HPLC (High Performance Liquid Chromatography) analysis method for poly-HRP and marker

Abstract

The invention relates to the technical field of biology, in particular to an HPLC (High Performance Liquid Chromatography) analysis method of poly-HRP and a marker thereof. The invention provides the HPLC detection method of the poly-HRP and / or the marker-labeled poly-HRP, the HPLC detection method can realize the polymerization degree of the marker-labeled poly-HRP and the detection of the marker or the HRP, and the HPLC detection method is simple, rapid, good in repeatability and convenient to control the difference between batches, so that the defects in the prior art are overcome, and the HPLC detection method has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a HPLC analysis method of polymerized HRP and its markers. Background Art

[0002] Horseradish peroxidase (HRP), also known as HRP, is a commonly used reporter enzyme molecule with a molecular weight of about 44 kDa. It is widely used in Western blot, ELISA, immunohistochemistry, chemiluminescence and other technologies. In the above technologies, HRP is usually directly or indirectly labeled on antibodies or antigens as a marker. When the antibody binds to the target molecule through a specific binding relationship, HRP can report the target by catalyzing substrates such as 3,3',5,5'-tetramethylbenzidine (TMB), 3,3'-diaminobenzidine (DAB), 3-amino-benzidine (luminol), etc. to form color or luminescent products.

[0003] In the field of molecular biology detection technology, it is often necessary to amplify the signal of mutual binding between molecules in order to achieve visualization and high-sensitivity sensing of the analysis target. Therefore, HRP is polymerized to form Poly-HRP, and then labeled to achieve signal amplification in conventional detection applications. The existing technology usually uses an HRP polymerization method based on an L-polylysine molecular skeleton, or a method of achieving HRP polymerization through a new branched molecular skeleton (hyperbranched amino polydimethylhydroxypropionate, PAMAM), so that each molecule of the conjugate contains a large amount of HRP (polymerized horseradish peroxidase), and is applied to multiple technologies, including ELISA, IHC, ICC, protein blotting or DNA / RNA in situ hybridization.

[0004] However, there is no unified method to detect the polymerization state of Poly-HRP and its markers, and it is impossible to control the consistency of the state after polymerization. In the patent "A method for preparing poly-HRP", it is only tested by electrophoresis, but the molecular weight of Poly-HRP is large and basically on the concentrated gel, which is difficult to separate, especially after being labeled with protein, the molecular weight is even larger, and electrophoresis cannot detect it at all. In "An antibody horseradish peroxidase marker and its preparation", there is no method to detect the marker for its application in enzyme-linked immunosorbent assay kits or chemiluminescence assay kits, and after polymerization, poly-HRP introduces more amino groups, resulting in changes in the spatial structure of Poly-HRP and its polymers, which increases the difficulty of analysis.

[0005] It can be seen that finding a fast and simple analytical method is still a problem that needs to be solved in this field. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide an HPLC analysis method for polymerized HRP and its markers.

[0007] The present invention provides an HPLC analysis method for poly-HRP and poly-HRP labeled with a marker, characterized in that after filtering the sample, HPLC is used for detection:

[0008] The detection conditions of the HPLC include:

[0009] Mobile phase A includes: sodium dihydrogen phosphate, disodium hydrogen phosphate and water

[0010] Mobile phase B includes: NaCl and water;

[0011] The elution procedure is as follows: the volume fraction of mobile phase A is 70%, running for 20 min; it is isocratic elution of mobile phase;

[0012] Furthermore,

[0013] In the mobile phase A, the concentration of the sodium dihydrogen phosphate is 0.02-0.05 mol / L, and the concentration of the sodium hydrogen phosphate is 0.02-0.05 mol / L; in a specific embodiment of the present invention, the concentration of the sodium dihydrogen phosphate is preferably 50 mol / L, and the concentration of the sodium hydrogen phosphate is preferably 50 mol / L;

[0014] The pH of the mobile phase A is 6.8-7.5, specifically 7.2.

