CN111073860B - Application of anti-shrimp arginine kinase monoclonal antibody in anti-allergic medicine and detection - Google Patents

Abstract

The invention discloses an application of an anti-shrimp arginine kinase monoclonal antibody in anti-allergic drugs and detection, wherein the anti-shrimp arginine kinase monoclonal antibody is secreted and generated by a hybridoma cell strain AK-3D6, and the hybridoma cell strain is named as a hybridoma cell strain AK-3D 6; the culture is preserved in China center for type culture Collection with the preservation date of 2019, 6 and 26 months and the preservation number of CCTCC NO: c2019128; the anti-shrimp arginine kinase monoclonal antibody can be combined with specific IgE (serum immunoglobulin E), so that the combination of arginine kinase and specific IgE is inhibited, allergy is relieved, and the anti-shrimp arginine kinase monoclonal antibody can be used as one of active ingredients or the only active ingredient to prepare medicines or health products for treating or relieving allergic reaction induced by shrimps and shrimp products. Can also be used for quantitatively detecting crustacean arginine kinase.

Description

Application of anti-shrimp arginine kinase monoclonal antibody in anti-allergic medicine and detection

Technical Field

The invention relates to the technical field of bioengineering, in particular to application of an anti-shrimp arginine kinase monoclonal antibody in anti-allergic drugs and detection.

Background

In recent years, food allergy (food) has attracted much attention from governments and scholars as a serious public health problem. The aquatic products are popular with consumers due to rich nutrition and delicious taste, and the market of the aquatic products tends to be complicated along with the rapid development of the aquatic product processing industry and the market globalization, so that the allergy incidence rate of the aquatic products is in a continuously rising trend. About 2.5% of people in the world have allergy to aquatic products, the allergy to aquatic products in Asian regions is particularly high, and about 40% of Asian children and 33% of Asian adult food allergy are caused by shellfish aquatic products such as shrimps and crabs. Allergy to crustacean aquatic products usually causes allergic symptoms such as skin red swelling, asthma, rhinitis and the like of patients, and in severe cases, the allergic symptoms are accompanied by collapse and shock, even the life is threatened, and the physical health and the life quality of allergic people are seriously affected.

Arginine Kinase (AK) is widely present in crustaceans such as shrimp and crab, is considered as one of the major allergens causing crustacean allergy, and can specifically react with 80% of serum IgE of crustacean allergic patients, and meanwhile, different crustacean arginine kinases have extremely high homology and severe cross-reactivity. At present, the sensitization of shrimp products is mainly reduced by avoiding eating the shrimp products or treating the shrimp products by high pressure, enzymolysis, ultrasonic waves and the like, so that the effect of preventing diseases is achieved, but the effect is not good.

Disclosure of Invention

In order to solve the above technical problems, the present invention aims to provide an anti-shrimp arginine kinase monoclonal antibody which can bind to specific IgE (serum immunoglobulin E) to inhibit the binding of arginine kinase to specific IgE and relieve allergy, and can be used as one of the active ingredients or the only active ingredient to prepare a drug or health product for treating or relieving allergic reactions induced by shrimp and shrimp products. Can also be used for quantitatively detecting crustacean arginine kinase.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides an application of an anti-shrimp arginine kinase monoclonal antibody in an antiallergic medicament, which is characterized by comprising the following steps: the anti-shrimp arginine kinase monoclonal antibody is secreted and produced by a hybridoma cell strain AK-3D6, and the hybridoma cell strain is named as a hybridoma cell strain AK-3D 6; deposited in China center for type culture Collection, address: the preservation date of Wuhan university in Wuhan, China is 6 months and 26 days in 2019, and the preservation number is CCTCC NO: c2019128; the monoclonal antibody is used as one of the active ingredients or the only active ingredient to prepare the medicine or health care product for treating or relieving the anaphylactic reaction induced by the shrimps and the shrimp products.

The invention provides an application of an anti-shrimp arginine kinase monoclonal antibody in detection, which is characterized by comprising the following steps:

the anti-shrimp arginine kinase monoclonal antibody is secreted and produced by a hybridoma cell strain AK-3D6, and the hybridoma cell strain is named as a hybridoma cell strain AK-3D 6; the culture is preserved in China center for type culture Collection with the preservation date of 2019, 6 and 26 months and the preservation number of CCTCC NO: c2019128; the anti-shrimp arginine kinase monoclonal antibody is used for quantitatively detecting crustacean arginine kinase.

