Canine albumin, preparation method and application thereof

Abstract

The present invention discloses canine albumin, a preparation method and an application thereof, wherein the preparation method comprises: using healthy canine serum or plasma as a raw material, performing a first chromatographic separation using an anion exchange chromatography column UniGel‑80Q to obtain a crude canine albumin product; (II) performing a second chromatographic separation using an anion exchange chromatography column QBestaroseFF to obtain purified canine albumin. The obtained whole albumin is then used as an immunogen to screen and obtain its specific nano-antibody. The present invention is further optimized on the basis of the applicant's early research, and can minimize the loss of albumin activity, significantly improve the purity and recovery rate of canine albumin, and the obtained product can be directly used for the preparation and screening of antibodies, and the corresponding nano-antibodies are obtained, which can be used for the detection of albumin products from different species.

Description

Technical Field

[0001] The present invention relates to the biological field, and further relates to canine albumin, a preparation method and an application thereof, and more specifically to canine albumin, a corresponding identification antibody, a detection kit and a specific application thereof. Background Art

[0002] Plasma albumin is a protein with the highest content, smallest molecule, highest solubility and most functions in plasma. The maintenance of plasma colloidal osmotic pressure mainly depends on albumin in plasma (plasma albumin constitutes about 75% to 80% of plasma colloidal osmotic pressure). The osmotic pressure generated by 1g of albumin is equivalent to the hemodynamic effect obtained by 20ml of plasma or 40ml of whole blood. In the clinical application of therapeutic protein drugs, attention should be paid to immunogenicity. Many international authoritative organizations have issued guidelines for the evaluation of immunogenicity of protein drugs. The US FDA recommends the use of multiple detection methods to evaluate the immunogenicity of antibody drugs. First, a screening method with high sensitivity, simple and fast operation, and high throughput should be established to conduct preliminary screening of clinical samples. Samples with positive results in the screening method need to be further confirmed by confirmation methods to determine whether the detection antibody specifically binds to the drug antibody.

[0003] Human serum albumin (HSA) is a single-chain non-glycosylated protein consisting of 585 amino acid residues with a relative molecular mass of 66.5×10 3 , the isoelectric point is 4.7-4.9. The primary sequence of canine serum albumin (CSA) shows that the protein is a single polypeptide composed of 584 amino acids. The paired sequence homology of CSA, HSA and BSA is high. The overall structure is very similar to that of HSA and other mammalian albumins. It consists of three topologically identical domains, each of which is divided into two subdomains. Since human serum albumin currently used in clinical practice is extracted from human plasma, the cost of human serum albumin is relatively high and it is widely used in the medical system. Therefore, in order to pursue high profits, many unscrupulous merchants use common canine albumin to prepare counterfeit goods in order to seek illegal profits. However, there is no simple detection method in the prior art that can effectively distinguish between the two. Summary of the invention

[0004] The present invention first provides a novel method for preparing canine albumin. The canine albumin obtained by the preparation method has not only high purity and high yield, but also can remove Escherichia coli endotoxin, greatly reduce the content of endotoxin in the product, and improve the safety of the product. The obtained protein can be directly applied to the screening and preparation process of monoclonal antibodies.

[0005] The above object of the present invention is achieved through the following technical solutions:

[0006] A method for preparing canine albumin comprises: (I) using healthy canine serum or plasma as a raw material and performing a first chromatography separation using an anion exchange chromatography column to obtain a crude canine albumin product; and (II) performing a second chromatography separation using an anion exchange chromatography column on the crude canine albumin product to obtain purified canine albumin.

[0007] Furthermore, the chromatographic separation method in step (I) is:

[0008] (1) The anion exchange chromatography column UniGel-80Q is equilibrated with 0.03 mol / L acetate buffer at pH 4.9-5.2 until the pH of the effluent is 4.9-5.2; (2) Canine serum or plasma is diluted with 8-10 times the amount of 0.03 mol / L acetate buffer at pH 4.9-5.2 and then loaded; (3) After loading, the anion exchange chromatography column is rinsed with 0.03 mol / L acetate buffer at pH 4.9-5.2 until the A280 absorption returns to the baseline level; (4) After rinsing, the chromatography column is eluted with 0.03 mol / L acetate buffer at pH 6.8-7.2 containing 0.4 mol / L NaCl, and the eluate (i.e., eluate (I)) is collected to obtain crude canine albumin.

