NEW METHOD AND KIT
20230243842 · 2023-08-03
Assignee
Inventors
Cpc classification
G01N33/543
PHYSICS
G01N2560/00
PHYSICS
International classification
Abstract
The disclosure relates to a method for measuring the amount of a target protein in body fluid. The method comprises preparing a sample suspected to comprise a target protein and adding a known amount of an isotope-labelled internal standard protein consisting of a fragment of the target protein. Furthermore, the method comprises bringing the sample into contact with a solid support comprising a binding agent. The target protein and the standard protein are thereafter digested to form a digested sample which is subjected to mass spectrometry, so as to determine the amount of the target protein in the sample by comparing with the standard protein. The disclosure further relates to a kit comprising at least one binding agent, at least one isotope-labelled internal standard protein, and instructions for carrying out the method.
Claims
1. A method for measuring the amount of a target protein in body fluid, the method comprising the following consecutive steps: preparing a sample suspected to comprise said target protein and comprising a known amount of an isotope-labelled internal standard protein, said standard protein consisting of a fragment of said target protein, bringing said sample into contact with a solid support comprising a binding agent, washing said solid support to remove unbound members of the sample, digesting said target protein and said standard protein to provide a digested sample, subjecting said digested sample to mass spectrometry, and determining the amount of said target protein in said sample by comparison with said standard, wherein said binding agent is capable of binding an epitope present in both said target protein and said standard protein.
2. The method according to claim 1, wherein said target protein is an actively secreted protein.
3. The method according to claim 1, wherein said target protein is selected from the group consisting of a cytokine, a chemokine, an interleukin, an interferon, a hormone, a neuropeptide, a growth factor, a receptor, a protein involved in transport, a protein involved in development, an enzyme, an enzyme inhibitor, a protein involved in the immune system, a protein involved in coagulation, a protein involved in the complement pathway, an acute phase protein and a cell adhesion protein.
4. The method according to claim 1, wherein said solid support is selected from the group consisting of a bead, such as a magnetic bead, and a column.
5. The method according to claim 1, wherein said digestion is carried out by means of a proteolytic enzyme.
6. The method according to claim 1, wherein the digested sample comprises at least one isotopically labeled standard peptide consisting of between 6 and 25 amino acids.
7. The method according to claim 1, wherein said standard protein comprises at least two cleavage sites for said proteolytic enzyme.
8. The method according to claim 1, wherein said standard protein is labelled with at least one isotope selected from the group consisting of .sup.15N, .sup.13C and/or .sup.18O.
9. The method according to claim 1, wherein said binding agent is an antibody or an antibody fragment.
10. The method according to claim 1, wherein said measuring comprises measuring the amount of at least two target proteins, such as three target proteins, such as four target proteins, such as five target proteins, such as six target proteins, such as seven target proteins, such as eight target proteins, such as nine target proteins, such as ten target proteins.
11. The method according to claim 1, wherein said at least one target protein suspected to be present in said sample, is present in said sample in a concentration of between 10.sup.−4 and 10.sup.−10 M, such as between 10.sup.−6 and 10.sup.−7 M.
12. The method according to claim 1, wherein said target protein and said standard protein bind to the binding agent with comparable affinity, at a ratio of K.sub.D values such as 1:1, such as 1:2, such as 1:3, such as 1:4, such as 1:5, such as 1:6, such as 1:7, such as 1:8, such as 1:9, such as 1:10, such as 1:11, such as 1:12, such as 1:13, such as 1:14, such as 1:15.
13. The method according to claim 1, wherein said epitope comprises at least 4 amino acids, such as 5 amino acids, such as 6 amino acids, such as 7 amino acids, such as 8 amino acids, such as 9 amino acids, such as 10 amino acids, such as 11 amino acids, such as 12 amino acids, such as 13 amino acids, such as 14 amino acids, such as 15 amino acids.
14. The method according to claim 1, wherein said method is preceded by a step of approximation of the amount of target protein by establishing a standard curve, such as a forward standard curve or a reverse standard curve.
15. A kit for carrying out the method according to claim 1, comprising at least one binding agent, at least one isotope-labelled internal standard protein, and instructions for carrying out the method.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0057]
[0058]
[0059]
EXAMPLES
Example 1
Selection of Binding Agents
Set of Target Proteins
[0060] Phage display selections was used to find binding agents having affinity for secretome targets using an in-house scFv library. For the development of binding agents, the following target proteins were used during the selection process: TNF, KLK3, REN, IL6, CRP, EPO, IL8, CLU, APOA1 and CGA. All proteins were produced and purified in-house.
