WO2016119639A1 - 检测重组蛋白提取物中残留杂蛋白的特异性抗体及检测试剂 - Google Patents

检测重组蛋白提取物中残留杂蛋白的特异性抗体及检测试剂 Download PDF

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WO2016119639A1
WO2016119639A1 PCT/CN2016/071740 CN2016071740W WO2016119639A1 WO 2016119639 A1 WO2016119639 A1 WO 2016119639A1 CN 2016071740 W CN2016071740 W CN 2016071740W WO 2016119639 A1 WO2016119639 A1 WO 2016119639A1
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protein
antibody
human serum
serum albumin
residual
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French (fr)
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杨代常
陈镇
刘静茹
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武汉禾元生物科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention belongs to the field of biotechnology, and particularly relates to an antibody and a detection method for identifying residual heteroproteins in protein preparations.
  • HCPs Host proteins
  • residual heteroproteins are a major impurity component of recombinant protein drugs and are important safety hazards for biological products. Excessive HCPs may cause a series of adverse reactions such as immunity, but even in the most stringent purification processes. Production under quality control, traces of HCPs are also inevitable. Sometimes even trace amounts of residual host proteins may cause a violent immune response, and its potential "adjuvant effect" may cause the body to produce antibodies to the drug, which in turn affects the efficacy of the drug. Quality control of residual heteroproteins is a top priority in biosafety assessment. Therefore, in the process development, these host residues should be removed as much as possible, and a rapid, accurate and sensitive HCP detection method should be established.
  • the identification and detection of host residual heteroproteins has always been a major problem in the development of recombinant protein drugs, especially for very high purity samples. Most of the previous studies have focused on the preliminary process research and provided necessary assistance for process development. However, the detection and analysis of the final sample often appears to be weak, and often only one or several high concentrations of host residual heteroproteins can be identified.
  • the first consideration is how to effectively reduce the interference of the target protein, and to achieve the concentration of the host residual protein in order to reach the detection range of the mass spectrometer.
  • the trace amount of the target protein will seriously interfere with and affect the accuracy of the detection. Therefore, how to prepare a specific antibody from the genetically engineered protein product is to establish a manuscript precision detection host residual protein. The essential.
  • the main techniques used for HCPs analysis include sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), high performance liquid chromatography, and immunoblotting, but usually the HCPs in the final product are in ppm.
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • ELISA enzyme-linked immunosorbent assay
  • Another object of the present invention is to provide an immunodetection reagent for detecting residual heteroprotein in a recombinant protein extract, and using the specific antibody prepared by the present invention as a capture antibody and a detection antibody in the immunodetection reagent The test was carried out by the double antibody sandwich method.
  • a specific antibody for specifically binding to a residual heteroprotein in a recombinant protein wherein the recombinant protein is a protein product which is recombined by a genetic engineering technique and expressed in a host, wherein Containing a protein of interest and a residual heteroprotein, characterized in that the antibody is prepared by the following method:
  • step 2) coupling the anti-first protein antibody obtained in step 1) with an affinity chromatography medium to prepare an anti-first protein antibody-immunoaffinity chromatography column;
  • step 4) using the total host residual heteroprotein obtained in step 3) as a second protein to immunize the animal to prepare an anti-second protein antibody;
  • the step of purifying the anti-first protein antibody and the anti-second protein antibody obtained by immunizing the animal may be further included after step 1) and step 4). And if necessary, steps 3) and 6) can be repeated to obtain a better effect of removing the protein of interest and the antibody against the first protein.
  • protein naturally occurring in an organism means a biologically active protein directly isolated from an organism.
  • recombinant protein refers to the application of genetic recombination technology to obtain a linker that can be translated into a protein of interest.
  • the recombinant vector of the gene fragment is then transferred to a host cell which can express the protein of interest to express a specific recombinant protein molecule.
  • residual heteroprotein refers to a protein derived from a non-target protein of a host cell during expression and isolation and purification of the recombinant protein.
  • Expression vectors for expressing recombinant proteins include prokaryotic cells such as E. coli, eukaryotic cells such as yeast, insect cells, and CHO cells, etc. Recombinant proteins can also be produced using animal expression systems or plant expression systems, such as breast or transgenic plants of transgenic animals. Endosperm or leaves.
  • a representative example of the invention is human serum albumin expressed by rice endosperm.
  • an enzyme-linked immunodetection reagent for quantitatively detecting residual heteroprotein in a genetically engineered recombinant protein extract which uses the specific antibody of the present invention as a capture antibody-coated solid phase carrier,
  • the specific antibody of the present invention is used as a detection antibody to bind to a label to prepare an enzyme conjugate;
  • a preferred detection reagent is an ELISA detection reagent using a double antibody sandwich method.
  • the present invention is a typical example of the preparation of recombinant human serum albumin expressed in transgenic rice endosperm, and provides an enzyme-linked immunosorbent assay reagent, wherein the characteristic antibody capable of specifically binding residual protein in the recombinant protein extract Prepared by the following method:
  • step i) the purified rabbit anti-human serum albumin polyclonal antibody prepared in step i) is coupled with an affinity chromatography medium to prepare an immunoaffinity chromatography column A;
  • step ii) using the immunoaffinity chromatography column A of step ii) to remove the target protein from the genetically engineered rice seed extract expressing recombinant human serum albumin, thereby preparing total residual protein of rice seeds, and performing 3 times of affinity chromatography. , completely remove the target protein, and collect the effluent containing the total residual protein of the rice seed;
  • affinity chromatography column B is prepared by coupling plasma-derived human serum albumin with an affinity chromatography medium
  • affinity chromatography medium described in step ii) and step v) is CNBr-activatived SepharoseTM 4 Fast Flow.
  • step i) comprises the steps of:
  • the purification described in step iv) comprises purifying a polyclonal antibody of the obtained antibody genetically engineered rice host heteroprotein by an IgG chromatography column.
  • a preferred capture antibody coating concentration may be 5 to 40 ⁇ g/ml, more preferably a capture antibody concentration of 20 ⁇ g/ml, and a detection antibody concentration of preferably 0.2 to 1.6 ⁇ g/ml, more preferably detection.
  • the antibody concentration was 0.8 ⁇ g/ml, and the antibody was detected by horseradish peroxidase labeling.
  • Biotin can also be combined with horseradish peroxidase, wherein the preferred capture antibody coating concentration is 2-16 ⁇ g/ml, the detection antibody concentration is 0.5 ⁇ g/ml, and the more preferred capture antibody concentration is 2 ⁇ g/ml.
  • the biotin labeling antibody is then bound to horseradish peroxidase.
  • the immunodetection reagent of the present invention preferably has a residual protein content of 2.5 ng/ml - 25 ng/ml in the recombinant protein extract detected.
  • the specific antibody of the present invention can specifically bind to the host residual heteroprotein in the recombinant protein extract, and the ELISA detection practice of the specific antibody of the present invention can quantitatively identify the residual heteroprotein in the recombinant protein extract for the recombinant protein product. Quality control has important uses and meanings.
  • Figure 1 is a technical roadmap for establishing an ELISA method for detecting residual host proteins in rice hosts
  • Figure 2 is a Western Blotting test of titer and coverage of anti-human serum albumin serum; wherein: 1: commercial rabbit anti-human serum albumin, 2: homemade rabbit anti-human serum albumin, M: Marker;
  • Figure 3 is a crude purification route diagram of rabbit seeds against total impurities in rice seeds
  • Figure 4 is a Native PAGE diagram of the purified antibody; wherein: 1 is an albumin antibody, M is a protein marker. A native PAGE of the purified antibody, 1 is an albumin antibody, and M is a protein marker.
  • Figure 5 is a measurement of antibody-coupled filler loading; wherein: left arrow: elution solution (ie, bound human serum albumin), right arrow: penetrating solution (ie, unbound impurity protein);
  • Figure 6 is a Western Blotting diagram of SDS-PAGE and specific antibody of a rice seed heteroprotein standard sample
  • Figure 7 is a preliminary purification route diagram of total impurity antiserum of rice seeds
  • Figure 8 is the antigen coverage and specificity analysis of the antibody; wherein: anti-impurity antibody SDS-PAGE (left) and Western blot (right) of impurities in each chromatography step; lanes 1-9 are samples of different purification steps .
  • M is a protein Marker
  • Figure 9 is a linear verification result of a standard curve of an ELISA detection reagent.
  • An exemplary embodiment describing a specific antibody and ELISA detection reagent of the present invention is recombinant human serum albumin (OsrHSA) expressed by genetically engineered rice endosperm cells.
  • the extract of recombinant human serum albumin contains plant source (rice)
  • the host's residual heteroprotein, the natural protein is human serum albumin.
