CN108732228B - Method for detecting vWF polymer in human blood coagulation factor VIII product - Google Patents
Method for detecting vWF polymer in human blood coagulation factor VIII product Download PDFInfo
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Abstract
The invention belongs to the technical field of biological medicine, and relates to a method for detecting von willebrand factor polymers in human blood coagulation factor FVIII products, which sequentially comprises the steps of glue preparation, electrophoresis, membrane and filter paper treatment, wet rotation, sealing, antibody incubation, color development and the like, and is characterized in that: the electrophoresis step is continuous vertical agarose electrophoresis, and a quantitative analysis step is added after the color development step to accurately analyze the quality of von willebrand factor polymers in the FVIII preparation. The quantitative analysis step is to calculate the relative content of each strip in the test sample and the mixed plasma by using gel analysis software, and to use the ratio of the relative content of each strip in the test sample to the relative content of each strip in the mixed plasma as a standard for evaluating the quality of the von willebrand factor multimer. The composition of von willebrand factor multimers in the FVIII preparation can be quantitatively analyzed, so that the quality of vWF in the FVIII preparation is evaluated, and a theoretical basis is provided for the therapy of vWD by the FVIII/vWF preparation.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to a method for detecting the composition of von Willebrand Factor (vWF) polymers in a Human blood coagulation Factor VIII product (Human Coagulantyactor VIII product, hereinafter abbreviated as F VIII product).
Background
The F VIII product is obtained by separating and purifying blood plasma of healthy people, and the blood plasma protein product taking the F VIII as a main medicinal component is mainly used for treating hemophilia A with congenital deficiency of the F VIII in clinic. vWF and FVIII are not easily separated during production, and most FVIII preparations contain vWF, so that the FVIII preparations are also used for treating von Willebrand disease, a hemorrhagic disease caused by decreased vWF amount or abnormal quality. vWF in the fviii preparation has mainly 2 functions: one is used as a carrier of the FVIII to protect the FVIII from being degraded, and the other is used for mediating the adhesion of blood platelets to a damaged blood vessel wall and inducing the blood platelets to aggregate, thereby playing a role in hemostasis. vWF exists in plasma in multimeric form and is classified into high molecular weight multimers (containing 11-20 dimers), medium molecular weight multimers (containing 5-10 dimers) and low molecular weight multimers (containing 1-5 dimers).
Research shows that although all molecular weight vWF has the F VIII carrier function, the hemostatic function of the vWF is related to the molecular weight of the vWF polymer, only the high molecular weight vWF polymer has stronger platelet binding capacity, only the medium molecular weight vWF polymer has lower platelet binding capacity, and the low molecular weight vWF polymer does not have the platelet binding capacity. Thus, FVIII preparations for the treatment of vWD must contain high and medium molecular weight vWF multimers. In assessing whether an FVIII preparation is useful for treating vWD, it is necessary to perform a vWF multimer assay on the FVIII preparation.
The traditional vWF multimer analysis method is usually analyzed by SDS-agarose electrophoresis and Western blot, and comprises the following steps: (1) discrete horizontal plate SDS-agarose electrophoresis was performed for approximately 6-8 hours. (2) Wet transfer is carried out, and the electrotransfer time is 12-20 h. (3) Primary antibody, secondary antibody incubation and substrate development took approximately 16 h. The traditional analysis method of vWF multimers is complex in operation, long in time, requires 3-5 days, and is not suitable for analyzing the composition of vWF multimers in FVIII preparations because quantitative analysis is not performed.
Disclosure of Invention
The invention aims to overcome the defects of the prior detection technology and establish a quick and sensitive method capable of quantitatively analyzing the composition of vWF polymers in an FVIII product, and the method is suitable for quantitatively analyzing the composition of the vWF polymers in the FVIII product; the technical problems that horizontal plate agarose electrophoresis consumes long time and discontinuous vertical plate agarose electrophoresis is very difficult to operate in the preparation process in the traditional method are solved.
The detection method for the vWF polymer in the human blood coagulation factor VIII product, which solves the technical problems, sequentially comprises the steps of glue preparation, electrophoresis, membrane and filter paper treatment, wet rotation, sealing, antibody incubation and color development, and is characterized in that: the electrophoresis step is continuous vertical agarose electrophoresis, and a quantitative analysis step is added after the color development step to accurately analyze the quality of the vWF polymer in the FVIII product. The quantitative analysis step is to calculate the relative content of each strip in the test sample and the mixed plasma by using gel analysis software, and taking the ratio of the relative content of each strip in the test sample to the relative content of each strip in the mixed plasma as a standard for evaluating the quality of the vWF multimer.
The method is continuous vertical plate agarose electrophoresis, is easy to operate and reduces gel preparation steps.
And a soaking and washing step is also arranged between the electrophoresis step and the membrane and filter paper treatment step, namely after the glue is taken out, the glue is soaked in 1mM beta-mercaptoethanol for 5min, then washed with PBS-T for 3 times, 5min each time, and placed in an electrophoresis buffer solution for 15 min.