The invention provides an application of the monoclonal antibody in inhibiting shrimp allergy information transfer, and the monoclonal antibody is used as one of active ingredients or the only active ingredient to prepare a medicament or health-care product for treating or relieving the allergic reaction induced by shrimps and shrimp products.

The invention has the beneficial effects that: the anti-shrimp arginine kinase monoclonal antibody can be combined with specific IgE (serum immunoglobulin E), so that the combination of arginine kinase and specific IgE is inhibited, and allergy is relieved. The medicine includes solution, syrup, injection, emulsion, capsule, etc. The medicine can be added with at least one of excipient, stabilizer, humectant, diluent, absorption enhancer, pH regulator and surfactant; if the health product is a health product, at least one of preservative, excipient, stabilizer, humectant, absorption enhancer, pH regulator and surfactant can be added. The anti-shrimp arginine kinase monoclonal antibody can also be used for quantitatively detecting crustacean arginine kinase, and solves the problem in arginine kinase detection.

Drawings

FIG. 1 is a schematic SDS-PAGE electrophoresis of naturally purified shrimp arginine kinase;

FIG. 2 is a schematic diagram showing the inhibitory effect of the antishrimp arginine kinase monoclonal antibody on the sensitization reaction of prawn;

FIG. 3 is an elution profile of Source15Q anion exchange purified arginine kinase;

FIG. 4 is a standard curve of quantitative determination of crustacean arginine kinase by double-enhanced ELISA based on nanogold and nanobagnetic beads;

FIG. 5 is a standard curve of quantitative determination of crustacean arginine kinase by immunofluorescence based on nanogold-quantum dot composite probe.

Detailed Description

The following detailed description of the present invention is provided in conjunction with the accompanying drawings, but it should be understood that the scope of the present invention is not limited to the specific embodiments. It should be noted that the experimental methods used in the following examples are all conventional methods unless otherwise specified; materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The anti-shrimp arginine kinase monoclonal antibody can be prepared into medicines of any administration mode and is formed by various medicines. The medicine includes solution, syrup, injection, emulsion, capsule, etc. The medicine may be added with at least one of excipient, stabilizer, humectant, diluent, absorption promoter, pH regulator and surfactant. If the health product is a health product, at least one of preservative, excipient, stabilizer, humectant, absorption enhancer, pH regulator and surfactant can be added.

Example 1

Preparation of shrimp arginine kinase:

(1) taking 50g of muscle of the penaeus vannamei boone, and removing the head, tail, shell and gut of the penaeus vannamei boone.

(2) The shrimp muscle was cut into paste with a small knife and dissolved in buffer A (50mM NaCl, 2mM NaHCO)₃10mM EDTA) was homogenized with a homogenizer and allowed to stand at 4 ℃ for 2 h.

(3) And (3) centrifuging the solution obtained after the standing in the step (2) at 8000r/min and 4 ℃ for 20min, taking supernate, adding 70% ammonium sulfate, and standing for 8h at 4 ℃.

(4) And (4) centrifuging the supernatant obtained after the standing in the step (3) at 8000r/min and 4 ℃ for 20min, taking the supernatant, adding 90% ammonium sulfate, and standing at 4 ℃ for 6 h.

(5) And (3) centrifuging the supernatant obtained in the step (4) at 8000r/min and 4 ℃ for 20min, taking a precipitate, and dissolving the precipitate in buffer B (20mM Tris-HCl, 1mM NaCl, pH8.0) to obtain an arginine kinase solution.