  • a method for expressing recombinant human serum albumin using rice endosperm cells as a bioreactor can be found, for example, in Chinese Patent Application No. 200510019084, which relates to a method for producing OsrHSA using rice endosperm cells as a bioreactor, or as in No. 201010597544.2,
  • a method involving the extraction of recombinant human serum albumin from rice seeds, No. 201010606635.8 relates to a method for isolating and purifying recombinant human serum albumin from rice seeds.
  • Human serum albumin human serum Albumin
  • FCA Freund's complete adjuvant
  • the plant-derived recombinant human albumin crude extract is provided by Wuhan Heyuan Biotechnology Co., Ltd.; (the preparation method refers to the above-referenced patent application)
  • Equlibration buffer 20 ⁇ M Na 2 HPO 4 ⁇ 12H 2 O + 4 ⁇ M NaH 2 PO 4 ⁇ 2H 2 O + 0.5 M NaCl pH 7.6;
  • Elution buffer 20 ⁇ M Na 2 HPO 4 ⁇ 12H 2 O+0.15M NaCl pH2.8
  • Saturated ammonium sulfate 50% saturated ammonium sulfate.
  • Membrane bag U650599PelliconXL5KD Millipore PXC005C50, 100% TCA, acetone;
  • Vacuum concentrating centrifuge (SpeedVac device, Thermo), modified (sequencing grade), Promega (Madison, WI)
  • the non-redundant protein database was downloaded from NCBI’s GenBank;
  • the detection method uses the whole impurity component of the crude extract of the genetic engineering rice extraction process, and the level of the rice seed host protein residue in the plant-derived recombinant human serum albumin is determined by the double antibody sandwich method; the detection antibody adopts HRP label and organism respectively. Prime label.
  • an immunochromatographic column for the preparation of anti-human serum albumin (natural protein, first protein) was used to remove recombinant human serum albumin (protein of interest) from the genetically engineered whole extract.
  • the remaining whole protein is used as a total residual heteroprotein (second protein) antigen
  • the rabbit is obtained a polyclonal antibody against the host total heteroprotein
  • the purified polyclonal antibody against the host total heteroprotein is used as a coating and detection antibody.
  • a sandwich ELISA quantification method was established using the immunizing antigen as a standard.
  • the serum was subjected to Western detection, and the anti-human serum albumin serum prepared by the experiment was also subjected to a 1:10000 dilution, and the commercially available anti-human serum albumin antibody (US Biological, A1327-30A)
  • the results of Western Blotting are shown in Figure 2.
  • the serum titer of anti-human serum albumin prepared is close to the purified titer of the market, and the polyclonal antibody can be effectively combined with human serum albumin.
  • the degradation fragment reaction indicated that the prepared antibody not only effectively removed the intact recombinant human serum albumin molecule, but also effectively removed the degradation fragment of human serum albumin, thereby improving the accuracy of identification of residual heteroprotein.
  • the crude serum was firstly purified, and the specific purification process is shown in FIG.
  • Crude purified antibody precipitated by secondary ammonium sulfate was reconstituted with 25 mM PBS. Dialysis of ammonium sulfate was removed by dialysis to adjust the pH to 7.6 with HCl for preparation of immunoaffinity chromatography.
  • the anti-human serum albumin antibody is subjected to crude purification and used for antigen immunoaffinity chromatography.
  • Human serum albumin, an antigen is first coupled to CNBr-activatived Sepharose TM 4Fast Flow.
  • the specific procedure of the coupling method refers to the manufacturer's instructions: first remove the filler's protective agent with 0-4 ° C HCl (pH 2-3) solution, with human serum albumin solution (pH 8.0): filler in a ratio of 1:2
  • the mixed coupling was carried out, and the mixture was allowed to stand at room temperature for 3 hours in the shaker, followed by addition of 0.1 M Tris-HCl (pH 8.0) buffer for 2 hours to block the unconjugated residue.
  • the human serum albumin-conjugated filler was repeatedly washed with a buffer (0.5 M NaCl, 0.1 M acetate, pH 3-4) and a buffer (0.5 M NaCl, 0.1 M Tris-HCl, pH 8-9).
  • a buffer 0.5 M NaCl, 0.1 M acetate, pH 3-4
  • a buffer 0.5 M NaCl, 0.1 M Tris-HCl, pH 8-9.
  • the finally prepared packing was packed into a chromatography column, and the immunoaffinity chromatography column was equilibrated with 3 column volume balance solution (20 ⁇ M Na 2 HPO 4 , 0.5 M NaCl pH 7.6) before loading, and eluted after loading.
  • the solution (20 ⁇ M Na 2 HPO 4 , 0.5 M NaCl pH 2.1) was used to elute the anti-human serum albumin antibody bound to the immunoaffinity chromatography medium, and the eluted column was subjected to rebalancing for the next purification. collect.
  • This experiment used a total of 700mg human serum albumin conjugated 6.5g CNBr-activatived Sepharose TM 4Fast Flow filler column is obtained contained approximately 100mg.
  • the antibody after antigen immunoaffinity chromatography was analyzed by 12% Native PAGE. The results showed that the purified antibody was of high purity (Fig. 4).
  • the purified antibody was subjected to BCA protein assay kits (BioCA). , Pierce, CA USA) determined that the total antibody amount was approximately 1.5 g.
  • the coupling process with antigen conjugated to human serum albumin procedure is determined.
  • the sample loading is increased, the non-adsorbed protein (penetrating solution) is increased, and the adsorbed human serum albumin remains unchanged, that is, the column is saturated and saturated.
  • the minimum sample load in the state is the load of the column.
  • the sample was loaded with recombinant human serum albumin (8 mg/ml), and the loading volume was gradually increased from 4 ml. As the loading volume increased, the elution volume gradually stabilized. When only the precipitation volume increased, this was confirmed.
  • the loading amount is the column load.
  • the loading volume of the plant-derived recombinant human serum albumin (8 mg/ml) was gradually increased from 4 ml to 24 ml.
  • the results showed that there was a dose-effect relationship between the binding of human serum albumin and the penetrating heteroprotein between 4 ml and 12 ml, that is, as the loading amount increased, the binding protein peak and the penetrating protein peak increased with the loading amount.
  • the increase is increased.
  • the saturation is basically reached, that is, the increase of the sample amount does not increase the peak of the bound protein, indicating that the sample volume of 12ml has reached saturation, and the packed volume of the immunochromatography resin is
  • the total loading of human serum albumin was 96 mg.
  • the recombinant human serum albumin genetically engineered rice raw rice was ground, and 0.1 g of rice flour was weighed, and an extraction buffer of 25 mM PBS, pH 7.5 (1:5) was added. Mix with a rotator for 60 min, centrifuge at 12,000 g, collect the supernatant, and perform anti-HSA immunoaffinity chromatography in an amount of 100 mg each to remove recombinant human blood. Albumin, isolated host heteroprotein.
  • the three-volume equilibration solution (20 ⁇ M Na 2 HPO 4 , 0.5 M NaCl, pH 7.6) was equilibrated on the coupled anti-HSA immunoaffinity column before loading, and the flow rate was 5 ml/min.
  • the solution was dialyzed and then eluted with an eluent (20 ⁇ M Na 2 HPO 4 , 0.5 M NaCl, pH 2.7).
  • an eluent (20 ⁇ M Na 2 HPO 4 , 0.5 M NaCl, pH 2.7).
  • the total residual protein solution of the host was collected, the amount of the sample was 100 mg once, and the penetrating solution was repeated twice to completely remove the human serum albumin from the total residual protein of the host.
  • the collected total residual protein of the host was concentrated by ultrafiltration in a 5 kD membrane package.
  • the collected total residual protein samples of the host were concentrated and determined by 12% SDS-PAGE to determine the impurity composition. (Refer to "Molecular Cloning" third
  • the prepared total residual heteroprotein antigen band of the host is clear, and the molecular weight range is wide.
  • the purified antigen standard is measured by the BCA method, and the protein concentration thereof is not less than 1.5 mg/ml.
  • the purified total residual protein of genetically engineered rice extract was used as antigen, and 4-6 months old pure male New Zealand white rabbits were used as immunized animals.
  • the primary immunization site was injected at multiple points in the lymph nodes and back, and the immune site was boosted. Multiple injections on the soles of the feet and the back; the initial immunization dose is 1.5 mg/ml total residual heteroprotein antigen, and an equal volume of Freund's complete adjuvant is added during the initial immunization to increase the immunogenicity of the antigen, and is strengthened 2 weeks after the initial immunization.
  • Immunization, booster immunization was 1 mg/ml total residual heteroprotein antigen, 3 times in total, 2 weeks apart.