[0015] In the mobile phase B, the concentration of NaCl is 1-2 mol / L, preferably 1.5 mol / L in the specific embodiment of the present invention;

[0016] The pH of the mobile phase B is 6.8-7.2, specifically 7.0.

[0017] The HPLC chromatographic column is a Bio SEC51000A chromatographic column; the inner diameter of the Bio SEC51000A chromatographic column is 7.8 mm, the column length is 300 mm, and the filler is bonded silica gel; the bonded silica gel includes C18, C8, CN and / or NH2 bonded silica gel; in a specific embodiment of the present invention, it is a fully porous bonded silica gel covered with a neutral hydrophilic layer; the particle size of the bonded silica gel is 5 μm.

[0018] The detection wavelength of the HPLC includes 280 and / or 403 nm.

[0019] The flow rate of the HPLC is 0.7-1.2 ml / min, preferably 0.8 ml / min.

[0020] The column temperature of the HPLC is 20-35°C, preferably 25°C.

[0021] The injection volume of the HPLC was 10 μl.

[0022] The concentration of the sample is 0.5-2 mg / ml, preferably 1 mg / ml.

[0023] The HPLC analysis method of the present invention can be used to calculate the proportion of the marker in the poly-HRP labeled with the marker, and the calculation method of the proportion of the marker in the poly-HRP labeled with the marker is:

[0024] Using the HPLC analysis method of the present invention, chromatograms of samples with detection wavelengths of 280 nm and 403 nm are obtained respectively;

[0025] Calculate the total peak area of ​​the samples with detection wavelengths of 280 nm and 403 nm;

[0026] The absorption peak of the sample with a detection wavelength of 403nm is the absorption peak of the HRP auxiliary group in the marker; the absorption peak of the sample with a detection wavelength of 280nm is the absorption peak of the protein component in the marker;

[0027] The ratio of the total peak area of ​​the sample at a detection wavelength of 403 nm to the total peak area of ​​the sample at a detection wavelength of 280 nm is used to obtain the ratio of the cofactor and protein components of the marker.

[0028] The HPLC analysis method of the present invention can evaluate the detection performance of poly-HRP and poly-HRP labeled with a marker, and the evaluation method is: matching the batch differences (activity, false positive, etc.) in the process of product application detection kit with the physical and chemical properties of horseradish peroxide labeled antibody, and finally establishing the quality standard of enzyme-labeled protein products to make up for the shortcomings of conventional inspection technology and reduce the application risk of enzyme-labeled protein products to the lowest level.

[0029] The prior art of Poly-HRP usually uses an HRP polymerization method based on an L-polylysine molecular skeleton, or a method of realizing HRP polymerization through a new branched molecular skeleton (hyperbranched amino polydimethylhydroxypropionate, PAMAM), or HRP polymers formed by other methods and marker-labeled poly-HRP. The present invention provides for the first time an HPLC analysis method for poly-HRP (Poly-HRP) or marker-labeled poly-HRP; the present invention obtains an HPLC analysis method for poly-HRP and / or marker-labeled poly-HRP by optimizing the mobile phase and the detection column, and the analysis method can be used for calculating the proportion of markers or poly-HRP in marker-labeled poly-HRP, and evaluating the detection performance of poly-HRP and / or marker-labeled poly-HRP. And the selection of the mobile phase detected in the present invention, the ratio of the mobile phase and the particle size of the detection column (compared with 500A and 1000A, 1000A is more suitable for the detection of the present invention) and other parameters complement each other and jointly complete the detection of poly-HRP and / or marker-labeled poly-HRP.

