Bevacizumab Fab antibody, detection reagent and preparation method and application thereof

Abstract

The application provides a bevacizumab Fab antibody, a detection reagent and a preparation method and application thereof, and belongs to the technical field of drug detection. The bevacizumab Fab antibody comprises a coating antibody Beva-7E1 and an enzyme-labeled antibody Beva-6C11; the coating antibody Beva-7E1 is produced by a hybridoma cell strain Beva-7E1 (the preservation number is CGMCC No.46305), and the enzyme-labeled antibody Beva-6C11 is produced by a hybridoma cell strain Beva-6C11 (the preservation number is CGMCC No.46306). The bevacizumab Fab antibody and the detection reagent thereof can be used for detecting the free blood drug concentration of bevacizumab with high specificity, high sensitivity, simplicity, rapidness and high automation degree.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug detection, and particularly relates to a bevacizumab Fab antibody, a detection reagent, a preparation method and application thereof. BACKGROUND

[0002] Bevacizumab, also known as Avastin, is a recombinant humanized monoclonal antibody, mainly used for the treatment of various types of cancer. Bevacizumab works by targeting vascular endothelial growth factor (VEGF), preventing its binding to the receptor on the cell surface, thereby inhibiting the formation and growth of tumor blood vessels (i.e. angiogenesis). This makes bevacizumab an anti-tumor drug, and its main uses include but are not limited to: colorectal cancer, non-small cell lung cancer, breast cancer, renal cell carcinoma, ovarian cancer, and glioblastoma. In order to ensure the effectiveness and safety of bevacizumab, monitoring of its blood concentration becomes crucial. Bevacizumab drugs that have bound to VEGF will be accelerated in clearance, so the results of free bevacizumab are more closely related to drug efficacy.

[0003] However, at present, bevacizumab blood concentration monitoring is rarely carried out in clinical practice, which is related to the cumbersome detection method of bevacizumab blood concentration and the lack of awareness of blood concentration monitoring among people. The detection and analysis of bevacizumab mainly includes ELISA and LC-MS method. Among them, the LC-MS method has a long analysis time and complex pretreatment operation, which is difficult to meet the requirements of high efficiency of clinical examination, and usually can only determine the total bevacizumab drug concentration; the ELSIA method has low automation degree and long test time, which cannot meet the requirements of high-throughput, rapid and accurate detection in clinical practice.

[0004] Immunological principles and fully automated chemiluminescence immunoassay are ideal methods for detecting bevacizumab drug concentration, but the development of paired monoclonal antibodies that specifically recognize bevacizumab is required. CN 108623684 A discloses a non-neutralizing monoclonal antibody that recognizes bevacizumab and its potential application in the determination of total bevacizumab; CN 108623688 A discloses a monoclonal antibody that neutralizes bevacizumab and its potential use in ADA detection. CN 111366655 A discloses a method using VEGF immunoaffinity bevacizumab followed by mass spectrometry detection, but this method involves many steps and a total detection time exceeding 16 hours. CN 113899894 A discloses a mass spectrometry-based bevacizumab detection scheme, which requires multiple steps including enzymatic digestion, resulting in a total detection time > 3.5 hours. These mass spectrometry methods are not suitable for high-throughput clinical applications. CN 115725588A discloses a bevacizumab drug detection method based on an electrochemical sensor and a DNA aptamer, but does not disclose the binding site adapted to bevacizumab. This method is suitable for rapid detection of a small number of samples, but is not suitable for the high-throughput detection needs of clinical central laboratories.

[0005] Therefore, there is currently a lack of highly specific, highly sensitive, simple, rapid, and highly automated methods for detecting bevacizumab free blood concentration. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a bevacizumab Fab antibody and detection reagent, which can detect bevacizumab free blood drug concentration with high specificity, high sensitivity, simplicity, speed and high degree of automation.

[0007] A bevacizumab Fab antibody is produced by hybridoma cell lines Beva-7E1 and Beva-6C11; the accession number of the hybridoma cell line Beva-7E1 is CGMCC No. 46305, and the accession number of the hybridoma cell line Beva-6C11 is CGMCC No. 46306.