  • Blood was collected within 7-10 days after the last booster immunization, placed in a test tube, allowed to stand at 37 ° C for 30 min, the blood cells were coagulated, and then placed in a refrigerator at 4 ° C overnight. The supernatant was collected by centrifugation the next day, and the supernatant was collected.
  • the antibody is purified and prepared. For specific purification methods, see the purification of the above-mentioned anti-human serum albumin polyclonal antibody.
  • the serum against the total residual protein of rice was crudely purified according to Figure 7, and then subjected to protein A affinity chromatography to obtain a polyclonal antibody mixture against total residual protein of rice, and then coupled with plasma-derived human serum albumin.
  • Affinity chromatography removes antibody components that recognize recombinant human serum albumin, and obtains specific antibodies that specifically recognize residual proteins in rice host residues.
  • the crude purification was carried out as above, and the purified serum residue of the residual residual protein was reconstituted with 25 mM PBS, and ammonium sulfate was removed by dialysis, and then the pH was adjusted to 7.6 with HCl to be used as an upper sample of protein A affinity chromatography.
  • the crude purified sample was further purified using protein A.
  • the chromatographic conditions were equilibration buffer 20 mM PBS pH 7.4, elution buffer 0.1 mM glycine-hydrochloric acid buffer pH 2.7, flow rate 2 ml/min.
  • the human serum albumin-conjugated affinity chromatography was repeated three times to completely remove the anti-HSA antibody in the specific antibody.
  • the specific method is the same as above, and the only difference is that the permeate is collected as the target-specific antibody solution.
  • the specific antibody prepared in Example 1 was HRP-labeled by sodium periodate method, that is, NaIO 4 was oxidized to HRP at a low pH, and the aldolase formed by oxidation of HRP by NaIO 4 was linked to the amino group of the antibody molecule to form an amino group.
  • Schiff base which can be further reduced with NaBH 4 (or ethanolamine) to form a stable enzyme Label the antibody.
  • the coupled antibody needs to remove the unencapsulated excess enzyme, and the unconjugated enzyme is removed by ammonium sulfate precipitation (pretreatment of the ammonium sulfate precipitation method with the above antibody), and the coupled antibody passes through OD208nm.
  • the total amount of bound antibody finally obtained was calculated by OD403nm detection. Calculated as follows:
  • Enzyme amount (mg/ml) OD403nm ⁇ 0.4
  • IgG amount (mg/ml) (OD280nm-OD403nm ⁇ 0.3) ⁇ 0.62
  • Example 1 3.5 mg of the antibody solution prepared in Example 1 was transferred to a dialysis bag, dialyzed against carbonate buffer (0.1 M carbonic acid, pH 9.4) overnight, and 1.5 mg of Biotin was added to the antibody solution. After mixing, the reaction was stirred at room temperature for 2 hours. Purification of the labeled product was carried out through a G-25 column to remove free biotin. The protein content was determined using an ultraviolet spectrophotometer.
  • the antibody to be labeled was a host residual heteroprotein ELISA capture primary antibody, 3.5 mg of the antibody solution to be labeled was transferred to a dialysis bag, and dialyzed against carbonate buffer (0.1 M carbonic acid, pH 9.4) overnight. 1.5 mg of biotin was added to the antibody solution, and the mixture was stirred and stirred at room temperature for 2 hours.
  • the labeled product was purified by G-25 column, and the free biotin was removed to prepare an ELISA detection secondary antibody.
  • the host protein ELISA was used to detect the concentration of the secondary antibody protein by BCA method, and the detection result should be no less than 1 mg/ml.
  • the influencing factors affecting the detection of the residual amount of the host heteroprotein were studied, respectively, 1) the concentration and purity of the impurity; 2) the capture primary antibody and the detection second Anti-antigen coverage and specificity; 3) Detection of antibody HRP labeling and biotin labeling selection and optimal ratio of capture antibody to detection antibody; 4) Determination range of sample.
  • the specific antibodies obtained by Western Blotting were detected by antigenic impurities. Most of the standard antigens and partially purified impurities were effectively detected by specific antibodies, and the results showed that the specific antibodies were prepared. Both pre- and post-products have good coverage.
  • the experiment first established an ELISA method based on HRP-labeled antibody.
  • the biotin-conjugated detection antibody concentration was 0.5 ⁇ g/ml, and the negative control absorbance value in the Blank behavior test.
  • the biotin-conjugated detection antibody concentration was 0.5 ⁇ g/ml, and each set of experiments was independently repeated 3 times.
  • the absorbance of the antigen was subtracted from the absorbance of the negative control, and the absorbance of the negative control in the behavioral test of Blank.
  • the concentration of the capture antibody is 4 ⁇ g/ml or more, the absorbance value of the antigen detection tends to be stable, and the concentration of the capture antibody coating at 4 ⁇ g/ml can satisfy the detection, but the background value is slightly higher than the above.
  • the OsrHSA stock solution (concentration: 200 mg/ml) was diluted and then measured, and the results are shown in the table.
  • concentration of OsrHSA in OsrHSA products is high, and there may be some influence on the determination of impurity content in ELISA. It can be seen from the results in the table that when the dilution ratio is in the range of 64-256 gradient, the measured value of the impurity content is basically in the same ratio relationship, and the dilution factor is too large, so that when the impurity content is close to the quantitative limit, the numerical deviation is large. Therefore, when the dilution gradient is 128 times, the measurement result is most reliable, that is, when the concentration of OsrHSA is preferably 3 mg/ml or less, the measured value of the impurity content in the product is more accurate and credible.
  • Negative sample human albumin, diluted 128 times with diluent
  • the actual limit of quantification is 1.25 ng/ml, the recovery of the assay results is between 75% and 125%, and the measured RSD is not greater than 25%.
  • Standard solutions were added to the sample solution at concentrations of 25 ng/ml, 10 ng/ml and 2.5 ng/ml to calculate the recoveries.
  • Spotting sequence The standard antigen spotting sequence is the same as 2.
  • the spiked samples were spiked with 25 ng/ml, spiked with 10 ng/ml, spiked with 2.5 ng/ml, and spiked with 0 ng/ml (negative control).
  • the standard product recovery rate is between 75% and 125%, and the RSD value between the recovery rates is less than 20%, in line with the standard.
  • Samples with linearity, precision, and accuracy that meet acceptable standards range from 2.5 ng/ml to 25 ng/ml.

