CN113845593B

CN113845593B - anti-alpha-SMA protein monoclonal antibody, cell line and application thereof

Info

Publication number: CN113845593B
Application number: CN202111254858.7A
Authority: CN
Inventors: 李恢波; 彭永辉; 王小亚; 邓晓; 陈惠玲
Original assignee: Fuzhou Maixin Biotech Co ltd
Current assignee: Fuzhou Maixin Biotech Co ltd
Priority date: 2021-10-27
Filing date: 2021-10-27
Publication date: 2023-03-10
Anticipated expiration: 2041-10-27
Also published as: CN113845593A

Abstract

The invention relates to the field of biological detection, and provides an anti-alpha-SMA protein monoclonal antibody, wherein the amino acid sequences of heavy chain and light chain variable regions of the monoclonal antibody are respectively the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO. 3. The inventor also provides a hybridoma cell line secreting anti-alpha-SMA protein, wherein the cell line is a mouse hybridoma cell line 8E5D12A9 with the preservation number of: CGMCC NO.22314. The monoclonal antibody resisting the alpha-SMA protein has high specificity and sensitivity, can specifically identify cells expressing the alpha-SMA protein, and is suitable for immunological detection, especially immunohistochemical detection.

Description

anti-alpha-SMA protein monoclonal antibody, cell line and application thereof

Technical Field

The invention relates to the field of biological detection, in particular to an anti-alpha-SMA protein monoclonal antibody, a cell line and application thereof.

Background

Actin is an intracellular protein which has contractile ability and can be polymerized into a cell with abundant microfilaments, and the microfilaments are essential to the structure of skeletal cells, the motility and the formation of cells, and at least 6 actin types, alpha-SMA types, are contained in vertebrate cells. Encoded by the ACTA2 gene, also known as alpha-Actin-2 (Actin Alp)ha 2, ACTA2). alpha-SMA (ACTA 2) (like all acts) is extremely conserved and found in almost all mammals. In humans, α -SMA (ACTA 2) is encoded by the ACTA2 gene at 10q22-q24, consists of 377 amino acid residues and has a relative molecular mass of 4.2X 10 ⁴ Widely distributed in various myotype cells.

alpha-SMA is a microfilament belonging to the actin family. It is a contractile muscle-specific isoserine protease and motility that controls cell morphology, and expresses smooth muscle cells primarily in blood vessels. In addition, expression of a-SMA is associated with myofibroblasts. Vascular smooth muscle cells are present in the tunica media of the vascular wall, are one of the main cellular components of the vascular wall, and are also important factors in determining vascular activity, vascular configuration and maintaining vascular tone. Actin is the basic component of cytoskeletal microfilaments, in which α -SMA is mainly distributed in muscle cells, while α -SMA specifically expressed in vascular smooth muscle is the protein most abundant in differentiated mature vascular smooth muscle cells. Thus, a-SMA can be used to reflect changes in vascular smooth muscle cell number and its contractile capacity, with actin content having a dramatic effect on the contractile capacity of VSMCs. In addition, α -SMA is also closely related to the fibrosis of various tissues in the body, such as cardiac muscle tissue, lung tissue, and kidney tissue. alpha-SMA is expressed in kidney, skin, tendon, heart, etc. injury, and is involved in the development of fibrosis, and is involved in TGF-. Beta.1. In the process of heart injury, the alpha-SMA can also promote the growth of atherosclerotic plaques through the ways of blood vessel growth regulation and the like, thereby participating in the occurrence and development process of coronary heart disease. Under the action of a large number of cytokines and growth factors, fibroblasts express a large amount of alpha-SMA and secrete a large amount of collagen, causing cardiac fibrosis.

The quaternary space structure of the alpha-SMA protein contains a plurality of bindable glycoprotein ligands, which can play a role in influencing the transcription or activation of cancer cell-related malignant tumor signal pathway proteins, aggravate the change of the biological properties of cancer cells, lead to the change of DNA amplification of the cancer cells or phosphorylation modification of oncogene-related proteins, and promote the invasion or metastasis of cancer cells of a patient. It is proved that the alpha-SMA protein can play a certain promoting role in promoting the clinical stage progress, cancer tissue infiltration or histological typing deterioration of various malignant tumors such as thyroid cancer, ovarian cancer or liver cancer. Alpha-smooth muscle actin (alpha-SMA) is the most common marker for smooth muscle, and has relatively high specificity and sensitivity to smooth muscle.

