Ceramide antibody or antigen binding fragment, quality control product for detecting ceramide antibody and application

Abstract

The invention provides a ceramide antibody or an antigen binding fragment, a quality control product for detecting the ceramide antibody and application, and belongs to the technical field of in-vitro diagnosis. The invention provides the ceramide antibody or the antigen binding fragment which can be used as a ceramide antibody chemiluminescence detection quality control product, and the detection accuracy and the detection efficiency can be improved when the ceramide antibody or the antigen binding fragment is combined with a matched ceramide antibody chemiluminescence detection kit. The invention also provides a quality control product for detecting the ceramide antibody. The quality control reagent is simple in component, easy to prepare, high in correlation, strong in specificity, good in stability, capable of being stored at 2-8 DEG C and convenient to transport. The quality control product can be used for indoor quality control, laboratory capability verification, detection system performance evaluation and the like of ceramide antibody detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis, and in particular relates to a ceramide antibody or antigen-binding fragment, a quality control product for detecting ceramide antibodies and applications thereof. Background Art

[0002] Ceramide (Cer) is a class of sphingolipid compounds composed of a sphingosine skeleton and plays an important role in the cell signal transduction process. The concentration of ceramide in the plasma membrane of healthy human cells is as low as μmol. The concentration can increase 10 times under stress or stimulation. Abnormal changes in ceramide have been found in a variety of pathological conditions. Ceramide antibodies in the body are positively correlated with ceramide content. Currently, ceramide antibodies are used as a joint diagnostic indicator of ceramide and are used in clinical auxiliary diagnosis, risk assessment and prognosis judgment of a variety of diseases.

[0003] At present, the chemiluminescence method is used to detect ceramide antibodies in clinical samples, which has the advantages of wide detection range and high sensitivity. However, there is a lack of ceramide antibody chemiluminescence detection quality control products with high specificity and stability on the market. Summary of the invention

[0004] The object of the present invention is to provide a ceramide antibody or antigen-binding fragment, a quality control product for detecting ceramide antibodies and applications. The ceramide antibody or antigen-binding fragment of the present invention can be used to prepare a ceramide antibody chemiluminescence detection quality control product with high specificity and stability.

[0005] The present invention provides a ceramide antibody or antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3; the light chain variable region comprises LCDR1, LCDR2 and LCDR3; the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NO.1 to SEQ ID NO.3; the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NO.4 to SEQ ID NO.6.

[0006] Preferably, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.7 or consists of the amino acid sequence shown in SEQ ID NO.7; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.8 or consists of the amino acid sequence shown in SEQ ID NO.8.

[0007] The present invention also provides a nucleic acid molecule encoding the ceramide antibody or antigen-binding fragment described in the above scheme, wherein the heavy chain variable region comprises the nucleotide sequence shown in SEQ ID NO.9 or consists of the nucleotide sequence shown in SEQ ID NO.9; the light chain variable region comprises the nucleotide sequence shown in SEQ ID NO.10 or consists of the nucleotide sequence shown in SEQ ID NO.10.

[0008] The present invention also provides an expression vector comprising the nucleic acid molecule described in the above scheme.

[0009] The present invention also provides a recombinant cell comprising the expression vector described in the above scheme.

[0010] The present invention also provides a quality control product for detecting ceramide antibodies, comprising the following components: the ceramide antibody or antigen-binding fragment described in the above scheme, HEPES or Tris-HCl buffer solution, fish skin gelatin, PEG 6000, sugar alcohol, sodium chloride, polyether alcohol and a preservative.

[0011] Preferably, the quality control product comprises components with the following concentrations: 0.1-10000 ng / mL ceramide antibody or antigen-binding fragment, fish skin gelatin with a weight concentration of 0.1%-10%, PEG 6000 with a weight concentration of 0.1%-5%, trehalose or sucrose with a weight concentration of 1%-10%, NaCl with a weight concentration of 0.1%-5%, polyether alcohol with a weight concentration of 0.1%-0.5% and preservative with a weight concentration of 0.1%-0.5%; the solvent of the quality control product comprises HEPES or Tris-HCl buffer solution with a concentration of 0.01-0.6 M.

[0012] Preferably, the preservative is Proclin 300 and / or sodium azide.

[0013] The present invention also provides the use of the quality control product described in the above scheme in preparing a ceramide antibody detection product or building a ceramide antibody detection platform.

[0014] Preferably, the final concentration of the quality control product in the ceramide antibody detection product is 0.1 to 1000 ng / mL.

