CN112557643B - Denatured IgG and rheumatoid factor immune antigen and preparation method thereof - Google Patents
Denatured IgG and rheumatoid factor immune antigen and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and particularly provides a denatured IgG and rheumatoid factor immune antigen and a preparation method thereof. According to the preparation method of the denatured IgG, the protein protective agent is added in the denaturation process, so that a certain distance is kept between IgG molecules, aggregation in the denaturation process is avoided, the IgG is denatured more fully, more antigen epitopes are exposed, and the antigen activity is improved. The method has the advantages of stable denaturation process, good monodispersity of the obtained denatured IgG, sufficient denaturation and high activity. Because the agglutination problem of the denatured IgG is well improved, the obtained rheumatoid factor immune antigen has better stability, the activity of the immune antigen is obviously improved, the batch difference is reduced, and the method is suitable for large-scale production.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a denatured IgG and rheumatoid factor immune antigen and a preparation method thereof.
Background
Rheumatoid Factor (RF) is an autoantibody with denatured IgG as a target antigen, and the main component is an IgM class antibody, and there are also IgG class, igA class, igD class, and IgE class. Studies have shown that the positive rate for rheumatoid factor is only 2% in normal persons and 5% in the elderly, and 80% in Rheumatoid Arthritis (RA) patients. Therefore, RF is of great value as an indicator of RA diagnosis.
At present, the research on the epitope of RF is not thorough, the epitope cannot be made into a recombinant antigen, the common method is to denature human IgG, the denaturation method comprises two means of chemical denaturation and physical denaturation, chemical denaturants are introduced into the chemical denaturation, a purification step is required to be added, and the amplification production is not facilitated; the physical denaturation method is simple, but the protein stability after denaturation is challenged, which brings much trouble to the application end, and particularly when the protein is applied to latex turbidimetric products, nonspecific agglutination reaction is easy to occur.
At present, the common latex enhanced turbidimetry method is to perform thermal denaturation on IgG and then couple the IgG to latex, but denatured IgG is unstable, a plurality of hydrophobic groups are exposed in the denaturation process, so that the IgG undergoes self-aggregation in the denaturation process, and a denatured polymer is unstable, so that a plurality of problems are brought to application, such as unstable reagents, easy aggregation of coupled latex and the like. In addition, direct heat denaturation is likely to cause aggregation of IgG molecules, and thus the heating time cannot be too long, but insufficient heating time results in insufficient denaturation of IgG molecules, resulting in a product not only having poor stability but also having low activity.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a preparation method of denatured IgG, which is used for relieving the technical problems that IgG agglutinates itself in the denaturation process and a denatured polymer is unstable in the prior art.
The second object of the present invention is to provide a denatured IgG produced by the above-mentioned production method.
The third purpose of the invention is to provide the application of the denatured IgG in preparing immune antigen.
The fourth purpose of the invention is to provide a preparation method of rheumatoid factor immune antigen, so as to relieve the technical problems of poor stability, easy agglutination and poor antigen activity of the rheumatoid factor immune antigen in the prior art.
The fifth purpose of the invention is to provide the rheumatoid factor immune antigen prepared by the preparation method.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a method for producing denatured IgG comprises denaturing IgG in the presence of a protein protecting agent to obtain denatured IgG.
Further, the protein protectant includes a disaccharide;
preferably, the disaccharide comprises sucrose, trehalose, maltose or lactose;
preferably, the final amount of the protein protectant is 10-50w/v%, more preferably 20-40w/v%.
Further, the method for denaturing IgG comprises heat denaturation, strong acid and strong base denaturation, guanidine hydrochloride denaturation or urea denaturation, preferably heat denaturation.
Further, the heat denaturation comprises: igG is heated and denatured in buffer solution containing protein protective agent, and then cooled and protected from light.