Biotinylation of Target Proteins
[0061] Each target protein was diluted to a final concentration of 0.25 μg/μl in a 1×PBS solution containing a 10 times molar excess of biotinylation reagent (EZ-Link Sulfo-NHS-LC-Biotin) and incubated for 1 h in a rotomixer at room temperature (RT). After incubation, the target protein was transferred into a pre-hydrated Slide-A-Lyzer® cassette and any remaining air in the cassette was removed. The Slide-A-Lyzer® was then incubated in dialysis buffer (1×PBS) for 2 hours on a magnetic stirrer at RT. The dialysis buffer was replaced after two hours and the Slide-A-Lyzer® cassette was subsequently incubated overnight on a magnetic stirrer at 4° C. The sample was retrieved from the Slide-A-Lyzer® and transferred to 1.5 ml tubes and the concentration of the sample was determined using absorbance measurements at 280 nm.
Evaluation of Biotinylation
[0062] In order to evaluate whether or not biotinylation had been successful, the target protein was subjected to a bead binding test. 100 μl magnetic beads (Dynabeads™ M-280 Streptavidin, SA beads) were washed thoroughly in PBST (1×PBS, 0.05% Tween20) and then resuspended in 100 μl PBST. 5 μg of biotinylated protein in 200 μl PBST was then incubated with 25 μl beads for 15 min in a rotomixer. The beads were separated from the supernatant and washed 4 times in 1,000 μl PBST and stored on ice. The supernatant was transferred to a new tube with 25 μl beads and this process was repeated a total of three times. The final supernatant was precipitated by addition of 1 ml of acetone and incubated at −20° C. for 60 min. The supernatant was then removed from the precipitate. The beads from the different fractions and the precipitate were then evaluated using SDS-PAGE to ensure sufficient biotinylation and retrieval.
Selection—Round 1
Selection
[0063] 40 μl SA beads per target protein were washed in PBST and then transferred to a 1.5 ml tube. The SA beads were then incubated in 200 pmol biotinylated target protein diluted in PBST to a total volume of 500 μl for 1 h on a rotomixer at RT. Additionally, 40 μl of beads were washed in PBST and incubated with 800 μl of phage library for 1 h in a rotomixer. The beads, incubated in target protein, were washed in 2×1 ml PBST and the supernatant from the beads incubated in the phage solution was mixed with the washed target protein beads and transferred to 96 well plate. The beads and phages were incubated on a KingFisher™ for 3 hours with slow mixing. The beads were then washed in 800 μl PBST 5×1 min with slow mixing and eluted using 500 μl elution buffer (0.25% trypsin, 0.02% Tween20) and 30 min incubation. The eluate was transferred to 1.5 ml tubes and 250 μl aprotinin was added to each tube (0.2 mg/ml).
Amplification
[0064] In order to amplify the eluted phages, 0.6 ml of the eluted solution was transferred to 50 ml of actively growing XL1-blue cells (Agilent Technologies, cat no 200249) and incubated for 30 min at 37° C. The cells were subsequently spun down, and the cell pellet was resuspended in 2 ml supernatant. The resuspended cells were spread on an agar plate supplemented with tetracycline (10 mg/ml), carbenicillin (100 mg/ml), kanamycin (25 mg/ml) and 1% glucose and incubated at 37° C. overnight (O/N).
[0065] The colonies on the agar plate were resuspended in 2×10 ml 2×YT Broth and OD600 was measured for the cell suspension. 45 ml medium (2×YT Broth, TET10/CARB100/GLU1%) was inoculated with the cell suspension to a final OD600 of 0.15-0.2. The cells were incubated at 37° C. until they reached an OD600 of 0.5. The cells were then infected with M13K07 helper phages and incubated for 1 h at 37° C. The cells were subsequently pelleted through centrifugation and the medium was discarded. The pellet was resuspended in 1 ml 2×YT Broth and transferred to baffled flask containing 45 ml 2×YT Broth (tetracycline (10 mg/ml), carbenicillin (100 mg/ml), kanamycin (25 mg/ml), and isopropyl β-d-1-thiogalactopyranoside (0.25 mg/ml)) and incubated O/N at 30° C.
Precipitation
[0066] The culture was transferred to a 50 ml tube and centrifuged at 10000 g and the supernatant was transferred to a 50 ml containing 10 ml PEG/NaCl (20% PEG8000, 2.5 M NaCl) and incubated on ice for 60 min before being centrifuged at 12000 g for 30 min at 4° C. The supernatant was then discarded, and the pellet was resuspended in 2 ml PBST. The resuspended pellet was centrifuged at 10000 g for ten minutes and the supernatant containing the phages was collected in 2 ml tubes.
Selection—Round 2
Selection
[0067] For the second selection round, 20 μl of SA beads per target protein were washed in PBST and transferred to a 1.5 ml tube and incubated in 100 pmol target protein diluted in PBST to a final volume of 500 μl and incubated on a rotomixer at RT for 1 h. The beads were washed with 2×1 ml PBST and 800 μl of the eluted phages from round 1 were added to the beads. The beads were incubated for 1 h at RT in a rotomixer. The beads were washed in 2×1 ml PBST and transferred to a 96-well plate. The beads and phages were incubated in a KingFisher™ for 1.5 hours with slow stirring and then washed 5×1 min with slow stirring in 800 μl PBST. The phages were then eluted with 500 μl elution buffer for 30 min with slow stirring and transferred to 1.5 ml tubes containing 250 μl aprotinin solution.