After electrophoresis is finished, the gel is treated by beta-mercaptoethanol, all bands on the gel are cracked into monomers, and then electrotransfer is carried out, so that the membrane transferring efficiency is improved.
vWF multimers having a molecular weight varying from 500kD to 5000kD may result in some low molecular weight vWF being transferred, i.e., across the membrane, while some high molecular weight vWF multimers are not transferred to the membrane, using the same electrotransfer time. Therefore, after electrophoresis, 1mM beta-mercaptoethanol is used for treatment, and beta-mercaptoethanol is used for cracking all vWF polymers with different molecular weights into vWF monomers and then carrying out membrane conversion, so that the membrane conversion time is shortened, and the membrane conversion effect is better. If the concentration of the beta-mercaptoethanol treatment is too high or the treatment time is too long, the transfer of the band to the membrane becomes blurred. PBS-T washing is to remove beta-mercaptoethanol on the gel, and can be washed for 5min for 3 times if the membrane transferring effect is affected without washing.
The preparation method of the gel comprises the steps of dissolving agarose in a buffer solution, heating to boil, pouring into a vertical glass plate, inserting a comb to manufacture a sampling hole, and standing for a period of time until the gel is solidified.
The agarose concentration is 1% -1.2%, the buffer solution is a continuous gel buffer solution, and the specific formula comprises: 30% glycerol, 50mM Tris, 0.384M Gly and 0.1% SDS. Continuous electrophoresis, only separation gel, no concentration gel, and 30% glycerol added in gel to achieve concentration effect. The continuous glue is easier to configure, operation steps are reduced, and meanwhile, the continuous glue has a good effect.
The continuous vertical electrophoresis step is to add the processed sample into a sample loading hole and then carry out electrophoresis in an electrophoresis buffer solution; the electrophoresis conditions are as follows: constant current 10mM,2-2.5h, until bromophenol blue comes out of the glass plate, ending the electrophoresis. The electrophoresis time is influenced by the size of the current, the current is overlarge, the electrophoresis time is short, the running-out strips are not a straight line, and the strips can be bent; too small a current, too long an electrophoresis time, and the band will diffuse.
The sample is a FVIII product to be detected and mixed plasma serving as a standard control; the fviii preparation to be tested and the pooled plasma were diluted with purified water to vWF: the Ag content is 1IU/ml, then the mixture is mixed with the sample loading buffer solution according to the proportion of 1:4, and finally bromophenol blue is added to ensure that the final concentration is 0.005%; the loading buffer included 9M urea, 4mM EDTA, 50mM Tris, 2% SDS, pH 6.8.
The pooled plasma was used as a standard reference to compare with the test samples to determine whether there was any abnormality in the vWF multimer composition in the fviii preparation.
The electrophoresis buffer is ice-cold wet-transfer buffer, namely the solution is placed at 4 ℃. The solution is placed at 4 ℃ for a period of time, and the effect is to cool the glue, reduce the heat generated in the film transferring process of the glue and improve the film transferring effect.
The electrophoresis buffer solution comprises 50mM Tris, 0.384M Gly and 0.1% SDS, and the pH value is 8.3; the wet transfer buffer solution in the wet transfer step comprises 2.5mM Tris,19.2mM Gly, 0.1% SDS and the pH is 8.8; wet-rotating at 120V for 1 h.
In the quantitative analysis step, the bands (medium molecular weight and high molecular weight vWF) after the 5 th band are analyzed together, and the vWF polymer composition in the FVIII preparation is analyzed by using the mixed plasma as a standard control, namely, the quality of the vWF polymers is evaluated by the ratio of the relative content of each band in the test sample to the relative content of each band in the mixed plasma, and the quality of the vWF polymers is reflected by percentage.
The bands following band 5 were analyzed as a whole to determine whether there was an abnormality in the composition of vWF multimers in the fvii preparation by using the ratio of the relative amount of each multimer in the test sample to the relative amount of each multimer in the pooled plasma, and this comparison increased the accuracy of the assay by increasing the quantification standard by quantitative analysis rather than by visual inspection of the bands alone.
The detection method of the invention is as follows:
(1) glue preparation: dissolving agarose in buffer solution, heating to boil, pouring into a vertical glass plate, inserting a comb to make a sample loading hole, standing for a period of time until the gel is solidified, and directly using or storing at low temperature;
(2) continuous vertical electrophoresis: adding the processed sample (including the FVIII product to be detected and thousands of mixed plasma serving as standard control) into a sample loading hole, and carrying out electrophoresis in an electrophoresis buffer solution under the following electrophoresis conditions: constant current 10mM,2-2.5h, until bromophenol blue comes out of the glass plate, ending the electrophoresis.