[0030] The present invention provides an HPLC detection method for poly-HRP and / or poly-HRP labeled with a marker. The HPLC detection method can detect the degree of polymerization of poly-HRP labeled with a marker, the marker or HRP. The HPLC detection method is simple, fast and has good repeatability, and is convenient for controlling batch differences, thereby supplementing the deficiencies of the prior art and having broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The chromatogram of polymerized HRP in Example 1 of the present invention is shown, wherein the blue is the Agilent standard marker, 1 corresponds to a marker size of 1340KD, 2 corresponds to a marker size of 670KD, 3 corresponds to a marker size of 300KD, 4 corresponds to a marker size of 150KD, 6 corresponds to a marker size of 45KD, 7 corresponds to a marker size of 17KD, and 8 corresponds to a marker size of 1KD; red is polymerized HRP;

[0032] Figure 2 The chromatogram of the poly-HRP marker (ploy-HRP-Ab) in Example 1 of the present invention is shown, wherein the blue is the Agilent standard marker, 1 corresponds to a marker size of 1340KD, 2 corresponds to a marker size of 670KD, 3 corresponds to a marker size of 300KD, 4 corresponds to a marker size of 150KD, 5 corresponds to a marker size of 45KD, 6 corresponds to a marker size of 17KD, and 7 corresponds to a marker size of 1KD; chromatogram of the poly-HRP marker (ploy-HRP-Ab);

[0033] Figure 3 Shows the reproducible chromatography of poly-HRP in Example 1 of the present invention;

[0034] Figure 4 The figure shows the reproducible chromatogram of the poly-HRP marker (ploy-HRP-Ab) in Example 1 of the present invention;

[0035] Figure 5 The chromatogram of the poly-HRP marker (ploy-HRP-Ab) in Example 2 is shown, wherein the red color is the 403 nm detection peak and the blue color is the 280 nm detection peak;

[0036] Figure 6 The chromatogram of the poly-HRP marker (ploy-HRP-Ab) in Example 3 is shown, wherein different colors represent different batches, and there are three batches in total;

[0037] Figure 7 The chromatogram of the poly-HRP marker (ploy-HRP-Ab) in Example 4 of the present invention is shown, wherein red is the sample; blue is the standard product, 1 corresponds to a Marker size of 1340KD, 2 corresponds to a Marker size of 670KD, 3 corresponds to a Marker size of 300KD, 5 corresponds to a Marker size of 150KD, 6 corresponds to a Marker size of 45KD, 7 corresponds to a Marker size of 17KD, and 8 corresponds to a Marker size of 1KD;

[0038] Figure 8 The chromatogram of the poly-HRP marker (ploy-HRP-Ab) in Example 5 of the present invention is shown, wherein red is the sample; blue is the standard product, 1 corresponds to a Marker size of 1340KD, 2 corresponds to a Marker size of 670KD, 3 corresponds to a Marker size of 150KD, 4 corresponds to a Marker size of 45KD, and 6 corresponds to a Marker size of 1KD.

[0039] Fig. 9 The chromatogram of the poly-HRP marker (ploy-HRP-Ab) in Example 6 of the present invention is shown, wherein red is the sample; blue is the standard product, 2 corresponds to a Marker size of 1340KD, 3 corresponds to a Marker size of 670KD, 5 corresponds to a Marker size of 150KD, 6 corresponds to a Marker size of 45KD, 7 corresponds to a Marker size of 17KD, and 9 corresponds to a Marker size of 1KD;

[0040] Fig.10The chromatogram of the poly-HRP marker (ploy-HRP-Ab) in Example 7 of the present invention is shown, wherein the red is the sample; the blue is the standard product, 1 corresponds to a Marker size of 1340KD, 2 corresponds to a Marker size of 670KD, 3 corresponds to a Marker size of 150KD, 4 corresponds to a Marker size of 45KD, and 7 corresponds to a Marker size of 17KD;

[0041] Fig.11 The chromatogram of the poly-HRP marker (ploy-HRP-Ab) in Example 8 of the present invention is shown, wherein the red is the sample; the blue is the standard product, 1 corresponds to a Marker size of 670KD, 2 corresponds to a Marker size of 150KD, 3 corresponds to a Marker size of 45KD, 5 corresponds to a Marker size of 17KD, and 7 corresponds to a Marker size of 1KD;