Further, the content of the IgG is 5-10mg/ml;
preferably, the buffer comprises a HEPES buffer, a MES buffer or a PBS buffer, more preferably a PBS buffer, still more preferably a PBS buffer of ph 7.3-7.5;
preferably, the conditions for heat denaturation include: heating at 56-65 deg.C for 10-120min, preferably 60-63 deg.C for 30-90min;
preferably, the cooling and shielding from light comprises: keeping away from light at 0-8 deg.C for at least 2h.
The denatured IgG obtained by the above-mentioned method for producing denatured IgG.
The application of the denatured IgG in preparing immune antigen.
A preparation method of rheumatoid factor immune antigen comprises the step of coupling an antigen carrier with the denatured IgG in the presence of a protein protective agent to obtain the rheumatoid factor immune antigen.
Further, the antigen carrier comprises latex, colloidal gold or magnetic beads;
preferably, the protein protectant comprises a disaccharide;
preferably, the disaccharide comprises sucrose, trehalose, maltose or lactose.
The rheumatoid factor immune antigen prepared by the preparation method of the rheumatoid factor immune antigen.
Compared with the prior art, the invention has the following beneficial effects:
according to the preparation method of the denatured IgG, the protein protective agent is added in the denaturation process, so that the denatured IgG is prevented from aggregating in the denaturation process, the stability of the denatured IgG is improved, the IgG is denatured more fully, more antigen epitopes are exposed, and the antigen activity is improved. The method has the advantages of stable denaturation process, good monodispersity of the obtained denatured IgG, sufficient denaturation, high activity, guaranteed batch difference, simple operation, low cost and convenience for large-scale production.
The preparation method of the rheumatoid factor immune antigen provided by the invention is to couple an antigen carrier and the denatured IgG provided by the invention in the presence of a protein protective agent to obtain the rheumatoid factor immune antigen. Because the agglutination problem of the denatured IgG is well improved, the obtained rheumatoid factor immune antigen has better stability, the activity of the immune antigen is obviously improved, the batch difference is reduced, and the method is suitable for large-scale production.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing the effect of different concentrations of sucrose on the denaturation of IgG in example 1;
FIG. 2 is a graph showing the effect of different denaturation times on denatured IgG activity in example 4;
FIG. 3 is a graph showing the effect of different denaturation temperatures on denatured IgG activity in example 5;
FIG. 4 is a graph showing the effect of different denaturation temperatures on denatured IgG activity in example 5;
FIG. 5 is a graph showing the effect of different denaturing buffers on denatured IgG activity in example 6;
FIG. 6 is a graph showing the effect of different concentrations of trehalose on denatured IgG in example 7.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.
Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.
A method for preparing denatured IgG comprises denaturing IgG in the presence of a protein protecting agent to obtain denatured IgG.
According to the preparation method of the denatured IgG, the protein protective agent is added in the denaturation process, so that the denatured IgG is prevented from aggregating in the denaturation process, the stability of the denatured IgG is improved, the IgG is denatured more fully, more antigen epitopes are exposed, and the antigen activity is improved. The method has the advantages of stable denaturation process, good monodispersity of the obtained denatured IgG, sufficient denaturation, high activity, simple operation, low cost and convenient large-scale production, and ensures the batch difference.
In a preferred embodiment, the protein protectant comprises a disaccharide, which may be, for example, sucrose, trehalose, maltose, lactose, and the like. Experiments show that the addition of a disaccharide according to the present invention can achieve a good anti-aggregation effect, and the disaccharide in the present invention is preferably sucrose, trehalose, maltose or lactose, and more preferably sucrose or trehalose.
In a preferred embodiment, the final amount of the protein protectant is 10-50w/v%, more preferably 20-40w/v%. It will be understood that "w/v%" in the final amount of protein protectant refers to the mg mass content of protein protectant per volume of denaturation reaction in ml of IgG in the denaturation reaction. The consumption of the protein protective agent is too small, and the anti-aggregation effect of the denatured IgG is poor; the use of the protein protectant is excessive, and the antigen activity of the denatured IgG is poor. The dosage of the protein protective agent is typically but not limited to 10w/v%, 20w/v%, 30w/v%, 40w/v% or 50w/v%, and the dosage of the protein protective agent is preferably 20-40w/v%, and the prepared denatured IgG has better performances such as stability, antigen activity and the like.
