Method for rapidly fishing phage library in real time

Abstract

The invention provides a method for fishing an antibody from a phage library, which can be quickly monitored in real time. Compared with a traditional bacteriophage library which needs three rounds of elutriation to enrich positive clones, the method disclosed by the invention has the advantages that the positive clones can be obtained by fishing in only one step, the combination and dissociation of a bacteriophage display antibody and an antigen can be monitored in real time, and low-affinity clones can be eliminated in time; and the screening success rate is quickly and accurately improved. The method comprises the following specific steps: curing an antigen to the surface of the BLI sensor; immersing into the bacteriophage library and fishing the bacteriophage display antibody; non-specific binding clones are removed under a high-intensity washing condition, and screening failure caused by high noise caused by high viscosity of bacteriophage is reduced; clone obtained through fishing is subjected to signal amplification, and the situation that measurement cannot be conducted due to a low response value is avoided; eluting to obtain a specifically bound clone on the sensor; and carrying out monoclonal phage ELISA verification on the clones obtained by fishing.

Description

Technical Field

[0001] This invention relates to an efficient, rapid, and sensitive antibody discovery technology, specifically to a method and application of using biological layer interference (BLI) technology to fish for phage-displayed antibody libraries. Background Technology

[0002] Biopharmaceuticals account for an increasingly larger proportion of innovative drugs, among which antibody drugs, such as monoclonal antibodies, multispecific antibodies, and ADCs, are becoming increasingly popular research topics. However, antibody discovery is the first hurdle restricting the rapid development of antibody drugs. Currently, antibody discovery pathways include hybridoma, single B cell screening, phage display antibody library panning, and AI-based antibody generation. Phage display antibody library panning, as a classic antibody discovery method, is simple to use and has a high tolerance for errors. The steps are: (1) incubate the phage library with the coated antigen; (2) wash and remove unbound phages; (3) elute the phages bound to the antigen; (4) infect E. coli with the help of helper phages to amplify the eluted candidate phages. The above steps (called panning) are repeated 2-3 times to enrich the phages bound to the target antigen. (5) infect E. coli with the phages obtained in the last round of panning and plate them to obtain positive phage clones. While the screening process is standardized, its drawback lies in the need for 2-3 rounds of antibody enrichment, requiring monitoring of the enrichment efficiency and affinity of the phage antibody library in each round. For different antigens, screening conditions need to be re-explored, making standardization difficult. Phage display antibody library screening methods typically use multi-well plates or immunotubes, which cannot provide real-time monitoring and feedback of the screening process. The phages obtained in the first round need to be infected with *E. coli* to prepare a second-round library. Enrichment efficiency must be calculated, and phage ELISA must be performed to compare the affinity of the input and output libraries before the success of this round of screening can be assessed. Exploring screening conditions for different antibodies is also necessary, resulting in a long and cumbersome process. Therefore, a sensitive and rapid phage antibody screening method is urgently needed. This invention designs a method for phage library "fishing" using BLI technology. This method monitors the binding and dissociation processes in real time, intervenes in the "fishing," and washes away the phage-displayed antibody process, obtaining high-affinity antibodies in a single round of "fishing."

[0003] BLI is a label-free technique that converts optical interference signals occurring on the surface of a biosensor into real-time response signals. BLI technology is commonly used for antibody affinity assays, but there is no precedent for its application to phage libraries. This is mainly because phages have high viscosity and complex composition, severely interfering with binding signals. Capturing weak binding signals amidst complex noise becomes extremely difficult. This invention attempts to improve noise reduction and signal amplification, successfully achieving one-step testing at the library level. It optimizes the traditional three-round phage display antibody panning to one round, reducing the original 6-7 day experimental cycle to one day, and improving the efficiency of antibody panning in a timely and accurate manner. Summary of the Invention

[0004] The technical problem to be solved by this invention is to overcome the problems of cumbersome and inefficient process in the current phage display antibody library selection, and to propose a method that can obtain high-affinity antibodies by real-time monitoring and one round of selection of phage display antibody libraries.

[0005] To address the aforementioned technical problems, this invention discloses a method and application for using a biosensor (e.g., a BLI sensor) to fish for bacteriophage-displayed antibody libraries. The method includes the following steps:

[0006] (1) Preparation of reagents and BLI instruments,

[0007] (2) The BLI sensor immobilizes the antigen.