Amplification
[0068] The phages were amplified by adding 350 μl of the eluate to 10 ml of actively growing (OD600: 0.5-0.7) XL1-blue cells (Agilent Technologies, cat no 200249) in a 50 ml tube and incubated at 37° C. for 30 min. Subsequently 10 ml of 2×YT Broth (TET10/CARB100/GLU1%) was added to the tube and it was incubated for 30 min at 37° C. with shaking at 180 rpm. The cells were then infected with M13K07 helper phages and incubated for 1 h at 37° C. The cells were then pelleted by centrifugation and the supernatant was discarded. The pellet was resuspended in 50 ml 2×YT Broth (TET10/CARB100/KAN25/IPTG0.25) and incubated O/N at 37° C. with shaking at 140 rpm.
[0069] The phages were precipitated as described above.
Selection—Round 3
[0070] For the third round, 800 μl of the precipitated phages from the second round was mixed with 50 pmol target protein and incubated in a rotomixer at RT for 1 h. Meanwhile, 20 μl SA beads per target protein were washed in 2×1 ml PBST. The target protein-phage suspension was transferred into a 96-well plate together with the beads and incubated in a KingFisher™ for 30 min with slow mixing. The beads were subsequently washed with 6×1 min with 800 μl PBST with medium mixing and then eluted over 30 min in elution buffer with slow mixing. The eluates were then transferred to 1.5 ml tubes with 250 μl aprotinin solution.
[0071] The phages were amplified as described for round 2 and then precipitated.
Selection—Round 4
[0072] For the final selection round, 800 μl of the precipitated phages from round three were incubated with 10 pmol target protein and then mixed with 20 μl washed beads in a 96-well plate, as described for round three. The mixture was incubated for 30 min with slow mixing in a KingFisher™ with slow mixing and the beads were subsequently washed 9×1 min in 800 μl PBST with medium mixing. The phages were eluted in 500 μl elution buffer over 30 min with slow mixing.
[0073] The phages from rounds three and four were then amplified as described for round two. 3 ml of each culture were saved for Miniprep purification.
Miniprep Purification
[0074] In order to prepare the genetic material for re-cloning into an scFv expression vector, minipreps were performed using the GeneJET Miniprep kit (Thermo Scientific, #K0503) according to the supplier's instructions. The material was eluted in a final volume of 50 μl and the concentration was determined using a NanoPhotometer NP80 (Implen GmbH; MOnchen, Germany) and stored at −20° C.
[0075] The genetic material was subsequently cloned into an expression vector for optimized production of scFv fused to a FLAG tag, and transformed for production of scFv. The cells were subsequently grown on agar plates and 47 colonies from round three and 48 colonies from round four were selected for each target protein and transferred to 96-well plates and incubated O/N.
Evaluation of Binding Agents
ELISA Evaluation of Binding Agents
[0076] In order to determine which binding agents (in this case scFv fragments) performed the best in terms of signal to noise, all selected binding agents were evaluated against their respective target protein and SA.
[0077] A 384-well plate was coated in SA by addition of SA diluted in PBS to a final concentration of 1 mg/ml and incubation O/N at 4° C. The solution was removed and the plate was subsequently washed in assay buffer (PBS, 0.5% BSA, 0.05% Tween20). Any remaining liquid was removed. The biotinylated target proteins were diluted ten times in assay buffer and were added to half of the wells of the plates. The plate was incubated and subsequently washed in assay buffer. Each clone of the different binding agents was subsequently added to four wells of the plates (two wells with the respective target protein and two wells with SA) and incubated before being washed. HRP-anti-FLAG antibody was added to each well and the plates were incubated before being washed. Substrate was subsequently added to all wells and after quenching, the plates were analyzed through a plate readout at 450 nm.
[0078] For each target protein, the binding agents showing the highest signal to noise were selected and the clones (a total number of 384 clones) were sent for DNA sequencing by Eurofin Genomics. All unique clones were subsequently transferred to new 96-well plates and used for further analysis.
Affinity Screening by Surface Plasmon Resonance (SPR)
[0079] An initial screening was performed on a BIAcore T200 system for which a CM5 chip with immobilized anti-FLAG antibody (#M8592, Sigma-Aldrich) was used for the analysis. For the analysis, the chip was flowed with target protein specific scFv from the bacterial supernatants to saturate the surface of the chip by binding to the FLAG tag present on all scFvs. The chip was thereafter flowed with 50 nM of non-biotinylated target protein. The chip was regenerated using a 10 mM glycine HCl buffer with a pH of 2.1. The binding agents which bound to the target protein with the highest affinity were selected for a more thorough kinetics analysis.
[0080] The same workflow as described above was used for determining the kinetic constants for the binding agents, but instead of flowing the chip with one concentration of target protein a four-fold dilution curve ranging from 0.3 nM to 80 nM was flowed across the chip. The kinetic constants were determined by fitting a 1:1 Langmuir binding model to the response curves.