(3) Membrane and filter paper treatment: cutting the PVDF membrane and the filter paper, soaking the PVDF membrane in methanol, placing the PVDF membrane in a wet-conversion buffer solution for electric conversion balance, and placing the filter paper in the wet-conversion buffer solution for soaking; the PVDF membrane is soaked in methanol to activate the membrane, and the glue, the membrane and the filter paper are soaked in the wet-conversion buffer solution so as to fill the ions in the electric conversion buffer solution on the glue and the membrane and improve the electric conversion efficiency.
The gel and membrane are balanced in the wet-transfer buffer for 15-30min, the filter paper is placed in the wet-transfer buffer for 5min, and the voltage is constant current or constant voltage, preferably constant voltage, and is kept constant.
(4) Wet rotation: sequentially arranging the filter paper, the glue, the membrane and the filter paper into a sandwich structure from top to bottom, putting the sandwich structure into a transfer tank, and adding a wet-transfer buffer solution; the ice box heat dissipation is put into to the in-process attention of electricity commentaries on classics, because wet commentaries on classics process can produce a lot of heat, if do not put into the cooling of ice box, the heat production is too much, and the resistance increases, and the electric current descends, influences the electricity and changes the effect.
(5) And (3) sealing: after electrophoresis is finished, taking out the PVDF membrane, washing the PVDF membrane by PBS-T, and then placing the PVDF membrane in a sealing solution for sealing for 1 h;
washing solution (PBS-T) is 0.02M PBS containing 0.1% Tween-20; the blocking solution was 0.02M PBS containing 5% BSA; wash 3 times for 5min each time.
(6) Primary antibody incubation: after washing the membrane with PBS-T, the membrane is incubated overnight at 37 ℃ for 2 hours or 4 ℃ and preferably overnight at 4 ℃ in a primary antibody diluted 1:3000 in an antibody diluent. If the temperature is selected to be room temperature or 37 ℃ overnight, the antigen-antibody reaction is too strong, so that the background color is too dark, and the strip observation is influenced.
(7) And (3) secondary antibody incubation: washing the membrane with PBS-T, placing the membrane in a secondary antibody diluted by 1:2000 of antibody diluent, and incubating for 1h at room temperature; the antibody dilution was 0.02M PBS-T with 0.5% BSA.
The primary antibody is anti-human vWF antibody, namely rabbit anti-human vWF antibody, mouse anti-human vWF antibody or anti-human vWF antibody of other species, or monoclonal antibody or polyclonal antibody.
(8) Color development: washing the membrane with PBS-T for 5 times, wherein after 5min each time, the membrane is fully contacted with the mixed solution in the luminescence kit, and capturing signals by using an ultrasensitive chemiluminescence imager; the side with the protein on the membrane faces downwards to fully contact with the mixed solution of the luminescence kit for 30s-2 min.
(9) Quantitative analysis: the relative amounts of each band, i.e., each multimer, of the test sample and pooled plasma were quantitatively analyzed by gel analysis software. And the vWF multimer composition in the fviii preparation was analyzed as a percentage using pooled plasma as a standard control.
The present invention uses quantitative analysis in analysis of vWF multimers with reduced or no visual observation, reduced subjectivity and error. And the bands after band 5 (i.e., medium and high molecular weight vWF) are quantified together as a whole for the present invention, and the presence of vWF multimer composition abnormalities (expressed as a percentage) in the fvii preparation is demonstrated by comparing pooled plasma as a standard reference with the test sample.
The method can quantitatively analyze the distribution of the vWF polymers in the detection sample, greatly reduce the complexity of operation, shorten the time and effectively ensure the authenticity and reliability of the test result.
Drawings
The invention will be described in further detail with reference to the following drawings and detailed description:
FIG. 1 is a graph showing the analysis of the results of the present invention
(wherein 1. domestic A factory FVIII products, 2. domestic B factory FVIII products, 3. domestic C factory FVIII products (60 times dilution), 4. domestic C factory FVIII products, 5. domestic D factory FVIII products, 6. foreign E factory FVIII products, 7. foreign F factory FVIII products (100 times dilution), 8. normal person mixed plasma);
FIG. 2 is a special attribute investigation diagram of the experiment III in the present invention
(wherein 1. domestic A manufacturer FVIII preparation, 2. normal human mixed plasma, 3. domestic B manufacturer FVIII preparation, 4. domestic C manufacturer FVIII preparation, 5. domestic D manufacturer FVIII preparation, 6. domestic E manufacturer FVIII preparation, 7. undiluted cold supernatant);
FIG. 3 is a graph showing that none of the test samples in the present invention was treated with β -mercaptoethanol;
FIG. 4 is an analysis chart showing the results of the test of the present invention in which the treatment time of pentabeta-mercaptoethanol is too long.
