Louis MO.
  • Bovine trypsin, cat. # 1 700002, used as a reference standard is from USP, Rockville MD.
  • Protein G chromatography cartridges, product #89926, EZ-Link ® Plus Activated Peroxidase Kit, product #31489, and Pierce Conjugate Purification Kit, product #44920 are from Pierce Biotechnology, Rockford IL.
  • Porcine pancreatic trypsin is prepared in 1 mM hydrochloric acid at a concentration of 200 ⁇ 9/ ⁇ .
  • the initial boost contains 200 ⁇ g trypsin, 0.5 ml Complete Freund's Adjuvant (CFA), and 0.5 ml 0.9% sodium chloride.
  • the second and third boosts are with 100 ⁇ g trypsin, 0.5 ml Incomplete Freund's Adjuvant (IFA), and 1 .0 ml 0.9% sodium chloride.
  • antisera are stored at -80 °C.
  • Capture proteins are prepared in 50 mM sodium bicarbonate, pH 9.6 at the indicated concentrations and 100 ⁇ are added to wells of ELISA plates and incubated overnight at 4 ⁇ . The wells are washed three times with 300 ⁇ TBST after each addition and incubation. All subsequent protein reagents are prepared in TBST and incubated at room temperature (about 19 to about 22 ⁇ C) for the times indicated, unless otherwise indicated.
  • TMB 2 substrate (SureBlueTM) is added, 100 ⁇ /well, and the reaction stopped by adding 100 ⁇ 1 N sulfuric acid. Absorbances at 450 nm (A450) are obtained within five minutes of stopping the reaction. Results are analyzed directly using SoftMax Pro software or after exporting into Excel spreadsheet.
  • porcine trypsin is immobilized to wells of an ELISA plate.
  • Stock solutions of porcine trypsin, at 1 mg/ml in 1 mM HCI (stored at -80 in aliquots) are diluted with bicarbonate buffer to 10 ⁇ g/ml and 100 ⁇ added to each well.
  • Goat antisera are diluted with TBST and 100 ⁇ added per well and incubated for one hour.
  • Bound goat IgG is detected using rabbit polyclonal antibody to goat IgG (heavy chains and light chains) conjugated to horseradish peroxidase (HRP) diluted 1 :5000 with TBST and 100 ⁇ /well is added for one hour. After washing with TBST, peroxidase activity is detected with TMB. [0053] To isolate IgG, 1 .0 ml of thawed antiserum is diluted with 4.0 ml 0.1 M sodium phosphate, pH 7.0 (NaP0 4 ).
  • a Protein G column is equilibrated with 10 ml NaP0 , then the diluted antiserum is passed through the column at a flow rate of about one drop per second, and fractions containing 20 drops (about 1 ml each) are collected.
  • the column is washed with 10 ml NaP0 4 , then eluted with 0.1 M sodium citrate, pH 3.0 and 20 drop fractions are collected directly into 1 .5 ml tubes containing 1 M Tris, pH 9.0. Portions of each fraction (300 ⁇ ) are transferred to wells of an ultraviolet (UV) transparent 96 well plate and A280 is determined.
  • UV ultraviolet
  • HRP horseradish peroxidase
  • goat IgG isolated from the Protein G column is buffer exchanged into 0.2 M sodium bicarbonate, pH 9.4 using a Pierce Zeba Desalt Spin Column.
  • the protein concentration is determined by UV absorbance as subsequently described, and 1 mg protein (551 ⁇ ) is conjugated using a Pierce EZ-Link Plus Activated Peroxidase Kit according to instructions.
  • the IgG-HRP is purified using a Pierce Conjugate Purification Kit according to instructions.
  • This method uses a nickel-chelated agarose column to bind IgG through a histidine-rich region in the Fc section of the IgG. Unconjugated H RP is thereby separated from the conjugate.
  • the column is activated with a solution containing nickel and the conjugate is then applied to the column, washed, and eluted with supplied Elution Buffer.
  • the Elution Buffer eluate from the column is desalted with the supplied desalting column using supplied PBS according to instructions.
  • Fractions (0.5 ml) are collected from the desalting column and analyzed for HRP activity by diluting 2 ⁇ of each fraction with 1 ml of supplied Tris-buffered saline (TBS), then mixing 5 ⁇ with 100 ⁇ SureBlue TMB in a 96 well plate for two minutes at room temperature and stopping the reaction with 100 ⁇ 1 N sulfuric acid. The absorbance is determined at A450.
  • Fractions containing peroxidase activity are pooled, and aliquots are prepared and stored at -80 °C.
  • Alpha-1 -antitrypsin as the capture protein is compared with IgG as the capture protein. The conditions are as follows: [0057]
  • G2032 IgG is the IgG fraction from antiserum; G2032 IgG-HRP is the same conjugated to horseradish peroxidase.
  • Figure 1 provides results of this exemplary study and compares trypsin ELISAs using IgG as the capture protein versus alpha-1 -antitrypsin (A-1 -AT) as the capture protein.
  • alpha-1 -antitrypsin as the capture protein in the disclosed method extends the range for the assay from about 75 ng/ml to almost 1 Mg/ ⁇ .
  • alpha-1 -antitrypsin as the capture protein in the disclosed method improves the assay sensitivity between 1 mg/ml trypsin - 1 0 ng/ml trypsin.
  • IgG as the capture protein produces a level response at higher concentrations of trypsin, thus restricting the range of the assay.
  • FIG. 1 The reactivity of antiserum to trypsin is determined.
  • Three goats are immunized with porcine trypsin.
  • the first test bleed for each provides high titers of reactivity against porcine trypsin.
  • Figures 2, 3, and 4 show the reactivities to a dilution of 1 :32,000 for the test and pre-bleed from goats # 2030, 2032 and 2033, respectively.
  • Figure 2 shows the titer of reactivity of antisera from goat #2030 against immobilized porcine trypsin.
  • Figure 3 shows the titer of reactivity of antisera from goat #2032 against immobilized porcine trypsin.
  • Figure 4 shows the titer of reactivity of antisera from goat #2033 against immobilized porcine trypsin.
  • the specificity of antiserum reactivity is determined by comparing its reactivity to bovine trypsin.