[0042] Fig.12 The chromatogram of the poly-HRP marker (ploy-HRP-Ab) of Example 9 of the present invention is shown, wherein red is the sample; blue is the standard product, 2 corresponds to a Marker size of 1340KD, 3 corresponds to a Marker size of 670KD, 4 corresponds to a Marker size of 300KD, 5 corresponds to a Marker size of 150KD, 6 corresponds to a Marker size of 45KD, 7 corresponds to a Marker size of 17KD, and 8 corresponds to a Marker size of 1KD. DETAILED DESCRIPTION

[0043] The present invention provides a HPLC analysis method for poly-HRP and its markers. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0044] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0045] Example 1 HPLC detection scheme 1

[0046] Chromatographic conditions: chromatographic column Bio SEC51000A particle size 5μm, column length 7.8×300mm; detection wavelength: 280nm; sample concentration: 1mg / ml, column temperature: 25℃, flow rate: 0.8ml / min, mobile phase A:B ratio of 70:30 (mobile phase A is sodium dihydrogen phosphate and disodium hydrogen phosphate solution, the concentrations of sodium dihydrogen phosphate and disodium hydrogen phosphate are 0.05mol / L respectively; mobile phase B is 1.5mol / L NaCl solution); running time: 20min.

[0047] Detection: Take an appropriate amount of test sample (L-polylysine polymer, Aladdin, catalog number P303255-100mg; or poly-HRP (L-polylysine polymer) labeled antibody (ploy-HRP-Ab)), accurately weigh, dissolve and dilute to a certain concentration of sample solution with mobile phase, the sample concentration is 1mg / ml), shake well, filter with 0.22μm filter membrane, accurately measure 10μl and inject into liquid chromatograph for high performance liquid chromatography analysis, record the chromatogram, see Figures 1 to 4 .

[0048] in Figures 1-2 The middle blue spectrum is the Agilent standard marker. The molecular weights from left to right are thyroglobulin dimer 1340KD, thyroglobulin (bovine) 670KD, γ-globulin dimer 300KD, γ-globulin 150KD, ovalbumin 45KD, myoglobin 17KD, and angiotensin 1KD. The standard corresponds to the sample molecular weight.

[0049] Among them, poly-HRP (L-polylysine polymer (poly-HRP), Aladdin, product number P303255-100 mg) has good repeatability, and the RSD is within 1.5%, see Table 1 for details.

[0050] Table 1. Poly-HRP (L-polylysine polymer) reproducibility

[0051]

[0052] The repeatability of poly-HRP (L-polylysine polymer) labeled antibodies is shown in Table 2, and the RSDs are all within 1.5%;

[0053] Table 2. Repeatability results of poly-HRP (L-polylysine polymer) labeled antibody (ploy-HRP-Ab)

[0054]

[0055]

[0056] Example 2 HPLC detection scheme 2

[0057] Chromatographic conditions: chromatographic column Bio SEC51000A particle size 5μm, column length 7.8×300mm; detection wavelength: 280 and 403nm; sample concentration: 1mg / ml, column temperature: 25℃, flow rate: 0.8ml / min, mobile phase A:B ratio of 70:30 (mobile phase A is sodium dihydrogen phosphate and disodium hydrogen phosphate solution, the concentrations of sodium dihydrogen phosphate and disodium hydrogen phosphate are 0.05mol / L respectively; mobile phase B is 1.5mol / L NaCl solution); running time: 20min.

[0058] Detection: Take an appropriate amount of test sample (poly-HRP-Ab), accurately weigh it, dissolve it with mobile phase and dilute it into a certain concentration of sample solution (sample concentration is 1 mg / ml), shake it well, filter it with a 0.22μm filter membrane, accurately measure 10μl and inject it into the liquid chromatograph for high performance liquid chromatography analysis, and record the chromatogram.

[0059] In poly-HRP-Ab (the antibody contained therein is CA199 antibody, IgG subtype 150KD), HRP is a glycoprotein with a molecular weight of 44,000KD, which is composed of a colorless enzyme protein and a dark brown iron porphyrin. Each HRP molecule contains a hemin IX as a cofactor, which has a maximum absorption peak at a wavelength of 403nm, and the enzyme protein without the cofactor has a maximum absorption at a wavelength of 275-280nm, while other proteins such as marker antibody Ab have no response at 403nm.