(6) And (3) carrying out gradient elution on the arginine kinase liquid by using a Source15Q anion exchange column and a 0.5MNaCl solution, and collecting an eluted product to obtain the arginine kinase for later use. SDS-PAGE electrophoresis of the purified shrimp arginine kinase shows that the protein has a band at 40kDa and the grey analysis purity is more than 90%, as shown in figure 1. It should be noted that the Source15Q anion exchange column has the characteristics of high flow rate and high loading capacity, and the particle size of its base frame is small, and is only 15 μm. Small particle size and high resolution, and is suitable for fine separation of protein. During the purification process, the protein with the size of 20.1kDa and the protein with the size of 40.1kDa cannot be separated by other chromatographic columns. Referring to the schematic of fig. 3, it can be seen that a distinct peak and several peaks with smaller peak values can be obtained. The collected solution was subjected to SDS-PAGE, and the lanes were in the order of: m, protein Marker; 1. ammonium sulfate precipitation of the product; 2. ion exchange chromatography peak 1 harvest; 3-5, collecting other peaks by ion exchange chromatography. The result showed that the peak a contained only protein having a molecular weight of 40.1 kDa. In order to enrich and obviously improve the purity of arginine kinase, in the step, for the setting of an elution program, the volume of the eluent is set to be 40mL, a 0.2MNaCl solution is adopted for carrying out 30mL linear elution, then a 10mL0.5MNaCl solution is adopted for carrying out gradient elution, and the arginine kinase with the purity of 99 percent is obtained by adjusting the elution program and combining a Source15Q anion exchange column, so that the preparation requirement of cell strains is met.

Example 2

Is used for preparing hybridoma cell strains secreting the AK-3D6 monoclonal antibody.

4-6 weeks old female Balb/c mice were taken and basal immunization of each mouse was performed by subcutaneous multiple injections of 100. mu. gAK protein emulsified in Freund's complete adjuvant for a total of 400. mu.L/mouse. A second boost was performed 20 days later by a subcutaneous multiple injection of 80. mu. gAK protein emulsified in Freund's incomplete adjuvant at a total of 400. mu.L/mouse. Third boost after 15 days, the procedure was the same as for the second boost. After 20 days, 120. mu. gAK protein was intraperitoneally injected, and after 72 hours, blood was taken from the orbit, the mouse was sacrificed, a cell suspension was prepared from the spleen, the cells were counted, sp2/0 (mouse myeloma cell) in a good growth state was taken from the number of 1/5 spleen cells, and after mixing and centrifugation, polyethylene glycol (Sigma) was added to fuse the two. In addition, equal volumes of feeder cells were added, mixed well and distributed in 96-well cell plates (200. mu.L/well), and cultured in 5% carbon dioxide incubator at 37 ℃. After 5 days, the medium is half reserved and changed, and after 10 days, the supernatant of the hybridoma cultured in the 96-well cell culture plate is detected by adopting an indirect enzyme-linked immunosorbent assay. The specific method comprises the following steps:

diluting AK protein with coating solution (final concentration of 1 μ g/mL), adding enzyme-labeled plate (Stannless Seisaku Bio-engineering Co., Ltd.) at 100 μ L/hole, coating at 4 deg.C for 12 hr, washing with washing solution for 1 time by DEM-3 type plate washing machine (Daan Gen Ltd of Zhongshan university); adding sealing liquid, sealing at 200 μ L/hole at 37 deg.C for 1 hr, and washing plate with plate washing machine for 1 time; adding cell culture supernatant to be detected, positive control serum and negative control sample, incubating at 100 μ L/well for 35min at 37 deg.C, and washing with washing solution for 3 times; adding goat anti-mouse IgG labeled with HRP (horseradish peroxidase), 100 mu L/well, incubating for 30 minutes at 37 ℃, and washing for four times by using a washing solution; adding 50 mu L of color development liquid A and 50 mu L of color development liquid B into each hole, after shading and developing for 10 minutes at 37 ℃, adding stop solution to stop reaction, and reading OD value after zero calibration of blank holes with the wavelength of 450nm of an enzyme labeling instrument at 50 mu L/hole. The relevant solution formulation is as follows:

for positive hybridoma clones, subcloning was performed by limiting dilution, individual cells were selected for culture and detected by indirect enzyme-linked immunosorbent assay (ELISA). And screening to obtain a monoclonal cell strain AK-3D6 after three times of subcloning.