[0009] Furthermore, the chromatographic separation method in step (II) is:

[0010] (1) After the anion chromatography column QBestaroseFF was equilibrated with a 0.03 mol / L acetate buffer having a pH value of 6.8 to 7.2 until the pH value of the effluent was 6.8 to 7.2, the pH of the eluate was adjusted to 6.8 to 7.2, and the sample was loaded;

[0011] (2) After loading, the column was washed with 0.03 mol / L acetate buffer at pH 6.8-7.2 until A280 absorption reached the baseline level, and then washed with 0.03 mol / L acetate buffer at pH 6.8-7.2 containing 0.1 mol / L NaCl. When A280 absorption returned to the baseline level, the column was eluted with 0.03 mol / L acetate buffer at pH 6.8-7.2 containing 0.4 mol / L NaCl, and the protein peak (i.e., eluate (II)) was collected to obtain purified canine albumin.

[0012] In order to achieve better results, the present invention can further concentrate the purified canine albumin (i.e., the eluate (II)) by ultrafiltration with an ultrafiltration membrane having a retention rate of 20 kDa until the protein concentration reaches more than 10%, and then slowly add sodium octanoate under stirring conditions to make the sodium octanoate content per gram of albumin 0.16 mmol to 0.18 mmol, adjust the pH to 6.4 to 7.4 with 0.5 mol / L sodium hydroxide solution, and then use pasteurization to inactivate viruses, and then prepare and package.

[0013] The virus inactivation by pasteurization is preferably carried out according to the following steps: sterilizing and filtering the dialyzed albumin product in a sterile container, heating the albumin solution to 60° C. for 10 hours, making the pH value of the product 6.4-7.4, and inactivating the virus under the conditions of 0.16 mmol-0.18 mmol of sodium octanoate per gram of albumin.

[0014] The second object of the present invention is to provide a canine albumin-specific antibody, which comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO: 1 and a light chain variable region having an amino acid sequence of SEQ ID NO: 3.

[0015] Another object of the present invention is to provide the use of the canine albumin and canine albumin monoclonal antibody in the preparation of canine albumin detection reagents.

[0016] Another object of the present invention is to provide a kit for detecting canine albumin, the kit comprising an effective amount of the monoclonal antibody described above and an effective amount of canine albumin; and supporting reagents for canine albumin detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 , wherein Lane 1: WB identification of canine albumin extracted in Example 1; M: marker, Lane 2 to Lane 5: WB identification of human serum albumin, bovine serum albumin, horse serum albumin, and porcine serum albumin; Lane 6: negative control.

[0018] Beneficial Effects

[0019] The present invention directly uses healthy canine serum or plasma as a raw material to prepare canine albumin with high purity and high yield. Compared with the existing preparation method, the preparation method of the present invention can minimize the loss of albumin activity, significantly improve the purity and recovery rate of canine albumin, and has simple steps, higher safety, and broad market application prospects. In addition, the present invention provides a monoclonal antibody that can specifically bind to canine albumin, which can be used to prepare canine albumin detection reagents, which is of great significance for the detection of blood products. Moreover, the advantages of the monoclonal antibody provided by the present invention over commercially available products are: 1. The antibody provided by the present invention has identified a clear antibody sequence, and the biological background is clear; 2. The antibody provided by the present invention has obvious specificity for canine albumin, and has no cross-reaction with human albumin, bovine, ovine, porcine, or horse blood albumin. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0021] Example 1 Preparation of canine albumin

[0022] 1. Preparation method

[0023] (1) Healthy dog ​​plasma or serum was melted, diluted with 10 times the amount of 0.03 mol / L acetate buffer with a pH value of 5.0, and the pH value was adjusted to 5.0 with 1.0 mol / L acetic acid, and filtered to clarify;