Disclosure of Invention

The inventor provides an anti-alpha-SMA protein monoclonal antibody, wherein the amino acid sequences of heavy chain and light chain variable regions of the monoclonal antibody are respectively the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO. 3.

Furthermore, the heavy chain and light chain variable region amino acid sequences of the monoclonal antibody are respectively encoded by the nucleotide sequences shown in SEQ ID NO.4 and SEQ ID NO. 5.

Furthermore, in the preparation process of the monoclonal antibody, an antigen used by an immunized mouse is a recombinant protein which is coded by a nucleotide sequence shown in SEQ ID NO.1 and is expressed by escherichia coli recombination.

Further, the monoclonal antibody specifically recognizes human α -SMA protein.

Further, the monoclonal antibody is produced by a hybridoma cell line with the preservation number of CGMCC NO.22314. The cell strain is a mouse hybridoma cell line 8E5D12A9, and is classified and named as: a mouse hybridoma cell line which has been deposited at 29.29.04 in 2021 at the center of China Committee for culture Collection of microorganisms and is addressed to the institute of microorganisms of the national academy of sciences, china, institute of sciences, no.3, west Lu 1, beijing, chaoyang, and the quarter.

Further, the anti-alpha-SMA protein is a mouse IgG1 subtype monoclonal antibody.

The inventor also provides a preparation method of the monoclonal antibody for resisting the alpha-SMA protein, and an antigen used by an immune mouse is a recombinant protein which is encoded by a nucleotide sequence shown in SEQ ID NO.1 and is expressed by escherichia coli recombination.

The inventor also provides a hybridoma cell line secreting anti-alpha-SMA protein, wherein the cell line is a mouse hybridoma cell line 8E5D12A9, the cell line is preserved in the China general microbiological culture Collection center with the preservation number: CGMCC NO.22314.

The inventor also provides the application of the anti-alpha-SMA protein monoclonal antibody in human alpha-SMA protein immunoassay.

Further, the immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.

The inventor further provides a human alpha-SMA protein immunodetection reagent, wherein the immunodetection reagent contains any one of the anti-alpha-SMA protein monoclonal antibodies as an effective component.

Different from the prior art, according to the technical scheme, according to the structure, antigenicity, hydrophilicity and hydrophobicity of amino acids and a secondary structure of alpha-SMA protein, the technical scheme selects amino acid fragments from 2 th to 300 th positions of the alpha-SMA protein and corresponding nucleotide fragments thereof, carries out recombinant expression through escherichia coli, immunizes mice, and obtains a monoclonal cell line 8E5D12A9 which efficiently secretes an anti-alpha-SMA protein monoclonal antibody through cell fusion, screening and cloning, and carries out in vitro culture on the monoclonal cell line 8E5D12A9, and obtains the anti-alpha-SMA protein monoclonal antibody secreted by the cell line. The antibody obtained by the scheme has high specificity and sensitivity, can specifically identify cells expressing the alpha-SMA protein, and is suitable for immunological detection, particularly immunohistochemical detection.

Drawings

FIG. 1 is a graph showing the results of electrophoresis of the α -SMA monoclonal antibody after purification in example 3; m is a molecular weight marker.

FIG. 2 is a graph showing the results of immunohistochemical staining for leiomyosarcoma; the left is 8E5D12A9 secreted alpha-SMA, the right is commercial alpha-SMA (1A 4).

Detailed Description

EXAMPLE 1 preparation of recombinant alpha-SMA protein fragments

1. Antigen fragment selection

The alpha-SMA protein sequence with the number P62736 was selected as the standard sequence from the Uniprot database (http:// www.uniprot.org). The invention selects amino acid fragments from 2 nd site to 300 th site of human alpha-SMA protein as antigen, and designs a specific upstream Primer 5'GGGATCCTGTGAAGAAGAAGATTCTACGG' (SEQ ID NO.6 restriction site, bamH I) and a downstream Primer of the alpha-SMA protein by using software Primer 5.0 according to a corresponding base sequence (SEQ ID NO. 1): 5'CCTCGAGAACGTTGTTTGCGTACAGATCT 3' (SEQ ID NO.7, restriction site, xho I), and a gene fragment SEQ ID NO.1 encoding amino acids 2 to 300.