Example 3

Preparation and purification of anti-shrimp arginine kinase monoclonal antibody, titer detection and specificity analysis

Healthy Balb/c male mice of 6-8 weeks old are taken, intraperitoneal injection is carried out on liquid paraffin, each 500 mu L of liquid paraffin is injected, 3 days later, the intraperitoneal injection is carried out on AK-3D6 monoclonal cells (about 1.2 multiplied by 106 cells/mouse), 7-9 days later, the abdomen of the mice is bulged, and ascites is collected. The agarose affinity medium protein A column (Nanjing Kingsley Biotech Co., Ltd.) was equilibrated with 50mL of an equilibration buffer PBS (pH 7.4) until the absorbance of the column was 0 as shown by a computer-aided nucleic acid protein detector (Shanghai West analytical Instrument Co., Ltd.). Ascites is centrifuged at 12000rpm for 5 minutes, the supernatant is collected and filtered by a 0.45 μ M filter, loaded and washed by PBS until the absorbance is 0, then eluted by 0.1M glycine (pH 3.0), the effluent is collected and neutralized by 500mM Tris-HCl (pH8.5) buffer until the pH is 7.0, and the monoclonal antibody AK-3D6 is obtained.

AK-3D6 monoclonal antibody titer detection: a96-well plate was coated with 1. mu.g/mL of AK protein carbonate buffer (pH9.5) at 4 ℃ overnight at 100. mu.L, and goat anti-mouse IgG-HRP (50ng/mL) was added to the plate to dilute the monoclonal antibody (1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000) in a gradient, whereby the titer of the purified monoclonal antibody (S/N >2.1) and the titer of the AK-3D6 monoclonal antibody were determined to be 1:16000, as shown in Table 1.

Monoclonal antibody specificity analysis: a96-well plate was coated with 1. mu.g/mL of carbonate buffer (pH9.5) of AK protein, BSA protein, fish parvalbumin, shrimp tropomyosin, 100. mu.L of 4 ℃ overnight, and antibody dilutions (1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000) were added to the 96-well plate, followed by addition of goat anti-mouse IgG-HRP (50ng/mL), and the results are shown in Table 1. As a result, it was confirmed that the AK-3D6 monoclonal antibody specifically recognizes only AK protein. In the embodiment, equilibrium agarose affinity medium Protein A chromatography is adopted, and compared with an ammonium sulfate precipitation method, the method has higher yield and purer product. The specific implementation process is as follows:

the agarose affinity medium Protein G column was equilibrated to room temperature, preheated for 20 minutes in a computer nucleic acid Protein detector (Shanghai Kagaku Kogyo Co., Ltd.), and washed with 10mmol/L of a PBS solution having a pH of 7.4 by passing through the column until the absorbance A of the computer nucleic acid Protein detector showed 0. After mouse ascites was centrifuged at 12000rpm for 5 minutes, the supernatant was applied to a 0.45um filter and then washed by column-washing with 10mmol/L of a pH 7.4 PBS solution until the absorbance A in a computer nucleic acid protein detector showed 0. Elution was performed with 0.1mol/L glycine solution at pH 3.0. And collecting the eluent, and adding 0.5mol/L Tris-HCl buffer solution with the pH value of 8.5 to neutralize the eluent to the pH value of 7.0, thereby obtaining the recombinant protein AK monoclonal antibody. Wherein:

preparing a glycine solution: 7.5g of glycine is dissolved in ultrapure water, the volume is adjusted to 800ml, and concentrated HCl 6-8 is added to adjust the pH value to 3.0.

Preparing a Tris-HCl buffer solution: 75.4g of Tris was dissolved in ultrapure water under stirring, and the volume was adjusted to 1000ml, and about 10ml of concentrated HCl was added to adjust the pH to 8.5.

Table 1: and (3) analyzing the titer and the specificity of the AK-3D6 monoclonal antibody.

Example 4

And (3) identifying the effect of the anti-shrimp arginine kinase monoclonal antibody on inhibiting shrimp sensitization.