[0008] (3) BLI sensor for phage display antibody display

[0009] (4) Wash the BLI sensor to remove non-specifically bound bacteriophages.

[0010] (5) Amplify the signal of the phage display antibody bound to the BLI sensor.

[0011] (6) Elute the phages bound to the BLI sensor.

[0012] (7) The bacteriophages obtained after elution were used to infect Escherichia coli, plated, and cultured.

[0013] (8) Select clones and verify them.

[0014] The specific operation of step (1) is as follows.

[0015] 1) Immerse the BLI sensor in PBST buffer (PBS containing 0.02% Tween 20) for at least 10 minutes to remove sucrose from the sensor surface. The preferred BLI sensor is a streptavidin (SA) sensor or a superstreptavidin sensor.

[0016] (SSA) sensor.

[0017] 2) Turn on the BLI instrument and warm it up for more than 30 minutes, and preset the fishing program.

[0018] 3) Prepare antigen (e.g., human Trop2) solutions using PBST, with an antigen concentration of [missing value].

[0019] The concentration is 1–100 μg / mL, preferably 5, 10, 15 or 20 μg / mL, and the label on the antigen is preferably biotin.

[0020] 4) Prepare a washing buffer, such as PBST+NaCl, with a sodium chloride concentration of at least [missing value].

[0021] 10 mM, preferably at least 50 mM, more preferably 100–1000 mM or

[0022] 150~800mM.

[0023] 5) Dilute the phage library with washing buffer. The amount of phage added should be at least 1, 10, 100, 1000 or 10000 times the library capacity, preferably 100-1000 times, and the volume should preferably be 200 μL.

[0024] 6) Dilute the anti-M13 antibody (HRP-conjugated) with wash buffer to a final concentration of [missing value].

[0025] 0.5 mg / mL or higher, preferably 1 mg / mL or 2 mg / mL, with a dilution factor of [missing value].

[0026] 100 to 10,000 times, preferably at least 500, 1,000 or 2,000 times.

[0027] 7) Prepare a 3,3′-diaminobenzidine (DAB) working solution using PBST; prepare a 3-amino-9-ethylcarbazole (AEC) working solution; and / or prepare a 4-aminoantipyrine:phenol working solution, wherein the ratio of 4-aminoantipyrine to phenol is 1-5:25, preferably 1-3:25, for example 1:25, 2:25, or 3:25, and the concentration of 4-aminoantipyrine is [missing information].

[0028] 1mM to 10mM, preferably at least 2mM.

[0029] 8) Prepare trypsin working solution using PBST at a concentration of 25–250 μg / mL.

[0030] 9) Following the preset procedure, add PBST, antigen, phage library, washing buffer, anti-M13 antibody (HRP-conjugated) working solution, and HRP substrate working solution to the corresponding wells of the 96-well plate.

[0031] The specific operation of step (2) is as follows.

[0032] 1) The BLI instrument is used to perform baseline operation by immersing the BLI sensor into the PBST well according to the preset program for 30–200 s, preferably at least 60 s, with a shaking speed of [missing information].

[0033] 200~1000rpm.

[0034] 2) The BLI instrument immerses the BLI sensor into the antigen well according to the preset program for a time of [time missing].

[0035] The shaking speed is 200-1000 rpm, with a shaking time of 300-1200 s, preferably 600 s, and the BLI signal is 0.2 nm or higher, preferably 1 nm, 1.5 nm, 2 nm, 2.5 nm or 3 nm or higher.

[0036] The specific operation of step (3) is as follows.

[0037] 1) The BLI instrument is used to perform baseline operation by immersing the BLI sensor into the PBST well according to the preset program for 30–200 s, preferably at least 60 s, with a shaking speed of [missing information].

[0038] 200~1000rpm.

[0039] 2) The BLI instrument immerses the BLI sensor in the phage library according to the preset program for 100-1800s, preferably 600-1000s, with a shaking speed of 200-1000rpm, preferably 400-800rpm, and monitors the BLI signal in real time.

[0040] The specific operation of step (4) is as follows.

[0041] 1) The BLI instrument immerses the BLI sensor in the washing buffer well according to the preset program for 100-1800s, with a shaking speed of 200-1000rpm. Multiple washing wells are set, such as 2-8, preferably 3, and the BLI signal is monitored in real time.