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Abstract

本发明提供一种特异性抗体，其可专一结合重组蛋白提取物中的残留杂蛋白。本发明还提供一种免疫检测试剂，该试剂用于检测重组蛋白提取物中的残留杂蛋白，将本发明制备的特异性抗体作为所述免疫检测试剂中的捕获抗体和检测抗体，用双抗体夹心法进行检测。

Description

检测重组蛋白提取物中残留杂蛋白的特异性抗体及检测试剂 技术领域
本发明属于生物技术领域，具体涉及鉴定蛋白制品中残留杂蛋白的抗体和检测方法。
背景技术
宿主蛋白(HCPs)(残留杂蛋白)是重组蛋白质药物的一个主要杂质成分，是生物制品的重要安全隐患，过多的HCPs可能会引起免疫等一系列不良反应，但即使在最严格的纯化工艺和质量控制下进行生产，微量的HCPs残留也不可避免。有时即使痕量的残留宿主杂蛋白就有可能引起剧烈的免疫反应，而且其潜在的“佐剂效应”也可能会引起机体对药物产生抗体，进而影响药物的疗效。对残留杂蛋白的质量控制是生物安全性评估的重中之重。因此，在工艺开发中，应尽量去除这些宿主残留蛋白，同时建立快速、准确且灵敏的HCPs检测方法。
宿主残留杂蛋白的鉴定与检测一直是重组蛋白药物发展过程中的一个重大难题，特别是对于极高纯度的样品，以往的研究大部分都是针对前期工艺的研究，为工艺发展提供必要的帮助，而对最终样品的检测分析往往显得无力，往往只能鉴定一个或几个高浓度的宿主残留杂蛋白。在残留杂蛋白鉴定的过程中，首先要考虑的就是如何有效减少目的蛋白的干扰，达到浓缩这些痕迹量的宿主残留蛋白，以达到质谱的检测范围内。同时，在宿主残留杂蛋白检测技术中，痕迹量的目的蛋白会严重干扰和影响检测的精度，因此如何从基因工程的蛋白制品制备具有专一性的抗体是建立稿精度检测宿主残留杂蛋白的关键。
在制备重组蛋白的质量控制中，如何从表达重组蛋白的提取液中分离制备没有与目的蛋白交叉反应的宿主残留杂蛋白是建立检测重组蛋白提取物的残留杂蛋白检测方法的关键，尤其是如何彻底地去除目的蛋白是一个非常棘手的技术难题，也是建立高精度的宿主残留杂蛋白检测方法的关键因素；目前国际上采用宿主残留杂蛋白的检测方法由于宿主细胞高效表达重组蛋白后，宿主蛋白的成分和种类均发生了较大变化，利用非基因工程宿主蛋白来建立重组蛋白的宿主杂蛋白检测方法具有极高的风险，也是目前还没很好地解决的技术难题。
用于HCPs分析的主要技术包括十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、高效液相色谱法和免疫印迹法，但是通常最终产品中HCPs含量都在ppm级，而上述方法的检测灵敏度和准确性明显不够，无法对高纯度的样品进行有效的HCPs定量分析。因此，酶联免疫吸附(ELISA)技术由于其高灵敏度和准确性被认为是行业内的黄金标准检测方法。到目前为止，绝大多数通过FDA批准认可的重组蛋白药物残留HCPs含量定量都是通过ELISA进行测 定，检测含量一般在1–100ppm之间。因而建立高精度的ELISA方法来检测宿主残留杂蛋白的关键在于具有专一性识别抗原(宿主残留杂蛋白)的特异性抗体。
检测残留杂蛋白的ELISA方法主要有两种，一种是利用商业化的试剂盒，但需要有成熟的商业化抗体，另一种是通过制备特异性抗体进行检测。
发明内容
本发明的一个目的在于提供一种特异性抗体，其可专一结合重组蛋白提取物中的残留杂蛋白。
本发明的另一个目的在于提供一种免疫检测试剂，该试剂用于检测重组蛋白提取物中的残留杂蛋白，将本发明制备的特异性抗体作为所述免疫检测试剂中的捕获抗体和检测抗体，用双抗体夹心法进行检测。
根据本发明的一方面，一种特异性抗体，用于专一结合重组蛋白中的残留杂蛋白，所述重组蛋白为将外源基因通过基因工程技术重组并在宿主中表达的蛋白质产物，其中含有目的蛋白和残留杂蛋白，其特征在于，通过下述方法制备所述抗体：
1)用第一种蛋白免疫动物，制备抗第一蛋白抗体，所述第一种蛋白为天然存在于生物体的蛋白，所述目的蛋白与第一种蛋白具有相同的蛋白质结构和性质；
2)将步骤1)获得的抗第一蛋白抗体与亲和层析介质偶联，制备抗第一蛋白抗体-免疫亲和层析柱；
3)用步骤2)制备的抗第一蛋白抗体-亲和层析柱去除重组蛋白中的目的蛋白，获得总宿主残留杂蛋白；
4)用步骤3)获得的总宿主残留杂蛋白作为第二种蛋白免疫动物，制备抗第二蛋白抗体；
5)将第一种蛋白与亲和层析介质偶联，制备第一种蛋白-亲和层析柱；
6)用步骤5)制备的第一种蛋白-亲和层析柱去除步骤4)制备的抗第二蛋白抗体中的痕迹量的抗第一种蛋白的抗体，获得能专一结合重组蛋白提取物中残留杂蛋白的抗体。
优选地，在步骤1)和步骤4)之后可以进一步包括纯化免疫动物获得的抗第一蛋白抗体和抗第二蛋白抗体的步骤。并且必要时可以重复步骤3)和步骤6)，以获得更好的去除目的蛋白和抗第一蛋白的抗体的效果。
在上述步骤中，术语“天然存在于生物体的蛋白”是指直接从生物体中分离的具有生物活性的蛋白质。
术语“重组蛋白”是指应用基因重组技术，获得连接有可以翻译成目的蛋白的 基因片段的重组载体，之后将其转入可以表达目的蛋白的宿主细胞从而表达特定的重组蛋白分子。
术语“残留杂蛋白”或者“宿主残留杂蛋白”是指在重组蛋白表达和分离纯化过程中来源于宿主细胞的非目的蛋白的蛋白质。
用于表达重组蛋白的表达载体包括原核细胞如大肠杆菌、真核细胞如酵母、昆虫细胞以及CHO细胞等，重组蛋白也可利用动物表达系统或植物表达体系产生，例如转基因动物的乳腺或者转基因植物的胚乳或叶片。本发明的一个代表性示例即通过水稻胚乳表达的人血清白蛋白。
根据本发明的另一方面，提供一种酶联免疫检测试剂，用于定量检测基因工程重组蛋白提取物中的残留杂蛋白，其使用本发明的特异性抗体作为捕获抗体包被固相载体，并用本发明的特异性抗体作为检测抗体与标记物结合制备酶结合物；优选的检测试剂为使用双抗体夹心法的ELISA检测试剂。
更具体的，本发明以转基因水稻胚乳表达的重组人血清白蛋白的制备为典型示例，提供一种酶联免疫检测试剂，其中所述能专一结合重组蛋白提取物中残留杂蛋白的特性抗体通过下述方法制备：
i)用人源血清白蛋白作为抗原免疫兔子，制备兔抗人血清白蛋白多克隆抗体并纯化；
ii)将步骤i)制备的纯化的兔抗人血清白蛋白多克隆抗体与亲和层析介质偶联，制备免疫亲和层析柱A；
iii)用步骤ii)的免疫亲和层析柱A从表达重组人血清白蛋白的基因工程水稻种子提取物中去除目的蛋白，进而制备水稻种子总残留杂蛋白，通过3次的亲和层析，彻底去除目的蛋白，收集含水稻种子总残留杂蛋白的流出液；
iv)用步骤iii)获得的含水稻种子总残留杂蛋白的流出液免疫兔子，获得抗基因工程水稻种子总杂蛋白的多克隆抗体并纯化；
v)以血浆来源人血清白蛋白与亲和层析介质偶联，制备亲和层析柱B；
vi)用步骤V)的亲和层析柱B对步骤iv)的获得的抗基因工程水稻种子总杂蛋白多克隆抗体进行亲和层析，通过3次的亲和层析，去除痕迹的抗重组人血清白蛋白的抗体，收集流出物为专一结合表达重组人血清白蛋白的基因工程水稻种子总提取物中残留杂蛋白的特异性抗体。
优选地，其中步骤ii)和步骤v)所述的亲和层析介质为CNBr-activatived SepharoseTM 4Fast Flow。
优选地，其中步骤i)所述的纯化包括下述步骤：
ia)硫酸铵沉淀粗纯化制备的兔抗人血清白蛋白多克隆抗体得到粗纯液；以及
ib)用proteinA亲和层析纯化粗纯液获得纯化的多克隆抗体。
其中步骤iv)所述的纯化包括用IgG层析柱纯化获得的抗体基因工程水稻宿主杂蛋白的多克隆抗体。
所述的免疫检测试剂中，优选的捕获抗体包被浓度可以在为5～40μg/ml，更优选捕获抗体浓度为20μg/ml，检测抗体浓度优选为0.2～1.6μg/ml，更优选的检测抗体浓度为0.8μg/ml，以辣根过氧化物酶标记检测抗体。
也可以将生物素与辣根过氧化物酶结合，其中优选的捕获抗体包被浓度为2～16μg/ml，检测抗体浓度为0.5μg/ml，更优选的捕获抗体浓度为2μg/ml，以生物素标记检测抗体后再与辣根过氧化物酶结合。
本发明的免疫检测试剂，其检测的重组蛋白提取物中残留杂蛋白含量优选为2.5ng/ml–25ng/ml。
本发明的特异性抗体可专一结合重组蛋白提取物中的宿主残留杂蛋白，利用本发明特异性抗体的ELISA检测实践可对重组蛋白提取物中的残留杂蛋白进行定量鉴定，对于重组蛋白产物的质量控制有重要的用途和意义。
附图简述
图1为检测水稻宿主残留杂蛋白的ELISA方法建立的技术路线图；
