Specific reversible capturing method for transplanted kidney exfoliated cells

Abstract

The invention discloses a specific reversible capture method for transplanted kidney exfoliated cells. The method comprises the following steps: (1) preparing a micro-fluidic chip; (2) conveying the magnetic beads of which the surfaces are modified with the EpCAM antibodies into a micro-fluidic chip, and then placing the micro-fluidic chip on a magnet for stabilization, so that the magnetic beads are uniformly and stably distributed in the micro-fluidic chip; (3) conveying peripheral blood containing the transplanted kidney exfoliated cells into the micro-fluidic chip, so that the transplanted kidney exfoliated cells are captured by the magnetic beads through the EpCAM antibodies modified on the surfaces of the magnetic beads; and (4) after the capture is completed, removing the magnet, and then introducing a release reagent into the micro-fluidic chip, so that the captured transplanted kidney exfoliated cells with the magnetic beads are released from the chip. The invention provides a promising solution for early diagnosis of kidney transplantation rejection, provides insight for improving the long-term survival rate of kidney transplantation, clarifies the mechanism of immunological rejection, and supports precise treatment of clinical kidney transplantation rejection.

Description

Technical Field

[0001] The invention belongs to the technical field of microfluidic chip applications, and particularly relates to a specific reversible capture method for exfoliated cells from transplanted kidneys. Background Art

[0002] End-stage renal disease (ESRD) is the advanced stage of chronic kidney disease (CKD), characterized by almost complete loss of renal function and a high morbidity rate. Dialysis and kidney transplantation are effective treatments for ESRD. Compared with dialysis, kidney transplantation (KT) can restore normal renal function, thereby significantly improving patients' quality of life and prolonging their life expectancy. KT is the most effective treatment for patients with end-stage organ failure and the best choice for ESRD patients. Although immunosuppressive therapy has effectively improved the treatment outcomes of kidney transplant recipients (KTRs), the long-term survival of kidney transplants remains a challenge. Transplant rejection is the main risk factor affecting the long-term survival of transplanted kidneys. Therefore, early detection and timely treatment of kidney transplant rejection have become key measures to reverse rejection.

[0003] The clinical diagnosis of renal transplant rejection primarily relies on renal biopsy and clinical indicators such as serum creatinine and urine output. Renal biopsy is the gold standard for diagnosing immune rejection, but it not only increases patient discomfort but also damages the transplanted kidney, potentially leading to infection and other complications. Clinical indicators play a crucial role in identifying and monitoring disease progression, enabling timely intervention. However, these indicators have limitations, such as low specificity and susceptibility to non-renal factors. In recent years, donor-derived cell-free DNA (dd-cfDNA) has shown great potential as a non-invasive biomarker for diagnosing transplant rejection. Rejection of the transplant leads to cell apoptosis, which releases large amounts of dd-cfDNA into the peripheral blood. Detection of dd-cfDNA can indicate the onset of organ transplant rejection. Next-generation sequencing has been shown to detect abnormal levels of dd-cfDNA in kidney transplant recipients (KTRs). For example, dd-cfDNA-based monitoring has demonstrated 88.9% sensitivity and 73.7% specificity for early rejection detection. However, due to the low absolute value of dd-cfDNA content, high degree of fragmentation, complex extraction procedures, and large background interference from recipient DNA, the detection accuracy is limited, there is a risk of misdiagnosis, and its clinical application is affected. Therefore, there is an urgent need to develop new low-invasive and highly sensitive methods to monitor the development of immune rejection in early renal transplant recipients.

[0004] In addition to dd-cfDNA, exfoliated cells (including endothelial cells and epithelial cells), as cells shed from the graft and enter the body fluids, have also been shown to be promising markers of immune rejection. Exfoliated cells carry actual information about the pathological state of kidney tissue and can be used to analyze the condition of transplanted organs. Analysis based on exfoliated cells will further provide clinical understanding of the mechanism of immune rejection and open up new avenues for the development of more non-invasive and effective clinical monitoring tools. At present, the detection of exfoliated cells in renal transplant rejection mainly relies on large amounts of urine samples (50-100mL). Patients with renal transplant rejection show symptoms of decreased urine volume and hematuria, which increases the difficulty of detection. Blood is an ideal clinical source for judging the immune rejection status of KTRs. Exfoliated cells in the blood are an ideal target for monitoring the status of the graft, but their number is limited. Each milliliter of peripheral blood contains a large number of background cells, including 10 6 White blood cells (WBCs) and 10 9 The number of red blood cells (RBCs) in the blood is very small, which brings great challenges to cell separation technology and subsequent analysis. Currently, several methods have been used to separate exfoliated cells, including immunomagnetic cell sorting and fluorescence-activated cell sorting (FACS).