The purified shrimp arginine kinase obtained in example 1 was dissolved in 100mmol/L sodium carbonate buffer (pH9.5) and diluted to 0, 0.25, 0.5, 1, 5, and 10. mu.g/mL, respectively. For coating, 6 parallel wells were made for each sample, and 100. mu.L of sample was added to each well of the microplate and allowed to coat overnight at 4 ℃. Washed 5 times with ultrapure water for 3min each time and patted dry. Add 200. mu.L blocking solution (PBS buffer containing 1% BSA) to each well, block for 1h at 37 ℃, wash and pat dry; adding 100 μ L of the anti-shrimp arginine kinase monoclonal antibody obtained in example 3 (the anti-shrimp arginine kinase monoclonal antibody is diluted to 9 μ g/mL with a blocking solution), incubating at 37 ℃ for 2h, washing, and patting to dry; adding crustacean allergic patient serum diluted by 100 times with confining liquid, incubating at 37 deg.C for 2h, washing, and patting to dry; HRP-labeled streptavidin diluted 2000-fold with blocking solution was added, incubated at 37 ℃ for 2h, washed and tapped dry. TMB was added to each well, incubated at 37 ℃ for 30min, and 50. mu.L of stop buffer (2mol/L sulfuric acid solution) was added to each well. Finally, the OD was measured at 450 nm.

Meanwhile, a sample without the addition of the anti-shrimp arginine kinase monoclonal antibody was used as a positive control. As shown in FIG. 2, the anti-shrimp arginine kinase monoclonal antibody of the present invention can inhibit the binding between natural arginine kinase and IgE, and has the effect of inhibiting shrimp sensitization reaction. Referring to FIG. 2, the control experiment shows that the antibody of the present example has an effect of alleviating allergy.

The anti-shrimp arginine kinase monoclonal antibody can be prepared into medicines of any administration mode and is formed by various medicines. The medicine includes solution, syrup, injection, emulsion, capsule, etc. The medicine may be added with at least one of excipient, stabilizer, humectant, diluent, absorption promoter, pH regulator and surfactant. If the health product is a health product, at least one of preservative, excipient, stabilizer, humectant, absorption enhancer, pH regulator and surfactant can be added.

It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

Example 5

Arginine Kinase (AK) is widely present in animals, plays a crucial role in metabolism, storage and utilization of bioenergy, and can cause IgE-mediated allergic reactions. Because of the high energy of the phosphorus-nitrogen bond and the easy hydrolysis under acidic conditions, the detection of arginine kinase is very challenging.

And quantitatively detecting the crustacean arginine kinase by a double-enhanced ELISA method based on nanogold and nanobagnetic beads.

(1) Nano gold-Ab₂Preparing a probe: adding 3 μ l of 0.1mol/L K into 1ml of 25nm nano gold solution₂CO₃The pH was adjusted to about 8.5. Then, 8. mu.l Ab with a concentration of 0.5mg/ml was added to 1ml of the nanogold solution₂(arginine kinase monoclonal antibody labeled HRP) and incubated at 25 ℃ for 1 h. Then 100. mu.l of 10% (w/v%) sodium chloride solution was added and incubated at 25 ℃ for 10 min. Then, 10. mu.l of 5% (w/v%) BSA solution was added thereto, incubated at 25 ℃ for 30min, centrifuged at 13000g for 20min, the supernatant was removed, and the precipitate was dissolved in Tris-HCl (pH8.5) to obtain AuNPs-Ab₂And (3) a probe.

(2) Preparation of magnetic bead-AK Probe: mu.g of the magnetic beads were mixed with 500. mu.l of 50mM PBS buffer (pH8.5) and 500. mu.l of glutaraldehyde, and incubated at 25 ℃ for 1h with exclusion of light. Then, the AK standard solution was added to the magnetic bead solution, and incubated at 25 ℃ for 2 hours to immobilize AK on the magnetic beads. The formed magnetic bead-AK probe was washed three times with 1ml of 50mM PBS buffer (pH 8.5). The magnetic bead-AK probe was mixed with 200. mu.l of 5% BSA solution and incubated at 25 ℃ for 1h to block non-specific binding sites.