[0015] The present invention provides a ceramide antibody or antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3; the light chain variable region comprises LCDR1, LCDR2 and LCDR3; the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO.1 to SEQ ID NO.3; the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are respectively shown as SEQ ID NO.4 to SEQ ID NO.6. The ceramide antibody or antigen-binding fragment provided by the present invention can be used as a quality control product for ceramide antibody chemiluminescence detection, and can be used in combination with a matching ceramide antibody chemiluminescence detection kit to improve detection accuracy and detection efficiency.

[0016] The present invention also provides a quality control product for detecting ceramide antibodies, comprising the following components: the ceramide antibody or antigen-binding fragment described in the above scheme, HEPES or Tris-HCl buffer solution, fish skin gelatin, PEG 6000, sugar alcohol, sodium chloride, polyether alcohol and preservative. In the present invention, the quality control product uses fish skin gelatin instead of traditional animal serum or animal serum albumin to play the role of protein protector, eliminating biosafety and ethical risks, and can obtain better stability results; at the same time, polyether alcohol is used as a surfactant to increase the dispersibility of protein components in the solution, so as to further improve the storage stability of the quality control product. The quality control product reagent of the present invention has simple components, is easy to prepare, has high correlation, strong specificity, good stability, can be stored at 2 to 8°C, and is easy to transport. The quality control product of the present invention can be used for indoor quality control, laboratory proficiency verification and detection system performance evaluation of ceramide antibody detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0018] Figure 1 It is a gel map of subtype identification of antibodies expressed by hybridoma cell line CER64754 and cloning of antibody variable regions according to an embodiment of the present invention; wherein lane 1 is DNA Marker, lane 2 is IgG2A heavy chain, lane 3 is IgG2B heavy chain, lane 4 is IgG3 heavy chain, lane 5 is IgG1 heavy chain, lane 6 is IgLambda1 light chain, lane 7 is IgLambda2 light chain, and lane 8 is IgKappa light chain. DETAILED DESCRIPTION

[0019] The present invention provides a ceramide antibody or antigen-binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3; the light chain variable region comprises LCDR1, LCDR2 and LCDR3; the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are respectively shown in SEQ ID NO.1 to SEQ ID NO.3; the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are respectively shown in SEQ ID NO.4 to SEQ ID NO.6.

[0020] In the specific implementation of the present invention, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.7 or consists of the amino acid sequence shown in SEQ ID NO.7; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.8 or consists of the amino acid sequence shown in SEQ ID NO.8.

[0021] In a specific implementation of the present invention, the ceramide antibody or antigen-binding fragment further comprises a heavy chain constant region and / or a light chain constant region; at least a portion of the heavy chain constant region is derived from a mammalian antibody; and at least a portion of the light chain constant region is derived from a mammalian antibody.

[0022] In the specific implementation of the present invention, the heavy chain constant region and the light chain constant region are derived from at least one of a murine antibody, an immune antibody, a primate antibody or a mutant thereof, and are further derived from a murine antibody or a mutant thereof.

[0023] In the specific implementation of the present invention, the ceramide antibody or antigen-binding fragment is a monoclonal antibody; the monoclonal antibody includes at least one of a full-length antibody Fv, a single-chain antibody and a Fab.

[0024] The ceramide antibody or antigen-binding fragment of the present invention has ceramide binding affinity and has the characteristics of high affinity and strong specificity, and can be used to prepare ceramide antibody detection quality control products, and used for clinical auxiliary diagnosis, risk assessment and prognosis judgment of various neurological diseases, filling the gap in the relevant domestic market.

[0025] The present invention also provides a nucleic acid molecule encoding the ceramide antibody or antigen-binding fragment described in the above scheme, wherein the heavy chain variable region comprises the nucleotide sequence shown in SEQ ID NO.9 or consists of the nucleotide sequence shown in SEQ ID NO.9; the light chain variable region comprises the nucleotide sequence shown in SEQ ID NO.10 or consists of the nucleotide sequence shown in SEQ ID NO.10.

[0026] The amino acid sequences and nucleotide sequences involved in the present invention are shown in Table 1.

[0027] Table 1 Amino acid sequence and nucleotide sequence

[0028]

[0029] The present invention also provides an expression vector comprising the nucleic acid molecule described in the above scheme.

[0030] The present invention has no particular limitation on the method for constructing the expression vector, and conventional construction methods in the art may be used.

[0031] The present invention also provides a recombinant cell comprising the expression vector described in the above scheme.

[0032] The present invention has no particular limitation on the method for constructing the recombinant cell, and conventional construction methods in the art may be used.

