CN110903388B - Method for removing cross reaction of meningococcal antiserum - Google Patents
Method for removing cross reaction of meningococcal antiserum Download PDFInfo
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- CN110903388B CN110903388B CN201911220600.8A CN201911220600A CN110903388B CN 110903388 B CN110903388 B CN 110903388B CN 201911220600 A CN201911220600 A CN 201911220600A CN 110903388 B CN110903388 B CN 110903388B
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- 238000001514 detection method Methods 0.000 abstract description 10
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1217—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Neisseriaceae (F)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
- C07K16/065—Purification, fragmentation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
Abstract
The invention provides a method for removing cross reaction of meningococcal antisera, which comprises the following steps: 1) Providing an antiserum directed against a specific meningococcal serogroup, wherein the antiserum comprises a desired antibody directed against a capsular polysaccharide of said specific meningococcal serogroup and interfering antibodies directed against capsular polysaccharides of other meningococcal serogroups; 2) Adding to said antisera at least one capsular polysaccharide of said other meningococcal serogroup; 3) When the antisera begins to change from clear to turbid, the addition of capsular polysaccharides from the other meningococcal serogroup is stopped and turbidity is removed. The method can effectively remove cross reaction, obtain specific purified meningococcal antiserum, and the obtained antiserum has stable quality, basically no cross reaction and higher titer, and can be used for all immunological detection of meningococcal vaccine.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to a method for removing cross reaction of meningococcal antisera.
Background
Epidemic cerebrospinal meningitis (epidemic cerebrospinal meningitis) is a respiratory infectious disease caused by meningococcal infection. Meningococci are gram-negative bacteria that can be divided into 13 serogroups, A, B, C, D etc., according to the chemical structure of their capsular polysaccharide, with A, B, C, Y and W135 groups being the main prevalent flora. About 50 ten thousand epidemic brain cases occur annually worldwide, with children being the main cause of serious threat to human health. Meningococcal vaccination plays an important role in preventing and controlling epidemic of epidemic meningitis in China, and according to different preparation processes, meningococcal vaccines comprise Meningococcal Polysaccharide Vaccine (MPV), meningococcal polysaccharide conjugate vaccine (MCV) and combined vaccine.
Polysaccharide antigen is the main antigen component of meningococcal vaccine, and polysaccharide content measurement is particularly important in quality control of vaccine, and in practical work, polysaccharide content of vaccine is often measured by adopting chemical or immunological method. The chemical method can only detect monovalent meningococcal vaccine (such as MPV-A), and the multivalent meningococcal vaccine (such as MPV-AC, MPV-ACYW, MCV-AC, MCV-ACYW135, MCV-AC-Hib) has serious cross interference among groups, and can only use highly specific immunological detection means such as rocket electrophoresis, rate turbidimetry and the like. However, these methods rely entirely on antibodies in serum for their antigenicity, and the presence of multiple polysaccharide antigens or/and carrier protein interference in the test sample requires high specificity in serum; some long-term tests (e.g., stability tests, compatibility tests) are run for up to 3 years or more, requiring good stability of the serum. Therefore, improvements to existing antiserum purification techniques are needed to make the detection of polysaccharide content of meningococcal vaccines easier and more efficient.
Disclosure of Invention
To solve the above-described problems of the prior art, the present invention provides a method for cross-reacting meningococcal antisera.
Specifically, the present invention provides:
(1) A method of cross-reacting meningococcal antisera comprising the steps of:
1) Providing an antiserum directed against a specific meningococcal serogroup, wherein the antiserum comprises a desired antibody directed against a capsular polysaccharide of said specific meningococcal serogroup and interfering antibodies directed against capsular polysaccharides of other meningococcal serogroups;
2) Adding to said antisera at least one capsular polysaccharide of said other meningococcal serogroup;
3) When the antisera begins to change from clear to turbid, the addition of capsular polysaccharides from the other meningococcal serogroup is stopped and turbidity is removed.
(2) The method according to (1), wherein in step 2) at least one capsular polysaccharide of said other meningococcal serogroup is added in a drop-wise manner
(3) The method of (1), wherein in step 2), the concentration of capsular polysaccharide of the other meningococcal serogroup added is greater than or equal to 5mg/mL.
(4) The method of (1), wherein steps 2) and 3) are repeated until the antisera is no longer clouded after the addition of the capsular polysaccharide of at least one of the other meningococcal serogroups.
(5) The method of (1), wherein in step 3), the turbidity is removed by centrifugation.