[0008] Preferably, the antibody comprises a coating antibody Beva-7E1 and an enzyme-labeled antibody Beva-6C11; the coating antibody Beva-7E1 is produced by the hybridoma cell line Beva-7E1, and the enzyme-labeled antibody Beva-6C11 is produced by the hybridoma cell line Beva-6C11.

[0009] The present invention also provides a bevacizumab free drug detection reagent, comprising the bevacizumab Fab antibody.

[0010] The present invention also provides a method for preparing the detection reagent, comprising the following steps:

[0011] Obtaining the magnetic microparticle working solution: Magnetic microparticles are diluted in borate buffer to obtain a magnetic microparticle solution. The coated antibody Beva-7E1 is added to the magnetic microparticle solution, and the mixture is shaken to obtain a magnetic microparticle-antibody solution. The supernatant of the magnetic microparticle-antibody solution is removed by magnetic aspiration, TBST buffer is added, and the reaction is carried out for 10-40 hours. The supernatant of the obtained solution is removed by magnetic aspiration again, and TBST buffer is added to obtain the magnetic microparticle working solution.

[0012] Obtaining the enzyme-labeled working solution: Alkaline phosphatase was diluted in PBS buffer to obtain a labeling solution; enzyme-labeled antibody Beva-6C11 was added to the labeling solution, and then a coupling agent was added to carry out a coupling reaction to obtain a solution containing the coupling agent; the solution containing the coupling agent was dialyzed with PBS buffer, and the obtained dialysate was diluted to obtain the enzyme-labeled working solution.

[0013] Preferably, the mass concentration of magnetic particles in the magnetic particle working fluid is 0.2-0.6 mg / mL.

[0014] Preferably, the mass ratio of alkaline phosphatase, enzyme-labeled antibody Beva-6C11, and coupling agent is 1:0.2-1:0.2-1; and the concentration of alkaline phosphatase in the enzyme-labeled working solution is 0.1-2 μg / mL. More preferably, the coupling agent is glutaraldehyde.

[0015] Preferably, it also includes the preparation of the indicator solution.

[0016] The present invention also provides the use of the detection reagent in the detection of bevacizumab or its biosimilar.

[0017] Preferably, magnetic microparticle luminescence assay is used to detect bevacizumab or its biosimilar; the reaction procedure of the magnetic microparticle luminescence assay is as follows: 5-100 μL of the sample to be tested is added to 20-100 μL of magnetic microparticle working solution and reacted for 1-30 min, then washed, then 20-100 μL of enzyme-labeled working solution is added, and the mixture is incubated at 37°C for 1-30 min, washed, and then an indicator solution is added for color development.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The bevacizumab Fab antibody of this invention comprises a coating antibody Beva-7E1 and an enzyme-labeled antibody Beva-6C11, which can accurately identify bevacizumab or its biosimilars, and has no cross-reactivity with a variety of endogenous proteins (such as human IgG protein and human albumin), exhibiting high specificity and effectively avoiding false positive results, thus providing a reliable basis for accurate clinical determination of bevacizumab drug concentration.

[0020] This invention can accurately measure bevacizumab concentrations as low as 0.5 μg / mL, demonstrating strong detection capabilities for trace amounts of drugs. This helps in early monitoring of drug concentration changes and timely adjustment of treatment plans.

[0021] The preparation method of the detection reagent of this invention is relatively simple, and the reaction procedure is concise when using magnetic microparticle luminescence detection. The entire detection process is short, from sample addition to obtaining results, and can be completed in a short time, meeting the clinical need for rapid detection and improving detection efficiency.

[0022] The detection reagent of this invention exhibits good precision across different concentration ranges, with stable, reliable, and highly reproducible results, providing accurate and consistent detection data for clinical use. It can be used not only to detect the original bevacizumab drug but also to accurately determine various bevacizumab biosimilars (such as Eresson, Prismin, and Probexi), making this detection reagent a promising candidate for clinical monitoring of bevacizumab-related drugs.

[0023] Instructions for the Preservation of Biological Materials

[0024] Hybridoma cell line Beva-7E1: Classified and named as hybridoma cell line Mus musculus The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 46305, and the deposit date is December 17, 2024.