And separating the PCR product by agarose gel electrophoresis, recovering, performing BamH I and Xho I double enzyme digestion on the recovered target gene and a plasmid vector pET28a for expression respectively, performing electrophoresis recovery again, and connecting by T4 DNA ligase. And transforming the connecting product into an escherichia coli competent cell Rosetta, selecting clone on a plate, inoculating, expanding and culturing, extracting plasmid DNA, and performing PCR identification. The clone with positive target gene shown by PCR is sequenced and analyzed, and the clone with completely correct sequence is used for the next step.

2. Expression and purification of recombinant alpha-SMA protein fragments

Take out the competent cell 100ul (BL 21) preserved at-80 deg.C, add 5ul alpha-SMA plasmid DNA after thawing, and stand on ice for 30min. After heat shock at 42 ℃ for 90 seconds and ice bath for 5min, 800. Mu.L of non-resistant LB medium was added. Recovering and culturing at 37 deg.C for 60min, plating, and culturing overnight. The transformed plate was picked up and single colonies were cultured in 4ml of LB liquid medium overnight at 37 ℃ and 200rpm, and then the cells were maintained.

4ul of the bacterial liquid is taken to be put into 4ml of LB liquid culture medium and cultured overnight for recovery. The cultured bacterial suspension was transferred to 200ml of LB liquid medium, mixed, cultured at 37 ℃ and 200rpm until OD =0.6-0.8, and then IPTG (0.5 mM) was added thereto to induce overnight at low temperature. The cells were collected by centrifugation in a 400ml large centrifuge bowl at 4000rpm for 10min and the supernatant was discarded. The precipitate was dispersed in 20-30ml of 10mM Tris-HCl (pH 8.0) and NaCl at a final concentration of 0.5M, and the cells were disrupted by sonication. After centrifugation at 12000rpm for 20 minutes, the centrifuged supernatant was applied to a Ni-NTA nickel column (QIAGEN) for purification, and then eluted with 10mM Tris-HCl (pH 7.0) (containing 0.5M sodium chloride) containing 50mM imidazole, 250mM imidazole and 500mM imidazole, respectively, to collect protein peaks, followed by electrophoresis for further use.

EXAMPLE 2 establishment of 8E5D12A9 hybridoma cell line

1. Immunization

The α -SMA protein fusion protein obtained in example 1 was emulsified by mixing with an equal volume of complete Freund's adjuvant (CFA, sigma Co.), and 18-20g of Balb/c mice (purchased from Wu's laboratory animal, fuzhou) were immunized by abdominal injection at a dose of 50 μ g/mouse. Thereafter, the booster was administered every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant (IFA, sigma Co.) at a dose of 25. Mu.g/mouse. The polyclonal titer of the anti-immunogen in the serum of the mice is detected by indirect ELISA (wavelength of 450 nm) 14 days after the 2 nd boosting immunization, the mice with the highest titer are injected by tail vein for impact immunization, and the antigen is uniformly mixed by PBS solution, and the dosage is 50 mu g/mouse.

2. Cell fusion:

aseptically preparing mouse spleen cell suspension with qualified immunity, mixing with mouse myeloma cell sp2/0 (ATCC) at a ratio of 5:1, centrifuging at 1000rpm for 10min, discarding supernatant, adding 1mL of PEG (Sigma) solution preheated to 37 ℃ from slow to fast within 1 min, and slightly rotating the centrifuge tube during the addition process to make the cells fully contact with PEG. Standing at room temperature for 90s, adding 4mL of serum-free DMEM (Hyclone company) culture medium preheated to 37 ℃ from slow to fast within 2min, then adding 10mL of preheated serum-free DMEM culture medium within 2min, finally adding the rest preheated serum-free DMEM culture medium within 2min, and fixing the volume to 50mL, wherein a centrifugal tube needs to be slowly shaken in the whole adding process to ensure uniform mixing, so that the damage to cells is reduced. Standing at room temperature for 10min, centrifuging (1000rpm, 5 min), discarding the supernatant, resuspending the cells in 10-20mLHAT (Sigma) medium, and diluting with HAT medium to a final concentration of 0.5X 10 ⁶ cells/mL, all solutions were transferred to 96-well plates at 200. Mu.L/well and labeled. Carefully transfer 96 well plates to 37 ℃,5% CO ₂ Culturing in an incubator. The growth state and potential pollution of cells are regularly checked, and the incubator is opened and closed as little as possible to ensure the stability of the culture environment. On day 5 post-fusion, plates were supplemented with HAT medium at 50. Mu.L/well.