[0005] However, these traditional methods often suffer from problems such as low cell recovery, poor purity, and cell damage. Therefore, there is an urgent need to develop new methods that can efficiently capture and gently release detached cells. Summary of the Invention

[0006] The present invention aims to overcome the defects of the prior art and provide a specific reversible capture method for transplanted kidney exfoliated cells.

[0007] The technical solutions of the present invention are as follows:

[0008] A method for specific reversible capture of exfoliated cells from transplanted kidneys, comprising the following steps:

[0009] (1) preparing a microfluidic chip, wherein the microfluidic chip is a fishbone-shaped chip, comprising an upper fishbone structure layer and a lower channel layer; the fishbone structure layer is provided with a plurality of spaced fishbone-shaped crossbeams, and the channel layer is provided with a plurality of evenly distributed support columns; the gaps between the support columns form fluid channels;

[0010] (2) delivering the magnetic beads modified with EpCAM antibodies to the microfluidic chip in step (1), and then placing the microfluidic chip on a magnet for stabilization, so that the magnetic beads are evenly and stably distributed inside the microfluidic chip;

[0011] (3) delivering peripheral blood containing exfoliated cells from the transplanted kidney into the microfluidic chip treated in step (2), so that the exfoliated cells from the transplanted kidney are captured by the magnetic beads through the EpCAM antibodies modified on the surface of the magnetic beads;

[0012] (4) After the capture is completed, the magnet is removed, and then a release reagent is introduced into the microfluidic chip to release the captured transplanted kidney exfoliated cells with magnetic beads from the chip.

[0013] In a preferred embodiment of the present invention, the geometric arrangement of the microfluidic chip is as follows: the height of the fishbone structure layer is 30-60 μm, and the height of the channel layer is 30-60 μm; the width of the fishbone beam is 80-120 μm, the height is 30-60 μm, and the spacing between adjacent fishbone beams is 80-120 μm; the height of the support column is 30-60 μm.

[0014] Further preferably, the material of the microfluidic chip is one or a combination of at least two of polydimethoxysilane, polymethyl methacrylate, polyethylene, polypropylene or polycarbonate; and the microfluidic chip is prepared by a photolithography template, an imprinting template or an injection molding template method.

[0015] In a preferred embodiment of the present invention, in step (3), the delivery rate is 1.8-2.2 mL / h.

[0016] In a preferred embodiment of the present invention, the release agent is 4.5%-5.5% BSA.

[0017] In a preferred embodiment of the present invention, the step (3) further comprises: fixing the captured exfoliated cells of the transplanted kidney, washing them with PBS, staining them with an antibody mixture and DAPI, and then imaging and identifying them with a fluorescence microscope.

[0018] Further preferably, the fixation in step (3) is performed by incubation with 4% PFA solution at room temperature, and the antibody mixture contains anti-EpCAM-PE, Alexa Fluor 488-labeled anti-PODXL and anti-CD45-APC.

[0019] In a preferred embodiment of the present invention, the step (4) further comprises: fixing the released exfoliated cells from the transplanted kidney and then identifying their origin by FISH analysis.

[0020] Further preferably, the fixation in step (4) is performed by incubating at room temperature using a mixed fixative, wherein the mixed fixative is prepared by mixing glacial acetic acid and methanol in a volume ratio of 1:3.

[0021] The beneficial effects of the present invention are:

[0022] 1. The present invention combines a dynamic reversible interface with bioaffinity properties, and can identify and recover exfoliated cells from kidney transplantation from peripheral blood. It can achieve early prediction of the rejection status of kidney transplant patients by counting exfoliated cells (ECs).

[0023] 2. The present invention designs a magnetic reversible affinity interface in the fishbone microfluidic chip to enhance the collision frequency between cells and anti-EpCAM antibodies, thereby improving the efficient capture and gentle release of target cells for subsequent analysis.