[0060] Table 3. Analysis of the ratio of protein to HRP auxiliary group in each component of the marker

[0061] Components time 403nm peak percentage 280nm peak percentage Cofactor / Protein Polymer 12.006 6.447 15.45 0.42 Oligomer 12.596 21.428 58.031 0.37 Naked enzyme 13.218 72.125 26.519 2.7

[0062] The results are as follows Figure 5 As shown in Table 3: red is 403nm, blue is 280nm; 403nm is the characteristic absorption peak of HRP prosthetic group. Through the analysis of 403nm and 280nm wavelengths, the proportion of protein and HRP prosthetic group in each component of the marker can be preliminarily known. As shown in Table 3, the absorbance ratio of naked enzyme (without marker, i.e. HRP monomer) in deionized water (0.5-1.0mg / ml) A403 / A275 enzyme is within 2.6-3.0. From the data in the table, the prosthetic group / protein of naked enzyme is 2.7, which is consistent with the conventional detection data, and the cross-linking ratio of HRP prosthetic group and protein calculated by LC-MS analysis is consistent with the results in Table 3.

[0063] Example 3 HPLC detection scheme 3

[0064] Chromatographic conditions: chromatographic column Bio SEC51000A particle size 5μm, column length 7.8×300mm; detection wavelength: 280nm; sample concentration: 1mg / ml, column temperature: 25℃, flow rate: 0.8ml / min, mobile phase A:B ratio of 70:30 (mobile phase A is sodium dihydrogen phosphate and disodium hydrogen phosphate solution, the concentrations of sodium dihydrogen phosphate and disodium hydrogen phosphate are 0.05mol / L respectively; mobile phase B is 1.5mol / L NaCl solution); running time: 20min.

[0065] Detection: Take an appropriate amount of test sample (poly-HRP-Ab), accurately weigh it, dissolve it with mobile phase and dilute it into a certain concentration of sample solution (sample concentration is 1 mg / ml), shake it well, filter it with a 0.22μm filter membrane, accurately measure 10μl and inject it into the liquid chromatograph for high performance liquid chromatography analysis, and record the chromatogram.

[0066] The results are as follows Figure 6 As shown: analysis of three batches of poly-HRP markers (three different batches of poly-HRP-Ab), among which the polymerization degrees of the three batches are quite different, among which the green batch has better performance in the kit, while the red batch has a higher polymerization degree and is prone to false positives, and the blue batch has a lower titer. Therefore, HPLC analysis can monitor the enzyme-labeled polymerization degree early to prevent batch differences (in the tumor project CA242 detection kit, the green sample (in the HPLC figure) has a higher titer and no false positives, so HPLC can be used to monitor the required polymerization degree and monitor batch differences).

[0067] Table 4. Degree of polymerization of three batches of poly-HRP labels

[0068] sample Degree of Polymerization Potency False Penis red high 1：3000 yes green middle 1：4000 no blue Low 1：1000 no

[0069] Example 4 HPLC detection scheme 4

[0070] Chromatographic conditions: chromatographic column Bio SEC51000A, particle size 5 μm, column length 7.8×300 mm; detection wavelength: 280 nm; sample concentration: 1 mg / ml, column temperature: 25°C, flow rate: 0.8 ml / min, mobile phase A: 10 mM PBS pH 7.2; running time: 20 min.

[0071] Detection: Take an appropriate amount of test sample (poly-HRP-Ab), accurately weigh it, dissolve it with mobile phase and dilute it into a certain concentration of sample solution (sample concentration is 1 mg / ml), shake it well, filter it with a 0.22μm filter membrane, accurately measure 10μl and inject it into the liquid chromatograph for high performance liquid chromatography analysis, and record the chromatogram, see Figure 7 .