Detailed Description
The invention will be described in detail with reference to the following detailed description and the accompanying drawings, in which the instruments and reagents used are as follows:
instruments and parameters: a Mini Trans-blot Cell (BIO-RAD); a small vertical electrophoresis cell (Mini Protein Tetra, BIO-RAD); gel imaging system (ImageQuant LAS 4000mini, GE); gel analysis software (ImageQuant TL, GE);
test reagents: HGT agarose (Seakem, FMC Bioproducts), PVDF membrane (Immobilon-P, Millipore), rabbit anti-human vwf polyclonal antibody (Dako), goat anti-rabbit conjugated HRP secondary antibody (Picea beijinensis), BSA, Tween-20, PBS buffer, chemiluminescent substrate kit (Seakem, FMC Bioproducts)
West Pico cheminescentSubstrate, Thermoscientific), bromophenol blue, urea, EDTA, Tris, glycine, methanol, β -mercaptoethanol, multi-aliquot mixed plasma.
Example 1
The detection method comprises the following steps:
(1) glue preparation: dissolving agarose in buffer solution, heating to boil, pouring into a vertical glass plate, inserting a comb to make a sample loading hole, standing for a period of time until the gel is solidified, and directly using or storing at low temperature; the agarose concentration is 1%, the buffer solution is a continuous gel buffer solution, and the specific formula comprises: 30% glycerol, 50mM Tris, 0.384M Gly and 0.1% SDS.
(2) Continuous vertical electrophoresis: adding the processed sample (including the FVIII product to be detected and thousands of mixed plasma serving as standard control) into a sample loading hole, and carrying out electrophoresis in an electrophoresis buffer solution under the following electrophoresis conditions: the flow was constant at 10mM for 2h until bromophenol blue exited the glass plate, and the electrophoresis was terminated.
The sample is a FVIII product to be detected and mixed plasma used as a standard control; the fviii preparation to be tested and the pooled plasma were diluted with purified water to vWF: the Ag content is 1IU/ml, then the mixture is mixed with the sample loading buffer solution according to the proportion of 1:4, and finally bromophenol blue is added to ensure that the final concentration is 0.005%; the loading buffer included 9M urea, 4mM EDTA, 50mM Tris, 2% SDS, pH 6.8.
(3) Membrane and filter paper treatment: cutting the PVDF membrane and the filter paper, soaking the PVDF membrane in methanol, placing the PVDF membrane in a wet-conversion buffer solution for electric conversion balance, and placing the filter paper in the wet-conversion buffer solution for soaking; the PVDF membrane is soaked in methanol to activate the membrane, and the glue, the membrane and the filter paper are soaked in the wet-conversion buffer solution so as to fill the ions in the electric conversion buffer solution on the glue and the membrane and improve the electric conversion efficiency.
The gel and membrane are equilibrated in the wet-transfer buffer for 15min, the filter paper is placed in the wet-transfer buffer for 5min, and the voltage is constant current or constant voltage, preferably constant voltage, and is kept constant.
(4) Wet rotation: sequentially arranging the filter paper, the glue, the membrane and the filter paper into a sandwich structure from top to bottom, putting the sandwich structure into a transfer tank, and adding a wet-transfer buffer solution; the ice box heat dissipation is put into to the in-process attention of electricity commentaries on classics, because wet commentaries on classics process can produce a lot of heat, if do not put into the cooling of ice box, the heat production is too much, and the resistance increases, and the electric current descends, influences the electricity and changes the effect.
(5) And (3) sealing: after electrophoresis is finished, taking out the PVDF membrane, washing the PVDF membrane by PBS-T, and then placing the PVDF membrane in a sealing solution for sealing for 1 h;
washing solution (PBS-T) is 0.02M PBS containing 0.1% Tween-20; the blocking solution was 0.02M PBS containing 5% BSA.
Washing for 5min for 3 times may cause strong background of the result on the film and affect the display of the final strip if the washing is insufficient. If the blocking time is not sufficient, the blocking effect is not satisfactory, and non-specific binding increases, which may cause a strong background of the result on the film and affect the display of the final band.
(6) Primary antibody incubation: after washing the membrane with PBS-T, the membrane is incubated overnight at 37 ℃ for 2 hours or 4 ℃ in a primary antibody diluted 1:3000 in an antibody dilution, preferably overnight at 4 ℃. If the temperature is selected to be room temperature or more than 37 ℃ overnight, the antigen-antibody reaction is too strong, so that the background color is too dark, and the strip observation is influenced.
(7) And (3) secondary antibody incubation: washing the membrane with PBS-T, placing the membrane in a secondary antibody diluted by 1:2000 of antibody diluent, and incubating for 1h at room temperature; the antibody dilution was 0.02M PBS-T with 0.5% BSA.
The primary antibody is anti-human vWF antibody, namely rabbit anti-human vWF antibody, mouse anti-human vWF antibody or anti-human vWF antibody of other species, or monoclonal antibody or polyclonal antibody. Monoclonal antibodies are antibodies directed against a specific site of vWF and polyclonal antibodies are antibodies directed against multiple sites of vWF, where polyclonal antibodies are selected because they capture more vWF.
The antigen-antibody reaction is weak and the band is unclear due to the fact that the concentration of the diluent is too low; the high dilution concentration easily causes the waste of the antibody and the deep background.