[0025] Hybridoma cell line Beva-6C11: Classified and named as hybridoma cell line Mus musculus The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 46306, and the deposit date is December 17, 2024. Attached Figure Description

[0026] Figure 1 SDS-PAGE electrophoresis image of the Fab 2 fragment;

[0027] Figure 2 Calibration curve for the magnetic microparticle luminescence method for detecting free bevacizumab in blood. Detailed Implementation

[0028] This invention provides a bevacizumab Fab antibody, produced by hybridoma cell lines Beva-7E1 and Beva-6C11; the hybridoma cell line Beva-7E1 has the accession number CGMCC No. 46305 and was deposited at the China General Microbiological Culture Collection Center on December 17, 2024, and the hybridoma cell line Beva-6C11 has the accession number CGMCC No. 46306 and was deposited at the China General Microbiological Culture Collection Center on December 17, 2024.

[0029] In this invention, the preferred antibodies include the coating antibody Beva-7E1 and the enzyme-labeled antibody Beva-6C11; the coating antibody Beva-7E1 is produced by the hybridoma cell line Beva-7E1, and the enzyme-labeled antibody Beva-6C11 is produced by the hybridoma cell line Beva-6C11.

[0030] The present invention also provides a bevacizumab free drug detection reagent, comprising the bevacizumab Fab antibody; preferably comprising a magnetic microparticle working solution containing the coated antibody Beva-7E1, and an enzyme-labeled working solution containing the enzyme-labeled antibody Beva-6C11; more preferably comprising an indicator solution, and more preferably the indicator solution being commercially available AMPPD and APS-5 substrates.

[0031] The present invention also provides a method for preparing the above-mentioned detection reagent, comprising the following steps:

[0032] 1. Obtaining the magnetic particle working fluid:

[0033] 1-1: Magnetic microparticles were diluted in borate buffer to obtain a magnetic microparticle solution. The coated antibody Beva-7E1 was added to the magnetic microparticle solution, and the mixture was shaken to obtain a magnetic microparticle-antibody solution.

[0034] 1-2: Remove the supernatant from the magnetic microparticle antibody solution by magnetic adsorption, add TBST buffer, and react for 10-40 h, preferably 16 h. Remove the supernatant from the obtained solution by magnetic adsorption, and add TBST buffer to obtain the magnetic microparticle working solution. Preferably, the mass concentration of magnetic microparticles in the magnetic microparticle working solution is 0.2-0.6 mg / mL, and more preferably 0.4 mg / mL.

[0035] As one possible implementation method, the steps are as follows:

[0036] 1-1: 50 mg Dynal beads M280 Tosyl magnetic beads were diluted in 2 mL of 50 mM BB pH 8.0 buffer, 1 mg of coated antibody Beva-7E1 was added, and the mixture was incubated at 37 °C with shaking for 8 h.

[0037] 1-2: Remove the supernatant with magnetic adsorption, add TBST (50mM Tris, 0.9wt% NaCl, 0.1wt% TW20, pH 7.4) and react at 37℃ for 12h; remove the supernatant with magnetic adsorption, add TBST, dilute to 0.4mg / mL, name it magnetic microparticle working solution, and store at 2~8℃ for later use.

[0038] 2. Obtaining the enzyme-labeled working solution:

[0039] 2-1: Dilute alkaline phosphatase in PBS buffer to obtain the labeled solution;

[0040] 2-2: Add enzyme-labeled antibody Beva-6C11 to the labeled solution and mix well, then add a coupling agent to carry out the coupling reaction to obtain a solution containing the coupling agent; preferably, the mass ratio of alkaline phosphatase, enzyme-labeled antibody Beva-6C11 and coupling agent is 1:0.2-1:0.2-1, more preferably 1:1:0.5; more preferably, the coupling agent is glutaraldehyde;

[0041] 2-3: Dialyze the solution containing the conjugate using PBS buffer, and dilute the resulting dialysate to obtain the enzyme-labeled working solution; preferably, dilute with a mixed buffer solution of 50 mM MES + 0.9% NaCl + 5 mg / mL BSA + 1 mM MgCl2 at pH 6.7; preferably, the concentration of the label in the enzyme-labeled working solution is 0.1-2 μg / mL, more preferably 1 μg / mL.