[0072] from Figure 7It can be seen that the protein adsorption is more serious, the response is low, and the peak shape is abnormal. The separation effect is poor compared with that of Implementation Case 1.

[0073] Example 5 HPLC Detection Scheme 5

[0074] Chromatographic conditions: chromatographic column Bio SEC51000A, particle size 5 μm, column length 7.8×300 mm; detection wavelength: 280 nm; sample concentration: 1 mg / ml, column temperature: 25°C, flow rate: 0.8 ml / min, mobile phase A is 50 mM PB pH 7.0, mobile phase B is isopropanol solution, mobile phase A: B = 90:10; running time: 20 min.

[0075] Detection: Take an appropriate amount of test sample (poly-HRP-Ab), accurately weigh it, dissolve it with mobile phase and dilute it into a certain concentration of sample solution (sample concentration is 1 mg / ml), shake it well, filter it with a 0.22μm filter membrane, accurately measure 10μl and inject it into the liquid chromatograph for high performance liquid chromatography analysis, and record the chromatogram, see Figure 8 .

[0076] from Figure 8 It can be seen that 10% isopropanol cannot weaken protein adsorption, the response is low, and the peak shape is abnormal. The separation effect is poor compared with Implementation Case 1.

[0077] Example 6 HPLC Detection Scheme 6

[0078] Chromatographic conditions: chromatographic column Bio SEC51000A, particle size 5 μm, column length 7.8×300 mm; detection wavelength: 280 nm; sample concentration: 1 mg / ml, column temperature: 25°C, flow rate: 0.8 ml / min, mobile phase A is 50 mM PB pH 7.0, mobile phase B is isopropanol solution, mobile phase A: B = 85:15; running time: 20 min.

[0079] Detection: Take an appropriate amount of test sample (poly-HRP-Ab), accurately weigh it, dissolve it with mobile phase and dilute it into a certain concentration of sample solution (sample concentration is 1 mg / ml), shake it well, filter it with a 0.22μm filter membrane, accurately measure 10μl and inject it into the liquid chromatograph for high performance liquid chromatography analysis, and record the chromatogram, see Fig. 9 .

[0080] from Fig. 9 It can be seen that 15% isopropanol can weaken protein adsorption, but high concentration of isopropanol will cause protein aggregation, and the molecular weight has exceeded the maximum separation range of the chromatographic column. The separation effect is poor compared with that of Implementation Case 1.

[0081] Example 7 HPLC Detection Scheme 7

[0082] Chromatographic conditions: chromatographic column Bio SEC51000A, particle size 5 μm, column length 7.8×300 mm; detection wavelength: 280 nm; sample concentration: 1 mg / ml, column temperature: 25°C, flow rate: 0.8 ml / min, mobile phase A is 50 mM PB pH 7.0, mobile phase B is 1.5 M NaCl solution, mobile phase A: B = 85:15; running time: 20 min.

[0083] Detection: Take an appropriate amount of test sample (poly-HRP-Ab), accurately weigh it, dissolve it with mobile phase and dilute it into a certain concentration of sample solution (sample concentration is 1 mg / ml), shake it well, filter it with a 0.22μm filter membrane, accurately measure 10μl and inject it into the liquid chromatograph for high performance liquid chromatography analysis, and record the chromatogram, see Fig.10 .

[0084] from Fig.10 It can be seen that adding salt can weaken protein adsorption, but the peak shape is abnormal and the separation effect is poor compared with Implementation Case 1.

[0085] Example 8 HPLC Detection Scheme 8

[0086] Chromatographic conditions: chromatographic column Bio SEC51000A, particle size 5 μm, column length 7.8×300 mm; detection wavelength: 280 nm; sample concentration: 1 mg / ml, column temperature: 25°C, flow rate: 0.8 ml / min, mobile phase A is 50 mM PB pH 7.0, mobile phase B is 1.5 M NaCl solution, mobile phase A: B = 40:60; running time: 20 min.