[0024] 3. The present invention utilizes changes in the magnetic field to achieve gentle and efficient release of exfoliated cells. By constructing a dynamic magnetic bead interface, changing the magnetic field enables the capture and lossless release of exfoliated cells. The released exfoliated cells are highly active and can be used for subsequent molecular analysis.

[0025] 4. This invention provides a promising solution for the early diagnosis of renal transplant rejection, provides insights into improving the long-term survival rate of renal transplants, clarifies the mechanism of immune rejection, and supports the precise treatment of clinical renal transplant rejection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The sample loading flow rate of the antibody-magnetic bead modified microfluidic chip in Example 2 of the present invention is optimized.

[0027] Figure 2 This shows the capture efficiency of the antibody-magnetic bead modified microfluidic chip in Example 2 of the present invention.

[0028] Figure 3 Shown in Example 3 of the present invention: (A) Representative immunofluorescence imaging showing EC and nonspecific WBC, scale bar = 25 μm; (B) Statistics of the number of EC isolated from clinical peripheral blood samples (**** indicates p-value less than 0.0001); (C) ROC curve analysis based on the number of EC between the SP group and the RP group; (D) Sensitivity and specificity curves of EC between the SP group and the RP group.

[0029] Figure 4 Figure 4 shows: (A) Schematic diagram of the FISH analysis procedure for chromosome analysis of released ECs; (B) FISH analysis of XY chromosomes (green represents X, red represents Y) in ECs from a KTR patient. Scale bar = 10 μm; (C) FISH analysis report for KTRs. P1, P3: male recipient, female donor; P2, P4, P5: female recipient, male donor (orange represents Y, blue represents X).

[0030] Figure 5The DEGs of renal single EC in the rejection group and the non-rejection group in Example 5 of the present invention are shown: (A) cluster heat map, (B) volcano map, (C) GO, and (D) KEGG. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further illustrated and described below through specific implementation methods in conjunction with the accompanying drawings.

[0032] Example 1 Preparation of Antibody-Magnetic Bead Modified Microfluidic Chip

[0033] In this embodiment, a fishbone-shaped microfluidic chip with eddy current effect is selected as the capture carrier. The specific production process is as follows:

[0034] (1) A fishbone-shaped microfluidic chip (1 cm wide and 4.5 cm long) was prepared according to the technical solution disclosed in CN110055158 A. The fishbone-shaped chip consists of two layers: a lower channel layer and an upper fishbone structure layer. The support columns of the channel layer are rectangular, and the channel layer is 50 μm high. In the fishbone structure layer, the fishbone beams are 100 μm wide, the interval between the two fishbone beams is also 100 μm, and the beam height is 50 μm. That is, the fishbone structure layer height is 50 μm, so the total flow channel height of the fishbone-shaped microfluidic chip is 100 μm.

[0035] (2) After the design of the fishbone microfluidic chip is completed, a photolithography mask is made and a photolithography mold is made using SU-8 3050 photoresist; then, dimethoxysilane (DMS) monomer and polymerization initiator are mixed in a mass ratio of 10:1 and poured into a glass culture dish containing a silicon wafer template. Bubbles are removed by negative pressure in a vacuum dryer. After there are no bubbles, it is placed on a 120°C heating plate and heated for 30 minutes to allow the PDMS to polymerize completely. It is then torn off and bonded to a supporting glass slide using a plasma bonding method. It is then placed in an oven at 80°C for 3 hours to ensure a firm bond. The fishbone microfluidic chip is obtained and stored at room temperature for later use.

[0036] (3) Take SA-MB (SA-coupled magnetic beads) solution, remove the supernatant after magnetic separation, add Biotin-EpCAM antibody (biotinylated EpCAM antibody) solution to the precipitate, incubate at 37°C for 30 minutes, perform magnetic separation again, remove the supernatant, add PBS equal to the supernatant volume to the precipitate to obtain the antibody-magnetic bead solution, and store at 4°C for later use.

[0037] (4) Take 20 μL of the antibody-magnetic bead solution prepared in step (3) and quickly inject it into the fishbone-type microfluidic chip prepared in step (2) from the inlet. Then stabilize it on a magnet for 30 minutes to ensure that the magnetic beads are evenly and stably distributed inside the fishbone-type microfluidic chip to obtain an antibody-magnetic bead modified microfluidic chip.