[0042] The specific operation of step (5) is as follows.

[0043] 1) The BLI instrument is programmed to immerse the BLI sensor in the anti-M13 antibody (HRP-conjugated) well for 100–1800 s, preferably 300 s, with a shaking speed of [missing information].

[0044] 200-1000 rpm, real-time monitoring of BLI signal.

[0045] 2) The BLI instrument is washed by immersing the BLI sensor in the washing buffer well according to the preset program for 100–1800 s, preferably 300 s, with a shaking speed of [missing information].

[0046] 200-1000 rpm, real-time monitoring of BLI signal.

[0047] 3) The BLI instrument is programmed to immerse the BLI sensor in the HRP substrate working fluid well for 100–1800 s, preferably 120 s, with a shaking speed of [missing information].

[0048] 200-1000 rpm, real-time monitoring of BLI signal.

[0049] The specific operation of step (6) is as follows.

[0050] 1) The BLI instrument immerses the BLI sensor in the trypsin working solution according to the preset program.

[0051] The time for drilling is 300–3600 s, preferably 2000–3000 s, and even more preferably...

[0052] 2300s, shaking speed of 200-1000rpm, preferably 500-1000rpm, with real-time monitoring of BLI signal.

[0053] 2) Add 10% AEBSF to the working well of the trypsin solution to stop elution and obtain the phage display antibody that binds to the antigen.

[0054] The specific operation of step (7) is as follows.

[0055] 1) Add an equal volume of logarithmic growth phase (OD600) to the phage elution buffer.

[0056] 0.4%) of Escherichia coli, preferably TG1, were cultured statically at 37℃.

[0057] 2) Spread all bacterial culture onto AG-2YT (containing Amp and glucose) solid culture medium.

[0058] Incubate overnight at 37°C, inverted. Amp concentration should be 50 μg / mL or higher, preferably 100 μg / mL. Glucose concentration should be 2% (w / v) or higher, preferably 2%.

[0059] The specific operation of step (8) is as follows.

[0060] 1) Select single clones and prepare phages for ELISA identification.

[0061] 2) Perform Sanger sequencing and analysis on ELISA-positive phage clones.

[0062] 3) Select sequences to construct vectors, express and purify them to obtain antibodies.

[0063] 4) Use a BLI instrument to determine the affinity between the antibody and the antigen.

[0064] In one embodiment, the wash buffer is prepared in PBS, PBST, TAE, TBE, or MOPS buffer. In one embodiment, the wash buffer further contains a cation. In one embodiment, the cation is a monovalent cation, such as sodium, potassium, or ammonium ions. In one embodiment, the cation is a divalent cation, such as calcium or magnesium ions. In one embodiment, the wash buffer further contains anions. In one embodiment, the anion is a monovalent anion, such as chloride, iodide, bromide, nitrate, nitrite, bicarbonate, bisulfate, bisulfite, or dihydrogen phosphate. In one embodiment, the anion is a divalent anion, such as carbonate, sulfate, sulfite, or hydrogen phosphate. In one embodiment, the anion is a trivalent anion, such as phosphate. In one embodiment, the wash buffer contains at least 10 mM, such as 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1200, 1500, 2000 mM or even higher concentrations of anions. In one embodiment, the wash buffer is prepared in PBST buffer and additionally contains 10-1000 mM, such as 150-800 mM, preferably 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 mM NaCl.

[0065] In one embodiment, the HRP substrate is 4-aminoantipyrine and phenol. In one embodiment, the ratio of 4-aminoantipyrine to phenol is 1-5:25, preferably 1-3:25, such as 1, 1.5, 2, 2.5, or 3:25. In one embodiment, the concentration of 4-aminoantipyrine is 1 mM to 10 mM, preferably at least 2 mM, such as 2, 3, 4, or 5 mM. In one embodiment, the HRP substrate working solution is prepared in a phosphate buffer. In one embodiment, the phosphate buffer contains one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate, such as two. In one embodiment, the concentration of the phosphate buffer is 0.01-1 M, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 M. In one embodiment, the phosphate buffer solution is composed of disodium hydrogen phosphate 20.7472 g / L, sodium dihydrogen phosphate 3.1167 g / L, and pH...