3. Cloning and ELISA screening positive hybridoma cells:

when the fused cell diameter is about 1-2mm, 50-200. Mu.L of culture supernatant is aspirated for the first cell selection (ELISA, IHC-P and other methods of detection), and HAT medium is added to the culture wells to 200. Mu.L. And (3) detecting the culture solution supernatant by ELISA, transferring all cell culture solution in the culture hole with the positive result obtained by detection to a 24-hole culture plate, supplementing HT medium, culturing for 3 days at a concentration of 2 mL/hole.

And repeatedly screening each cell strain in the 24-pore plate, and removing the culture well cells which are not positive results to obtain the culture well cells with better positive results. Performing subclone screening on the positive well cells obtained from the 24-well culture plate by limiting dilution method, namely adding cell sap obtained by limiting dilution method into 96-well culture plate, and transferring to CO ₂ Culturing in an incubator for 11 days, and repeating cell screening when the diameter of the cloned cell is 1-2 mm. According to the detection result, 4 well-grown monoclonal positive culture wells are selected from each subcloned cell strain, and transferred to a 24-well plate for continuous culture. And after a period of time, screening the positive clone cell strain obtained by cloning in the 24-pore plate again, namely the hybridoma cell strain 8E5D12A9 secreting the specific monoclonal antibody. The cell strain is transferred into a T-75 culture flask to be amplified to a logarithmic growth phase for seed preservation or subsequent experiments are carried out.

EXAMPLE 3 preparation of monoclonal antibodies by in vitro culture

1. In vitro culture

After obtaining the stable hybridoma cell line, obtaining the monoclonal antibody by adopting an in vitro culture method.

The hybridoma cells were cultured in DMEM complete medium containing 10% fetal bovine serum, and then centrifuged at low speed to collect culture supernatant, which was stored at 4 ℃ for further use.

2. Purification of monoclonal antibodies

Antibody purification using rProtein A sepharose Fast Flow (GE) affinity column: (1) loading the column, loading a proper amount of purchased ProteinA filler into a gravity chromatography column, and washing the column with an equilibrium buffer (0.1M Tris solution containing 1.5M NaCl, pH 7.0) until the column is balanced; (2) loading, namely adding cell supernatant filtered by a 0.22-micron filter membrane into a packed chromatographic column, and controlling the flow rate to be 1 drop/second; (3) balancing, and washing the sample solution to balance by using a balance buffer solution after the sample solution is loaded; (4) eluting, adding elution buffer (0.1M citric acid solution, pH4.5) to wash the column, and collecting eluate; (5) regenerating, adding an equilibrium buffer solution to wash the column to balance after the elution is finished, washing the column with 2 times of column volume of 20% ethanol, and storing at 4 ℃. And finally, identifying the purity of the antibody by adopting an SDS-PAGE method. The result is shown in figure 1, the polyacrylamide gel electrophoresis picture of the purified alpha-SMA monoclonal antibody has the purity of more than 95 percent, and the concentration of the antibody measured by an ultraviolet micro-spectrophotometer method reaches more than 3.0 mg/mL.