[0038] Example 2 Optimization of capture conditions and verification of capture efficiency of antibody-magnetic bead-modified microfluidic chip

[0039] 0.3 mL of calcein-stained HK2 and CEM cells were loaded onto an antibody-magnetic bead-modified microfluidic chip (prepared in Example 1) modified with IMBs (placed on a magnet) at different flow rates (0.5, 1.0, 2.0, 3.0, and 4.0 mL / h). The number of cells loaded onto the antibody-magnetic bead-modified microfluidic chip was simultaneously counted. After loading, uncaptured cells were washed away with PBS (PBS wash flow rate of 0.2 mL / h). The number of HK2 and CEM cells captured within the antibody-magnetic bead-modified microfluidic chip was then counted to calculate the capture efficiency (capture efficiency (%) = number of captured cells / number of loaded cells × 100%).

[0040] The results are as follows Figure 1 As shown, it was found that when the flow rate was 2.0 mL / h, the capture rate could reach more than 90% while the CEM nonspecific adsorption efficiency was only about 4%, so this flow rate was subsequently selected as the experimental condition.

[0041] Next, this example uses a flow rate of 2.0 mL / h as the sample flow rate to investigate the capture efficiency of the microfluidic antibody-magnetic bead modified microfluidic chip on model cells in the blood. Using calcein pre-stained cells, 20, 50, 100, 150, and 200 cells were introduced into the antibody-magnetic bead modified microfluidic chip, and the number of captured cells was counted under an inverted fluorescence microscope. The results are shown in Figure 2. Figure 2 As shown, the antibody-magnetic bead-modified microfluidic chip achieved a capture efficiency of over 85% in blood, while the nonspecific adsorption efficiency was only about 5%. Therefore, the antibody-magnetic bead-modified microfluidic chip prepared in Example 1 can achieve efficient capture of target cells and can be used to analyze exfoliated cells in the peripheral blood of renal transplant patients.

[0042] Example 3 Clinical sample detection

[0043] To evaluate clinical applicability, in this example, 1 mL of peripheral blood sample was introduced into the antibody-magnetic bead-modified microfluidic chip prepared in Example 1 at a flow rate of 2.0 mL / h for cell capture.

[0044] To fix the captured cells, a 4% PFA solution was introduced and incubated at room temperature for 15 minutes. After washing with PBS buffer, the cells were stained with an antibody cocktail for 1 hour, which included 5 μg / mL of anti-EpCAM-PE, 5 μg / mL of Alexa Fluor 488-labeled anti-PODXL, and 5 μg / mL of anti-CD45-APC. Subsequently, the cell nuclei were stained with 2 μg / mL of DAPI (in PBS). Finally, the captured cells were imaged and identified under a Nikon Ti-U fluorescence microscope.

[0045] The results are as follows Figure 3 As shown in a cohort of 35 kidney transplant recipients, 8-73 EC cells were captured per milliliter of whole blood. In contrast, only 0 to 1 EC cell per milliliter of blood was detected in non-transplanted individuals (including patients with nephritis and healthy controls). Figure 3 B). The results showed that EC cells were only present in transplant recipients and may have originated from the donor organ. This example also observed a significant difference in cell number between stable recipients and rejection patients (P<0.0001). The sensitivity and specificity curves showed that 35.0 ECs / mL could be used as a diagnostic cutoff value to distinguish between SP and RP groups ( Figure 3 D). In addition, the present embodiment carried out receiver operating characteristic curve (ROC curve) analysis based on Kid-Chip detection result to evaluate its sensitivity and specificity. The area under the ROC curve (AUC) of EC count was 1.0, indicating that it is a good indicator for predicting renal transplant rejection status ( Figure 3 C).

[0046] Example 4 Identification of the Source of Exfoliated Cells

[0047] After the capture was completed according to the technical solution of Example 3, the magnet was removed and 300 μL of 5% BSA was introduced into the antibody-magnetic bead modified microfluidic chip to release the magnetic bead-cell complex, thereby achieving the capture and enrichment of renal transplant exfoliated cells.