[0066] 7.2–7.6. In one embodiment, the 4-aminoantipyrine:phenol working solution is a 0.1 M phosphate buffer (disodium hydrogen phosphate 20.7472 g / L, sodium dihydrogen phosphate 3.1167 g / L, pH 7.2–7.6) containing 2 mM 4-aminoantipyrine, 25 mM phenol, and 0.8 mM H2O2. Attached Figure Description

[0067] Figure 1 Using washing buffer with different concentrations of salt ions, the BLI sensor was used to fish for phages to display antibodies, and the sensor signal response results were obtained.

[0068] Figure 2 Using different HRP substrates, the BLI sensor was used to fish for phages to display antibodies, and the sensor signal response results were observed.

[0069] Figure 3 : BLI sensor for phage display antibody library, sensor signal response results.

[0070] Figure 4 Comparison of the screening efficiency (phage ELISA positivity rate) of the method of this invention with that of traditional screening methods.

[0071] Figure 5 Example: Affinity test results of antibody and antigen. Detailed Implementation

[0072] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Specific details not specified in the examples are performed under conventional conditions in the art or conditions recommended by the manufacturer.

[0073] Example 1: Effect of different concentrations of salt ion washing buffer on antibody signal displayed by phage.

[0074] (I) Experimental Methods

[0075] 1. Preparation of reagents and BLI instruments

[0076] 1.1. The BLI sensor (Sartorius, SSA Biosensor) was placed in PBST (containing 0.02%...

[0077] Soak the sensor in Tween 20 PBS buffer for at least 10 minutes to remove sucrose from the sensor surface.

[0078] 1.2. Power on the BLI instrument (Sartorius, Octet RED384) and preheat for 30 minutes.

[0079] The experiment was conducted with a pre-set program, setting up three experimental groups and three corresponding control groups.

[0080] 1.3. Dilute human Trop-2 (Bipsys, biotin, histidine tag) to 10 μg / mL with PBST.

[0081] 1.4. Prepare washing buffers with different salt ion concentrations: Washing buffer A (PBST + 50mM NaCl), Washing buffer B (PBST + 300mM NaCl), and Washing buffer C.

[0082] (PBST+800mM NaCl).

[0083] 1.5. 30 μL of the phage display library (a self-built alpaca natural nanobody library, with a capacity of 3E+9 pfu, a titer of 1E+14 pfu / mL, and an insertion rate of 96%) stock solution was separately...

[0084] Dilute with 470 μL of washing buffers A, B, and C to obtain the working solution for the phage display library.

[0085] The amount of bacteriophages added was 1000 times the library capacity.

[0086] 1.6. Dilute the anti-M13 antibody (HRP-conjugated) with washing buffers A, B, and C, respectively.

[0087] (Sino Biological, 11973-MM05T-H) 1000 times, to obtain the working solution of anti-M13 antibody.

[0088] 1.7. Preparation of 1X 3,3′-diaminobenzidine (DAB) using PBST (Sigma,

[0089] D4293) working fluid.

[0090] 1.8. Following the preset procedure, take the antigen, phage library, washing buffer, and anti-M13.

[0091] 200 μL each of the antibody (HRP-conjugated) working solution and the DAB working solution were added to the corresponding wells of a 96-well plate (Greiner, 655209).

[0092] 2. Using the BLI instrument to fish for phage display antibodies

[0093] 2.1. Following the preset procedure, the BLI instrument takes 6 BLI sensors and immerses them in the PBST wells for baseline operation for 60 seconds, with a shaking speed of 1000 rpm. At this time, the BLI signal is recorded as 0 nm.

[0094] 2.2. The BLI instrument was programmed to immerse the three experimental BLI sensors into the antigen wells and the three control BLI sensors into the PBST wells for a set time.

[0095] At 600s and a shaking speed of 1000rpm, the BLI instrument detected an increase in the thickness of the biological layer of the BLI sensor in the experimental group, and the BLI signal rose to 3nm, indicating that the antigen was successfully immobilized on the sensor.

[0096] 2.3. The BLI instrument performs baseline operation by immersing the BLI sensor into the PBST well according to the preset procedure for 60 seconds, with a shaking speed of 1000 rpm.