图2为抗人血清白蛋白血清的滴度与覆盖度的Western Blotting检验；其中：1:商业购买兔抗人血清白蛋白，2：自制兔抗人血清白蛋白，M:Marker；
图3为水稻种子抗总杂质兔血清的粗纯化路线图；
图4为纯化后抗体的Native PAGE图；其中：1为白蛋白抗体，M为蛋白marker.纯化后抗体的Native PAGE图，1为白蛋白抗体，M为蛋白marker。
图5为抗体偶联填料载量的测定；其中：左向箭头：洗脱溶液(即结合的人血清白蛋白)，右向箭头：穿透溶液(即未结合杂质蛋白)；
图6为水稻种子杂蛋白标准品样品的SDS-PAGE与专一性抗体的Western Blotting图；
图7为水稻种子总杂质抗血清的初步纯化路线图；
图8为抗体的抗原覆盖度及专属性分析；其中：抗杂质抗体对各层析步骤的杂质的SDS-PAGE(左)和Western blot图(右)；泳道1-9为不同纯化步骤的样品。M为蛋白Marker；
图9为ELISA检测试剂的标准曲线的线性验证结果。
具体实施方式
下面通过对优选实施例的描述，详细说明但不限制本发明。
描述本发明的特异性抗体和ELISA检测试剂的一个示范性实施方式是用基因工程水稻胚乳细胞表达的重组人血清白蛋白(OsrHSA)。在重组人血清白蛋白的提取物中除目的蛋白(重组人血清白蛋白)外，包含来自植物源(水稻) 宿主的残留杂蛋白，天然蛋白为人源血清白蛋白。
利用水稻胚乳细胞作为生物反应器表达重组的人血清白蛋白的方法，可以参见如中国专利申请No.200510019084，其涉及利用水稻胚乳细胞作为生物反应器生产OsrHSA的方法，或者如No.201010597544.2，其涉及从水稻种子中提取重组人血清白蛋白的方法，No.201010606635.8涉及从水稻种子中分离纯化重组人血清白蛋白的方法。
以下实施例所使用的材料和设备如无特别说明均为市售商业购买。
人血清白蛋白(human serum Albumin)Sigma；
弗氏完全佐剂(Freund’s complete adjuvant,FCA)Sigma F-5881；
弗氏不完全佐剂(Freund’s incomplete adjuvant,FIA)Sigma F-5506；
新西兰兔(New Zealand rabbits)中科院武汉病毒所；
植物源重组人血白蛋白粗提液由武汉禾元生物科技有限公司提供；(制备方法参考上述引用的专利申请)
抗人血清白蛋白抗体(Anti-Human Serum Albumin antibody)abcam ab2406；
用于多克隆抗体的纯化与偶联的试剂：
层析填料：CNBr-activatived SepharoseTM 4Fast Flow；
层析仪：AKATA Purifier 100UPC,Bio-rad Duoflow；
载量测定：BCA法测定提取溶液蛋白含量(ThermoScientific)；
Equlibration buffer:20μM Na2HPO4·12H2O+4μMNaH2PO4·2H2O+0.5MNaCl pH7.6；
Elution buffer:20μM Na2HPO4·12H2O+0.15M NaCl pH2.8
饱和硫酸铵：50％饱和硫酸铵。
用于免疫亲和层析与NanoLC-ESI-MS/MS的设备：
膜包：U650599PelliconXL5KD Millipore PXC005C50，100％TCA，丙酮；
Agilent 1200系列快速高分辨液相色谱仪(Rapid Resolution Liquid Chromatography,Agilent Technologies,Waldbronn,Germany)，
真空浓缩离心机(SpeedVac device，Thermo)，胰蛋白酶(modified,sequencing grade)，Promega(Madison,WI)
四级离子陷阱质谱分析仪Thermo(Palo Alto,CA)；
非冗余蛋白数据库下载于NCBI’s GenBank；
所有在蛋白酶解和HPLC用到的化学试剂均来自Sigma(St.Louis,MO,USA)。
【实施例1】特异性抗体制备
1、技术路线
由于目前还没有可用于检测水稻宿主残留杂蛋白的ELISA商业试剂盒。为 了开发适用水稻表达体系制备的重组人血清白蛋白提取物的检测方法，在重组蛋白水稻胚乳细胞过量表达时，产生内质网胁迫，而从导致一些内源蛋白的组分发生一些改变，为了更准确地检测宿主蛋白，本检测方法利用基因工程水稻种子全杂质，建立了具有灵敏度高、重复性好的检测方法，用于水稻种子生物反应器系列产品的检测和质量控制。
本检测方法应用基因工程水稻的提取工艺的粗提取物的全杂质成分，采用双抗体夹心法测定植物源重组人血清白蛋白中的水稻种子宿主蛋白残留量水平；检测抗体分别采用HRP标记和生物素标记。
实验技术路线如图1所示，实验制备抗人血清白蛋白(天然蛋白，第一种蛋白)的免疫层析柱，用于去除基因工程全提取物中的重组人血清白蛋白(目的蛋白)，剩余的全蛋白作为总残留杂蛋白(第二种蛋白)抗原，免疫兔子获得抗宿主总杂蛋白的多克隆抗体，纯化后的抗宿主总杂蛋白的多克隆抗体作为包被和检测抗体，并以免疫抗原作为标样，建立夹心ELISA定量方法。
2、抗人血清白蛋白多克隆抗体制备
取20只成年新西兰兔，每4周注射一次人源的人血清白蛋白，共注射3次，在最后一次注射后的7-10天后放血采集血液。将收集血液在37℃恒温箱中放置30分钟，在4℃放置过夜。将血凝块从管壁上拨落，转移至塑料离心管中，在4℃下10,000g离心10分钟，收集上清液即为血清，血清样品随即用抗IgG的免疫亲和层析对血清中的抗体进行进一步纯化，获得高纯度抗人血清白蛋白抗体，纯化后的抗体在-20℃冰箱保存以备用。
表1抗人血清白蛋白多克隆抗体制备流程
免疫次数 抗原配制 注射位置 WB检测
初次免疫 1.0mg HSA+0.8ml H2O+0.8ml FCA 淋巴结与背部下皮 －
一次加强 0.8mg HSA+0.8ml H2O+0.8ml FIA 脚底与背部下皮 +
二次加强 0.8mg HSA+0.8ml H2O+0.8ml FIA 脚底与背部下皮 +
在第三次免疫后一个星期，制备血清经过Western检测，同样在1:10000稀释度的条件下，实验制备的抗人血清白蛋白的血清与市场销售的抗人血清白蛋白抗体(US Biological，A1327-30A)进行比较，Western Blotting结果如图2所示，制备的抗人血清白蛋白的血清效价跟市场经过纯化后的效价接近，制备多克隆抗体还能有效地与人血清白蛋白的降解片段反应，表明制备的抗体不仅能有效地去除完整重组人血清白蛋白分子，而且能有效地去除人血清白蛋白的降解片段，因而可以提高对残留杂蛋白鉴定的精确度。
3、抗人血清白蛋白多克隆抗体的纯化
为了消除任何血清不纯物对检测结果的影响，先对抗血清进行粗纯化，具体纯化的过程见图3。经二级硫酸铵沉淀的粗纯化抗体用25mM PBS复溶，经过 透析去除硫酸铵用HCl调节pH值至7.6，用于免疫亲和层析的制备样品。
抗人血清白蛋白抗体经粗纯化后，用于抗原免疫亲和层析。人血清白蛋白抗原首先偶联至CNBr-activatived SepharoseTM 4Fast Flow。偶联方法具体流程参照厂家说明书为：先用0-4℃的HCl(pH 2-3)溶液去除填料的保护剂，以人血清白蛋白溶液(pH8.0)：填料按照1:2的比例进行混合偶联，混合后摇床里室温放置3小时，随后加入0.1M Tris-HCl(pH 8.0)缓冲液放置2小时，对未偶联残基进行封闭。封闭结束后用缓冲液(0.5M NaCl,0.1M acetate,pH 3-4)和缓冲液(0.5M NaCl,0.1M Tris-HCl,pH 8–9)反复清洗人血清白蛋白偶联的填料，最后制备的填料装入层析柱，上样前采用3倍柱体积的平衡液(20μM Na2HPO4,0.5MNaCl pH7.6)对免疫亲和层析柱进行平衡，上样后用洗脱液(20μM Na2HPO4,0.5M NaCl pH 2.1)对结合在免疫亲和层析填料的抗人血清白蛋白抗体进行洗脱收集，洗脱后的层析柱经过再平衡进行下一次的纯化收集。
本试验共利用700mg人血清白蛋白偶联了6.5g CNBr-activatived SepharoseTM 4Fast Flow填料，获得层析柱载量约为100mg。对抗原免疫亲和层析后的抗体通过12％的Native PAGE分析，结果显示纯化的抗体具有较高纯度(图4)，纯化后的抗体经过BCA蛋白定量试剂盒(BCA Protein assay kits,Bio Rad,Pierce,CA USA)测定，总抗体量约为1.5g。