(8) Color development: washing with PBS-T for 5 times, each for 5min, contacting the membrane with the mixed solution in the luminescence kit, and capturing the signal with an ultrasensitive chemiluminescence imager; the side with the protein on the membrane faces downwards to fully contact with the mixed solution of the luminescence kit for 30s-2 min.
(9) Quantitative analysis: the relative amounts of each band, i.e., each multimer, of the test sample and pooled plasma were quantitatively analyzed by gel analysis software. And the vWF multimer composition in the fviii preparation was analyzed as a percentage using pooled plasma as a standard control.
Example 2
The detection method comprises the following steps:
(1) glue preparation: dissolving agarose in buffer solution, heating to boil, pouring into a vertical glass plate, inserting a comb to make a sample loading hole, standing for a period of time until the gel is solidified, and directly using or storing at low temperature; the agarose concentration is 1.2%, the buffer solution is a continuous gel buffer solution, and the specific formula comprises: 30% glycerol, 50mM Tris, 0.384M Gly and 0.1% SDS.
(2) Continuous vertical electrophoresis: adding the processed sample (including the FVIII product to be detected and thousands of mixed plasma serving as standard control) into a sample loading hole, and carrying out electrophoresis in an electrophoresis buffer solution under the following electrophoresis conditions: constant current 10mM,2.5h, until bromophenol blue runs out of the glass plate, ending the electrophoresis.
The sample is a FVIII product to be detected and mixed plasma serving as a standard control; the fviii preparation to be tested and the pooled plasma were diluted with purified water to vWF: the Ag content is 1IU/ml, then the mixture is mixed with the sample loading buffer solution according to the proportion of 1:4, and finally bromophenol blue is added to ensure that the final concentration is 0.005%; the loading buffer included 9M urea, 4mM EDTA, 50mM Tris, 2% SDS, pH 6.8.
(3) And (3) a soaking and washing step, namely after the gel is taken out, soaking the gel in 1mM beta-mercaptoethanol for 5min, washing the gel with PBS-T for 3 times, 5min each time, and placing the gel in an electrophoresis buffer solution for 15 min.
(4) Membrane and filter paper treatment: cutting the PVDF membrane and the filter paper, soaking the PVDF membrane in methanol, placing the PVDF membrane in a wet-conversion buffer solution for electric conversion balance, and placing the filter paper in the wet-conversion buffer solution for soaking; the PVDF membrane is soaked in methanol to activate the membrane, and the glue, the membrane and the filter paper are soaked in the wet-conversion buffer solution so as to fill the ions in the electric conversion buffer solution on the glue and the membrane and improve the electric conversion efficiency.
The gel and membrane are equilibrated in the wet-transfer buffer for 30min, the filter paper is placed in the wet-transfer buffer for 5min, and the voltage is constant current or constant voltage, preferably constant voltage, and is kept constant.
(5) Wet rotation: sequentially arranging the filter paper, the glue, the membrane and the filter paper into a sandwich structure from top to bottom, putting the sandwich structure into a transfer tank, and adding a wet-transfer buffer solution; the ice box heat dissipation is put into to the in-process attention of electricity commentaries on classics, because wet commentaries on classics process can produce a lot of heat, if do not put into the cooling of ice box, the heat production is too much, and the resistance increases, and the electric current descends, influences the electricity and changes the effect.
(6) And (3) sealing: after electrophoresis is finished, taking out the PVDF membrane, washing the PVDF membrane by PBS-T, and then placing the PVDF membrane in a sealing solution for sealing for 1 h;
washing solution (PBS-T) is 0.02M PBS containing 0.1% Tween-20; the blocking solution was 0.02M PBS containing 5% BSA.
PBS-T wash 3 times for 5min each.
(7) Primary antibody incubation: after washing the membrane with PBS-T, the membrane is incubated overnight at 37 ℃ for 2 hours or 4 ℃ in a primary antibody diluted 1:3000 in an antibody dilution, preferably overnight at 4 ℃.
(8) And (3) secondary antibody incubation: washing the membrane with PBS-T, placing the membrane in a secondary antibody diluted by 1:2000 of antibody diluent, and incubating for 1h at room temperature; the antibody dilution was 0.02M PBS-T with 0.5% BSA.
The primary antibody is anti-human vWF antibody, namely rabbit anti-human vWF antibody, mouse anti-human vWF antibody or anti-human vWF antibody of other species, or monoclonal antibody or polyclonal antibody.
(9) Color development: washing the membrane with PBS-T for 5 times, each time for 5min, fully contacting the membrane with the mixed solution in the luminescence kit, and capturing signals by using an ultrasensitive chemiluminescence imager; the side with the protein on the membrane faces downwards to fully contact with the mixed solution of the luminescence kit for 30s-2 min.