Evaluation of Target Specificity
[0081] To assess the specificity of the binding agents an ELISA was used. The plates were prepared as described above, but instead of evaluating each binder against its respective target protein and SA, the binding agents were evaluated against all target proteins used in the selection process. Only binding agents showing high specificity towards their target protein were selected.
Screening for Protein Fragment Binding Agents
[0082] In order to assess the ability of the binding agents to bind recombinant protein standards, the affinity of the binding agents to different protein fragments from each respective protein target was evaluated by SPR as described above. Binding agents that were able to bind both target protein and recombinant protein fragments with similar affinity were used for co-capture of target protein and protein fragments for subsequent mass spectrometry (MS) readout.
Capture for MS Readout
Capturing and Digestion for MS Readout
[0083] Protein A-coupled beads were coated with the obtained binding agents for the targets APOA1, IL6 and IL8. 15 μl Protein A-coupled beads (Pierce/Thermo Fisher) per target protein were washed three times in 500 μl washing buffer (0.1 μg/μl casein hydrolysate, 0.3% (w/v) Chaps, PBS). The washing buffer was removed, and the beads were resuspended in 30 μl binding buffer (0.5 μg/μl casein hydrolysate, 0.3% (w/v) Chaps, 1×PBS). The beads were subsequently incubated with 1.2 μg of the respective binding agent in individual tubes and diluted to a final volume of 100 μl in binding buffer. The samples were incubated for 30 min at RT in a rotomixer. The supernatant was removed, and the beads were resuspended in 30 μl binding buffer. Beads coated with binding agent, i.e. “binding agent coated beads” had thus been obtained.
[0084] For the capture of target proteins and stable isotope labeled (Protein Recombinant Isotope Standard (PRecIS)) protein fragments of APOA1, IL6 and IL8, 10 μl of each of the binding agent coated beads were transferred to tubes with 20 μl pre-aliquoted binding buffer in duplicates. The amount of added binding agents was adjusted based on the endogenous amount of target proteins in order to decrease the dynamic range of captured molecules. The relative concentration between APOA1, IL6 and IL8 binding agents was 10:1:1 Additionally, a negative sample consisting of washed bare beads was prepared.
[0085] A mixture of the target proteins and the corresponding isotope-labelled standard proteins (PRecIS) in binding buffer (Table 2) was added to each tube. The samples were diluted to a final volume of 200 μl in binding buffer and incubated for 1 h at RT in a rotomixer. The supernatant was subsequently removed. The beads were thereafter washed twice in 500 μl 1× wash buffer and the supernatant was discarded. 10 μl of 10 mM dithiothreitol in 50 mM ammonium bicarbonate was added to the beads and the samples were incubated at 56° C. for 30 min. 1 μl of 500 mM 2-chloroacetamide was subsequently added to the tubes and the samples were incubated at RT, shielded from light, for 30 min. SOLu-Trypsin (#EMS0004, Sigma Aldrich) was thereafter added to the samples to a final amount of 0.5 μg and the samples were incubated O/N at 37° C. The digestion was quenched by addition 10% formic acid (FA) to a final concentration of 0.5% FA and the supernatant was transferred to a HPLC vial for analysis by MS.
TABLE-US-00002 TABLE 2 Target protein and standard isotope-labelled protein mixture composition. Protein Concentration (uM) APOA1 10 IL6 1 IL8 1 PRecIS APOA1 10 PRecIS IL6 1 PRecIS IL8 1
Mass Spectrometry Analysis
[0086] For the MS analysis of captured proteins digested into peptides, samples were analyzed using a TSQ Altis (Thermo Fisher) coupled to a Dionex Ultimate 3000 liquid chromatography (LC) system (Thermo Fisher) equipped with a cartridge type trap column (160434, Thermo Fisher) and a 15 cm analytical EASY-spray column (ES806A, Thermo Scientific) with a mobile phase consisting of solvent A (3% acetonitrile (ACN), 0.1% FA) and solvent B (95% ACN, 0.1% FA). For the analysis, 10 μl of each sample was loaded onto the column and separated over a 15 min long run with a flow rate of 3 μl/min. The gradient used for the separation of peptides was as follows: 1% solvent B for 0.75 min, 1-30% solvent B for 9.25 min, 30-95% solvent B for 0.1 min, followed by a cycling between 0% and 95% solvent B three times during 3 min, finally followed by a re-equilibration of the column at 1% solvent B for 1.5 min. The MS was operated in an unscheduled SRM mode, monitoring the 10 most intense product ions for all tryptic peptides present both in the PRecIS protein fragments and the target proteins, with a dwell time of 0.5 ms. All data analysis was performed in Skyline Targeted Mass Spec Environment (University of Washington).