[0087] Detection: Take an appropriate amount of test sample (poly-HRP-Ab), accurately weigh it, dissolve it with mobile phase and dilute it into a certain concentration of sample solution (sample concentration is 1 mg / ml), shake it well, filter it with a 0.22μm filter membrane, accurately measure 10μl and inject it into the liquid chromatograph for high performance liquid chromatography analysis, and record the chromatogram, see Fig.11 .

[0088] from Fig.11 It can be seen that high salt will lead to protein instability, abnormal peak shape, and the separation effect is poor compared with Implementation Case 1.

[0089] Example 9 HPLC Detection Scheme 9

[0090] Chromatographic conditions: chromatographic column Bio SEC5500A, particle size 5 μm, column length 7.8×300 mm; detection wavelength: 280 nm; sample concentration: 1 mg / ml, column temperature: 25°C, flow rate: 0.8 ml / min, mobile phase A is 50 mM PB pH 7.0, mobile phase B is 1.5 M NaCl solution, mobile phase A: B = 70:30; running time: 20 min.

[0091] Detection: Take an appropriate amount of test sample (poly-HRP-Ab), accurately weigh it, dissolve it with mobile phase and dilute it into a certain concentration of sample solution (sample concentration is 1 mg / ml), shake it well, filter it with a 0.22μm filter membrane, accurately measure 10μl and inject it into the liquid chromatograph for high performance liquid chromatography analysis, and record the chromatogram, see Fig.12 .

[0092] from Fig.12 It can be seen that the protein polymerization degree is large, exceeding the separation range, and the separation effect is poor compared with implementation case 1.

[0093] Conclusion: Comparison of the results of the above Examples 1 to 8 shows that the method of the present invention is suitable for the detection of poly-HRP and its markers, and has a fast analysis speed and good detection effect; the final preferred conditions of the method of the present invention are: chromatographic column: Bio SEC51000A, particle size 5 μm, column length 7.8×300 mm; detection wavelength: 280 nm; sample concentration: 1 mg / ml, column temperature: 25°C, flow rate: 0.8 ml / min, mobile phase A is 50 mM PB pH 7.0, mobile phase B is 1.5 M NaCl solution, and the ratio of A to B is 70:30; running time: 20 min.

[0094] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A HPLC analysis method for poly-HRP and / or poly-HRP labeled with a marker, characterized in that: After filtering the sample, HPLC was used for detection: The detection conditions of the HPLC include: Mobile phase A includes: sodium dihydrogen phosphate, disodium hydrogen phosphate and water; Mobile phase B includes: NaCl and water; The elution procedure was as follows: the volume fraction of mobile phase A was 70% and the run time was 20 min.

2. The HPLC detection method according to claim 1, characterized in that In mobile phase A, the concentration of sodium dihydrogen phosphate is 0.02-0.05 mol / L, and the concentration of disodium hydrogen phosphate is 0.02-0.05 mol / L.

3. The HPLC detection method according to claim 1 or 2, characterized in that The pH of the mobile phase A is 6.8-7.

5.

4. The HPLC detection method according to claim 1, characterized in that In the mobile phase B, the concentration of NaCl is 1-2 mol / L.

5. The HPLC detection method according to claim 1 or 4, characterized in that The pH of the mobile phase B is 6.8-7.

2.

6. The HPLC analysis method according to claim 1, characterized in that The HPLC chromatographic column is a BioSEC51000A chromatographic column; the inner diameter of the Bio SEC51000A chromatographic column is 7.8 mm, the column length is 300 mm, and the filler is bonded silica gel; the particle size of the bonded silica gel is 5 μm.

7. The HPLC analysis method according to claim 1, characterized in that The detection wavelength of the HPLC includes 280 and / or 403 nm.

8. The HPLC analysis method according to claim 1, characterized in that The flow rate of the HPLC is 0.7-1.2 ml / min.

9. The HPLC analysis method according to claim 1, characterized in that The column temperature of the HPLC is 20-35°C.

10. The HPLC analysis method according to claim 1, characterized in that The concentration of the sample is 0.5-2 mg / ml.

Images