  • Bovine and porcine trypsin are immobilized to an ELISA plate at a concentration of 1 g/well and the three goat antisera are tested up to a dilution of 1 :512,000 for each.
  • Figures 5A, 5B, 6A, 6B, 7 A and 7B show the reactivity of all three antisera are substantially higher against porcine trypsin.
  • Figures 5A and 5B show antisera reactivity titer from goat #2030 against immobilized bovine and porcine trypsin.
  • Figures 6A and 6B show antisera reactivity titer from goat #2032 against immobilized bovine and porcine trypsin.
  • Figures 7 A and 7B show antisera reactivity titer from goat #2033 against immobilized bovine and porcine trypsin. The higher reactivity from porcine trypsin as compared to bovine trypsin is understandable given that the immunizing trypsin is of porcine origin.
  • Reactivity to trypsin of a second test bleed is determined as follows. A second test bleed is collected from each goat at day 224 and the antisera from both test bleeds from each animal are tested in the trypsin ELISA against porcine trypsin. Figures 8, 9, and 10 show comparative titers for both bleeds for each goat with the antisera concentration expressed as the log of the dilution.
  • test bleed #2 is selected based on its having obtained the highest level or reactivity against porcine trypsin, as demonstrated.
  • the IgG fraction from goat #2032, test bleed #2 is isolated using a Protein G chromatography column. The antiserum is applied to the column, which is then washed and the IgG eluted as described.
  • the elution profile is shown in Figure 11.
  • the unbound fraction is not collected.
  • the IgG isolation procedure is also applied to goat #2033, test bleed #2, in which case both the unbound and the bound and eluted fraction containing IgG are collected.
  • Figure 12 shows the Protein G profile for goat #2033 antisera.
  • the IgG fraction from goat #2032 has a higher level of reactivity against porcine trypsin than goat #2033 IgG. This is consistent with the relative titers that are observed with the unfractionated antisera.
  • the unbound fraction for goat #2033 has substantial reactivity, indicating that not all the IgG bound to the column.
  • G2032 IgG, 2.0 ml is buffer exchanged into 0.2 M sodium bicarbonate, pH 9.4 using a Pierce Zeba Desalting Spin Column. After buffer exchange, the protein concentration is determined at A280 using purified bovine gamma G prepared in bicarbonate buffer as a standard.
  • Figure 14 shows the standard curve for A280 for various concentrations of bovine gamma G prepared in bicarbonate buffer.
  • G2032 IgG (bicarbonate) is conjugated to HRP as previously described. After the conjugation reaction, the IgG-HRP is purified by metal affinity
  • G2032 IgG as the capture protein and G2032 IgG-HRP for detection.
  • G2032 IgG (bicarbonate) is immobilized to an ELISA plate at 0 g/well, 0.5 g/well, 1 .0 g/well, and 5.0 g/well.
  • Porcine trypsin at concentrations ranging from 0 ng/well to 500 ng/well are incubated on the plate for one hour, washed, and then treated with G2032 IgG-H RP at a 1 :500 dilution for one hour. After washing, TMB is added for one hour at 37 °C, the reaction is stopped with acid, and A450 is determined.
  • the results are shown in Figure 17.
  • the activity although substantial, levels off at a relatively low concentration of trypsin, providing a range of approximately 1 ng/ml to 50 ng/ml.
  • An ELISA is developed using a trypsin inhibitor as the capture protein and G2032 IgG-HRP for detection.
  • a naturally occurring trypsin inhibitor used as the capture protein while simultaneously serving to inactivate the proteolytic activity of the enzyme, extends the assay range and allows for more extensive binding by the secondary detection antibody.
  • Alpha-1 -antitrypsin is immobilized in the same experiment as described above at corresponding amounts of protein per well, and simultaneously treated with porcine trypsin, detection antibody, and TMB substrate. The results are shown in Figure 18.
  • alpha-1 -antitrypsin as the capture protein, the curve does not tend to plateau at higher trypsin concentrations, as occurred using IgG as the capture protein.
  • the method using alpha-1 -antitrypsin as the capture protein is not as sensitive from 1 mg/ml trypsin to about 10 ng/ml trypsin compared to the method using IgG as the capture protein.
  • An ELISA is developed using alpha-1 -antitrypsin as the capture protein.
  • a format using a commercially available secondary enzyme conjugate (rabbit anti-goat IgG-HRP) in place of the G2032 IgG-HRP is evaluated.
  • Alpha-1 -antitrypsin is used as the capture protein, trypsin is added, followed by G2032 IgG, which is then detected using rabbit anti-goat IgG-HRP.
  • alpha-1 -antitrypsin is immobilized at 5 g/well, then the plate is incubated for one hour with trypsin from 0 ng/ml to 500 ng/ml.
  • Figure 1 For comparison, data from Figure 17 using G2032 IgG as the capture protein at 5 g/well, versus data from Figure 19 using alpha-1 -antitrypsin as the capture protein at 5 g/well are plotted together in Figure 1 , which compares results from an ELISA detecting trypsin using alpha-1 -antitrypsin as the capture protein using an IgG-horseradish peroxidase secondary antibody at a dilution of 1 :500 to results from an ELISA detecting trypsin using alpha-1 -antitrypsin as the capture protein at 5 g/well detected with rabbit anti-goat IgG-horseradish peroxidase at a dilution of 1 :5000.
  • the format and conditions are summarized as follows: [0074]
  • alpha-1 -antitrypsin as the capture protein in this format extends the range for the assay.
  • Using alpha-1 -antitrypsin as the capture protein in this format enhances assay sensitivity between 1 mg/mL trypsin - 1 0 ng/ml trypsin (absorbance difference of 0.3228 for alpha-1 -antitrypsin vs. 0.2881 for IgG).