4、抗人血清白蛋白抗体偶联亲和层析填料的载量测定
纯化后的抗体按照同样偶联方法偶联至CNBr-activatived SepharoseTM 4Fast Flow，偶联的流程跟抗原人血清白蛋白的偶联流程相同。偶联完后对层析柱进行载量的确定，在增加上样量的情况下，非吸附蛋白(穿透液)增加，吸附的人血清白蛋白保持不变，即柱子已饱和，在饱和状态下最低上样量即为柱子的载量。以含植物源重组人血清白蛋白(8mg/ml)上样，上样体积从4ml逐步增加，随着上样体积增加，当洗脱体积逐渐平稳，当只有析出体积增加时，即可确定此上样量即为柱子载量。
从图5可以看出，植物源重组人血清白蛋白(8mg/ml)上样体积从4ml逐步增加到24ml。结果显示：在上样量4ml至12ml之间结合的人血清白蛋白和穿透的杂蛋白具有量效关系，即随着上样量增加，结合蛋白峰和穿透蛋白峰随着上样量的增加而增加，当上样量达到12ml时，基本达到饱和，即增加上样量并没有增加结合蛋白的峰值，表明12ml的上样量已经达到饱和，在该免疫层析树脂的装柱体积下，捕获人血清白蛋白的总载量为96mg。
5、宿主总残留杂蛋白抗原的制备
重组人血清白蛋白基因工程水稻原料大米经过研磨，称取米粉0.1g，加入提取缓冲液25mM PBS,pH7.5,(1:5)。用旋转仪混匀60min，12,000g离心，收集上清液后，以每次100mg的量进行anti-HSA免疫亲和层析，去除重组人血 清白蛋白，分离宿主杂蛋白。
上样前先对偶联好的anti-HSA免疫亲和层析柱进行三倍体积的平衡液(20μM Na2HPO4,0.5M NaCl,pH7.6)进行平衡，流速为5ml/min,收集穿透液，然后用洗脱液(20μM Na2HPO4,0.5M NaCl,pH2.7)进行洗脱。上样过程中，收集的宿主总残留杂蛋白溶液，一次上样量为100mg，穿透液重复过柱两次，以彻底地去除宿主总残留杂蛋白中的人血清白蛋白，经过三次免疫层析收集的宿主总残留杂蛋白经过5kD膜包的超滤浓缩。收集的宿主总残留杂蛋白样品经浓缩后分别经12％的SDS-PAGE跑胶确定杂质组成。(参照《分子克隆》第三版)。
从图6结果可看出，所制备的宿主总残留杂蛋白抗原条带清晰，分子量范围广泛，用BCA法测纯化出来的抗原标准品，其蛋白浓度不低于1.5mg/ml。
6、抗总宿主杂蛋白多克隆抗体的制备过程
以基因工程水稻提取物的经纯化后的总残留杂蛋白为抗原，取4-6个月龄的纯种雄性新西兰大白兔作为免疫动物，初次免疫部位在淋巴结和背部多点注射，加强免疫部位在脚底和背部多点注射；初次免疫剂量为1.5mg/ml总残留杂蛋白抗原，初次免疫时加入等体积弗氏完全佐剂，以提高抗原的免疫原性，初次免疫后2周后进行加强免疫，加强免疫为1mg/ml总残留杂蛋白抗原，共3次，每次间隔2周。最后一次加强免疫后7-10天内采血,装于试管中，于37℃静置30min，使血球凝固，再置于4℃冰箱过夜，第二天离心收集上清液，并对上清液中的抗体进行纯化制得。具体纯化方法参见上述抗人血清白蛋白多克隆抗体的纯化。
抗水稻总残留杂蛋白的血清按照图7进行粗纯化，再经过protein A亲和层析，获得抗水稻总残留杂蛋白的多克隆抗体混合液，再用血浆来源的人血清白蛋白偶联的亲和层析，去除可以识别重组人血清白蛋白的抗体成分，获得专一识别水稻宿主残留杂蛋白的特异性抗体。粗纯化的过程同上，纯化后的抗残留杂蛋白的血清沉淀用25mM PBS复溶，经过透析去除硫酸铵，然后用HCl调节pH至7.6，用作protein A亲和层析的上样品。选用protein A对粗纯化后的样品进一步的纯化，层析条件为平衡缓冲液20mM PBS pH 7.4，洗脱缓冲液0.1mM甘氨酸－盐酸缓冲液pH 2.7，流速2ml/min。再经重复三次的人源血清白蛋白偶联的亲和层析，以彻底地去除特异性抗体中的anti-HSA抗体，具体方法同上，唯一区别为收集穿透液为目标特异性抗体溶液。
【实施例2】ELISA检测试剂制备
1、特异性抗体的HRP的偶联
采用过碘酸钠法对实施例1制备的特异性抗体进行HRP标记，即在低PH下使NaIO 4氧化HRP，HRP经NaIO 4氧化后形成的醛化酶可与抗体分子的氨基相连，形成斯夫氏碱，后者可进一步用NaBH 4(或乙醇胺)还原生成稳定的酶 标记抗体。具体步骤如下：标记步骤:
(1)称取20mgHRP溶解于4ml 0.3M碳酸氢盐(pH8.1)；
(2)于上述溶液中加入0.4ml新配的0.1％的FDNP溶液，室温下搅拌1小时；
(3)加4ml 0.5M NaIO 4,室温搅拌30min,待溶液变为黄绿色；
(4)加4ml乙二醇，室温搅拌1小时；
(5)将上述溶液装入透析袋中，用0.01M pH9.5的醋酸钠缓冲液透析，4℃过夜；
(6)加入20mg抗体到15ml HRP溶液，室温搅拌2-3小时；
(7)加入20mg NaBH4混匀，再置4℃2小时；
(8)装入透析袋中，对0.15M PH7.4PBS透析，4℃过夜。
偶联后的抗体需要去除未被偶联的多余酶，采用硫酸铵沉淀法去除未被偶联上的酶(硫酸铵沉淀方法同上面的抗体的预处理)，偶联完后的抗体经过OD208nm和OD403nm检测计算最终获得的偶联的抗体总量。计算公式如下：
酶量(mg/ml)＝OD403nm×0.4
IgG量(mg/ml)＝(OD280nm-OD403nm×0.3)×0.62
2、Biotin与特异性抗体的偶联
3.5mg由实施例1制备的抗体溶液移入透析袋中，用碳酸盐缓冲液(0.1M碳酸，pH 9.4)透析过夜，加入1.5mg的Biotin于抗体溶液中。混匀后常温搅拌反应2小时。过G-25柱进行标记产物的纯化，去除游离生物素。蛋白含量用紫外分光光度计进行测定。
3、鉴定宿主残留杂蛋白ELISA方法的二抗制备过程
待标记的抗体为宿主残留杂蛋白ELISA捕获一抗，将3.5mg待标记的抗体溶液移入透析袋中，用碳酸盐缓冲液(0.1M碳酸，pH 9.4)透析过夜。加入1.5mg的生物素于抗体溶液中，混匀后常温搅拌反应2小时。过G-25柱进行标记产物的纯化，去除游离生物素后制得ELISA检测二抗。用BCA法检测宿主蛋白ELISA检测二抗蛋白浓度，检测结果应不低于1mg/ml。
【实施例3】ELISA检测影响因素的确定：
为了检测由本发明制备的抗体在检测宿主残留杂蛋白的可行性，对影响宿主杂蛋白残留量检测的影响因素进行了研究，分别对1)杂质的浓度与纯度；2)捕获一抗与检测二抗的抗原覆盖度与特异性；3)检测抗体HRP标记与生物素标记的选择及捕获抗体与检测抗体最佳配比；4)样品的测定范围。
我们对各检测影响因素进行实验确定后得出结果，杂质的浓度为2.5mg/ml，且在Western Blotting中用检测二抗检出单一条带；捕获一抗和检测二抗均对抗原具有很好的覆盖度且与目的蛋白OsrHSA(水稻胚乳表达的重组人血清白蛋 白)间无交叉反应；最终确定的生物素体系中，捕获抗体包被浓度为2μg/ml，检测抗体浓度为0.5μg/ml，最低检测限可降低至1.25ng/ml；将酶标板包被抗体且封闭制成干板后本底值更低；稀释液使用PBS+0.1％BSA，检测结果更加准确；产品稀释比例为128倍时，结果最为可信。
【实施例4】特异性抗体的抗原覆盖度：
用Western Blotting法对获得的特异性抗体用抗原杂质检测，不管是标样抗原杂质还是部分纯化后的杂质，绝大部分都能有效地被特异性抗体检测，结果表明所制备的特异性抗体对前期产品和后期产品都具备很好的覆盖度。
交叉反应:在SDS-PAGE上，人血清白蛋白的带非常的明显(66.5kD),但Western Blotting结果显示，所有的层析样品包括最终样品没有重组人血清白蛋白对应的条带显示，表明所制备的特异性抗体不存在重组人血清白蛋白的交叉反应，图8。
【实施例5】检测抗体标记HRP或生物素的影响
为了比较HRP标记抗体与生物素标记抗体在本检测中的适用性，比较了两种标记抗体在灵敏度，检测限及本底上的区别。
基于HRP标记的检测抗体ELISA抗体浓度优化
实验首先建立了基于HRP标记抗体的ELISA方法，从
表2可以看出，在抗原浓度为250、50、10ng/ml条件下，HRP捕获抗体量在20-40μg/ml之间没有明显差异，而在20μg/ml时，OD值明显高于10μg/ml和5μg/ml，同样检测抗体在1.6与0.8μg/ml之间没有明显差异，而0.8μg/ml与0.4、0.2μg/ml则出现明显不同。在抗原浓度为2ng/ml时，OD检测值在各个浓度的捕获和检测抗体浓度下都很低，没有规律可循，可能是由于本身的检测限度所限。基于以上实验结果，我们最终选择捕获抗体浓度为20μg/ml，检测抗体浓度为0.8μg/ml(1:5000)。