[0048] In this example, FISH staining was performed on the exfoliated renal transplant cells released above according to the protocol provided by the X / Y chromosome FISH kit. Briefly, the released cells were fixed in 200 μL of fixative (composed of a mixture of glacial acetic acid and methanol in a 1:3 ratio) at room temperature for 15 minutes and then incubated at -20°C for 30 minutes. The cell suspension was centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. Approximately 5 μL of the cell mixture was applied to a polylysine-coated glass slide to prepare a cell smear, and this process was repeated until all the solution was used up. The slide was incubated in a 65°C oven overnight, and then washed in 2×SSC buffer, 70%, 90% and 100% ethanol solutions for 2 minutes each. After air drying, the FISH probe mixture was added to the cell smear, denatured at 78°C for 2 minutes, and hybridized with the X / Y chromosome probe in a dark room at 37°C for approximately 48 hours. Afterwards, the slides were sequentially immersed in 0.3% NP-40 / SSC solution (72°C, 2 min), 0.1% NP-40 / 2×SSC solution (30 sec), and 70%, 90%, and 100% ethanol solutions (2 min each), and finally dried in the dark. Finally, the cell nuclei were stained with 10 μL of nuclear staining solution, coverslipped, and observed under a Nikon Ni-U upright fluorescence microscope.

[0049] In this example, samples from 5 kidney transplant recipients with sex-mismatched donors were collected and ECs were isolated from their peripheral blood. Subsequently, fluorescence in situ hybridization (FISH) analysis was performed to detect the XY chromosomes in their chromosomes ( Figure 4 A). In male recipients from female donors (P1 and P3) and female recipients from male donors (P2, P4, and P5), donor and recipient cells exhibited opposite chromosome expression patterns. Specifically, recipient cells from P1 and P3 expressed both the X and Y chromosomes (green signal for X and red signal for Y), whereas donor cells expressed only the X chromosome. Notably, EpCAM+ / PODXL+ / CD45- nuclear cells (ECs) from P1 and P3 showed positive signals only for the X chromosome, whereas EpCAM- / PODXL- / CD45+ nuclear cells (WBCs) showed positive signals for both the X and Y chromosomes ( Figure 4 B and C), indicating that ECs were donor-derived. In contrast, the chromosome expression patterns of ECs and WBCs observed in P2, P4, and P5 were opposite to those in P1 and P3. FISH analysis results showed that ECs were donor-derived, further validating the accuracy of the antibody-magnetic bead-modified microfluidic chip prepared in Example 1 in capturing exfoliated cells from renal transplants.

[0050] Example 5 Single Cell Analysis

[0051] According to Example 4, after the exfoliated cells were captured from the peripheral blood, the cells were released and stained with a fluorescent antibody mixture at room temperature for 1 hour, followed by nuclear staining with 1× Hoechst for 10 minutes. Single-cell transcriptome amplification was performed according to the Smart-seq2 method proposed by Picelli et al. By picking a single exfoliated cell (EC) and immediately transferring it to a 200 μL thin-walled tube containing 4 μL lysis buffer (including 0.95 μL 0.4% Triton X-100, 0.5 μL 40U / μL Takara recombinant RNase inhibitor, 1 μL 10mM dNTPs and 1 μL 10 μM oligo-dT). The cells were lysed at 72°C for 3 minutes to fully lyse. Immediately thereafter, 5.7 μL of reverse transcription (RT) reaction solution (including 10 U / μL Takara recombinant RNase inhibitor, 10 U / μL ThermoFisher Superscript II reverse transcriptase, 1 μM TSO primer, 1 M betaine, 1× RT buffer, 6 mM MgCl2, and 5 mM DTT) was added and reacted at 42°C for 90 min, followed by inactivation of the RT enzyme at 70°C for 15 min. Finally, 15 μL of PCR reaction mixture (including 12.5 μL 2× KAPA HiFi Mix, 0.25 μL 10 μM IS PCR primer, and 2.25 μL DEPC water) was added and amplified for 25 cycles in a PCR thermal cycler to prepare a single EC transcriptome sample.