[0033] In the specific implementation process of the present invention, the recombinant cells are cultured under conditions suitable for protein expression and secretion to obtain the ceramide antibody or antigen-binding fragment described in the above scheme.

[0034] The present invention also provides a quality control product for detecting ceramide antibodies, comprising the following components: the ceramide antibody or antigen-binding fragment described in the above scheme, HEPES or Tris-HCl buffer solution, fish skin gelatin, PEG 6000, sugar alcohol, sodium chloride, polyether alcohol and a preservative.

[0035] In the specific implementation process of the present invention, the quality control product includes the following components at the following concentrations: 0.1-10000ng / mL ceramide antibody or antigen binding fragment, fish skin gelatin with a weight concentration of 0.1%-10%, PEG 6000 with a weight concentration of 0.1%-5%, trehalose or sucrose with a weight concentration of 1%-10%, NaCl with a weight concentration of 0.1%-5%, polyether alcohol with a weight concentration of 0.1%-0.5%, and preservative with a weight concentration of 0.1%-0.5%; the solvent of the quality control product includes HEPES or Tris-HCl buffer solution with a concentration of 0.01-0.6M. In the specific implementation process of the present invention, the quality control product is fixed to the preparation volume by volume using HEPES or Tris-HCl buffer solution with a concentration of 0.01-0.6M.

[0036] In the specific implementation of the present invention, the preservative is Proclin 300 and / or sodium azide.

[0037] In a specific implementation of the present invention, the concentration of the ceramide antibody or antigen-binding fragment in the quality control product is 25 ng / mL, 100 ng / mL, 500 ng / mL or 1000 ng / mL.

[0038] The quality control product of the present invention does not contain animal serum or animal serum albumin, eliminating biosafety and ethical risks, and has simple ingredients and low cost; moreover, the quality control product of the present invention is simple to prepare, has good stability, can be stored in liquid form, and is easy to transport; in addition, the quality control product of the present invention is suitable for a magnetic particle chemiluminescence platform, and can be used for indoor quality control of ceramide antibody detection, laboratory proficiency verification, detection system performance evaluation, etc.

[0039] In one embodiment of the present invention, the quality control product is composed of the following components: ceramide antibody or antigen-binding fragment, 0.05M Tris-HCl buffer solution, 2% fish skin gelatin by weight, 1% PEG 6000 by weight, 2% trehalose by weight, 0.9% sodium chloride by weight, 0.1% polyether alcohol by weight and 0.1% sodium azide by weight.

[0040] In another embodiment of the present invention, the quality control product is composed of the following components: ceramide antibody or antigen-binding fragment, 0.05M Tris-HCl buffer solution, 5% fish skin gelatin by weight, 1% PEG 6000 by weight, 2% trehalose by weight, 0.9% sodium chloride by weight, 0.1% polyether alcohol by weight and 0.1% sodium azide by weight.

[0041] In another embodiment of the present invention, the quality control product is composed of the following components: ceramide antibody or antigen-binding fragment, 0.05M Tris-HCl buffer solution, 2% fish skin gelatin by weight, 1% PEG 6000 by weight, 2% trehalose by weight, 0.9% sodium chloride by weight, 0.5% polyether alcohol by weight and 0.1% sodium azide by weight.

[0042] In the specific implementation process of the present invention, the Tris-HCl buffer solution was purchased from Shanghai Test and is numbered 30188260; the fish skin gelatin was purchased from Merck Sigma and is numbered G7041; the PEG 6000 was purchased from Solarbio and is numbered P8250; the trehalose was purchased from Merck Sigma and is numbered PHR1344; the sodium chloride was purchased from Shanghai Test and is numbered 10019308; the polyether alcohol was purchased from Wokai Bio and is numbered A33525; and the sodium azide was purchased from Shanghai Test and is numbered 80115518.

[0043] In the specific implementation of the present invention, the final protein concentration of the ceramide antibody or antigen-binding fragment in the quality control product can be determined according to usage requirements.

[0044] The components of the quality control product are similar or the same as the matrix of the patient sample, and are used as a detection scale to check the performance of the analytical instrument or method, and to verify the performance of the analytical instrument or method. The quality control product of the present invention has high specificity and stability.

[0045] In the specific implementation of the present invention, the preparation method of the quality control product comprises the following steps:

[0046] Fish skin gelatin, PEG 6000, trehalose, sodium chloride and polyether alcohol solution are sequentially added to a HEPES or Tris-HCl buffer solution to obtain a mixed solution; the ceramide antibody or antigen-binding fragment is mixed with the mixed solution, filtered and sterilized to obtain a quality control product.