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Abstract

L'invention concerne un procédé et une trousse pour la mise en œuvre d'un essai par immunosorbant lié à une enzyme (ELISA) pour détecter un analyte catalytiquement actif. Un inhibiteur catalytique est utilisé en tant qu'agent primaire de liaison ou de capture dans l'essai dans des conditions telles que l'analyte lié conserve une activité immunologique pour lier un anticorps contre l'analyte, en faisant suivre par un agent secondaire de liaison ou de capture, mais son activité enzymatique est inhibée par l'inhibiteur catalytique. La détection de l'analyte est par conséquent non compromise par l'activité catalytique de l'analyte dégradant l'agent secondaire de liaison ou de capture.
PCT/US2012/039915 2011-06-02 2012-05-30 Essai par immunosorbant lié à une enzyme pour la quantification de trypsine résiduelle dans un échantillon biologique WO2012166755A1 (fr)

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CN113092758A (zh) * 2021-04-01 2021-07-09 山西集创生物科技有限公司 用于检测胰蛋白酶类似物TrypLE的双抗夹心ELISA检测试剂盒及检测方法与应用

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US4753875A (en) * 1981-11-02 1988-06-28 Ryan James W Method for assaying proteases with tagged proteinaceous inhibitors
GB2259362A (en) * 1991-09-07 1993-03-10 Biosyn Limited Bio immunoassay for proteases
WO2001038560A2 (fr) * 1999-11-22 2001-05-31 American Red Cross Nouvelle methode permettant de detecter la presence d'une forme d'enzyme fonctionnellement active dans des echantillons biologiques et kit

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CN113092758A (zh) * 2021-04-01 2021-07-09 山西集创生物科技有限公司 用于检测胰蛋白酶类似物TrypLE的双抗夹心ELISA检测试剂盒及检测方法与应用

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