[0097] 2.4. Following the preset program, the BLI instrument immerses the BLI sensor in the wells of the phage display antibody library diluted with washing buffers A, B, and C, respectively, for 600 seconds, with a shaking speed of 400 rpm.

[0098] In the experimental group, the antigen-captured phage on the BLI sensor displayed antibodies, and the BLI signal increased.

[0099] 2.5. The BLI instrument washes the BLI sensor by immersing it in the corresponding washing buffer well according to the preset program. The shaking speed is 1000 rpm. There are 3 washing wells, and the washing times are 150s, 300s and 150s respectively. The phages that are not specifically bound to the sensor are washed off, which is reflected as a decrease in the BLI signal.

[0100] 2.6. The BLI instrument immerses the BLI sensor in the anti-M13 antibody (HRP-conjugated) well according to the preset program for 300 seconds, with a shaking speed of 1000 rpm. The anti-M13 antibody binds to the phage on the sensor, and the BLI signal increases.

[0101] 2.7. The BLI instrument is washed by immersing the BLI sensor in the corresponding washing buffer well according to the preset program for 300 seconds, with a shaking speed of 1000 rpm.

[0102] 2.8. The BLI instrument is set up by immersing the BLI sensor in the DAB working fluid orifice according to the preset program for 120 seconds, with a shaking speed of 1000 rpm. The HRP catalyst on the sensor reacts with the DAB.

[0103] The H2O2 reaction produces a brown precipitate on the sensor, thickening the biolayer and resulting in an increase in the BLI signal, indicating that the BLI sensor has captured specific phage display antibodies.

[0104] (II) Experimental Results

[0105] The amplified signal value was obtained by subtracting the BLI signal from the control group's BLI signal in the experimental group. The experimental results are shown in Table 1 and... Figure 1 .

[0106] Table 1: Effects of different washing buffers on the amplification of antibody signals from phage display on the BLI sensor

[0107] experimental group BLI signal value, nm Washing buffer A 0.7 Washing Buffer B 1.0 Washing buffer C 0.22

[0108] Experimental results showed that the wash buffer group B had the highest amplification signal, indicating that at this salt concentration (PBST + 300mM NaCl), the BLI sensor achieved the highest efficiency in displaying antibodies against specific phages and washing non-specifically bound phages. The high salt concentration group (PBST + 800mM NaCl) also showed a relatively high amplification signal, indicating that this salt concentration of wash buffer could also be used for screening. The low salt concentration group (PBST + 50mM NaCl) had an amplification signal below 0.3nm, indicating failure in displaying antibodies against phages.

[0109] Example 2: Effects of different HRP substrates on signal amplification of phage display antibody library using BLI sensor

[0110] (I) Experimental Methods

[0111] 1. Preparation of reagents and BLI instruments

[0112] 1.1. Refer to section “1.1” in Example 1.

[0113] 1.2. Refer to section “1.2” in Example 1.

[0114] 1.3. Refer to section “1.3” in Example 1.

[0115] 1.4. Prepare washing buffer B according to section “1.4” in Example 1.

[0116] 1.5. Referring to section “1.5” in Example 1, the phage display library was diluted with washing buffer B to obtain the working solution of the phage display library.

[0117] 1.6. Dilute anti-M13 antibody (HRP-conjugated) with wash buffer B (Sino

[0118] Biological, 11973-MM05T-H) 1000 times, to obtain anti-M13 antibody working solution.

[0119] 1.7. Referring to section 1.7 of Example 1, prepare 1X DAB working solution; prepare 1X AEC (Vector, SK-4200) working solution according to the instructions; prepare 4-aminoantipyrine:phenol working solution in 0.1M phosphate buffer (disodium hydrogen phosphate).

[0120] 20.7472 g / L, sodium dihydrogen phosphate 3.1167 g / L, pH 7.2~7.6 (Merck,

[0121] Dissolve 2 mM 4-aminoantipyrine (Merck, A4382), 25 mM phenol (Merck, W322318), and 0.8 mM H2O2 (Merck, HX0636) in P4417 to obtain the 1X working solution.

[0122] 1.8. Referring to section “1.8” in Example 1, different HRP substrates are added to the corresponding well plates.

[0123] 2. Using the BLI instrument to fish for phage display antibodies

[0124] 2.1. Refer to section “2.1” in Example 1.

[0125] 2.2. Refer to section “2.2” in Example 1.