[0047] In the specific implementation of the present invention, the solvent of the polyether alcohol solution includes anhydrous ethanol.

[0048] In the specific implementation process of the present invention, when fish skin gelatin, PEG 6000, trehalose, sodium chloride and polyether alcohol solution are added to HEPES or Tris-HCl buffer solution in sequence, each component is fully mixed before adding the next component.

[0049] The present invention also provides the use of the quality control product described in the above scheme in preparing a ceramide antibody detection product or building a ceramide antibody detection platform.

[0050] In the specific implementation of the present invention, the ceramide antibody detection product includes a ceramide antibody chemiluminescent detection reagent or kit; the ceramide antibody chemiluminescent detection reagent or kit includes a ceramide antibody magnetic particle chemiluminescent detection reagent or kit.

[0051] In the specific implementation of the present invention, the ceramide antibody detection platform includes a ceramide antibody chemiluminescence detection platform; the ceramide antibody chemiluminescence detection platform includes a ceramide antibody magnetic particle chemiluminescence detection platform.

[0052] In the specific implementation process of the present invention, the final concentration of the quality control product in the ceramide antibody detection product is 0.1-1000 ng / mL.

[0053] In the specific implementation process of the present invention, the quality control product is used in combination with a ceramide antibody chemiluminescence kit.

[0054] In order to further illustrate the present invention, a ceramide antibody or antigen-binding fragment, a quality control product for detecting ceramide antibodies and applications provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0055] In the following examples, C14 ceramide (Merck, 860525P) was coupled with KLH as a ceramide immunogen, mice were immunized, and hybridoma cells were prepared; C14 ceramide (Merck, 860525P) was coupled with BSA as a ceramide detector, immunized mice and hybridoma cells were screened, and the immune effect was detected.

[0056] Example 1

[0057] 1. Large-scale preparation of monoclonal antibodies

[0058] 1. According to the characteristics of the antigen, the immunization scheme was selected. BALB / c mice aged 6 to 8 weeks were selected for the following immunization procedure: for the first immunization, 25 μg of ceramide immunogen was mixed with an equal amount of Freund's complete adjuvant, and injected subcutaneously at multiple points after emulsification; 14 days after the first immunization, 12.5 μg of ceramide immunogen was mixed with an equal amount of Freund's incomplete adjuvant, and emulsified for booster immunization. 14 days after the second immunization, 12.5 μg of ceramide immunogen was mixed with an equal amount of Freund's incomplete adjuvant, and emulsified for booster immunization. 14 days after the third immunization, blood was collected and serum was separated. ELISA plates were coated with 1 μg / mL ceramide detector, and indirect ELISA tests were performed to determine the serum titer.

[0059] The results showed that the titer of the prepared mouse antiserum was greater than 72,000. The typical immune mouse serum test results are shown in Table 2.

[0060] Table 2 Detection results of serum from immune mice

[0061] Serum dilution multiple <![CDATA[OD 450 ]]> 100 1.674 300 1.644 900 1.574 2700 1.448 8100 1.287 24300 0.994 72900 0.437 blank 0.124

[0062] 2. Carry out cell fusion in the above immunized mice. The specific process is as follows:

[0063] 1) After removing the eyeballs of the mice and collecting blood, the mice were killed by dislocation and placed in a 70% alcohol bottle for 2 min. The mice were then fixed on a foam board in a biosafety cabinet, the abdominal skin was untied to find the spleen, which was removed with forceps and gently crushed in a 200-mesh stainless steel filter. The cells were gently rinsed with DMEM culture medium (Thermo, 11965092), and then centrifuged at 200×g for 10 min at room temperature. The supernatant was discarded and set aside.

[0064] 2) When preparing feeder cells, mice were killed by dislocation and placed in a 70% alcohol bottle for 2 minutes. The mice were then fixed on a foam board in a biosafety cabinet, the abdominal skin was untied, PBS was drawn up with a syringe and gently injected into the subperitoneum, and the liquid containing the feeder cells was washed out from the other side. The mixture was then centrifuged at 200×g for 10 minutes at room temperature, and the supernatant was discarded for later use.