In a preferred embodiment, the method of denaturing IgG comprises heat denaturation, strong acid and strong base denaturation, guanidine hydrochloride denaturation or urea denaturation, preferably heat denaturation. The method for denaturing IgG in the invention can be conventional methods which are currently used, and comprises but is not limited to thermal denaturation, strong acid and strong base denaturation, guanidine hydrochloride denaturation or urea denaturation, but the method is more preferably thermal denaturation, and the thermal denaturation method does not need purification, thereby simplifying the operation steps.
In a preferred embodiment, the thermal denaturation comprises: igG is heated and denatured in buffer solution containing protein protective agent, and then cooled and protected from light.
In a preferred embodiment, the IgG content is 5-10mg/ml. Where "mg/ml" means mg IgG per ml of buffer containing the protein protectant, the IgG content is typically, but not limited to, 5mg/ml, 6mg/ml, 7mg/ml, 8mg/ml, 9mg/ml, or 10mg/ml.
In a preferred embodiment, the buffer comprises HEPES buffer, MES buffer or PBS buffer, more preferably PBS buffer, even more preferably ph7.3-7.5PBS buffer. The research of the invention finds that the buffer solution for thermal denaturation can be HEPES buffer solution, MES buffer solution or PBS buffer solution, wherein the effect of PBS with pH7.4 is the best.
In a preferred embodiment, the conditions for heat denaturation comprise: heating at 56-65 deg.C for 10-120min, preferably at 60-63 deg.C for 30-90min. The heating temperature is too low, and IgG cannot be denatured; when the heating temperature is too high, protein agglutination of IgG is easy to occur. Too little heating time, insufficient denaturation of IgG; when the heating time is too long, protein aggregation of IgG is likely to occur. The heating temperature is typically, but not limited to, 56 deg.C, 57 deg.C, 58 deg.C, 59 deg.C, 60 deg.C, 61 deg.C, 62 deg.C, 63 deg.C or 65 deg.C, and the heating time is typically, but not limited to, 10min, 30min, 50min, 70min, 90min or 120min.
In a preferred embodiment, the cooling and light shielding comprises: and keeping away from light at 0-8 ℃ for at least 2h.
The present invention provides a denatured IgG produced by the above production method. The denatured IgG has good stability and sufficient exposure of antigen epitope.
The application of the denatured IgG provided by the invention in preparing immune antigen.
The preparation method of the rheumatoid factor immune antigen comprises the step of coupling an antigen carrier with the denatured IgG provided by the invention in the presence of a protein protective agent to obtain the rheumatoid factor immune antigen. The invention well improves the agglutination problem of denatured IgG, so the obtained rheumatoid factor immune antigen has better stability, the activity of the immune antigen is obviously improved, the batch difference is reduced, and the invention is suitable for large-scale production.
In preferred embodiments, the antigen carrier comprises latex, colloidal gold, or magnetic beads.
In a preferred embodiment, the protein protectant comprises a disaccharide, which may be, for example, sucrose, trehalose, maltose, lactose, and the like.
The invention finally provides the rheumatoid factor immune antigen prepared by the preparation method.
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever.
The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.
Latex (manufactured by JSR corporation);
human IgG (purchased from satellite Wu Guangming);
EDC 1-Ethyl- (3-dimethylaminopropyl) carbonyl diimine hydrochloride (available from Shanghai Aladdin Biotech Co., ltd.);
NHS: n-hydroxysuccinimide (available from Shanghai Aladdin Biotechnology Ltd.).