[0052] After amplification, the cDNA product was purified using VAHTS DNA Clean Beads at a volume ratio of 0.6 (VVAHTS DNA Clean Beads / VSample) and eluted with 20 μL of DEPC-treated water to obtain cDNA fragments approximately 700-1000 bp in length. The purified cDNA was quantified using a Qubit kit. Finally, 5 ng of cDNA was used to prepare sequencing libraries for the Illumina NovaSeq X Series sequencer using a DNA library preparation kit (Vazyme, TD502) according to the manufacturer's instructions.

[0053] like Figure 5 As shown, single-cell RNA sequencing results confirmed significant transcriptional differences between exfoliated cells (ECs) in the rejection and non-rejection groups, leading to their different clinical responses and further revealing the potential regulatory mechanisms of increased cell shedding during renal transplant rejection. The results suggest that counting exfoliated cells in peripheral blood can be used as a noninvasive alternative method for early monitoring of transplant rejection.

[0054] Overall, this example combines the count of exfoliated cells in the transplanted kidney with the assessment of rejection status, providing a multidimensional platform for assessing renal transplant prognosis. This strategy facilitates the early diagnosis and differential diagnosis of post-transplant rejection, enabling timely and effective intervention and treatment for renal transplant recipients, thereby reducing the risk of renal function loss associated with immune rejection.

[0055] The above description is merely a preferred embodiment of the present invention and therefore cannot be used to limit the scope of the present invention. In other words, equivalent changes and modifications made within the scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for specific reversible capture of exfoliated cells from transplanted kidneys, characterized by: The steps include: (1) preparing a microfluidic chip, wherein the microfluidic chip is a fishbone-shaped chip, comprising an upper fishbone structure layer and a lower channel layer; the fishbone structure layer is provided with a plurality of spaced fishbone-shaped crossbeams, and the channel layer is provided with a plurality of evenly distributed support columns; the gaps between the support columns form fluid channels; (2) delivering the magnetic beads modified with EpCAM antibodies to the microfluidic chip in step (1), and then placing the microfluidic chip on a magnet for stabilization, so that the magnetic beads are evenly and stably distributed inside the microfluidic chip; (3) delivering peripheral blood containing exfoliated cells from the transplanted kidney into the microfluidic chip treated in step (2), so that the exfoliated cells from the transplanted kidney are captured by the magnetic beads through the EpCAM antibodies modified on the surface of the magnetic beads; (4) After the capture is completed, the magnet is removed, and then a release reagent is introduced into the microfluidic chip to release the captured transplanted kidney exfoliated cells with magnetic beads from the chip.

2. The specific reversible capture method according to claim 1, wherein: The geometric arrangement of the microfluidic chip is as follows: the height of the fishbone structure layer is 30-60 μm, the height of the channel layer is 30-60 μm; the width of the fishbone beam is 80-120 μm, the height is 30-60 μm, and the spacing between adjacent fishbone beams is 80-120 μm; the height of the support column is 30-60 μm.

3. The specific reversible capture method according to claim 2, wherein: The material of the microfluidic chip is one or a combination of at least two of polydimethoxysilane, polymethyl methacrylate, polyethylene, polypropylene or polycarbonate; the microfluidic chip is prepared by a photolithography template, an imprinting template or an injection molding template method.

4. The specific reversible capture method according to claim 1, wherein: In the step (3), the delivery speed is 1.8-2.2 mL / h.

5. The specific reversible capture method according to claim 1, wherein: The release reagent is 4.5%-5.5% BSA.

6. The specific reversible capture method according to any one of claims 1 to 5, characterized in that: The step (3) further comprises: fixing the captured exfoliated cells of the transplanted kidney, washing them with PBS, staining them with an antibody mixture and DAPI, and then imaging and identifying them with a fluorescence microscope.

7. The specific reversible capture method according to claim 7, wherein: The fixation in step (3) is performed by incubating with 4% PFAa solution at room temperature. The antibody mixture contains anti-EpCAM-PE, Alexa Fluor 488-labeled anti-PODXL and anti-CD45-APC.

8. The specific reversible capture method according to any one of claims 1 to 5, characterized in that: The step (4) further comprises: fixing the released exfoliated cells of the transplanted kidney and then identifying their origin by FISH analysis.

9. The specific reversible capture method according to claim 8, wherein: The fixation in step (4) is performed by incubating at room temperature using a mixed fixative, wherein the mixed fixative is prepared by mixing glacial acetic acid and methanol in a volume ratio of 1:3.

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