[0065] 3) 2.0×10 7 FO myeloma cells and 2.0×10 8 The spleen cells were mixed, centrifuged at 200 × g for 10 min, the supernatant was discarded, and the mixture was gently shaken to mix. In a 37°C water bath, 1 mL of a 50% PEG-1450 (Merk, P1458) aqueous solution was added dropwise within 90 s, followed by 20 mL of DMEM culture medium. The mixture was centrifuged at 200 × g for 10 min, the supernatant was discarded, and the mixture was washed repeatedly. The mixture was centrifuged at 200 × g for 10 min, the supernatant was discarded, and hybridoma cells were obtained. The cells were plated into 10 96-well culture plates with 150 μL per well.

[0066] 4) Add 10,000 feeder cells / well to the 10 96-well cell culture plates, 100 μL per well, mark the plates, and place them in a 37°C incubator containing 5% CO. 2 Culture in a cell culture incubator, add HAT screening medium (Merk, H0262) on the second day, and then use HAT selection culture for 1-2 days. A large number of tumor cells will die, and the tumor cells will disappear after 3-4 days. The hybrid cells will form small colonies. After 7-10 days of HAT screening culture medium, it should be replaced with HT culture medium (Merk, H0137), and then maintained for 2 weeks, and then replaced with DMEM medium with 20% FBS (ExCell, FSP500) to continue culture. During the above selection culture period, when the hybridoma cells cover 1 / 10 of the bottom of the well, you can start to detect specific antibodies and screen out the desired hybridoma cell lines. During the selection culture period, half of the culture medium is generally replaced every 2-3 days.

[0067] 3. Determine the optimal coating amount of ceramide detector by orthogonal experiment. 0.5, 1.0, 2.0, 4.0 μg of ceramide detector were coated on 96-well plates, and 6 wells of each concentration were set as 3 positive and 3 negative. Square titration was performed with positive serum of ceramide-immunized mice at different dilutions, and negative serum of unimmunized mice was used as negative control. 96-well ELISA plates were coated with 0.5 μg ceramide immunogen per well and incubated at 4°C overnight; washed twice with PBST; 200 μL of 1% BSA in PBS was added to each well, blocked at room temperature for 2 hours, and then placed on trifold paper and patted dry; sample addition: add 0.1 mL of the sample to be tested to the reaction well, incubate at 37°C for 1 hour, and then wash, and make blank wells (no sample), negative control wells and positive control wells at the same time. Add 0.1 mL of newly diluted antibody to each reaction well, incubate at 37°C for 1 hour, and then wash 3 times. Add enzyme-labeled secondary antibody: Add 0.1 mL of freshly diluted enzyme-labeled antibody to each reaction well. Incubate at 37°C for 1 hour and wash 3 times. Add substrate solution for color development: Add 0.1 mL of TMB substrate solution to each reaction well and let stand at room temperature for 10 minutes. Add 1 M H 2 SO 4 0.1 mL. Measure the OD value to determine the result, and use an enzyme marker to measure the absorbance at 450 nm (A 450 ). A positive result was defined as 2.1 times or more of the negative control OD value (calculated after adjusting the blank control well to zero). A hybridoma cell monoclone resistant to ceramide was selected.

[0068] According to the above method, the positive hybridoma cells screened were subcloned, and the original wells were diluted with HAT selection medium by limiting dilution method and redistributed into 96-well culture plates, and then the morphology and number of cells were observed. The cells were adjusted to 3-10 cells / mL. Take the cell culture plate with the feeder cell layer prepared the day before, and add 100 μL of diluted cells to each well. Incubate at 37°C and 5% CO 2 Culture in a static incubator. Change the medium on the 7th day, and every 2-3 days thereafter. Cell clones can be seen on the 8th to 9th day, and the antibody activity can be detected in time. Move the cells in the positive wells to a 24-well plate for expansion and culture. Each clone should be frozen as soon as possible, and finally the clone number CER64754 hybridoma cell line was selected for antibody production.