(10) Quantitative analysis: the bands after band 5 (medium and high molecular weight vWF) were analyzed together and the vWF multimer composition in the fvii preparation was analyzed using pooled plasma as a standard control, i.e., the relative amount of each band in the test sample to the relative amount of each band in pooled plasma was used to assess the quality of vWF multimers, expressed as a percentage, reflecting the quality of vWF multimers.
Example 3
And (3) testing the sample: and (3) redissolving the FVIII product according to the product specification, standing for at least 20min after redissolving, and waiting until the sample is completely dissolved. The samples were diluted with pure water to vWF: the Ag content is 1 IU/ml. The FVIII preparation was a lyophilized powder, dissolved in water. Preparing various solutions:
continuous gel buffer: 60ml of glycerol, 1.21g of Tris, 5.76Gly and 0.2g of SDS were dissolved in 200ml of pure water;
loading buffer solution: 54.04g of urea, 0.15g of EDTA, 0.61g of Tris, 2g of SDS in 100ml of pure water;
electrophoresis buffer solution: 12.1g Tris, 57.6g Gly,2g SDS in 2L pure water;
wet-transfer buffer: 0.303g Tris,1.44g Gly, 1g SDS in 1L pure water;
washing solution (PBS-T): 0.02M PBS containing 0.1% Tween-20;
sealing liquid: 0.02M PBS 5% BSA;
antibody dilution: 0.02M PBS-T contained 0.5% BSA.
Preparing a test article: the sample or mixed plasma diluted 1.2 is mixed with the loading buffer at a ratio of 1:4, and finally bromophenol blue is added to give a final concentration of 0.005%.
The preparation method comprises the following specific steps:
(1) glue preparation: 1% agarose: dissolving 0.2g HTG in 20ml continuous gel buffer solution, heating to boil with microwave oven, pouring into 2 vertical glass plates, rapidly inserting into comb, making sample hole, standing for 30min until the gel solidifies, and storing at 4 deg.C for 3 days or directly using.
(2) Continuous vertical electrophoresis: adding the processed sample into a sample loading hole, wherein the sample loading amount is 6 ul/hole, and the electrophoresis conditions are as follows: constant current 10mM,2.2h, until bromophenol blue runs out of the glass plate, ending the electrophoresis. Vertical plate agarose gel saves time compared to horizontal plate agarose gel.
(3) After the gel was removed, it was soaked in 1mM β -mercaptoethanol for 5min, washed with PBS-T for 3 times, 5min each time, and then placed in ice-cold wet-transfer buffer for 15 min.
(4) Membrane and filter paper treatment: the PVDF membrane and the filter paper are cut into pieces with the same size as the gel, the PVDF membrane is soaked in methanol for 1-2min, placed in ice-cold wet-transfer buffer for 15min, and the filter paper is placed in the wet-transfer buffer for 5 min.
(5) Wet rotation: the filter paper, the membrane and the glue are arranged into a sandwich structure and placed into a transfer tank as follows: from negative to positive (from top to bottom): and (3) filter paper, glue, membrane and filter paper, adding a wet-turning buffer solution, and carrying out wet turning at 120V for 1 h. During the electricity rotation process, the ice box is placed for heat dissipation.
The electrophoresis buffer comprises 50mM Tris, 0.384M Gly, 0.1% SDS, and the pH is 8.3; the wet-transfer buffer solution in the wet-transfer step comprises 2.5mM Tris,19.2mM Gly, 0.1% SDS, the pH is 8.8, and 120V wet-transfer is carried out for 1 h.
(6) And (3) sealing: after electrophoresis, the PVDF membrane is taken out, the PBS-T membrane is washed for 3 times, each time for 5min, and the membrane is placed in a sealing solution to be sealed for 1 h.
(7) Primary antibody incubation: the membrane was washed 3 times with PBS-T for 5min each and incubated overnight at 4 ℃ with primary antibody (rabbit anti-human vwf polyclonal antibody) diluted 1:3000 in antibody dilution.
(8) And (3) secondary antibody incubation: the membrane was washed 3 times with PBS-T for 5min each and incubated for 1h at room temperature with a secondary antibody (goat anti rabbit linked HRP) diluted 1:2000 with antibody dilution.
(9) Color development: and (3) washing the membrane with PBS-T for 5 times, 5min each time, mixing reagents Luminol/Enhancer and Stable Peroxide1:1 in the luminescence kit, namely dripping the mixed liquid on a preservative film, fully contacting the mixed liquid with the PVDF membrane protein surface facing downwards for 2min, and capturing a signal by using Image Quant Las 4000 mini.
(10) And (3) calculating: quantitative analysis was added and the relative content of each band (each multimer) of the test sample and pooled plasma was analyzed using Image Quant TL software. Since the high molecular weight polymer bands are not easily separated completely, the bands after band 5 are analyzed together. And comparing the polymer distribution of the mixed plasma and the test sample, and expressing the polymer distribution in percentage.
Experimental results and analysis, as in figure 1.