[0126] 2.3. Refer to section “2.3” in Example 1.

[0127] 2.4. The BLI instrument was set up by immersing the BLI sensor in the phage display antibody library well diluted with washing buffer B for 600 seconds and shaking at 400 rpm. In the experimental group, the antigen captured the phage display antibody on the BLI sensor, and the BLI signal increased.

[0128] 2.5. The BLI instrument washes the BLI sensor by immersing it in the B well of the washing buffer according to the preset program. The shaking speed is 1000 rpm. There are 3 washing wells, and the washing times are 150s, 300s and 150s respectively. The phages that are not specifically bound to the sensor are washed off, which is reflected as a decrease in the BLI signal.

[0129] 2.6. Refer to section “2.6” in Example 1.

[0130] 2.7. Refer to section “2.7” in Example 1.

[0131] 2.8. The BLI instrument immerses the BLI sensor in DAB, AEC, and according to the preset program.

[0132] 4-Aminoantipyrine working solution well, time: 120s, shaking speed:

[0133] At 1000 rpm, HRP bound to different sensors catalyzed DAB, AEC, and...

[0134] The 4-aminoantipyrine reaction produced brown, red, and red precipitates on the sensor, respectively, and the biolayer thickened, which was reflected as an increase in the BLI signal, indicating that the BLI sensor had captured specific phage display antibodies.

[0135] (II) Experimental Results

[0136] The amplified signal value was obtained by subtracting the BLI signal from the control group's BLI signal in the experimental group. The experimental results are shown in Table 2 and... Figure 2 .

[0137] Table 2: Effects of different HRP substrates on the amplification of antibody signals displayed by phage display on the BLI sensor

[0138] experimental group BLI signal value, nm 4-Aminoantipyrine:phenol 1.26 DAB 0.88 AEC 0.57

[0139] Experimental results showed that signal amplification of phage-displaying antibodies obtained by the BLI sensor was achieved with the 4-aminoantipyrine:phenol group, DAB group, and AEC group all exceeding 0.3 nm. All three HRP substrates can be used for phage-displaying antibody capture using the BLI sensor, with the 4-aminoantipyrine:phenol group exhibiting the highest amplification.

[0140] Example 3: Comparison of BLI sensor-based phage display antibody library and traditional screening methods (I) Experimental method for BLI sensor-based phage display antibody library

[0141] 1. Referring to sections “1” and “2” in Example 1, the phage display antibody library was diluted with washing buffer B and the non-specifically binding phage was washed, and the signal was amplified using 4-aminoantipyrine:phenol.

[0142] 1.1. Immerse the amplified BLI sensor in the trypsin working solution well for 2300 s with shaking at 1000 rpm. The phage display antibody binds to the gIII protein via a lysine-arginine link. Trypsin cleaves this site, releasing the phage display antibody that specifically binds to the antigen. A continuous decrease in the BLI signal indicates that the phage has been successfully eluted into the solution.

[0143] 1.2. Add 2 μL of 10% AEBSF (Shanghai Sangon Biotech Co., Ltd.) to the trypsin working solution well.

[0144] C510022) terminates the elution and yields phages that have bound to the antigen.

[0145] 1.3. Add an equal volume of Escherichia coli TG1 (Beyotime, D0389) in the logarithmic growth phase (OD600 = 0.4) to the phage eluent and incubate at 37°C for 30 minutes for infection.

[0146] 1.4. Spread all bacterial culture onto AG-2YT (containing 100 μg / mL Amp, 2% glucose).

[0147] Solid culture medium, inverted, incubated overnight at 37°C.

[0148] 2. Verify the phages obtained by BLI fishing.

[0149] 2.1. Preparation of monoclonal bacteriophages

[0150] 2.1.1. Pick a single colony from the overnight culture plate and add it to 0.2 mL of Amp-2YT (containing...)

[0151] Incubate 100 μg / mL Amp medium at 37°C with shaking at 220 rpm until the OD600 reaches 0.4 (bacterial concentration of 4.0E+8 / mL). Add 1.6E+9 helper phages (Thermo, 18311019) according to a multiplicity of infection (MOU) of 20 for superinfection for 30 minutes. Then add 50 μg / mL kanamycin and incubate at 30°C with shaking.

[0152] Incubate overnight at 220 rpm.