表2不同抗原浓度在不同捕获抗体及检测抗体组合下OD450吸光值比较
Figure PCTCN2016071740-appb-000001
Figure PCTCN2016071740-appb-000002
基于生物素标记的检测抗体ELISA抗体浓度优化
在建立基于HRP标记抗体的ELISA方法时，比较50ng/ml、12.5ng/ml、2.5ng/ml、1.25ng/ml抗原浓度在不同捕获抗体及检测抗体组合下OD450吸光值，由表3结果可看出，抗原浓度为1.25ng/ml仍有阳性信号，且所使用的捕获抗体浓度也较低，但在捕获抗体浓度高于2本底值μg/ml时，检测本底值大于0.2过高。因而需找到最佳的检测抗体与捕获抗体浓度组合，由
表4不同抗原浓度50ng/ml、12.5ng/ml、2.5ng/ml、1.25ng/ml在2μg/ml捕获抗体+0.5μg/ml检测抗体组合及4μg/ml捕获抗体+0.5μg/ml检测抗体组合OD450吸光值比较可看出，当用2μg/ml捕获抗体进行包被，用0.5μg/ml检测抗体进行检测时，本底最低，且重复性较好。
表3不同抗原浓度在不同捕获抗体及检测抗体组合下OD450吸光值比较
Figure PCTCN2016071740-appb-000003
生物素偶联检测抗体浓度为0.5μg/ml，Blank行为实验中阴性对照吸光值。
表4检测抗体与捕获抗体组合试验
Figure PCTCN2016071740-appb-000004
生物素偶联检测抗体浓度为0.5μg/ml，每组实验独立重复3次。抗原吸光值为减去阴性对照后吸光值，Blank行为实验中阴性对照吸光值。
通过比较基于HRP标记与生物素标记的检测抗体ELISA方法数据，发现，由于生物素标记体系中，检测抗体孵育完成后，需辣根酶标记链亲和素进一步进行交叉反应从而显色，因此生物素标记体系中，检测信号进一步放大，灵敏度提高，HRP标记体系中，2ng/ml抗原无阳性检测信号；而生物素体系中，捕获抗体及检测抗体浓度降低的情况下，1.25ng/ml抗原有稳定的阳性检测信号，灵敏度较HRP体系的5ng/ml提高到1.25ng/ml。
生物素标记检测抗体ELISA方法中，捕获抗体浓度为4μg/ml以上时，抗原检测吸光值趋于稳定，捕获抗体包被浓度在4μg/ml即可满足检测，但本底值稍高，高于0.2，不利于低浓度抗原检测数据的稳定；当捕获抗体浓度降低为2μg/ml时，除高浓度抗原吸光值稍有下降外，低浓度抗原吸光值较为稳定，且显著高于空白对照+3×SD，OsrHSA中杂质含量较低，低本底值及更稳定的低浓度样品检测对于方法的稳定性更为重要，故最终选择2μg/ml的捕获抗体进行包被，0.5μg/ml的检测抗体进行检测。
【实施例6】样品测定范围
为了确定样品的测定范围，将OsrHSA原液(浓度为200mg/ml)进行倍比稀释后进行测定，结果如表所示。OsrHSA产品中OsrHSA浓度较高，在ELISA中对杂质含量测定可能存在一定影响。由表中结果可以看出，当其稀释比例为64-256梯度范围内时，杂质含量测定值基本呈同等倍比关系，稀释倍数过大，导致杂质含量接近定量限时，数值偏差会较大。故稀释梯度为128倍时，测定结果最为可信，即OsrHSA浓度最好为3mg/ml以下时，产品中杂质含量测定值较为准确可信。
表5样品倍比稀释测定值
Figure PCTCN2016071740-appb-000005
【实施例7】ELISA方法验证
1、专属性
阳性样品：1.25ng/ml标准品
阴性样品：人血白蛋白，用稀释液稀释128倍
空白：样品稀释液
点样序列：阳性样品、阴性样品、空白每块ELISA干板上各点样6个复孔，独立重复3次
专属性验证结果：
可接受标准：阳性样品有信号，OD值大于空白OD平均值+5×SD；阴性样品无信号，OD值与空白接近
结果：3次独立重复实验均显示，阳性样品OD450大于空白OD+5×SD；阴性样品无阳性信号，OD值与空白值接近，符合标准规定
表6宿主蛋白残留量测定方法专属性验证结果
Figure PCTCN2016071740-appb-000006
2、线性
方法：以吸收值(OD)对抗原标准品的浓度(Concentration)做4-parameter模式的标准曲线。标准曲线的R2应不得小于0.99，结果如图9.
点样序列：
表7线性验证点样序列
Figure PCTCN2016071740-appb-000007
Figure PCTCN2016071740-appb-000008
表8宿主蛋白残留量测定方法线性验证结果
Figure PCTCN2016071740-appb-000009
3、定量限
方法：将抗原标准品溶液稀释至最低可准确定量的浓度，点样测试。
点样序列：定量限溶液点样6个复孔，试验独立重复3次
定量限度样品：1.25ng/ml标准品抗原，点样6个复孔，独立重复3次(n＝18)
定量限验证结果：
可接受标准：实际定量限为1.25ng/ml，测定结果的回收率在75％-125％之间，测得值的RSD不得大于25％
定量限为1.25ng/ml，测定结果的回收率为96.5％，且测得值的RSD(n＝18)＝16.7％，符合标准规定
表9宿主蛋白残留量测定方法定量限度验证结果
Figure PCTCN2016071740-appb-000010
4、重复性
方法：将同一样品稀释后重复点样，计算结果间的RSD。
点样序列:标准品点样序列同2。
供试品批号：201310035、201310036、201310037。
供试品点样序列：各批次供试品稀释后点样9个复孔(3个复孔为一组，板内重复性，n＝9)，独立重复3次(板间重复性，n＝9)。
重复性验证结果:
可接受标准：
各批次样品板内测定结果(n＝9)与板间测定结果(n＝9)的RSD值不大于20％
结果：各批次样品板内测定结果(n＝9)及板间测定结果(n＝9)的RSD值均小于20％，符合标准规定
表10宿主蛋白残留量测定方法重复性验证结果
Figure PCTCN2016071740-appb-000011
5、准确度
方法：样品溶液中添加浓度为25ng/ml、10ng/ml和2.5ng/ml的标准品溶液，计算加标回收率。
点样序列：标准品抗原点样序列同2。
加标样品：加标25ng/ml、加标10ng/ml、加标2.5ng/ml、加标0ng/ml(阴性对照)。
加标样品点样序列：各浓度加标样品每块板各点样3个复孔，独立重复3次。
回收率计算公式：
回收率％＝(加标样品测定值-未加标样品测定值)/标准品理论添加量×100％。准确度验证结果
可接受标准：回收率在75％-125％之间，回收率间的RSD不得超过20％ 结果：
标准品添加回收率均在75％-125％之间，回收率间的RSD值均小于20％，符合标准规定
表11宿主蛋白残留量测定方法准确度验证结果
Figure PCTCN2016071740-appb-000012
6、精密度
方法：准确度项下各回收率间的RSD来确定方法的精密度。
点样序列：同2
精密度计算方法：各浓度加标样品回收率间RSD值(n＝9)。
精密度验证结果：可接受标准：各浓度回收率间的RSD值不得超过20％(n＝27)。
结果：各浓度回收率间的RSD值为8.3％(n＝27)，小于20％，符合标准规定
表12宿主蛋白残留量测定方法精密度验证结果
Figure PCTCN2016071740-appb-000013
7、范围
线性、精密度和准确度都符合可接受标准的样品测定范围为：2.5ng/ml–25ng/ml。

Claims (17)

  1. 一种特异性抗体，用于专一结合基因工程重组蛋白提取物中的残留杂蛋白，所述重组蛋白提取物中含有目的蛋白和残留杂蛋白，其特征在于，通过下述方法制备所述特异性抗体：
    1)用第一种蛋白免疫动物，制备抗第一蛋白抗体，所述第一种蛋白为天然存在于生物体的目的蛋白，所述目的蛋白与第一种蛋白具有相同的蛋白质结构和性质；
    2)将步骤1)获得的抗第一蛋白抗体与亲和层析介质偶联，制备抗第一蛋白抗体-免疫亲和层析柱；
    3)用步骤2)制备的抗第一蛋白抗体-亲和层析柱去除重组蛋白提取物中的目的蛋白，获得总宿主残留杂蛋白；