(6) The method according to (1), wherein in step 2), the capsular polysaccharide of the other meningococcal serogroup added is a purified capsular polysaccharide.
(7) The method of (1), wherein the particular meningococcal serogroup is one of a meningococcal W135 serogroup and a meningococcal Y serogroup, and the other meningococcal serogroup is the other of the meningococcal W135 serogroup and the meningococcal Y serogroup.
(8) The method of any one of (1) - (7), wherein the method further comprises: 4) The meningococcal antisera from which the turbidity was removed was subjected to sterile filtration.
Compared with the prior art, the invention has the following advantages and positive effects:
the method provides that the capsular polysaccharide of other meningococcal serogroups is added to remove cross reaction of the meningococcal antisera, so that the specific meningococcal antisera is obtained, and the problem of detecting the content of the meningococcal vaccine polysaccharide is solved. The method can effectively remove cross reaction, obtain specific purified meningococcal antiserum, and the obtained antiserum has stable quality, basically no cross reaction and higher titer, and can be used for all immunological detection of meningococcal vaccine.
Compared with the traditional method for adding bacterial liquid to remove cross reaction, the method provided by the invention is efficient, stable, strong in controllability and simple and convenient. In particular, more effective addition can be realized, the stability is stronger, the meningococcal bacterial liquid is stored at 4 ℃ in the form of suspension, the batch-to-batch difference is large, the stability is only a few months, and the added capsular polysaccharide can be stored at-70 ℃ in the form of vacuum freeze-drying, and the stability is more than 5 years.
In addition, the invention further provides that the capsular polysaccharide is added in a dropwise manner, so that very excellent absorption and purification/cross reaction removal effects can be unexpectedly obtained, the cross reaction of antisera can be completely removed, and the serum specificity is greatly enhanced, thereby having better application value and being applicable to all immunological detection of meningococcal vaccines. And the addition amount of the polysaccharide is easier to control, and the capsular polysaccharide can be just removed from nonspecific components in a dropwise manner without excessive addition.
Detailed Description
The invention is further illustrated by the following description of specific embodiments, which are not intended to be limiting, and various modifications or improvements can be made by those skilled in the art in light of the basic idea of the invention, but are within the scope of the invention without departing from the basic idea of the invention.
In order to solve the above problems in the prior art, the inventors of the present invention propose that the preparation of stable, cross-reactive meningococcal antisera is a key factor in controlling the quality of meningococcal vaccines. However, few methods for absorption purification/cross-reaction of meningococcal antisera have been reported, and although many other methods for absorption purification/cross-reaction of pathogenic bacteria have been reported and studied, some disadvantages still remain. For example, the classical pneumococcal antiserum absorption purification/de-cross reaction method is described in the literature "LUND E, HENRICHSEN J.laboratory diagnostics, serology and epidemiology of Streptococcus pneumonia [ M ]// BERGAN T, NORRIS JR.methods in microbiology New York: academic Press,1978:242-261", in which a corresponding type of pneumococcal bacterial liquid is added in empirically unequal volumes to absorb non-specific components in the serum. The method is not complicated, but the preparation process of the additive pneumococcal bacterial liquid is quite time-consuming and labor-consuming: the pneumococcal strain is planted on a solid culture medium, then a lawn is scraped and cultured by a liquid culture medium, the lawn is harvested when the lawn grows to a proper OD, the bacterium is inactivated, the supernatant is removed by centrifugation, the collected bacterium is washed twice by normal saline, and then the bacterium concentration is diluted to a proper concentration by a turbidimetry method. In addition, the preparation of the pneumococcal bacterial liquid of the additive inevitably has batch-to-batch difference, and the bacterial liquid is stored at 4 ℃ and has poor stability.
The inventors of the present invention have innovatively proposed to cross-react meningococcal antisera by way of the addition of capsular polysaccharides by thorough research and experimentation and by way of smart conception.
Thus, the present invention provides a method of cross-reacting meningococcal antisera, the method comprising the steps of:
1) Providing an antiserum directed against a specific meningococcal serogroup, wherein the antiserum comprises a desired antibody directed against a capsular polysaccharide of said specific meningococcal serogroup and interfering antibodies directed against capsular polysaccharides of other meningococcal serogroups;
2) Adding to said antisera at least one capsular polysaccharide of said other meningococcal serogroup;
3) When the antisera begins to change from clear to turbid, the addition of capsular polysaccharides from the other meningococcal serogroup is stopped and turbidity is removed.