[0153] 2.1.2. The supernatant of the overnight culture contained monoclonal bacteriophages. Centrifuged at 4000 rpm.

[0154] After 20 minutes, remove the supernatant and place it in a 4°C refrigerator for later use.

[0155] 2.2. Monoclonal phage ELISA

[0156] 2.2.1. Add 100 μL of 2 μg / mL antigen (human Trop-2) to the wells of an ELISA plate (Thermo, 442404), place at 4°C overnight, and coat the wells with the antigen.

[0157] 2.2.2. Clean the wells with PBST three times.

[0158] 2.2.3. Add 300 μL of blocking buffer (PBST containing 2% BSA) and block at room temperature.

[0159] 1 hour.

[0160] 2.2.4. Clean the wells with PBST three times.

[0161] 2.2.5. Add 30 μL of blocking buffer to the well plate, then add 70 μL of monoclonal phage.

[0162] Add 70 μL of blocking buffer to the positive and negative control wells, mix well, and incubate at room temperature for 1 h. 2.2.6. Wash the wells 6 times with PBST.

[0163] 2.2.7. Add anti-M13 antibody (HRP-conjugated) (Sino Biological, 11973-

[0164] MM05T-H working solution (dilution factor 5000).

[0165] 2.2.8. Clean the wells with PBST 10 times.

[0166] 2.2.9. Add 100 μL of colorimetric solution (Beyotime, PO206) and incubate at room temperature for 10 min.

[0167] 2.2.10. OD370 was measured using an ELISA reader (BioTek, SYNERGY H1).

[0168] 2.2.11. Clones with an OD370 value higher than 1 are defined as positive phages.

[0169] (II) Traditional phage display antibody panning assay method

[0170] 1. Add 2 mL of human Trop-2 antigen to a 5 mL immunoassay tube (Thermo, 444202).

[0171] (40 μg / tube, coating buffer is PBS), add 2 mL BSA to the control tube in parallel.

[0172] (40 μg / tube, coating solution is PBS), coating overnight at 4℃ and 50 rpm by rotation.

[0173] 2. Clean and seal the immunotherapy tubes.

[0174] 3. Clean the immunotherapy tube, then add 2 mL of phage display antibody library (3E+9pfu) in a volume of 1000 times the volume of the library, and incubate at room temperature and 50 rpm for 1 hour.

[0175] 4. Discard the liquid in the immunoassay tube, and rinse the immunoassay tube 20 times, rotating it at 50 rpm each time.

[0176] 5 minutes.

[0177] 5. Discard the liquid in the immunosorbent assay tube, remove as much residual liquid as possible, add 1 mL of 250 μg / mL trypsin solution, and elute by rotating at 50 rpm for 30 min at room temperature.

[0178] 6. Add 10 μL of 10% AEBSF to stop elution, and transfer the solution in the immunoassay tube to a new 1.5 mL centrifuge tube. This is the first round of phage elution buffer.

[0179] 7. Infect E. coli with the phage eluent, dilute and plate.

[0180] 8. Select single clones for phage ELISA verification. The experimental method is the same as in (I).

[0181] (III) Experimental Results

[0182] The BLI sensor was used to display a phage antibody library once, and single clones were selected for phage ELISA validation. The results are shown in Table 3. Figure 3 and 4 .

[0183] Table 3: One-step panning of phage display antibody library using BLI sensor, and ELISA readings of monoclonal phages (G12 and H12 wells are negative controls).

[0184]

[0185] A phage display antibody library was panned once using immunotubes, and single clones were selected for phage ELISA validation. The results are shown in Table 4. Figure 4 .

[0186] Table 4: ELISA readings of monoclonal phages after one round of screening of the phage display antibody library (wells A1 and B1 are negative controls).

[0187]

[0188] Example 4: Determination of the affinity of BLI sensor for phage display antibodies

[0189] (I) Experimental Methods

[0190] 1. Sanger sequencing was performed on phage ELISA-positive clones.

[0191] 1.1. Performed according to the standard Sanger sequencing procedure.

[0192] 2. Antibody expression

[0193] 2.1. Select antibody sequences and construct vectors and express antibodies according to standard procedures.

[0194] 3. Determine antibody affinity

[0195] 3.1. Determine the affinity between the antibody and the antigen according to the BLI supplier's standard procedure.