(3) AK solution was serially diluted to 1, 2.5, 5, 10, 20, 40ng/ml as a working solution. And respectively incubating the activated magnetic beads with AK working solution and 5% BSA to obtain the magnetic bead-AK probe. Then, the magnetic bead-AK probe is sequentially contacted with Ab₁(arginine kinase monoclonal antibody, AK-3D6 monoclonal antibody obtained in example 3) and Nanogold-Ab₂The probe was incubated at 37 ℃ for 45 min. However, the device is not suitable for use in a kitchenThen, 100. mu.l of TMB solution was added, incubated at 37 ℃ for 15min in the absence of light, and then 50. mu.l of 2M sulfuric acid solution was added to terminate the reaction, and the absorbance was read at 450 nm. And (3) drawing a standard curve by taking the concentration of arginine kinase as an abscissa and the light absorption value as an ordinate. The result is shown in fig. 4, where the regression equation is 0.06853x +0.5768R²0.9909, the limit of detection was 3.91ng/ml (S/N3).

Example 6

Quantitative detection of crustacean arginine kinase by immunofluorescence based on nanogold-quantum dot composite probe

(1) Nano gold-Ab₁Preparing a probe: adding 2 μ l of 0.1mol/L K into 1ml of 30nm nano-gold solution₂CO₃The pH was adjusted to about 8.5. Then, 10. mu.l of Ab with a concentration of 0.05mg/ml was added to 1ml of the nanogold solution₁(arginine kinase monoclonal antibody, AK-3D6 monoclonal antibody obtained in example 3), and incubated at 25 ℃ for 1 h. Then, 10. mu.l of 5% (w/v%) BSA solution was added thereto, incubated at 25 ℃ for 1h, centrifuged at 13000g for 20 minutes, the supernatant was removed, and the precipitate was dissolved in Tris-HCl (pH7.52) to obtain nanogold-Ab₁And (3) a probe.

(2) Quantum dot-Ab₂Preparing a probe: mu.L of 5mg/mL quantum dots were mixed with 10. mu.L of 10 mg/mL EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) buffer, followed by addition of 100. mu.L of 0.005mg/mLAb₂(another arginine kinase mab bound to antibody 1 at a different site) was incubated at 25 ℃ for 2h in the absence of light. Excess EDC was removed by 3 ultrafiltration (3990g, 20 min). Purified quantum dot-Ab₂The probe was stored in a refrigerator at 4 ℃.

(3) AK standard solutions were serially diluted to 0, 1, 5, 10, 50, 100,500 and 1000ng/mL as working solutions. Mixing nano gold-Ab₁The probes were incubated with different concentrations of AK working solutions for 10 min. Then, quantum dot-Ab is added₂The probe is sequentially connected with AK and nano-Au-Ab₁The probe was incubated at 25 ℃ for 30 min. Then, the fluorescence signal was measured at an excitation wavelength of 360nm and a collection wavelength of 520 nm. A standard curve was drawn with the logarithmic value of the arginine kinase concentration as the abscissa, the fluorescence value of the blank working solution as F0, and the fluorescence intensity F/F0 as the ordinate. ResultsAs shown in fig. 5, the regression equation is-0.117 x-0.2381R²0.9909, the limit of detection was 9.40ng/ml (S/N3).

Claims (2)

1. An application of an anti-shrimp arginine kinase monoclonal antibody in preparing an antiallergic medicament is characterized in that:

the anti-shrimp arginine kinase monoclonal antibody is secreted and produced by a hybridoma cell strain AK-3D6, and the hybridoma cell strain is named as a hybridoma cell strain AK-3D 6; the culture is preserved in China center for type culture Collection with the preservation date of 2019, 6 and 26 months and the preservation number of CCTCC NO: c2019128;

the monoclonal antibody is used as one of the active ingredients or the only active ingredient for preparing the medicine for treating or relieving the anaphylactic reaction induced by the shrimps and the shrimp products.

2. An application of an anti-shrimp arginine kinase monoclonal antibody in preparing a reagent for detecting crustacean arginine kinase is characterized in that:

the anti-shrimp arginine kinase monoclonal antibody is secreted and produced by a hybridoma cell strain AK-3D6, and the hybridoma cell strain is named as a hybridoma cell strain AK-3D 6; the culture is preserved in China center for type culture Collection with the preservation date of 2019, 6 and 26 months and the preservation number of CCTCC NO: c2019128;

the anti-shrimp arginine kinase monoclonal antibody is used for preparing a reagent for quantitatively detecting crustacean arginine kinase.

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