Compared with the method for absorbing non-specific components in serum by using bacterial liquid, the method provided by the invention is efficient, stable, strong in controllability, simple and convenient, and particularly, more effective addition can be realized, and the additive capsular polysaccharide is a necessary condition and main factor of meningococcal virulence; (2) the meningococcal bacterial liquid has stronger stability, is stored at 4 ℃ in the form of suspension, has large batch-to-batch difference and has stability for only months, and the polysaccharide (such as refined polysaccharide) is stored at-70 ℃ in the form of vacuumizing and freeze-drying, and has stability for more than 5 years.
On the effect of absorbing, purifying and removing cross reaction, the method can effectively remove the cross reaction to obtain specific meningococcal antiserum, and the obtained antiserum has stable quality, basically no cross reaction and higher potency and can be used for all immunological detection of meningococcal vaccine.
The antisera of the present invention against a particular meningococcal serogroup may be obtained using techniques known in the art, or may be obtained by commercially available means. The capsular polysaccharides of meningococcus of each serogroup used may also be obtained using techniques known in the art, or may be obtained by commercial means.
The present invention may not be limited to the form of the capsular polysaccharide added, which may be in solid or liquid form. The present invention may also not limit the concentration of capsular polysaccharide added.
However, further, the present invention proposes to add the capsular polysaccharide in a drop-wise manner. By dropwise adding capsular polysaccharide, the invention unexpectedly discovers that the excellent purification effect can be realized, the cross reaction of antisera can be completely removed, and the serum specificity is greatly enhanced, so that the invention has excellent application value and can be used for all immunological detection of meningococcal vaccines. And the addition amount of the polysaccharide is easier to control, and the nonspecific ingredients can be just removed in a dropwise manner without excessive addition.
Thus, more preferably, in step 2) the capsular polysaccharide of at least one of the other meningococcal serogroups is added in a drop-wise manner.
The term "drop-wise" as used herein has a generally accepted meaning in the art, typically about 50 μl in one drop.
Preferably, the concentration of capsular polysaccharides of the other meningococcal serogroups added is greater than or equal to 5mg/mL. When the capsular polysaccharide of the other meningococcal serogroup is added below this concentration, the antisera obtained will be diluted more and of lower titer.
Preferably, the capsular polysaccharide of the other meningococcal serogroup added is a purified polysaccharide. The refined polysaccharide is prepared by culturing bacterial strain in a proper culture medium, sterilizing, extracting capsular polysaccharide antigen, purifying, and lyophilizing. Different from crude sugar, each biochemical detection result and molecular size of refined sugar meet the related requirements of the current version of the pharmacopoeia of the people's republic of China and European pharmacopoeia on corresponding polysaccharide, and the refined sugar is a raw material of polysaccharide vaccine.
The present invention is not limited by theory, but it is believed that when capsular polysaccharides from other meningococcal serogroups are added to the antisera, they react with the corresponding antibodies in the antisera that cross-react, forming a precipitate, and thus the antisera is immediately observed to change from clear to turbid. The invention advantageously adopts a small number of times of adding capsular polysaccharide, and when turbidity occurs, the capsular polysaccharide of other meningococcal serogroups is stopped to remove turbidity; this is repeated a number of times.
In the method of the invention, steps 2) and 3) are preferably repeated until after addition of the capsular polysaccharide of at least one of the other meningococcal serogroups, the meningococcal antisera is no longer clouded.
Meningococci are known to fall into 13 serogroups, A, B, C, D, Y, W, 135 etc. It is known, for example, that there is a cross-reaction between antisera directed against Y and against the W135 serogroup, whereas they do not cross-react with other serogroups.
Thus, in one embodiment of the invention, when the antiserum is an antiserum against the W135 serogroup, there is a cross-reaction with the Y serogroup, requiring the addition of a capsular polysaccharide of the Y serogroup to the antiserum; and vice versa. In light of the spirit of the present invention, it will be appreciated by those skilled in the art that when other serogroups are present in the antisera than the one to which they are directed, it is desirable to add capsular polysaccharides of the other serogroups to the antisera.
In light of the spirit of the present invention, those skilled in the art will appreciate that when there is a cross-reaction with a plurality of other meningococcal serogroups in the antisera directed against a particular meningococcal serogroup, it is desirable to add capsular polysaccharides from the corresponding plurality of other meningococcal serogroups to that antisera. For example, capsular polysaccharides from a corresponding plurality of other meningococcal serogroups are added simultaneously, and the resulting turbidity is removed.