[0069] 5. 8-week-old BALB / C mice were intraperitoneally injected with 0.5 mL of Freund's incomplete adjuvant. Two weeks later, 1×10 6Hybridoma cells with clone number CER64754 can produce ascites 7 to 10 days after inoculation. Closely observe the health status and ascites signs of the animals. When ascites is as much as possible and before the mice die, kill the mice and use a dropper to suck the ascites into a test tube. 5 to 10 mL of ascites can be obtained from one mouse. Ascites can also be extracted with a syringe and can be collected repeatedly several times. The ascites obtained was centrifuged at 3000×g for 10 minutes, the upper layer of fat and the bottom precipitate were discarded, the supernatant was collected and distributed at -20℃, and after the ascites supernatant was thawed and equilibrated to room temperature, 1 / 10 volume of 1M Tris-HCl PH8.0 was added to adjust the sample pH to 8.0. The protein G affinity column was balanced with 20 column volumes of 100 mM Tris-HCl at pH = 8.0, and the ascites supernatant after adjusting the pH to 8.0 was loaded onto the column, followed by washing with 20 column volumes of 100 mM Tris-HCl at pH = 8.0, and finally eluted with 100 mM Glycine-HCL pH 2.5 (i.e., ceramide antibody CER64754, which may be referred to as antibody CER64754 or CER64754). The antibody eluate was added to a concentrator tube (Millipore, UFC801008, 10K) and centrifuged in a centrifuge (Xiangyi, L550) at room temperature at 3000 × g for 20 min. The solution was centrifuged in batches until the volume of the solution reached 1 mL / concentrator tube (2 tubes), 4 mL of 10 mM PBS pH = 7.4 buffer was added, and centrifugation at room temperature at 3000 × g was continued for 20 min. Repeat centrifugation 3 times to make the antibody buffer 10mM PBS pH = 7.4, add 10mM PBS pH = 7.4 to a total volume of 10mL. Finally, 2mL / tube of concentrated antibody solution was dispensed into centrifuge tubes and stored at -80°C. The antibody concentration was determined using a BCA kit (Solabo, PC0020), and the concentration of the purified monoclonal antibody was determined to be 1.832mg / mL.

[0070] 2. Antibody titer determination

[0071] The titer of ceramide antibody CER64754 was detected by indirect ELISA. The ceramide detector was diluted with PBS and coated in a 96-well ELISA plate at 0.2μg / mL and 100μL / well. After overnight at 4℃, the ELISA plate was washed twice with PBST solution, 300μL / well each time, and patted dry after washing. The ELISA plate was blocked with 1% BSA PBS solution, 200μL / well, at room temperature for 2h, and patted dry after blocking; ceramide antibody CER64754 diluted to 20ng / mL with PTB was added, reacted in a 37℃ incubator for 1h, and the ELISA plate was washed twice with PBST solution, 300μL / well each time. Patted dry after washing. HRP-labeled goat anti-mouse antibody diluted 5000 times with PTB was added, reacted in a 37℃ incubator for 1h, and the ELISA plate was washed twice with PBST solution, 300μL / well each time. Patted dry after washing. Add TMB substrate solution, 100 μL / well, react at room temperature for 10 min, add 100 μL / well 1M H 2 SO 4 The reaction was terminated. The absorbance (A450) was measured at 450 nm using an ELISA reader. The results are shown in Table 3, and it can be seen that the titer of the purified antibody is greater than 1.7 million.

[0072] Table 3 Antibody titer determination results

[0073]

[0074]

[0075] 3. Sequence Analysis of Monoclonal Antibodies

[0076] (1) Monoclonal antibody subtype identification

[0077] Hybridoma cell line CER64754 was cultured in a 10 cm diameter cell culture dish (37°C, 5% CO) using DMEM medium (GIBCO, #C11995500BT) supplemented with 10% serum. 2 After 7 days of culture, the cells were transferred to a 15 mL centrifuge tube and 4 × 10 6 After centrifugation at 200×g for 5 min, the supernatant was discarded, and the centrifuge tube was inverted to drain the liquid in the tube. The cells in the tube were synthesized into cDNA using the reverse transcription kit (Qiagen, 74134) of QIAGEN Company.

[0078] The antibody subtype was determined by PCR using antibody subtype-specific primers. The PCR primer information is shown in Table 4, and the antibody information is shown in Table 5. The above-synthesized cDNA was used as a PCR reaction template. PCR reaction solution system: TAKARAEx Taq (5U / μL, TAKARA, RR001B), 0.25μL; 10×Ex Taq Buffer, 5μL; dNTP mixture (2.5mM each), 4μL; template cDNA, 1μL; upstream primer (100μM), 1μL; downstream primer (100μM), 1μL; double distilled water was added to a total volume of 50μL. PCR reaction temperature program: 94℃, 5min pre-denaturation, temperature cycle 30 times (94℃, 1min, 57℃, 1min, 72℃, 1min), 72℃, 10min extension. After the reaction, 10 μL of each PCR product was loaded on 1% agarose gel for electrophoresis, and the order of samples added to each lane is shown in Table 5. The subtype of the antibody can be inferred from the PCR product results. The monoclonal antibody CER64754 obtained in the present invention has a heavy chain of IgG1 and a light chain of kappa.

[0079] Table 4 PCR primer information

[0080]

[0081]

[0082] Note: S=C or G, M=A or C, R=A or G, W=A or T.