Example 2
Determination of Affinity to Target Proteins and Corresponding Standard Proteins
[0087] A set of eight target proteins were selected for assay development based on clinical relevance and reference concentration in plasma. They are listed in Table 3, sorted by increasing plasma concentration reference range for the corresponding protein.
TABLE-US-00003 TABLE 3 Proteins selected for recombinant antibody generation. Protein Modification during selection Reference concentration 1. TNF chemically biotinylated 14 ng/l 2. IL6 chemically biotinylated 5.1 ng/l 3. IL8 chemically biotinylated 6 ng/l 4. KLK3 chemically biotinylated NA 5. CRP chemically biotinylated 1.6 mg/l 6. REN chemically biotinylated 690 ng/l 7. CLU chemically biotinylated 110 mg/l 8. APOA1 chemically biotinylated 1.4 g/l
[0088] Phage display selections were performed to find binding agents for the target proteins. An in-house scFv library was used as described in Example 1. The biotinylated recombinant proteins of Table 3 were subjected to western blot analysis in order to verify their molecular weight prior to phage display selection.
[0089] Successfully biotinylated target proteins were bound to magnetic streptavidin beads and used for phage display selection in a four round setup. Selected binding phages (successfully enriched clones) were eluted using trypsin/aprotinin and re-cloned into a scFv expression vector.
[0090] Binding of the scFv clones from the phage selection was verified by ELISA. Positive scFv clones were selected for surface plasmon resonance (SPR) measurement towards their target protein. Furthermore, those clones were in parallel screened towards isotope-labelled internal standard proteins corresponding to their target protein. The identity of the scFv binding agents and their binding constants were determined using a kinetic SPR setup (Table 4).
Results
[0091] In total, six scFv binding agents towards IL8, eight towards IL6, three towards KLK3 and two binding agents towards APOA0 that successfully recognized the two different forms of their target were obtained. That is, the obtained scFv binding agents recognized their target standard protein as well as its corresponding isotope-labelled internal standard protein.
[0092] The affinity of the binding agent denoted “IL8-12” towards a corresponding isotope-labelled standard protein was determined to be 78 nM, while the affinity of the same binding agent towards the IL8 target protein was more than 3 times lower, determined to be 250 nM (Table 4, entry 5).
TABLE-US-00004 TABLE 14 Binding agents recognizing both a protein and its corresponding fragment Ka Ka Kd ratio KD ratio scFv clone (l/Ms) Kd (1/s) KD (M) (1/Ms) Kd (1/s) KD (M) (Target: (Target: name Antigen Target Target Target PRecIS PRecIS PRecIS PRecIS) PRecIS) AB-IL8-1 IL8 4.9 × 10.sup.6 4.1 × 10.sup.-2 8.4 × 10.sup.-9 4.0 × 10.sup.4 1.3 × 10.sup.-2 3.1 × 10.sup.-7 3.15 0.03 AB-IL8-5 IL8 1.8 × 10.sup.3 1.2 × 10.sup.-3 6.7 × 10.sup.-7 1.5 × 10.sup.4 4.3 × 10.sup.-3 2.8 × 10.sup.-7 0.28 2.39 AB-IL8-7 IL8 4.0 × 10.sup.3 1.8 × 10.sup.-3 4.4 × 10.sup.-7 3.1 × 10.sup.4 1.0 × 10.sup.-2 5.1 × 10.sup.-7 0.18 0.86 AB-IL8-9 IL8 8.2 × 10.sup.4 1.4 × 10.sup.-2 1.7 × 10.sup.-7 3.1 × 10.sup.4 4.1 × 10.sup.-3 1.3 × 10.sup.-7 3.41 1.31 AB-IL8-12 IL8 3.7 × 10.sup.4 9.4 × 10.sup.-3 2.5 × 10.sup.-7 1.1 × 10.sup.9 8.5 × 10.sup.1 7.8 × 10.sup.-8 1.1 × 10.sup.-4 3.21 AB-IL8-14 IL8 3.4 × l0.sup.5 1.0 × 10.sup.-2 3.0 × 10.sup.-8 1.6 × 10.sup.4 7.8 × 10.sup.-3 4.9 × 10.sup.-7 1.28 0.06 AB-IL6s-3 IL6 4.0 × 10.sup.5 9.8 × 10.sup.-5 2.5 × 10.sup.-10 1.0 × 10.sup.5 6.0 × 10.sup.