In step 3), the turbidity may be removed by centrifugation. Suitable centrifugal rotational speeds and times may be employed as desired.
Preferably, the capsular polysaccharide of the other meningococcal serogroup added is a purified polysaccharide.
In a particular embodiment of the invention, the particular meningococcal serogroup is one of a meningococcal W135 serogroup and a meningococcal Y serogroup, and the other meningococcal serogroup is the other of the meningococcal W135 serogroup and the meningococcal Y serogroup.
In a preferred embodiment, the method of the present invention further comprises: 4) The meningococcal antisera from which the turbidity was removed was subjected to sterile filtration. For example, a 0.2 μm filter is used for the sterilization filtration.
The present invention is further illustrated or described by way of examples which are not to be construed as limiting the scope of the invention.
Examples
Unless otherwise indicated, the experimental procedures, materials, and conditions used in the examples below were all conducted using conventional experimental procedures, materials, and conditions in the art. The antiserum and capsular polysaccharide (hereinafter also referred to simply as polysaccharide) in the examples are all from all responsible companies in the Lanzhou biological research, and the related detection method (rate nephelometry) is conventional operation, specifically, a positive control (5.0 mug/mL of X-valent polysaccharide mixed solution) and a negative control (positive control without the polysaccharide in the serogroup) are detected by a rate nephelometer with antiserum before and after treatment, and the corresponding reaction turbidity value is obtained after the detection. Serum crossover = negative control/positive control x 100%.
Example 1: method for absorbing, purifying and/or removing cross reaction of W135 group meningitis antiserum
To 50mL of the cross-reactive W135 meningococcal antiserum, 5mg/mL of the Y-group meningococcal polysaccharide solution was added dropwise (about 50. Mu.L per drop), and after mixing, 1844g was centrifuged for 10 minutes to collect the supernatant. The above steps are repeated until the supernatant is clear and transparent. Sterilizing and filtering the obtained supernatant with a film of 0.2 μm, subpackaging, and storing in a refrigerator at-70 to-80 ℃.
Before and after the absorption and purification/cross reaction removal of meningococcal antisera, the meningococcal antisera are identified by a rate nephelometry, and the result is as follows: before the W135 meningococcal antiserum is treated by the method, the antiserum crossing rate is up to 56.10%; the cross-over rate of antisera after treatment was reduced to 1.46%. It was confirmed that the cross-reaction of the W135 meningococcal antisera was completely removed after the treatment by the above method, and the serum specificity was greatly enhanced (titer 16). That is, the group W135 meningococcal antisera specifically reacts only with the group W135 polysaccharide, and does not cross-react with other polysaccharides.
Example 2: method for absorbing, purifying and/or removing cross reaction of Y-group meningitis antiserum
To 50mL of the Y meningococcal antiserum having cross reaction, 5mg/mL of the W135 meningococcal polysaccharide solution was added dropwise (about 50. Mu.L per drop), and after mixing, 1844g was centrifuged for 10 minutes to collect the supernatant. The above steps are repeated until the supernatant is clear and transparent. Sterilizing and filtering the obtained supernatant with a film of 0.2 μm, subpackaging, and storing in a refrigerator at-70 to-80 ℃.
Before and after the absorption and purification/cross reaction removal of meningococcal antisera, the meningococcal antisera are identified by a rate nephelometry, and the result is as follows: before the Y-group meningococcal antiserum is treated by the method, the antiserum crossing rate is as high as 61.98%; the cross-over rate of antisera after treatment was reduced to 0.96%. It was confirmed that the cross-reactions of the Y-group meningococcal antisera were all removed after treatment by the above method, and the serum specificity was greatly enhanced (titer 20). That is, the group Y meningococcal serum reacts specifically only with the group Y polysaccharides and does not cross-react with other group polysaccharides.
Example 3: method for absorbing, purifying and/or removing cross reaction (adding polysaccharide in non-drop-by-drop mode) of Y-group meningitis antiserum
To two 10mL of the Y-group meningococcal antiserum having a cross reaction, 0.5mL and 1.0mL of 5mg/mL of W135-group meningococcal polysaccharide solution were added in a non-dropwise manner, and after mixing, 1844g was centrifuged for 10 minutes to collect supernatants, and the obtained supernatants were sterilized and filtered with 0.2 μm membranes, respectively.