Example 1 Effect of sucrose concentration on denaturation Effect
1. Preparing 0w/v%, 10w/v%, 20w/v%, 30w/v%, 40w/v%, 50w/v% sucrose solutions with 10mmol/L PBS (pH 7.4), respectively;
2. IgG (50 mg/ml) was diluted to 10mg/ml with the buffer solution in step 1, respectively;
3. heating the solution in the step 2 in a constant temperature water bath box at 63 ℃ for 60min;
4. placing the heated solution in the step 3 in an ice water bath, and carrying out light-proof treatment for 2h;
5. the solution in step 4 was observed for the presence of turbidity, and the results are shown in FIG. 1, in which the sucrose concentration was 0w/v%, 10w/v%, 20w/v%, 30w/v%, 40w/v%, and 50w/v%, in the order from left to right.
As is clear from the results shown in FIG. 1, when the sucrose concentration is less than 30w/v%, aggregation is likely to occur in the denaturation step.
Example 2 denatured IgG conjugate latex
The latex coupling was carried out without the occurrence of a turbid solution in example 1.
The coupling scheme is as follows:
1. taking 160 μ L10% polystyrene carboxyl latex with particle diameter of 188nm, and diluting to 2ml with 10mmol/L HEPES (PH 7.5) buffer solution;
2. slowly adding 50 μ L (10 mg/ml) EDC solution and 50 μ L (10 mg/ml) NHS solution into step 1, stirring at room temperature for 20min, and adding 10ml HEPES (pH 7.5) buffer solution at 10 mmol/L;
3. mu.l of the denatured IgG solution which did not become turbid in example 1 was diluted with 200mmol/L urea (final concentration) and 0.05% BSA (final concentration);
4. slowly adding the diluted denatured IgG solution in the step (3) into the step (2), and stirring at room temperature for reaction for 3 hours;
5363 and centrifuging at 5.15000rpm for 40min, and ultrasonically resuspending with 10mmol/L PBS (containing 0.2v/v% Tween 20,0.1w/v% BSA,10w/v% sucrose), thus obtaining the RF detection reagent 2.
EXAMPLE 3 Effect of different sucrose concentrations on denatured IgG Activity
And (3) detecting an instrument: a Mirui BS480 full-automatic biochemical analyzer;
experimental reagent: using 50mmol/L glycine buffer solution as RF detection reagent 1, and combining with RF detection reagent 2 in example 2 to form a cost test detection reagent;
calibration products: diluting preparation is carried out by using RF patient sample serum;
detecting parameters: see Table 1
And (3) detection results: see table 2, in which the data are the measured reactivity values, obtained by multiplying the absorbance (absorbance) by 10000. As can be seen from Table 2, the heated IgG, after dilution with 10mmol/L PBS buffer containing 30w/v% sucrose, was the most active after coupling the reagents.
TABLE 1
| Sample size (μ l) | 2.5 |
| RF detection reagent 1 (ul) | 200 |
| RF detection reagent 2 (ul) | 50 |
| Detection wavelength (nm) | 660 |
| Read point 1 | 51-51 |
| Read point 2 | 65-65 |
TABLE 2
Example 4 Effect of denaturation time on denatured IgG Activity
1. Diluting human IgG with 10mmol/L PBS containing 30% sucrose to obtain solution 1, wherein the final concentration of human IgG is 10mg/ml;
2. heating the solution 1 in water bath at 63 deg.C for 10, 20, 30, 60, 90, and 120min to obtain solution 2;
3. the solution 2 was diluted to 1mg/ml with 10mmol/L PBS (containing 0.2v/v% Tween 20,5w/v% sucrose, 0.1w/v% BSA) to prepare RF detection reagent 2;
4. 10mmol/L PBS (containing 0.5% Tween 20, 500mmol/L NaCl,5% PEG 6000) was used as RF detection reagent 1;
5. a detection instrument: a Mirui BS480 full-automatic biochemical analyzer;
6. detecting parameters: see table 3 for details;
7. and (3) detection results: see table 4 for details, wherein the data is the measured reactivity value obtained by multiplying the absorbance (absorbance) by 10000. As can be seen from Table 4 and FIG. 2, the reaction trend was the best when the heating time was 60 min.