[0196] (II) Experimental Results

[0197] Antibody-antigen affinity was fitted using a 1:1 model; the experimental results are shown in Table 5. Figure 5 .

[0198] Table 5: Results of Affinity Constant Detection between Selected Antibodies and Antigens

[0199] Antibody number KD(M) kon(1 / Ms) koff(1 / s) Ab1 7.09E-08 1.68E+05 1.19E-02 Ab2 1.78E-08 2.05E+05 3.64E-03 Ab3 8.23E-08 2.50E+05 2.06E-02 Ab4 7.35E-08 3.18E+05 2.34E-02 Ab5 1.63E-09 1.05E+05 1.71E-04

[0200] The results showed that the phage display antibody positivity rate obtained by the one-step BLI sensor method was 80% (clones with an OD value greater than 1 were defined as positive clones), while the proportion of positive clones obtained by one round of panning in the immune tubes was 0. The one-step BLI sensor method completes the phage collection within 2 hours and is automated by the machine; the traditional panning method requires antigen coating one day in advance and multiple manual washings during the panning stage, with a single experimental cycle exceeding 1.5 days.

[0201] This invention optimizes the washing buffer based on the previously disclosed BLI sensor-based method for phage display antibody detection. The highest recovery rate of specific phage display antibodies was achieved in PBST containing 300 mM NaCl. Secondly, a signal amplification step was added to the detection process, significantly improving the method's sensitivity. For antigens with low abundance of specific phage display antibodies, adding a signal amplification step greatly increases the success rate of detecting specific phage display antibodies. Furthermore, the signal amplification substrate was screened; 4-aminoantipyrine:phenol amplified the signal 1.4 times that of DAB, further improving the method's sensitivity.

Claims

1. A method for fishing antibodies specifically binding to an antigen from a phage display antibody library, comprising the following steps: (1) antigen immobilization immobilizing the antigen to the surface of a BLI sensor, optionally, the BLI detection signal is 0.2 nm or more, preferably 3 nm; (2) antibody fishing immersing the BLI sensor into the phage display antibody library for fishing, optionally, the time is 100-1800 s, and the shaking speed is 200-1000 rpm; (3) non-specific antibody washing immersing the BLI sensor into a plurality of washing wells containing high-salt washing buffer in sequence for washing, optionally, the time is 100-1800 s, the shaking speed is 1000 rpm, the number of washing wells is 2-8, preferably 3; (4) signal amplification immersing the BLI sensor into a staining well containing HRP-labeled anti-phage antibody working solution and HRP substrate working solution in sequence, optionally, the time is 100-1800 s, and the shaking speed is 200-1000 rpm; (5) specific antibody elution immersing the sensor into an elution well containing trypsin working solution, optionally, the time is 300-3600 s, and the shaking speed is 200-1000 rpm.

2. The method of claim 1, further comprising the following step: (6) specific antibody verification performing monoclonal ELISA verification on the eluted phage display antibody.

3. The method of claim 1, wherein, The antigen is immobilized to the surface of the BLI sensor by a biotin-streptavidin system (e.g. biotin-streptavidin system), an amino coupling system, or a Protein A / G-Fc system.

4. The method of claim 3, wherein, The BLI sensor is a streptavidin (SA) sensor or a super-streptavidin (SSA) sensor, and the antigen is biotinylated.

5. The method of claim 1, wherein, The input amount of the phage display antibody library is at least 1, 10, 100, 1000, or 10000 times, for example, 100-1000 times, of the library capacity.

6. The method of claim 1, wherein, The high-salt washing buffer has a salt ion concentration of 100-1000 mM, preferably 150-800 mM.

7. The method of claim 6, wherein, The salt ion of the high-salt washing buffer is sodium or potassium ion.

8. The method of claim 1, wherein, The HRP substrate is (1) 3,3'-diaminobenzidine (DAB), (2) 3-amino-9-ethylcarbazole (AEC), or (3) 4-aminoantipyrine and phenol.

9. The method of claim 8, wherein, The ratio of 4-aminoantipyrine to phenol is 1-3:25, and the concentration of 4-aminoantipyrine is 1-10 mM, preferably 2 mM or more.

10. The method of claim 1, wherein, The concentration of the trypsin working solution is 10-250 μg / mL.

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