-3 5.9 × 10.sup.-8 0.02 0.00 AB-IL6s-4 IL6 1.4 × 10.sup.5 4.8 × 10.sup.-4 3.5 × 10.sup.-9 2.6 × 10.sup.5 1.5 × 10.sup.-3 5.8 × 10.sup.-8 0.32 0.06 AB-IL6s-6 IL6 1.7 × 10.sup.5 5.0 × 10.sup.-4 3.0 × 10.sup.-9 9.9 × 10.sup.4 4.9 × 10.sup.-3 4.9 × 10.sup.-8 0.10 0.06 AB-IL6s-9 IL6 3.7 × 10.sup.5 5.5 × 10.sup.-4 1.5 × 10.sup.-9 1.1 × 10.sup.5 3.7 × 10.sup.-3 3.5 × 10.sup.-8 0.15 0.04 AB-IL6s-10 IL6 2.1 × 10.sup.5 2.2 × 10.sup.-4 1.0 × 10.sup.-9 9.9 × 10.sup.4 4.4 × 10.sup.-3 4.4 × 10.sup.-8 0.05 0.02 AB-IL6s-12 IL6 5.7 × 10.sup.4 2.6 × 10.sup.-4 4.5 × 10.sup.-9 7.8 × 10.sup.4 4.2 × 10.sup.-3 5.4 × 10.sup.-8 0.06 0.08 AB-IL6s-16 IL6 2.1 × 10.sup.5 5.8 × 10.sup.-4 2.7 × 10.sup.-9 1.1 × 10.sup.5 4.6 × 10.sup.-3 4.1 × 10.sup.-8 0.13 0.07 AB-IL6s-18 IL6 1.8 × 10.sup.5 6.5 × 10.sup.-4 3.6 × 10.sup.-9 9.6 × 10.sup.4 3.6 × 10.sup.-3 3.8 × 10.sup.-8 0.18 0.09 AB-KLK3-1 KLK3 2.3 × 10.sup.5 4.2 × 10.sup.-3 1.9 × 10.sup.-8 1.2 × 10.sup.9 9.0 × 10.sup.1 7.6 × 10.sup.-8 4.7 × 10.sup.-5 0.25 AB-KLK3-5 KLK3 2.9 × 10.sup.4 5.9 × 10.sup.-3 2.0 × 10.sup.-7 7.5 × 10.sup.3 5.3 × 10.sup.-3 7.1 × 10.sup.-7 1.11 0.28 AB-KLK3-15 KLK3 2.4 × 10.sup.5 1.1 × 10.sup.-2 4.5 × 10.sup.-8 1.1 × 10.sup.5 1.2 × 10.sup.-2 1.1 × 10.sup.-7 0.92 0.41 AB-KLK3-1 KLK3 2.3 × 10.sup.5 4.2 × 10.sup.-3 1.9 × 10.sup.-8 8.5 × 10.sup.4 1.0 × 10.sup.-2 1.2 × 10.sup.-7 0.42 0.16 AB-KLK3-5 KLK3 2.9 × 10.sup.4 5.9 × 10.sup.-3 2.0 × 10.sup.-7 3.8 × 10.sup.4 3.4 × 10.sup.-2 9.1 × 10.sup.-7 0.17 0.22 AB-KLK3-15 KLK3 2.4 × 10.sup.5 1.1 × 10.sup.-2 4.5 × 10.sup.-8 7.4 × 10.sup.4 8.0 × 10.sup.-3 1.1 × 10.sup.-7 1.38 0.41 AB-APOA1-6 APOA1 2.5 × 10.sup.4 4.1 × 10.sup.-3 1.7 × 10.sup.-7 8.0 × 10.sup.4 3.8 × 10.sup.-2 4.8 × 10.sup.-7 0.11 0.35 AB-APOA1-25 APOA1 4.2 × 10.sup.6 3.4 × 10.sup.-1 7.9 × 10.sup.-8 1.1 × 10.sup.5 4.3 × 10.sup.-2 4.0 × 10.sup.-7 7.91 0.20
Example 3
Co-Capture of Target Proteins and Corresponding Standard Proteins in a Complex Background
Preparation of Mixture of Target Proteins and Corresponding Standard Proteins
[0093] A pool of target proteins and corresponding standard proteins was established by pooling 10 pmol secretome APOA1 (30736 Da), 1 pmol secretome IL8 (11766 Da) and 1 pmol secretome IL6 (23470 Da) into a buffer comprising 1% casein hydrolysate, 0.3% (w/v) Chaps, and 1×PBS. To this mixture, the corresponding standard proteins were pooled in equimolar amounts (10 pmol PRecIS APOA1, HPRR3450265; 1 pmol PRecIS IL6, HPRR330007; and 1 pmol PRecIS IL8, HPRR2700195; Edfors et al (2019), J Proteome Res 18(7):2706-2718).
[0094] In addition, a sample of the mixture was spiked into a complex background of bovine serum albumin (BSA). This mixture served as a baseline for the following experimental procedure.
Preparation of Beads Comprising Binding Agent
[0095] 60 μl Protein A (Thermo Scientific) magnetic beads were washed three times with 500 μl wash buffer (0.1 μg/μl casein hydrolysate, 0.3% (w/v) Chaps, 1×PBS). Supernatant was removed after first collecting the beads using a magnetic stand and beads were re-dissolved in 120 μl binding buffer (0.5 μg/μl casein hydrolysate, 0.3% (w/v) Chaps, 1×PBS). A total of 30 μl beads were split into four separate tubes (for the binding agents towards IL6, IL8, APOA1 and negative control, respectively). The negative control consisted of bare beads with no binding agent. A total of 1.2 μg binding agent (anti-APOA1, anti-IL6 and anti-IL8 scFv (denoted IL8-5)), respectively, was immobilized onto the Protein A magnetic beads, by incubation for 30 minutes. Excess buffer was removed after first collecting the beads using a magnetic stand.