EXAMPLE 4 characterization of monoclonal antibodies

1. Subtype identification

The antigen proteins were diluted to 1. Mu.g/mL coated ELISA plates, 100. Mu.L per well, coated overnight at 4 ℃, the liquid was decanted, the plates were washed 3 times with PBS containing 0.05% Tween (PBS-T), 200. Mu.L of blocking solution (PBS-T solution containing 2% BSA) was added per well, and incubated at 37 ℃ for 1h. The liquid was decanted and washed 3 times with PBS-T. 0.1mL of culture supernatant of hybridoma cell line diluted 5-fold was added to each well and incubated at 37 ℃ for 1 hour. The liquid was decanted and washed 3 times with PBS-T. Using a confining liquid 1: HRP-labeled goat anti-mouse (kappa, lambda, igM, igG1, igG2a, igG2b, igG) diluted 400 ₃ IgA) antibody (Southern Biotech) was added in an amount of 0.1mL per well and incubated at 37 ℃ for 1 hour. The liquid was decanted and washed 3 times with PBS-T. 100 mu LTMB (Hiroshi, huzhou) substrate (A, B equal volume mixed solution) is added into each hole for color development, the reaction is carried out for 15min at room temperature, 50 mu L of 1N HCl solution is added into each hole to stop the color development reaction, and then an enzyme-labeling instrument is used for measuring the OD value at the wavelength of 450 nm. The results show that the monoclonal antibody of the invention is an IgG1 type murine monoclonal antibody.

2. Determination of affinity constant

alpha-SMA protein was coated at a concentration of 100. Mu.g/ml, 100. Mu.l/well, coated overnight at 4 ℃ and washed 3 times with PBS-T. Add 200. Mu.l of blocking solution to each well and block at 37 ℃ for 1h, wash 3 times with PBS-T. The monoclonal antibody purified in example 4 was diluted to the following concentrations (unit: ng/mL): 2000. 500, 125, 62.5, 31.25, 15.625, 3.125, 0.625, incubation at 37 ℃ for 1h, PBS-T wash 3 times. HRP-labeled goat anti-mouse secondary antibody 1:5000 dilution, 100. Mu.l per well, incubation at 37 ℃ for 1h, PBS-T wash 3 times. Mu.l of TMB (Hiroshi Biotech, inc., england, huzhou) color developing solution was added to each well, and the reaction was stopped by adding 100. Mu.l of 1.0N hydrochloric acid solution for color development for 13 min. Measuring wavelength 450nm with enzyme-linked immunosorbent assayAnd (4) light absorption value. And drawing a curve of OD values corresponding to the antibody dilution times, and finding out the antibody concentration A corresponding to 1/2 of the platform OD value. The affinity constant was calculated to be 6.26X 10 using the following formula ⁹ 。

Example 5 tissue chip staining and characterization

1. Tissue wax block preparation

HE section staining was performed on the sample tissue to determine the tumor lesion site. And (5) circling the lesion site and preparing to punch. When the receptor wax block is manufactured, a plastic frame is placed on a mould, melted paraffin (the melting point is 56-58 ℃) is poured into the mould, the tissue block is placed into wax liquid in the mould, then a proper amount of wax liquid is added to completely embed the tissue block in the wax liquid, the mould is placed into a refrigerator with the temperature of-20 ℃ for 6min after being cooled to room temperature, the wax block is taken out of the mould, and the section is sliced or placed into the refrigerator with the temperature of 4 ℃ for storage and standby. Slicing continuously, slicing to 4 μm thickness, rinsing the slices in 40% ethanol, naturally spreading, transferring the slices into 45 deg.C warm water, spreading for 30 s, mounting the slices with 2% APES acetone solution treated glass slide, baking the obtained tissue chip in 60 deg.C oven for 2 hr, taking out, cooling at room temperature, and storing in-4 deg.C refrigerator.

2. IHC staining and analysis

Conventional xylene dewaxing was performed 3 times for 6 minutes each time with 100%, 95%, 85% gradient ethanol hydration for 3 minutes each time and finally tap water rinse. Antigen retrieval was performed and the sections were then placed in a wet box and washed 3 x 3min with PBS. Dropwise adding 3%H ₂ O ₂ Incubate for 10min and wash with PBS for 3 × 3 min. The PBS was spun off and the peroxidase blocker was added dropwise and incubated for 10 minutes at room temperature. Spin-drying the section, dripping primary antibody diluted in a proper proportion (the dilution proportion of the antibody is designed according to the concentration of the antibody in the first dilution) and incubating for 1 hour at room temperature (25 ℃), washing for 3X 3 minutes by PBS, dripping secondary antibody and incubating for 30 minutes at room temperature, washing for 3X 3 minutes by PBS, throwing away PBS, and developing for 3-10 minutes by using a freshly prepared DAB developing solution. Lignum sappanThe element was counterstained for 20 seconds and PBS turned blue. Dehydration was carried out in a gradient of 85% (3 min), 95% (3 min), 100% (3 min) and 100% (3 min) in order, and finally two times xylene was cleared for 10min, followed by sealing with neutral gum.