[0083] Table 5 Antibody information

[0084]

[0085] (2) Sequencing of the variable region (V region) of the hybridoma cell line CER64754 antibody

[0086] The variable region (V region) of the antibody of the hybridoma cell line CER64754 in step (1) of this embodiment obtained after PCR amplification was cut from the agarose gel and extracted using a DNA extraction kit (Qiagen, 74134). The extracted DNA fragment was linked to the pEASY-T1 cloning vector and transformed into Trans1-T1 competent cells (Transgen, CT101-1). The transformed bacterial colonies were picked into LB culture medium and DNA sequencing was performed after overnight culture. The nucleic acid sequence of the heavy chain V region of the antibody (CER64754) for recognizing ceramide provided by the present invention is shown in SEQ ID NO.9, and the nucleic acid sequence of the light chain V region is shown in SEQ ID NO.10.

[0087] Example 2 Preparation of Ceramide Antibody Quality Control

[0088] Prepare a ceramide antibody control consisting of the following components:

[0089] Ceramide antibody (prepared according to Example 1), 0.05M Tris-HCl buffer solution (30188260, Shanghai trial), 2% fish skin gelatin (G7041, Merck Sigma) at a weight concentration, 1% PEG 6000 (P8250, Solarbio) at a weight concentration, 2% trehalose (PHR1344, Merck Sigma) at a weight concentration, 0.9% sodium chloride (10019308, Shanghai trial), 0.1% polyether alcohol (A33525, Wokai Bio) at a weight concentration, and 0.1% sodium azide (80115518, Shanghai trial) at a weight concentration.

[0090] Example 3

[0091] Prepare a ceramide antibody control consisting of the following components:

[0092] Ceramide antibody (prepared according to Example 1), 0.05M Tris-HCl buffer solution, 5% fish skin gelatin by weight, 1% PEG 6000 by weight, 2% trehalose by weight, 0.9% sodium chloride by weight, 0.1% polyether alcohol by weight and 0.1% sodium azide by weight.

[0093] Example 4

[0094] Prepare a ceramide antibody control consisting of the following components:

[0095] Ceramide antibody (prepared according to Example 1), 0.05M Tris-HCl buffer solution, 2% fish skin gelatin by weight, 1% PEG 6000 by weight, 2% trehalose by weight, 0.9% sodium chloride by weight, 0.5% polyether alcohol by weight and 0.1% sodium azide by weight.

[0096] Comparative Example 1

[0097] Prepare a ceramide antibody control consisting of the following components:

[0098] Ceramide antibody (prepared according to Example 1), 0.05M Tris-HCl buffer solution, 2% by weight bovine serum albumin (YH(B)-0500, Yuanhengshengma), 1% by weight PEG 6000, 2% by weight trehalose, 0.9% by weight sodium chloride, 0.1% by weight polyether alcohol and 0.1% by weight sodium azide.

[0099] Comparative Example 2

[0100] Change the composition ratio. Prepare a ceramide antibody control consisting of the following components:

[0101] Ceramide antibody (prepared according to Example 1), 0.05M Tris, 2% fish skin gelatin by weight, 1% PEG 6000 by weight, 2% trehalose by weight, 0.9% sodium chloride by weight, 0.1% Tween 20 (T8220, Solarbio) by weight, and 0.1% sodium azide by weight.

[0102] The ceramide antibody quality control products described in Examples 2 to 4 and Comparative Examples 1 to 2 above were prepared according to the following steps:

[0103] (1) dissolving the polyether alcohol in anhydrous ethanol to completely dissolve the polyether alcohol to obtain a mixed solution;

[0104] (2) adding fish skin gelatin or bovine serum albumin, trehalose, sodium chloride, PEG 6000, sodium azide, Tween 20, polyether alcohol and anhydrous ethanol mixed solution to the Tris-HCl buffer solution in a stirring state, and mixing them thoroughly to obtain a mixed solution, and then adding ceramide antibody to the mixed solution obtained in step (1), and adding the next material after each material is fully mixed. After the material is completely dissolved, the mixed solution is filtered and sterilized to obtain a mixed solution to be tested;

[0105] (3) Using a magnetic particle chemiluminescence kit on a chemiluminescence instrument, the concentration of the mixed solution to be tested was determined, and the final concentration of the ceramide antibody was determined. Finally, ceramide antibody quality control products with final concentrations of 25 ng / mL, 100 ng / mL, 500 ng / mL and 1000 ng / mL were obtained, and the ceramide antibody quality control products of each concentration were respectively packaged and stored.