Co-Capturing
[0096] 10 μl of the prepared pool of target proteins and corresponding standard proteins in casein background were added to each respective tube of beads comprising binding agent, together with 190 μl binding buffer. Target proteins and corresponding standard proteins were captured by incubation for 1 h at RT on a rotor mixer. The supernatant was then removed, and the beads were washed once with 500 μl 1× wash buffer (0.01 μg/μl casein hydrolysate, 0.03% (w/v) Chaps, 0.1×PBS) and once with 500 μl 0.1× wash buffer.
Digestion and Analysis
[0097] Captured proteins were reduced on the beads following the addition of 10 μl 10 mM DTT in 50 mM ammonium bicarbonate and incubated for 30 minutes at 56° C. The proteins were alkylated following addition of 2-chloroacetamide (CAA) to a final concentration of 50 mM and incubated in the dark for 30 minutes at RT. Digestion was performed by the addition of 0.5 μg trypsin to the sample. The sample was incubated overnight at 37° C. The reaction was quenched with formic acid to a final concentration of 0.5% (v/v). The beads were removed using a magnet, and the peptide digest was analyzed using LC-MS/SRM after addition on BSA.
Results
[0098] The mass spectrometry read-out revealed that the binding agent anti-IL8 scFv successfully co-captures the target protein together with its corresponding standard protein (PRecIS) (
[0099]
[0100] In addition, the ratio between target protein and corresponding PRecIS was quantified for the pool of target proteins (APOA1, IL6 and IL8 and corresponding PRecIS) diluted in BSA, and quantified without capturing (“Mix”). The “Mix” can be considered as the baseline for this experimental procedure. The same ratio was quantified for a non-diluted pool of the target proteins APOA1, IL6 and IL8 and corresponding PRecIS without capturing (“Target”). The “Target” represents a pool of non-diluted recombinant proteins and serves as a positive control.
[0101] Each protein name on the x-axis in
CONCLUSION
[0102] This experiment shows that IL8 and PRecIS-IL8 were specifically captured in the co-capturing using an anti-IL8 scFv, compared to anti-APOA1, anti-IL6 and the negative control. Thus, binding agents generated towards full length protein sequences can co-capture proteins with identical and or similar epitopes presented on their surface. The difference in affinity of the generated scFv binding agent for the target protein and the internal standard, respectively, resulted in less effective capturing of the PRecIS relative to its full-length recombinant protein (secretome). However, it does not matter which of the target protein or the PRecIS that binds with higher affinity, as long as one knowns the difference in affinity. As discussed in the general sections of the disclosure, this effect can be accounted for if the difference in efficiency is known. To conclude, such pull-down or co-capturing experiments can therefore successfully be used for quantitative proteomics.
Itemized Listing of Embodiments
[0103] 1. A method for measuring the amount of a target protein in body fluid, the method comprising the following consecutive steps: [0104] preparing a sample suspected to comprise said target protein and comprising a known amount of an isotope-labelled internal standard protein, said standard protein consisting of a fragment of said target protein, [0105] bringing said sample into contact with a solid support comprising a binding agent, [0106] washing said solid support to remove unbound members of the sample, [0107] digesting said target protein and said standard protein to provide a digested sample, [0108] subjecting said digested sample to mass spectrometry, and [0109] determining the amount of said target protein in said sample by comparison with said standard, wherein
[0110] said binding agent is capable of binding an epitope present in both said target protein and said standard protein.
[0111] 2. The method according to item 1, wherein said target protein is a soluble protein.
[0112] 3. The method according to item 2, wherein said target protein is a water soluble protein.
[0113] 4. The method according to any one of the preceding items, wherein said target protein is an actively secreted protein.
[0114] 5. The method according to any one of the preceding items, wherein said secretory protein is a protein secreted into blood.
[0115] 6. The method according to any one of the preceding items, wherein said target protein is an FDA qualified biomarker.
[0116] 7. The method according to any one of the preceding items, wherein said target protein is selected from the group consisting of a cytokine, a chemokine, an interleukin, an interferon, a hormone, a neuropeptide, a growth factor, a receptor, a protein involved in transport, a protein involved in development, an enzyme, an enzyme inhibitor, a protein involved in the immune system, a protein involved in coagulation, a protein involved in the complement pathway, an acute phase protein and a cell adhesion protein.
[0117] 8. The method according to any one of the preceding items, wherein said solid support is selected from the group consisting of a bead, such as a magnetic bead, and a column.
[0118] 9. The method according to any one of the preceding items, wherein said digestion is carried out by means of a proteolytic enzyme.
[0119] 10. The method according to item 9, wherein said proteolytic enzyme is trypsin.