[0042] As one possible implementation method, the steps are as follows:

[0043] 2-1: Dissolve 1 mg of ALP in 1 mL of PBS;

[0044] 2-2: Add 0.5 mg of enzyme-labeled antibody Beva-6C11, mix well, add 10 μl of 50% glutaraldehyde solution, and mix at room temperature for 2 h;

[0045] 2-3: Dialyze into PBS, then dilute to 1 μg / mL with 50 mM MES, 0.9% NaCl, 5 mg / mL BSA, 1 mM MgCl2, pH 6.7, and name it the enzyme-labeled working solution.

[0046] 3. Obtaining the indicator solution.

[0047] The present invention also provides the application of the detection reagent in the detection of bevacizumab or its biosimilars, preferably the biosimilars include Eressor, Pulsatilla, Probex, Dystos, Hembetaz, Betaine, Boyonuo, Ancoda, and Ambes.

[0048] The present invention preferably uses magnetic microparticle luminescence assay to detect bevacizumab or its biosimilar; a more preferred magnetic microparticle luminescence assay reaction procedure is as follows: 5-100 μL of the sample to be tested is added to 20-100 μL of magnetic microparticle working solution, reacted for 1-30 min, washed, then 20-100 μL of enzyme-labeled working solution is added, incubated at 37°C for 1-30 min, washed, and then an indicator solution is added for color development; more preferably, 10 μL of the sample to be tested (automatically diluted 20 times with PBS) is added to 50 μL of magnetic microparticle working solution, reacted for 5 min, washed, then 50 μL of enzyme-labeled working solution is added, incubated at 37°C for 5 min, washed, and then AMPPD luminescence solution is added for color development.

[0049] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0050] Example 1

[0051] Preparation of Bevacizumab (Fab 2) fragment immunogen

[0052] 1. Dilute 20 mg of bevacizumab antibody with 20 mM PB7.4 buffer to 5 mg / mL, for a total volume of 4 mL;

[0053] 2. Add 1 mL of IdeZ protease affinity column (Beijing Deoping Biotechnology), incubate at 37℃ for 30 min, centrifuge at 2000 rpm for 10 min, and collect the supernatant;

[0054] 3. The supernatant reaction product was passed through a Protein A column to adsorb unreacted full-length antibody and cleaved Fc fragments, and then collected for flow-through.

[0055] 4. The concentration was determined using OD280, and the product was stored frozen at -20℃.

[0056] 5. Perform SDS-PAGE analysis; the results are as follows: Figure 1 As shown, the full-length bevacizumab is not completely cut, but after adsorbing impurity fragments with Protein A, (Fab)2 fragments are obtained.

[0057] Example 2

[0058] Preparation of Bevacizumab (Fab 2) fragment antibody

[0059] Using hybridoma cell fusion technology, referring to "Development and characterization of mousehybridomas", the steps are as follows:

[0060] The bevacizumab (Fab)2 fragment antigen prepared in Example 1 was diluted to 1 mg / mL with PBS buffer, and an equal volume of Freund's complete adjuvant was added. After complete emulsification, mice were immunized for the first time at a dose of 0.1 mg / mouse. Four weeks later, 1 mg of the bevacizumab (Fab)2 fragment antigen prepared in Example 1 was mixed with an equal volume of Freund's incomplete adjuvant and emulsified. Mice were then immunized for the second time at a dose of 0.1 mg / mouse. Three days later, the spleen cells from the second immunization were fused with Sp2 / 0 cells. The supernatant of the fused cells was coated with 2 μg / mL of bevacizumab and the titer of the supernatant was determined. Clones with positive titers were then subjected to competitive screening.

[0061] Competitive screening involved coating ELISA 96-well plates with 2 μg / mL bevacizumab, followed by the addition of 1 μg / mL VEGF protein and fusion cell supernatant. Clones showing strong positive titers were selected, while weakly positive or negative clones were also competitively screened. Five competitive anti-bevacizumab idiotype antibodies were ultimately identified, as shown in Table 1.