[0024] (2) The anion chromatography column (UniGel-80Q, purchased from Suzhou Nanovita Technology Co., Ltd.) was equilibrated with 0.03 mol / L acetate buffer at a pH of 5.0 until the pH of the effluent was 5.0, and then the diluted filtered serum or plasma sample was loaded;

[0025] (3) After loading, rinse with 0.03 mol / L acetate buffer at pH 5.0 until A280 absorption returns to the baseline level, and then elute the column with 0.03 mol / L acetate buffer (pH 6.8) containing 0.4 mol / L NaCl. This eluate (Ⅰ) is the crude canine albumin;

[0026] (4) The anion chromatography column (QBestaroseFF, purchased from Boglon (Shanghai) Biotechnology Co., Ltd.) was equilibrated with 0.03 mol / L acetate buffer at pH 7.0 until the pH of the effluent was 7.0, and the pH of the eluate (I) was adjusted to 7.0 with NaOH and loaded with the sample;

[0027] (5) After loading, wash thoroughly with 0.03 mol / L acetate buffer at pH 7.0 until A280 absorption reaches the baseline level, then wash the column with 0.03 mol / L acetate buffer (pH 7.0) containing 0.1 mol / L NaCl to remove the impurities bound to the cation column. When A280 absorption returns to the baseline level, elute the target protein with 0.03 mol / L acetate buffer (pH 6.8) containing 0.4 mol / L NaCl, and collect the protein peak. This eluate (II) is the purified canine albumin.

[0028] (6) The collected eluate (II) was concentrated by ultrafiltration using an ultrafiltration membrane with a retention rate of 20 kDa until the protein concentration reached more than 10%, and then sodium octanoate was slowly added under stirring to make the sodium octanoate content per gram of protein 0.16 mmol, and the pH was adjusted to 7.0 with 0.5 mol / L sodium hydroxide solution.

[0029] (7) The dialyzed albumin product was sterilized and filtered into a sterile container, and the albumin solution was heated to 60°C for 10 hours to achieve a pH value of 7.0 and a sodium octanoate content of 0.16 mmol per gram of albumin for virus inactivation.

[0030] (8) Preparation and packaging: The protein concentration, purity and endotoxin content of the prepared canine albumin were determined according to the methods specified in the Chinese Veterinary Pharmacopoeia and the Chinese Pharmacopoeia. The final test product had a canine albumin content of 115 mg / ml, a purity of 99%, an endotoxin content of 3 EU / ml, and an albumin recovery rate of 40 g / L.

[0031] Example 2 Preparation of canine albumin antibody library

[0032] (1) Amplify the established natural single-domain antibody phage library: add 100 μL of glycerol bacterial library to 2×YT medium, add 20 MOI helper phage when OD600=0.5, let stand for 30 min, centrifuge and resuspend the precipitate with 2×YT medium, culture for another 1 h, add antibiotics and culture for 16 h before centrifugation. The supernatant is precipitated with pre-cooled PEG-NaCl (1 / 4 volume) and resuspended in 1 mL PBS to obtain the amplified single-domain antibody library;

[0033] (2) Immunotube panning: 50 μg / tube of canine albumin obtained in Example 1 was coated on an immunotube overnight, the coating solution was removed and the tubes were washed three times, blocked with 2 mL of BSA (1%) for 2 h, washed three times with PBST, 100 μL of the single domain antibody library amplified in step (1) was added as the primary antibody, incubated at 37° C. for 2 h, washed three times with PBST, eluted with Glycine-HCl (pH 2.2), and the eluate was adjusted to pH 7.4 with Tris-HCl to obtain the first round of natural single domain antibody library for panning;

[0034] (3) Amplifying the first round natural single domain antibody library obtained in step (2) according to step (1) to obtain the first round natural single domain antibody resuspended library, and then repeating the immunotube panning step (2), except that the primary antibody is added to 100 μL of the amplified first round natural single domain antibody resuspended library, and finally obtaining the second round natural single domain antibody library for panning;

[0035] (4) The second round natural single domain antibody library obtained in step (3) is amplified according to step (1) to obtain the second round natural single domain antibody resuspended library, and then the immunotube panning step (2) is repeated, except that the primary antibody is added to 100 μL of the amplified second round natural single domain antibody resuspended library, and finally the third round natural single domain antibody library is obtained.