TABLE 3
| Sample size (. Mu.l) | 15 |
| RF detection reagent 1 (ul) | 250 |
| RF detection reagent 2 (ul) | 50 |
| Detection wavelength (nm) | 340 |
| Read point 1 | 49-49 |
| Read point 2 | 82-82 |
TABLE 4
Example 5 Effect of denaturation temperature on denatured IgG Activity
1. 30w/v% sucrose was added with 10mmol/L PBS (pH 7.4);
2. diluting IgG (50 mg/ml) to 10mg/ml with the buffer in step 1;
3. equally dividing the solution in the step 2 into 4 parts, each 5ml, and respectively heating in a constant temperature water bath box at 56 ℃, 60 ℃, 63 ℃ and 65 ℃ for 60min;
4. placing the solution heated in the step 3 in an ice water bath, and carrying out light-proof treatment for 2h;
5. diluting the solution obtained in the step 4 with 10mmol/L PBS (containing 0.2v/v% Tween 20,5w/v% sucrose, 0.1w/v% BSA) to 1mg/ml to prepare an RF detection reagent 2;
6. using 10mmol/L PBS (containing 0.5% Tween 20, 500mmol/L NaCl,5% PEG 6000) as RF detection reagent 1;
7. a detection instrument: a Mirui BS480 full-automatic biochemical analyzer;
8. detecting parameters: see table 3 for details;
9. and (3) detection results: specifically, see Table 5, in which the data are the measured reactivity values obtained by multiplying the absorbance (absorbance) by 10000. As can be seen from Table 5, FIG. 3 and FIG. 4, the reaction tendency was the best when the heating time was 60 min.
TABLE 5
Example 6 Effect of denaturing buffer on denatured IgG Activity
1. Respectively preparing PBS, PB, HEPES and MES solutions containing 30w/v% of sucrose;
2. diluting human IgG (50 mg/ml) to 10mg/ml with the buffer solution in the step 1;
3. putting the solution in the step 2 into a water bath kettle at 63 ℃, and heating for 60min;
4. putting the solution heated in the step 3 in an ice water bath, and keeping out of the sun for 2 hours;
5. diluting the solution obtained in the step 4 with 10mmol/L PBS (containing 0.2v/v% Tween 20,5w/v% sucrose, 0.1w/v% BSA) to 1mg/ml to prepare an RF detection reagent 2;
6. using 10mmol/L PBS (containing 0.5% Tween 20, 500mmol/L NaCl,5% PEG6000) as RF detecting reagent 1;
7. a detection instrument: mirui BS480 full-automatic biochemical analyzer;
8. detecting parameters: see table 3 for details;
9. the results are shown in Table 6 and FIG. 5; as can be seen from Table 6 and FIG. 5, the reactivity and the trend of the curve of the PBS buffer were the best;
TABLE 6
Example 7 Effect of protein protecting Agents on denatured IgG
1. Preparing trehalose solutions containing 0w/v%, 10w/v% and 30w/v% with 10mmol/L PBS (pH 7.4);
2. IgG (50 mg/ml) was diluted to 10mg/ml with the buffer solution in step 1, respectively;
3. heating the solution in the step 2 in a constant temperature water bath box at 63 ℃ for 60min;
4. placing the solution heated in the step 3 in an ice water bath, and carrying out light-proof treatment for 2h;
5. the solution in step 4 was observed for turbidity, and the results are shown in FIG. 3, with trehalose concentrations of 0w/v%, 10w/v%, and 30w/v%, in order from left to right.
As is clear from the results shown in FIG. 6, when the trehalose concentration is less than 30w/v%, aggregation is likely to occur in the denaturation step.