[0120] 11. The method according to any one of the preceding items, wherein the digested sample comprises at least one isotopically labeled standard peptide consisting of between 6 and 25 amino acids.
[0121] 12. The method according to any one of the preceding items, wherein said standard protein is a recombinant protein.
[0122] 13. The method according to any one of items 1-11, wherein said standard protein is a synthetic protein.
[0123] 14. The method according to any one of the preceding items, wherein said standard protein comprises at least two cleavage sites for said proteolytic enzyme.
[0124] 15. The method according to any one of the preceding items, wherein said standard protein is labelled with at least one isotope selected from the group consisting of .sup.15N, .sup.13C and/or .sup.18O.
[0125] 16. The method according to any one of the preceding items, wherein said binding agent is an antibody or an antibody fragment.
[0126] 17. The method according to item 16, wherein said antibody or antibody fragment is a monoclonal antibody or fragment thereof.
[0127] 18. The method according to item 16, wherein said antibody or antibody fragment is a polyclonal antibody or fragment thereof.
[0128] 19. The method according to any one of items 16-18, wherein said antibody fragment is an scFv.
[0129] 20. The method according to any one of items 1-16, wherein said binding agent is an antibody mimetic.
[0130] 21. The method according to any one of the preceding items, wherein said measuring comprises measuring the amount of at least two target proteins, such as three target proteins, such as four target proteins, such as five target proteins, such as six target proteins, such as seven target proteins, such as eight target proteins, such as nine target proteins, such as ten target proteins.
[0131] 22. The method according to item 21, wherein said at least two target proteins are present in said sample at a relative concentration of at least 10× in difference, such as 100× in difference, such as 1000× in difference, such as 10 000× in difference, such as 100 000× in difference, such as 1000 000× in difference, such as 10 000 000× in difference.
[0132] 23. The method according to any one of the preceding items, wherein said at least one target protein suspected to be present in said sample, is present in said sample in a concentration of between 10.sup.−4 and 10.sup.−10 M, such as between 10.sup.−6 and 10.sup.−7 M.
[0133] 24. The method according to any one of the preceding items, wherein said target protein and said standard protein bind to the binding agent with comparable affinity, at a ratio of KD values such as 1:1, such as 1:2, such as 1:3, such as 1:4, such as 1:5, such as 1:6, such as 1:7, such as 1:8, such as 1:9, such as 1:10, such as 1:11, such as 1:12, such as 1:13, such as 1:14, such as 1:15.
[0134] 25. The method according to any one of the preceding items, wherein said epitope comprises at least 4 amino acids, such as 5 amino acids, such as 6 amino acids, such as 7 amino acids, such as 8 amino acids, such as 9 amino acids, such as 10 amino acids, such as 11 amino acids, such as 12 amino acids, such as 13 amino acids, such as 14 amino acids, such as 15 amino acids.
[0135] 26. The method according to any one of the preceding items, wherein said epitope is linear.
[0136] 27. The method according to any one of the preceding items, wherein said body fluid is selected from the group consisting of plasma, serum, cerebrospinal fluid, urine, dry blood spots and saliva.
[0137] 28. The method according to any one of the preceding items, wherein said body fluid is from a mammal, e.g. human.
[0138] 29. The method according to any one of the preceding items, wherein said method is preceded by a step of approximation of the amount of target protein by establishing a standard curve, such as a forward standard curve or a reverse standard curve.
[0139] 30. The method according to any one of the preceding items, wherein said step of digesting said target protein and said standard protein is preceded by a step of eluting said target protein and said standard protein.
[0140] 31. The method according to any one of the preceding items, wherein said step of subjecting said digested sample to mass spectrometry is preceded by a step of liquid chromatography.
[0141] 32. The method according to any one of the preceding items, wherein said standard protein is added to said sample in an amount approximately equal to the amount of said target protein suspected to be present in that sample.
[0142] 33. The method according to any one of the preceding items, wherein said mass spectrometry is selected from the list consisting of tandem mass spectrometry with data dependent acquisition mode, tandem mass spectrometry with data independent acquisition mode and tandem mass spectrometry with selective reaction monitoring mode.
[0143] 34. A kit for carrying out the method according to any one of the preceding items, comprising
[0144] at least one binding agent,
[0145] at least one isotope-labelled internal standard protein, and
[0146] instructions for carrying out the method.
[0147] 35. The kit according to item 34, wherein a target protein and said standard protein bind to said binding agent with comparable affinity, such as at a ratio of KD values such as 1:1, such as 1:2, such as 1:3, such as 1:4, such as 1:5, such as 1:6, such as 1:7, such as 1:8, such as 1:9, such as 1:10, such as 1:11, such as 1:12, such as 1:13, such as 1:14, such as 1:15.
[0148] 36. The kit according to any one of items 34 or 35, wherein said binding agent is a monoclonal antibody.
[0149] 37. The kit according to any one of items 34-36, wherein said binding agent is an scFv fragment.