[0062] Table 1. Competitive anti-bevacizumab idiotype antibodies screened.

[0063]

[0064] The screening of paired antibodies was performed using a checkerboard method. 2 μg / mL of anti-bevacizumab idiotype antibody was coated onto 96-well ELISA plates. 0 or 20 ng / mL of bevacizumab was added, and the plates were reacted for 30 min. After washing three times with PBST, 2 μg / mL of anti-bevacizumab idiotype antibody-HRP conjugate was added, and the plates were reacted for 30 min. After washing three times with PBST, TMB chromogenic buffer was added, and the results were read using a microplate reader. One paired antibody was obtained, as shown in Table 2.

[0065] Table 2

[0066]

[0067] The results showed that Beva-7E1 / Beva-6C11 exhibited a good low signal-to-noise ratio. Therefore, this pair was subsequently used to establish a method for detecting bevacizumab in free blood.

[0068] Example 3

[0069] Establishment of a chemiluminescence method for detecting bevacizumab in blood using magnetic microparticles

[0070] (1) 50 mg of Dynal beads M280 Tosyl magnetic beads were diluted in 2 mL of 50 mM BB pH 8.0 buffer, and 1 mg of anti-Beva-7E1 antibody obtained in Example 2 was added. After mixing, the mixture was shaken at 37 °C for 8 h.

[0071] Remove the supernatant using magnetic adsorption, add TBST (50mM Tris, 0.9wt% NaCl, 0.1wt% TW20, pH 7.4) and react at 37℃ for 12h.

[0072] Remove the supernatant using magnetic adsorption, add TBST, dilute to 0.4 mg / mL, name it Magnetic Microparticle Working Solution, and store at 2-8℃ for later use.

[0073] (2) Dissolve 1 mg ALP in 1 mL PBS, add 0.5 mg anti-bevacizumab Beva-6C11 antibody, mix well, add 10 μl of 50% glutaraldehyde solution, mix at room temperature for 2 h; dialyze into PBS, dilute to 1 μg / mL with 50 mM MES, 0.9% NaCl, 5 mg / mL BSA, 1 mM MgCl2, pH 6.7, and name it enzyme-labeled working solution.

[0074] (3) The detection was performed using the Danda DF200i chemiluminescence immunoassay analyzer. Reaction procedure: 10 μL of sample (automatically diluted 20 times with PBS) was added to 50 μL of magnetic microparticle working solution and reacted for 5 min. After washing, 50 μL of enzyme-labeled working solution was added and incubated at 37℃ for 5 min. After washing, AMPPD luminescent solution was added for color development.

[0075] Example 4

[0076] Standard curves were prepared using human serum matrix from individuals who had not received bevacizumab treatment, and calibrators of different concentrations of bevacizumab (0-0.5-2-5-20-50 ug / mL) were prepared as shown in Table 3. Figure 2 As shown.

[0077] Table 3. Calibration curve data of bevacizumab free plasma concentration using magnetic microparticle luminescence method

[0078]

[0079] from Figure 2 It can be seen that the calibration curve obtained by the magnetic particle luminescence method is: a four-parameter equation: Y=(AD) / [1+(X / C)B]+D; where: A=12739.811; B=1.18757592; C=64.79017513; D=4528878.2229; and the correlation coefficient R0 is... 2 0.998.

[0080] Precision was determined by repeatedly measuring the low and high values ​​10 times each, and the CV results were calculated according to SD / Mean, as shown in Table 4.

[0081] Table 4 Precision Results

[0082]

[0083] As can be seen from Table 4, the magnetic microparticle luminescence assay reagent for bevacizumab blood concentration determination prepared using Beva-7E1 / Beva-6C11 paired antibodies has high precision.

[0084] Different concentrations of cross-reaction reagents were added to bevacizumab samples at concentrations of 5 μg / mL and 50 μg / mL to conduct cross-reaction, and the cross-reaction rate was calculated. The results are shown in Table 5.

[0085] Table 5 Cross-reactivity results

[0086]

[0087] Cross-reactivity rate = (Measured concentration - Theoretical concentration) / Concentration of added cross-reactive agent × 100%;

[0088] The results in Table 5 show that the magnetic microparticle luminescence method for determining bevacizumab blood concentration prepared in this invention has no cross-reaction with various endogenous proteins and possible combined drugs, and has high specificity.