The immunohistochemical staining results were divided into: positive and negative. Positive expression must be at a specific antigenic site in cells and tissues to be considered positive. Under the condition that the tissue staining distribution is clear and the cell positioning is accurate, the staining result is further divided according to the difference of staining intensity, which is as follows:

1. the sample is weakly positive; marked "+";

2. the sample is moderately positive; marked "+";

3. the sample is highly positive; marked as "+ + +".

4. The sample was negative and marked "-".

3. And (3) detecting a sample:

the anti-alpha-SMA protein monoclonal antibody (8E 5D12A 9) prepared by the invention and the commercial anti-alpha-SMA protein monoclonal antibody (1A 4) are used for synchronous detection on 52 cases of leiomyoma and 9 cases of leiomyosarcoma, and the results are shown in the table

The result shows that the staining location of the anti-alpha-SMA protein monoclonal antibody (8E 5D12A 9) is accurate, the staining is clear, no non-specific staining exists, the background is clean, and the specificity of the anti-alpha-SMA protein monoclonal antibody (8E 5D12A 9) is strong. In 52 cases of leiomyoma and 9 cases of leiomyosarcoma, the positive rate of the anti-alpha-SMA protein monoclonal antibody (8E 5D12A 9) is equivalent to that of the commercial antibody, but the number of positive cells and the positive intensity are higher than those of the control reagent, which indicates that the differential diagnosis of the anti-alpha-SMA protein monoclonal antibody (8E 5D12A 9) on leiomyoma and leiomyosarcoma is more sensitive than that of the commercial antibody, and the risk of missed detection can be avoided. FIG. 2 is a graph of immunohistochemical staining results for leiomyosarcoma in 1 of the cases, with secreted α -SMA (8E 5D12A 9) on the left and commercially available α -SMA (1A 4) on the right.

And the anti-alpha-SMA protein monoclonal antibody (8E 5D12A 9) and the control antibody (1A 4) are adopted to carry out synchronous detection on 30 normal tissue chips, and the positive and negative results of the samples are consistent, which shows that the specificity of the antibody on the commercial tissues is equivalent to that of the commercial antibody.

The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectal, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (young child), skin, bone marrow, peripheral nerve, lung, mesothelial cells.

Sequence listing

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Claims

1. The monoclonal antibody for resisting alpha-SMA protein is characterized in that the amino acid sequences of heavy chain and light chain variable regions of the monoclonal antibody are respectively the amino acid sequences shown in SEQ ID NO.2 and SEQ ID NO. 3.

2. The monoclonal antibody of claim 1, wherein the heavy and light chain variable region amino acid sequences of the monoclonal antibody are encoded by the nucleotide sequences set forth in SEQ ID No.4 and SEQ ID No.5, respectively.

3. The monoclonal antibody of claim 1, wherein the antigen used for immunizing a mouse in the preparation process of the monoclonal antibody is a recombinant protein encoded by the nucleotide sequence shown in SEQ ID No.1 and recombinantly expressed by Escherichia coli.

4. The monoclonal antibody of claim 1, which specifically recognizes human α -SMA protein.

5. The monoclonal antibody of claim 1, wherein the monoclonal antibody is produced by a hybridoma cell line having a collection number of CGMCC No.22314.

6. The monoclonal antibody of claim 1, wherein said anti-a-SMA protein is a mouse IgG1 subtype monoclonal antibody.

7. A hybridoma cell line for secreting an anti-alpha-SMA protein monoclonal antibody is a mouse hybridoma cell line 8E5D12A9, and is preserved in the China general microbiological culture Collection center with the preservation number: CGMCC NO.22314.

8. An immunoassay reagent for human α -SMA protein, characterized in that the immunoassay reagent contains the monoclonal antibody against α -SMA protein according to any one of claims 1 to 6 as an active ingredient.