Before and after the absorption and purification/cross reaction removal of meningococcal antisera, the meningococcal antisera are identified by a rate nephelometry, and the result is as follows: before the Y-group meningococcal antiserum is treated by the method, the antiserum crossing rate is as high as 61.98%; the cross-over rate of antisera after the treatment of 0.5mL of polysaccharide solution is reduced to 28.33%, which indicates that the cross reaction is not completely removed by the addition; the cross rate of antisera was reduced to 0.88% after treatment with 1mL polysaccharide solution, but the specific reaction of the group Y meningococcal serum with the group Y polysaccharide was also significantly reduced (titer was only 4).
Claims (10)
1. A method of cross-reacting meningococcal antisera comprising the steps of:
1) Providing an antiserum against a meningococcal W135 serogroup, wherein the antiserum comprises a desired antibody against a capsular polysaccharide of said meningococcal W135 serogroup and an interfering antibody against a capsular polysaccharide of meningococcal Y serogroup;
2) Adding only capsular polysaccharides of the meningococcal Y serogroup to the antisera to remove cross-reactions;
3) Stopping the addition of capsular polysaccharide of the meningococcal Y serogroup when the antiserum starts to change from clear to turbid, and removing turbidity;
wherein in step 2) the capsular polysaccharide of the meningococcal Y serogroup is added in a drop-wise manner, 50 μl per drop;
wherein the turbidity value of the antisera is detected with a rate turbidimeter before step 2), as well as after step 2) and before step 3);
wherein in step 2) the concentration of capsular polysaccharide of meningococcal Y serogroup added is greater than or equal to 5mg/mL;
wherein the meningococcal antisera cross-reacted by the method has an antisera cross-over rate of 1.46% and a titer of 16.
2. The method of claim 1, wherein steps 2) and 3) are repeated until after the capsular polysaccharide of the meningococcal Y serogroup is added, the antisera is no longer clouded.
3. The method of claim 1, wherein in step 3), the turbidity is removed by centrifugation.
4. The method according to claim 1, wherein in step 2) the capsular polysaccharide of the meningococcal Y serogroup added is a purified capsular polysaccharide.
5. The method of any one of claims 1-4, wherein the method further comprises: 4) The meningococcal antisera from which the turbidity was removed was subjected to sterile filtration.
6. A method of cross-reacting meningococcal antisera comprising the steps of:
1) Providing an antiserum against a meningococcal Y serogroup, wherein the antiserum comprises a desired antibody against a capsular polysaccharide of said meningococcal Y serogroup and an interfering antibody against a capsular polysaccharide of meningococcal W135 serogroup;
2) Adding only capsular polysaccharides of the meningococcal W135 serogroup to the antisera to remove cross-reactions;
3) Stopping the addition of capsular polysaccharide of the meningococcal W135 serogroup when the antisera starts to change from clear to turbid, and removing turbidity;
wherein in step 2) the capsular polysaccharide of the meningococcal W135 serogroup is added in a drop-wise manner, 50 μl per drop;
wherein the turbidity value of the antisera is detected with a rate turbidimeter before step 2), as well as after step 2) and before step 3);
wherein in step 2) the concentration of capsular polysaccharide of the meningococcal W135 serogroup added is greater than or equal to 5mg/mL;
wherein the meningococcal antisera cross-reacted by the method has an antisera cross-over rate of 0.96% and a titer of 20.
7. The method of claim 6, wherein steps 2) and 3) are repeated until the antisera is no longer clouded after the capsular polysaccharide of the meningococcal W135 serogroup is added.
8. The method of claim 6, wherein in step 3), the turbidity is removed by centrifugation.
9. The method according to claim 6, wherein in step 2) the capsular polysaccharide of the added meningococcal W135 serogroup is a purified capsular polysaccharide.
10. The method of any one of claims 6-9, wherein the method further comprises: 4) The meningococcal antisera from which the turbidity was removed was subjected to sterile filtration.
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| NZ622900A (en) * | 2001-01-23 | 2015-11-27 | Sanofi Pasteur Inc | Multivalent meningococcal polysaccharide-protein conjugate vaccine |
| EP2977759A1 (en) * | 2014-07-25 | 2016-01-27 | Serum Institute Of India Ltd. | Highly sensitive immunoassay for rapid quantification of meningococcal capsular polysaccharide antigens |
| CN106399257A (en) * | 2016-06-22 | 2017-02-15 | 云南沃森生物技术股份有限公司 | Monoclonal antibody capable of resisting group A meningococcal capsular polysaccharide conjugate, hybridoma cell strain and applications |
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