[0089] Example 5

[0090] Various biosimilars of bevacizumab, including Eritrexate, Prismidine, Probex, Datox, Henbeta, Betaine, Boyonuo, Ancoda, and Ambes, were prepared at concentrations of 5 μg / mL and 50 μg / mL using serum matrix from individuals who had not received bevacizumab treatment. The concentrations were then measured using the bevacizumab blood drug concentration assay reagent of this invention. The results are shown in Table 6.

[0091] Table 6. Results of assays for bevacizumab biosimilars

[0092]

[0093] The results showed that the deviation between the measured concentration and the theoretical concentration of the nine biosimilars was <±10%, indicating that the reagent of the present invention can be used to determine a variety of bevacizumab biosimilars.

[0094] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A hybridoma cell line combination for generating bevacizumab Fab antibodies to detect free blood drug concentration, characterized in that, The hybridoma cell line combination is hybridoma cell line (Mus musculus) Beva-7E1 and hybridoma cell line (Mus musculus) Beva-6C11; the accession number of hybridoma cell line Beva-7E1 is CGMCC No. 46305, and the accession number of hybridoma cell line Beva-6C11 is CGMCC No. 46306.

2. A bevacizumab Fab antibody produced from the hybridoma cell line combination of claim 1, characterized in that, The antibody includes a coating antibody Beva-7E1 and an enzyme-labeled antibody Beva-6C11; the coating antibody Beva-7E1 is produced by the hybridoma cell line Beva-7E1, and the enzyme-labeled antibody Beva-6C11 is produced by the hybridoma cell line Beva-6C11.

3. A reagent for detecting bevacizumab free drug, characterized in that, It contains the bevacizumab Fab antibody as described in claim 2.

4. A method for preparing the detection reagent according to claim 3, characterized in that, Includes the following steps: Obtaining the magnetic microparticle working solution: Magnetic microparticles are diluted in borate buffer to obtain a magnetic microparticle solution. The coated antibody Beva-7E1 is added to the magnetic microparticle solution, and the mixture is shaken to obtain a magnetic microparticle-antibody solution. The supernatant of the magnetic microparticle-antibody solution is removed by magnetic aspiration, TBST buffer is added, and the reaction is carried out for 10-40 hours. The supernatant of the obtained solution is removed by magnetic aspiration again, and TBST buffer is added to obtain the magnetic microparticle working solution. Obtaining the enzyme-labeled working solution: Alkaline phosphatase was diluted in PBS buffer to obtain a labeling solution; enzyme-labeled antibody Beva-6C11 was added to the labeling solution, and then a coupling agent was added to carry out a coupling reaction to obtain a solution containing the coupling agent; the solution containing the coupling agent was dialyzed with PBS buffer, and the obtained dialysate was diluted to obtain the enzyme-labeled working solution.

5. The preparation method according to claim 4, characterized in that, The mass concentration of magnetic particles in the working fluid is 0.2-0.6 mg / mL.

6. The preparation method according to claim 4, characterized in that, The mass ratio of alkaline phosphatase, enzyme-labeled antibody Beva-6C11, and coupling agent is 1:0.2-1:0.2-1; the concentration of alkaline phosphatase in the enzyme-labeled working solution is 0.1-2 μg / mL.

7. The preparation method according to claim 4 or 6, characterized in that, The coupling agent is glutaraldehyde.

8. The preparation method according to claim 4, characterized in that, It also includes the preparation of indicator solutions.

9. The use of the detection reagent according to claim 3 in the detection of bevacizumab or its biosimilar.

10. The application according to claim 9, characterized in that, The magnetic microparticle luminescence assay was used to detect bevacizumab or its biosimilar. The reaction procedure for the magnetic microparticle luminescence assay was as follows: 5-100 μL of the sample to be tested was added to 20-100 μL of magnetic microparticle working solution and reacted for 1-30 min. After washing, 20-100 μL of enzyme-labeled working solution was added and incubated at 37°C for 1-30 min. After washing, indicator solution was added for color development.

Images