Lane 4 (domestic manufacturer D as an example analysis: 1 result analysis): the relative content of each band (each multimer) in the test sample and the pooled plasma was analyzed using Image Quant TL software, and the quality of vWF multimers was assessed by the ratio of the relative content of each band in the test sample to the relative content of each band in the pooled plasma:
1) manufacturer D relative content of each band: a first tape: 7.8%, second band: 10%, third strip: 12.7%, fourth band: 12.3%, fifth band: 10.7%, sixth band: 46.5 percent.
2) Relative content of each band in pooled plasma: a first tape: 6%, second strip: 9.1%, third strip: 12%, fourth band: 12.3%, fifth band: 10.1%, sixth band: 50.5 percent.
3) The ratio of the relative amount of each band in the test sample to the relative amount of each band in the pooled plasma, expressed as a percentage: a first tape: 7.8%/6% ═ 130%, second band: 10%/9.1% ═ 110%, third band: 12.7%/12% ═ 106%, four bands: 12.3%/12.3% ═ 100%, fifth band: 10.7%/10.1%: 106%, and the sixth 46.5%/50.5%: 92%.
Through analysis, the ratio of the 6 th band reflecting the vWF polymer with the molecular weight and the high molecular weight in the F VIII product of the D manufacturer to the blood plasma is 92 percent and is less than 100 percent, which indicates that the content of the vWF polymer with the molecular weight and the high molecular weight polymer in the F VIII product of the D manufacturer is lower than that of the normal human blood plasma.
Test one: repeatability of
The purpose is as follows: the precision of the same test sample in the measurement range is determined in the same test.
The method comprises the following steps: the test article was repeatedly loaded on the same gel 10 times according to the procedure of example 3.
As a result: relative content of the 1 st band: 6.3%, standard deviation: 1.7 percent and Coefficient of Variation (CV)27.2 percent;
relative content of lane 2: 9.6%, standard deviation: 1.9 percent and Coefficient of Variation (CV)19.3 percent;
relative content of band 3: 12.2%, standard deviation: 1.6 percent and Coefficient of Variation (CV)12.7 percent;
relative content of 4 th band: 12.3%, standard deviation: 1.1% and Coefficient of Variation (CV) 9.2%;
relative content of band 5: 11.2%, standard deviation: 1.0% and Coefficient of Variation (CV) 8.5%;
relative content of band 6: 48.4%, standard deviation: 3.7 percent and Coefficient of Variation (CV)7.7 percent;
acceptance criteria: the coefficient of variation of the repeated determination result is not more than 30%.
And (2) test II: intermediate precision
The purpose is as follows: the precision between the results measured in the same laboratory at different times by different analysts at different lots using the same equipment was investigated.
The method comprises the following steps: at various times, the procedure was as described in example 3, and the assay was repeated on 3 agarose gels (i.e., three experiments were performed at various times), the procedure being as described above for the reproducibility check test.
As a result: average relative content of 1 st band: 7.3%, standard deviation: 1.2 percent and Coefficient of Variation (CV)16.4 percent;
average relative content of band 2: 9.9%, standard deviation: 1.5 percent and Coefficient of Variation (CV)15.2 percent;
average relative content of 3 rd band: 12.3%, standard deviation: 1.2 percent and Coefficient of Variation (CV)9.8 percent;
average relative content of 4 th band: 11.7%, standard deviation: 1.1% and Coefficient of Variation (CV) 9.4%;
average relative content of band 5: 10.6%, standard deviation: 1.4 percent and Coefficient of Variation (CV)13.2 percent;
average relative content of band 6: 48.2%, standard deviation: 4.2 percent and Coefficient of Variation (CV)8.7 percent;
acceptance criteria: the coefficient of variation of the assay results within each condition was not more than 30%. Therefore, the detection method of the invention has repeatability and small error.
And (3) test III: specificity
The purpose is as follows: and (4) inspecting whether other components in the product influence the determination result.
The method comprises the following steps: the vWF multimer assay was performed using undiluted cold supernatant (very low levels of vWF and FVIII and normal other plasma constituents) as a sample, and the procedure was as in example 3.
The results are shown in FIG. 2: no bands indicate that no non-specific immune response was elicited by other components in the preparation.
Experiment four
The details are as in example 3, wherein after removing no "gel", the gel was soaked in 1mM β -mercaptoethanol for 5min, washed 3 times with PBS-T for 5min each, and then placed in ice-cold wet-transfer buffer for 15min "step.
The detection result is shown in fig. 3, and the corresponding content cannot be displayed in the graph.
Experiment five
The specific contents are as in example 3, wherein the soaking step is "after the gel is taken out, soaking in 1mM beta-mercaptoethanol for 10min, washing with PBS-T for 3 times, 5min each time, and standing in ice-cold wet-transfer buffer for 15 min".
The detection result is shown in FIG. 4, and the corresponding content cannot be shown in the figure because the treatment time of the beta-mercaptoethanol is too long.