[0036] Example 3 ELISA Identification of Canine Albumin Monoclonal Antibodies

[0037] (1) Amplification of single positive clones selected for antibody expression by shaking: The natural single domain antibody library selected in the third round in Example 2 was inoculated in 2×YT medium. When OD600nm=0.5, 20MOI helper phage was added and the mixture was allowed to stand for 30 min. After centrifugation, the precipitate was resuspended in 2×YT medium and cultured for another 1 h. Then, the precipitate was spread on a 2×YT plate containing antibiotics and cultured overnight. The next day, 40 single colonies were selected and inoculated in 2×YT medium. When OD600=0.5, 20MOI helper phage was added and the mixture was allowed to stand for 30 min. After centrifugation, the precipitate was resuspended in 2×YT medium and cultured again. IPTG was added to induce expression for 8 h.

[0038] (2) ELISA identification: 100 μL / well of canine albumin obtained in Example 1 was coated on an ELISA plate overnight, the coating solution was removed and then washed 3 times, blocked with 200 μL / well BSA (3%) for 2 h, washed 3 times with PBST, and 100 μL / well of the single domain antibody library amplified in step (1) was added as the primary antibody (library construction vector with M13), incubated at 37°C for 2 h, washed 3 times with PBST, and M13-HRP was added as the secondary antibody. After termination, the OD450nm value was detected, and a value 3 times higher than the OD450nm value of the control group was judged as positive. Finally, 3 strains of canine albumin-specific single domain antibodies obtained in Example 1 were obtained, which were named CSAnb-1-3 respectively.

[0039] Example 4 Specificity Identification of Canine Albumin Monoclonal Antibody CSAnb-1-3

[0040] Protein electrophoresis: Dilute canine albumin, human albumin (70024-90-7, Hubei Rishengchang New Material Technology Co., Ltd.), bovine serum albumin (BTN131070, Biolab), porcine serum albumin (XV-XQ-1049, Shanghai Xuanya Biotechnology Co., Ltd.), and horse serum albumin (ABX585581, Abbexa) obtained in Example 1 to 60 μg with 6 x SDS protein electrophoresis loading buffer, boil at 100°C for 5 min, and pre-stain with 2 μL of Marker. Load the samples into the SDS-PAGE gel loading wells in a certain order, perform constant voltage electrophoresis at 90V for the concentrated gel and 120V for the separation gel for about 1 hour, and stop the electrophoresis when the blue dye reaches the bottom of the gel;

[0041] Transfer: Place the gel on a nitrocellulose membrane (NC membrane), and place 3 Whatman 3mm filter papers on each side. Soak the above items in the transfer electrophoresis buffer for 15 minutes to remove the bubbles left on the filter membrane. Install the electrotransfer device in order, and place 3 filter papers, gel, NC membrane, and 3 filter papers on the negative electrode plate in turn, ensuring that each layer is accurately aligned (from bottom to top), and remove the bubbles between the layers, mark the position, and close the anode plate. 2mA / cm 2 Transfer the membrane under constant flow for 2 hours. Western blot identification: First block with 5% skim milk powder at 4°C overnight; then add purified canine albumin single-domain antibodies CSAnb-1-3 with His tag (5% skim milk diluted 1:3000) and incubate the membrane at room temperature for 3 hours; then wash the membrane three times with PBST (Tween is 1‰), 10 minutes each time; then add goat anti-His Dy800 antibody (5% skim milk diluted 1:20000), incubate at room temperature in the dark for 40 minutes; finally use Odyssey's 700 and 800 fluorescence channels for scanning and identification. From them, select the single-domain antibody CSAnb-3 ( Figure 1, among which, the protein band of human serum albumin is about 72kD, which is basically the same as that of dog; the protein band of bovine serum albumin is about 66kD; the protein band of horse serum albumin is about 1.6kD; the protein band of porcine serum albumin is about 69kD). The plasmids were extracted from the corresponding bacterial liquid and sent to BGI for sequencing using universal primers of plasmid vectors. The gene sequences of each clone were analyzed using the sequence alignment software MEGA 6.0, and the sequence of CSAnb-3 was finally obtained as follows:

[0042] MAGVQLVESGGLVGQPGSLSCACARTAS GIIYTMRTIS WYRQAPKERRELVASI SGGSGTYSY ADTVSRKGRFTIDNKNTVYLQMNSKPLEDTAFIYCH QRSRYLGRYWDS WGQPTQVHVSS (SEQ ID NO. 1), wherein the underlined portion is CDR1-3.