Example 8 inter-batch Difference
Carrying out process repeatability verification on the condition that turbidity does not occur in the example 1, namely the condition that the sucrose concentration is 30w/v%, carrying out latex coupling on the denatured raw materials of 4 batches, and comparing the batch-to-batch difference;
the coupling scheme is as follows:
1. taking 160 μ L10% polystyrene carboxyl latex with particle diameter of 188nm, and diluting to 2ml with 10mmol/L HEPES (PH 7.5) buffer solution;
2. slowly adding 50 μ L (10 mg/ml) EDC solution and 50 μ L (10 mg/ml) NHS solution into step 1, stirring at room temperature for 20min, and adding 10ml HEPES (pH 7.5) buffer solution at 10 mmol/L;
3. taking 70 mu L of denatured IgG solution of different batches, adding 200mmol/L urea (final concentration) and 0.05 percent BSA (final concentration) for dilution;
4. slowly adding the diluted denatured IgG solution obtained in the step (3) into the step (2), and stirring at room temperature for reaction for 3 hours;
5363 and centrifuging at 5.15000rpm for 40min, and ultrasonically resuspending with 10mmol/L PBS (containing 0.2v/v% Tween 20,0.1w/v% BSA,10w/v% sucrose), thus obtaining the RF detection reagent 2.
6. A detection instrument: a Mirui BS480 full-automatic biochemical analyzer;
7. experimental reagent: 50mmol/L glycine buffer was used as RF detection reagent 1;
8. a calibration product: preparing by dilution with serum of an RF patient sample;
9. detecting parameters: see Table 1
10. And (3) detection results: as can be seen from Table 7, 4 batches of denatured IgG were produced repeatedly and the reaction was tested after coupling the latex with a batch-to-batch CV of less than 10% and with good batch-to-batch reproducibility.
TABLE 7
| |
0 | 11.5 | 18.5 | 40 | 85 | 150 | Blank space |
| 20190104 | 55.1 | 600 | 1065 | 2161 | 4692 | 7155 | 5503 |
| 20190301 | 47.2 | 556 | 988 | 2142 | 4810 | 7509 | 5398 |
| 20190808 | 47.8 | 655 | 1066 | 2101 | 4360 | 7169 | 5605 |
| 20190919 | 52.8 | 598 | 958 | 2058 | 4583 | 7244 | 5536 |
| Mean value | 50.725 | 602.25 | 1019.25 | 2115.5 | 4611.25 | 7269.25 | 5510.5 |
| SD | 3.848268 | 40.59865 | 54.79279 | 45.78573 | 191.4391 | 164.5405 | 86.20325 |
| CV | 8％ | 7％ | 5％ | 2％ | 4％ | 2％ | 2％ |
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (9)
1. The application of the preparation method of the denatured IgG in the preparation of the rheumatoid factor immune antigen is characterized in that the preparation method comprises the following steps of thermally denaturing the IgG in the presence of sucrose or trehalose to obtain denatured IgG;
the final amount of sucrose or trehalose is 30w/v%, and the heat denaturation comprises: heat denaturation of IgG in a buffer containing sucrose or trehalose, including PBS buffer or PB buffer; the conditions for heat denaturation include: heating at 60-65 deg.C for 10-120min.
2. The use according to claim 1, wherein the thermal denaturation comprises: and cooling and protecting from light after the heating denaturation.
3. The use according to claim 2, wherein the IgG is present in an amount of 5-10mg/ml.
4. The use of claim 1, wherein the buffer is PBS buffer, ph 7.3-7.5.
5. The use according to claim 1, wherein the heat denaturation condition is heating at 60-63 ℃ for 30-90min.
6. Use according to claim 2, wherein said cooling and protecting from light comprises: and keeping away from light at 0-8 ℃ for at least 2h.
7. A method for preparing a rheumatoid factor immune antigen, which is characterized in that an antigen carrier is coupled with the denatured IgG obtained by the preparation method in the application of any one of claims 1~6 in the presence of sucrose or trehalose to obtain the rheumatoid factor immune antigen.
8. The method according to claim 7, wherein the antigen carrier comprises latex, colloidal gold, or magnetic beads.
9. The rheumatoid factor immune antigen prepared by the preparation method of claim 7 or 8.
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