The quality of the vWF polymer is quantitatively analyzed by the steps of gel preparation, continuous vertical agarose electrophoresis, membrane and filter paper treatment, wet conversion, sealing and the like, and by calculating the proportion of each strip in the test sample and the mixed plasma and calculating the ratio of the relative content of each strip in the test sample to the relative content of each strip in the mixed plasma. The invention can quantitatively analyze the composition of the vWF polymer in the FVIII product, thereby evaluating the quality of vWF in the FVIII product and providing a theoretical basis for the use of the FVIII/vWF product in the treatment of vWD.
While the foregoing shows and describes the fundamental principles and principal features of the invention, together with the advantages thereof, the foregoing embodiments and description are illustrative only of the principles of the invention, and various changes and modifications can be made therein without departing from the spirit and scope of the invention, which will fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (6)
1. A detection method of von Willebrand factor polymers in human blood coagulation factor VIII products sequentially comprises the steps of glue preparation, electrophoresis, membrane and filter paper treatment, wet rotation, sealing, antibody incubation and color development, and is characterized in that: the electrophoresis is continuous vertical agarose electrophoresis, and a quantitative analysis step is added after the color development step so as to accurately analyze the quality of von willebrand factor polymers in the human blood coagulation factor VIII product; the quantitative analysis step is to calculate the relative content of each strip in the test sample and the mixed plasma by using gel analysis software, and the ratio of the relative content of each strip in the test sample to the relative content of each strip in the mixed plasma is used as a standard for evaluating the quality of the von willebrand factor polymer;
the electrophoresis is to add the processed sample into a sample loading hole and carry out electrophoresis in an electrophoresis buffer solution; the electrophoresis conditions are as follows: constant current is 10mA, 2-2.5h, until bromophenol blue comes out of the glass plate, and electrophoresis is finished;
a soaking and washing step is also arranged between the electrophoresis step and the membrane and filter paper treatment step, namely after the gel is taken out, the gel is soaked in 1mM beta-mercaptoethanol for 5min, then washed with PBS-T for 3 times, 5min each time, and placed in ice-cold wet-conversion buffer solution for 15 min;
in the quantitative analysis step, the bands after the 5 th band in the test sample are analyzed together, and the composition of the von Willebrand factor multimer in the human blood coagulation factor VIII preparation is analyzed by using the mixed plasma as a standard control, namely, the quality of the von Willebrand factor multimer is evaluated by the ratio of the relative content of each band in the test sample to the relative content of each band in the mixed plasma, and the quality of the von Willebrand factor multimer is reflected by percentage.
2. The method of claim 1 for detecting von willebrand factor multimers in a human factor viii preparation, wherein: the gel is prepared by dissolving agarose in buffer solution, heating to boil, pouring into a vertical glass plate, inserting a comb to make a sample loading hole, and standing for a period of time until the gel is solidified.
3. The method of claim 2 for detecting von willebrand factor multimers in a human factor viii preparation, wherein: the agarose concentration is 1% -1.2%, the buffer solution is a continuous gel buffer solution, and the specific formula comprises: 30% glycerol, 50mM Tris, 0.384M Gly and 0.1% SDS.
4. The method of claim 1 for detecting von willebrand factor multimers in a human factor viii preparation, wherein: the sample is a human blood coagulation factor VIII product to be detected and mixed plasma serving as a standard control; diluting a human blood coagulation factor VIII product to be detected and mixed plasma with pure water until the content of von willebrand factor antigen (vWF: Ag) is 1IU/ml, mixing the product with a sample buffer solution according to the proportion of 1:4, and finally adding bromophenol blue to enable the final concentration to be 0.005%; the loading buffer comprises 9M urea, 4mM EDTA, 50mM Tris, 2% SDS, and the pH of the loading buffer is 6.8.
5. The method of claim 1 for detecting von willebrand factor multimers in a human factor viii preparation, wherein: the specific formula of the electrophoresis buffer solution comprises 50mM Tris, 0.384MGly and 0.1% SDS, and the pH value of the electrophoresis buffer solution is 8.3; the specific formula of the wet-transfer buffer comprises 2.5mM Tris,19.2mM Gly and 0.1% SDS, and the pH value of the wet-transfer buffer is 8.8.
6. The method of claim 1 for detecting von willebrand factor multimers in a human factor viii preparation, wherein: the developing step is that PBS-T washes the membrane for 5 times, 5min each time; then the membrane is fully contacted with the mixed solution in the luminescence kit, and an ultrasensitive chemiluminescence imager is adopted to capture signals; the side with the protein on the membrane faces downwards to fully contact with the mixed solution of the luminescence kit for 30s-2 min.
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| US7214479B2 (en) * | 2000-10-26 | 2007-05-08 | Wisconsin Alumni Research Foundation | E. coli O157:H7 C1-INH-binding protein and methods of use |
| CN103539856A (en) * | 2013-11-05 | 2014-01-29 | 广西大学 | Preparation, detection and application of polyclonal antibody of Yam mild mosaic virus |
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