[0043] Example 5 Identification of the properties of canine albumin monoclonal antibody CSAnb-3

[0044] Determination of antibody titer:

[0045] Add 100 microliters of canine albumin dissolved in PBS buffer at a concentration of 2 μg / ml to the wells of a 96-well ELISA plate and place it at 4°C overnight. The next day, wash the 96-well ELISA plate coated with canine albumin twice with a wash solution (PBS buffer containing 0.05% Tween). Then, block the ELISA plate with a blocking solution (1% BSA / wash solution) at room temperature for two hours. Pour off the blocking solution. The antibody was serially diluted 1:10, 100 μl of each dilution of the antibody was added to a 96-well plate, incubated at room temperature for 2 hours, the supernatant was discarded, the plate was washed twice with a washing solution (PBS buffer containing 0.05% Tween), and then 100 μl of alkaline phosphatase-labeled goat anti-mouse IgG antibody was added to the wells of the 96-well ELISA plate, incubated at room temperature for 1 hour, washed twice with a washing solution, and 100 μl of a color reaction substrate (4-nitrophenyl phosphate disodium salt hexahydrate) was added. The color was developed at room temperature for 30 minutes, and the light absorption value was read at a wavelength of 410 nanometers. The highest dilution multiple is the titer of the antibody. The experimental results show that the titer of the anti-canine albumin monoclonal antibody of the present invention is 1:10 5 .

[0046] Affinity determination:

[0047] 96-well ELISA plate was coated with 2 μg / ml canine albumin. Then, the ELISA plate was blocked with blocking solution (1% BSA / washing solution) at room temperature for two hours. Canine albumin monoclonal antibody CSAnb-3 was first incubated with canine albumin of different concentrations, and then the incubation solution was added to the canine albumin-coated 96-well ELISA plate, incubated at room temperature for 2 hours, the supernatant was poured out, and the plate was washed twice with washing solution (PBS buffer containing 0.05% Tween), and then 100 microliters of alkaline phosphatase-labeled goat anti-mouse IgG antibody was added to the small wells of the 96-well ELISA plate, incubated at room temperature for 1 hour, washed twice with washing solution, and 100 microliters of color reaction substrate was added. The color was developed at room temperature for 30 minutes, and the light absorption value was read at a wavelength of 410 nanometers. The affinity of the antibody is calculated using the formula A0 / (A0-A)=1+K / a0, where A0 is the OD value of canine albumin at 0, A is the OD value of canine albumin at a0, and K is the reciprocal of the affinity constant. The test results show that the affinity of the canine albumin monoclonal antibody CSAnb-3 of the present invention is approximately 1x10 -8 M.

[0048] The above specific examples are used to illustrate the present invention, which is only used to help understand the present invention and is not intended to limit the present invention. For those skilled in the art, according to the concept of the present invention, some simple deductions, modifications or substitutions can be made.

Claims

1. A single domain antibody specific for canine albumin, the amino acid sequence of which is shown in SEQ ID NO.1: MAGVQLVESGGLVGQPGSLSCACAARTASGIIYTMRTISWYRQAPKERRELVA SISGGSGTYSYADTVSRKGRFTIDNKNTVYLQMNSKPLEDTAFIYCHQRSRYL GRYWDSWGQPTQVHVSS.

2. Use of the canine albumin-specific single-domain antibody as claimed in claim 1 in preparing a kit for detecting albumin products from various genera.

3. A kit for detecting albumin products from various genera, comprising the specific single domain antibody against canine albumin as claimed in claim 1.

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