[0106] Example 7 Stability Evaluation

[0107] The stability of the quality control products with a final concentration of 100 ng / mL of ceramide antibodies prepared in Examples 2 to 4 and Comparative Examples 1 to 2 was evaluated. Accelerated thermal stability at 37°C, the quality control products of 100 ng / mL prepared in Examples 2 to 4 of the present invention and Comparative Examples 1 to 2 were stored at 37°C, and one bottle was taken for detection on the 0th day, the 1st day, the 3rd day, the 7th day, the 10th day, and the 14th day, respectively, using a fully automatic chemiluminescence instrument and a matching ceramide antibody quantitative detection kit for determination, each bottle was measured 3 times, and the relative deviation between the mean and the target value was calculated, relative deviation = [(mean value - target value) / target value] × 100%, the results are shown in Table 6.

[0108] Table 6 Determination results of ceramide antibody concentration of different quality control products at 37℃ stability

[0109]

[0110] The experimental results in Table 6 show that the ceramide antibody quality control products prepared in Examples 2 to 4 of the present invention can be stable for 14 days at 37°C, and the activity difference detected by the ceramide antibody quality control products is within the range of ±5%. After the ceramide antibody quality control products prepared in Comparative Examples 1 to 2 of the present invention were stored at 37°C for a period of time, the relative deviation between the measurement results and the target value exceeded the range of ±5%. This shows that compared with the addition of bovine serum albumin and Tween 20, the selection of fish skin gelatin and polyether alcohol in the quality control reagent of the present invention plays an important role. The use of the preparation method in this patent can make the quality control products prepared by the present invention have good accelerated thermal stability at 37°C.

[0111] The stability of the 1000 ng / mL ceramide antibody quality control product in Examples 2 to 4 and the 1000 ng / mL ceramide antibody quality control product in Comparative Examples 1 to 2 was further evaluated and compared. The results were similar to those of the 100 ng / mL quality control product. Examples 2 and 3 had the best stability at 37°C.

[0112] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A ceramide antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, characterized in that: The heavy chain variable region includes HCDR1, HCDR2 and HCDR3; the light chain variable region includes LCDR1, LCDR2 and LCDR3; the amino acid sequences of HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.1 to SEQ ID NO.3, respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.4 to SEQ ID NO.6, respectively.

2. The ceramide antibody or antigen-binding fragment according to claim 1, characterized in that The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO.7 or consists of the amino acid sequence shown in SEQ ID NO.7; the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.8 or consists of the amino acid sequence shown in SEQ ID NO.

8.

3. A nucleic acid molecule encoding the ceramide antibody or antigen-binding fragment of claim 1 or 2, characterized in that: The heavy chain variable region comprises the nucleotide sequence shown in SEQ ID NO.9 or consists of the nucleotide sequence shown in SEQ ID NO.9; the light chain variable region comprises the nucleotide sequence shown in SEQ ID NO.10 or consists of the nucleotide sequence shown in SEQ ID NO.

10.

4. An expression vector, characterized in that: Comprising the nucleic acid molecule of claim 3.

5. A recombinant cell, characterized in that Comprising the expression vector according to claim 4.

6. A quality control product for detecting ceramide antibodies, characterized in that: The invention comprises the following components: the ceramide antibody or antigen-binding fragment according to claim 1 or 2, HEPES or Tris-HCl buffer solution, fish skin gelatin, PEG 6000, sugar alcohol, sodium chloride, polyether alcohol and a preservative.

7. The quality control product according to claim 6, characterized in that: The quality control product comprises components with the following concentrations: 0.1-10000 ng / mL ceramide antibody or antigen-binding fragment, fish skin gelatin with a weight concentration of 0.1%-10%, PEG 6000 with a weight concentration of 0.1%-5%, trehalose or sucrose with a weight concentration of 1%-10%, NaCl with a weight concentration of 0.1%-5%, polyether alcohol with a weight concentration of 0.1%-0.5% and a preservative with a weight concentration of 0.1%-0.5%; the solvent of the quality control product comprises a HEPES or Tris-HCl buffer solution with a concentration of 0.01-0.6 M.

8. The quality control product according to claim 7, characterized in that: The preservative is Proclin 300 and / or sodium azide.

9. Use of the quality control product according to any one of claims 6 to 8 in preparing a ceramide antibody detection product or building a ceramide antibody detection platform.

10. The use according to claim 9, characterized in that: The final concentration of the quality control product in the ceramide antibody detection product is 0.1-1000 ng / mL.

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