    4)用步骤3)获得的总宿主残留杂蛋白作为第二种蛋白免疫动物，制备抗第二蛋白抗体；
    5)将目的蛋白与亲和层析介质偶联，制备目的蛋白-亲和层析柱；
    6)用步骤5)制备的目的蛋白-亲和层析柱去除步骤4)制备的抗第二蛋白抗体中的抗第一种蛋白的抗体，获得能专一结合重组蛋白提取物中残留杂蛋白的抗体。
  2. 权利要求1所述的特异性抗体，其特征在于在步骤1)和步骤4)之后进一步包括纯化免疫动物获得的抗第一蛋白抗体和抗第二蛋白抗体的步骤。
  3. 权利要求1所述的特异性抗体，其特征在于必要时重复步骤3)和步骤6)。
  4. 权利要求1所述的特异性抗体，其特征在于所述重组蛋白通过植物表达系统表达。
  5. 权利要求4所述的特异性抗体，其特征在于所述重组蛋白为通过水稻胚乳细胞表达的人血清白蛋白，所述第一种蛋白为人血清白蛋白，所述目的蛋白为基因工程重组人血清白蛋白。
  6. 权利要求1～5任一项所述的特异性抗体在制备免疫检测试剂中的应用，其中所述免疫检测试剂用于检测基因工程重组蛋白提取物中的残留杂蛋白。
  7. 一种酶联免疫检测试剂，用于定量检测基因工程重组蛋白提取物中的残留杂蛋白，其特征在于用专一结合重组蛋白提取物中的残留杂蛋白的特异性抗体作为捕获抗体包被固相载体，以及用专一结合重组蛋白提取物中的残留杂蛋白的特异性抗体作为检测抗体与标记物结合制备酶结合物；
    其中所述重组蛋白提取物中含有目的蛋白和残留杂蛋白，通过下述方法制备所述特异性抗体：
    1)用第一种蛋白免疫动物，制备抗第一蛋白抗体，所述第一种蛋白为天然 存在于生物体的蛋白，所述目的蛋白与第一种蛋白具有相同的蛋白质结构和性质；
    2)将步骤1)获得的抗第一蛋白抗体与亲和层析介质偶联，制备抗第一蛋白抗体-免疫亲和层析柱；
    3)用步骤2)制备的抗第一蛋白抗体-亲和层析柱去除重组蛋白提取物中的目的蛋白，获得总宿主残留杂蛋白；
    4)用步骤3)获得的总宿主残留杂蛋白作为第二种蛋白免疫动物，制备抗第二蛋白抗体；
    5)将目的蛋白与亲和层析介质偶联，制备目的蛋白-免疫亲和层析柱；
    6)用步骤5)制备的目的蛋白-免疫亲和层析柱去除步骤4)制备的抗第二蛋白抗体中的抗第一种蛋白的抗体，获得能专一结合重组蛋白中残留杂蛋白的特异性抗体。
  8. 权利要求7所述的酶联免疫检测试剂，其特征在于所述标记物选自辣根过氧化物酶和/或生物素。
  9. 权利要求7所述的酶联免疫检测试剂，其特征在于所述重组蛋白为通过水稻胚乳细胞表达的人血清白蛋白，所述第一种蛋白为人血清白蛋白，所述目的蛋白为基因工程重组人血清白蛋白。
  10. 权利要求9所述的酶联免疫检测试剂，其特征在于所述能专一结合重组蛋白提取物中残留杂蛋白的特性抗体通过下述方法制备：
    i)用人源血清白蛋白作为抗原免疫兔子，制备兔抗人血清白蛋白多克隆抗体并纯化；
    ii)将步骤i)制备的纯化的兔抗人血清白蛋白多克隆抗体与亲和层析介质偶联，制备免疫亲和层析柱A；
    iii)用步骤ii)的免疫亲和层析柱A从表达重组人血清白蛋白的基因工程水稻种子提取物中分离和制备水稻种子总杂蛋白，通过亲和层析去除重组人血清白蛋白，收集含总水稻种子总杂蛋白的流出液；
    iiii)用步骤iii)获得的含总杂蛋白的流出液免疫兔子，获得抗基因工程水稻种子总杂蛋白的多克隆抗体并纯化；
    v)以重组人血清白蛋白与亲和层析介质偶联，制备亲和层析柱B；
    iv)用步骤V)的亲和层析柱B对步骤iv)的获得的抗基因工程水稻的种子总杂蛋白多克隆抗体进行亲和层析，去除痕迹的抗重组人血清白蛋白的抗体，收集流出物为专一结合表达重组人血清白蛋白的基因工程水稻种子总提取物中总杂蛋白的特异性抗体。
  11. 权利要求9所述的酶联免疫检测试剂，其中步骤ii)和步骤v)所述的亲和层析介质为CNBr-activatived SepharoseTM 4Fast Flow。
  12. 权利要求9所述的酶联免疫检测试剂，其中步骤i)所述的纯化包括下述步骤：
    ia)硫酸铵沉淀粗纯化制备的兔抗人血清白蛋白多克隆抗体得到粗纯液；以及
    ib)用proteinA亲和层析纯化粗纯液获得纯化的多克隆抗体。
  13. 权利要求9所述的酶联免疫检测试剂，其中步骤iv)所述的纯化包括用IgG层析柱纯化获得的抗体基因工程水稻宿主蛋白的多克隆抗体。
  14. 权利要求9所述的免疫检测试剂，其中所述捕获抗体包被浓度为5～40μg/ml，检测抗体浓度为0.2～1.6μg/ml，以辣根过氧化物酶标记检测抗体。
  15. 权利要求14所述的免疫检测试剂，其中所述捕获抗体浓度为20μg/ml，检测抗体浓度为0.8μg/ml.
  16. 权利要求9所述的免疫检测试剂，其中所述捕获抗体包被浓度为2～16μg/ml，检测抗体浓度为0.5μg/ml，以生物素标记检测抗体后再与辣根过氧化物酶结合。
  17. 权利要求9所述的免疫检测试剂，其中所述重组蛋白提取物中残留杂蛋白含量为2.5ng/ml–25ng/ml。
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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CN113461807A (zh) * 2021-07-30 2021-10-01 杭州博岳生物技术有限公司 一种抗体纯化过程中配基脱落的复合物去除方法
CN113817686A (zh) * 2020-06-18 2021-12-21 上海医药工业研究院 一种杂交瘤细胞株及其分泌的单克隆抗体和应用
CN113960318A (zh) * 2021-09-28 2022-01-21 苏州赛分科技股份有限公司 一种基于酶联免疫的重组蛋白a测定方法及试剂盒
CN117169518A (zh) * 2023-11-03 2023-12-05 赛德特(北京)生物工程有限公司 T淋巴细胞制剂中的cd3抗体残留物的检测方法和试剂盒
CN117192107A (zh) * 2023-09-11 2023-12-08 福建基诺厚普生物科技有限公司 一种工艺特异性宿主细胞蛋白残留的检测方法及试剂盒
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Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112858682B (zh) * 2019-11-27 2023-06-02 广东唯实生物技术有限公司 用于尿微量白蛋白的检测试纸条及其制备方法、检测试剂盒和检测方法
CN112798792A (zh) * 2020-12-29 2021-05-14 东曜药业有限公司 一种检测cho细胞宿主蛋白残留的试剂盒及检测方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1896239A (zh) * 2005-07-13 2007-01-17 杨代常 利用水稻胚乳细胞作为生物反应器生产重组人血清白蛋白

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102532254B (zh) * 2010-12-24 2015-06-24 武汉禾元生物科技股份有限公司 一种从水稻种子中分离纯化重组人血清白蛋白的方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1896239A (zh) * 2005-07-13 2007-01-17 杨代常 利用水稻胚乳细胞作为生物反应器生产重组人血清白蛋白

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JIA Q. ET AL.: "Determining the host cell protein impurities in recombinant human granulocyte colony stimulating factor preparation", CHINESE JOURNAL OF BIOCHEMICAL PHARMACEUTICS, vol. 25, no. 04, 31 August 2004 (2004-08-31) *

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