CN106749652A - A kind of polyclonal antibody of aureus peptide glycan - Google Patents
A kind of polyclonal antibody of aureus peptide glycan Download PDFInfo
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- CN106749652A CN106749652A CN201710147906.XA CN201710147906A CN106749652A CN 106749652 A CN106749652 A CN 106749652A CN 201710147906 A CN201710147906 A CN 201710147906A CN 106749652 A CN106749652 A CN 106749652A
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- Prior art keywords
- polyclonal antibody
- peptide glycan
- immune
- antibody
- peptide
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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
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
-
- 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
Abstract
The present invention provides a kind of polyclonal antibody of aureus peptide glycan antigen.The step of obtaining the polyclonal antibody has：The peptide glycan prepared as starting strain using staphylococcus aureus obtains polyclonal antibody as immunogene, immunizing rabbit.It is final that purifying preparation is carried out to the anti-Staphylococcus aureus peptide glycan polyclonal antibody for obtaining using saturated ammonium sulfate salting out method and immunoaffinity chromatography.Using the present invention prepare polyclonal antibody potency is high, purity is high, and recognize other gram-positive bacteria peptide glycans while nonrecognition Gram-negative bacteria peptide glycan, it is the polyclonal antibody of the anti-Staphylococcus aureus peptide glycan antigen that domestic the first is prepared using immunoaffinity chromatography method, it is with polyclonal antibody sensitivity advantage high, the good advantage of monoclonal antibody specificity, two kinds of antibody are matched, set up detection gram-positive bacteria to lay the foundation, had broad application prospects in medical treatment and scientific research field.
Description
Technical field
The present invention relates to antibody and preparation method thereof technical field, specifically a kind of aureus peptide glycan it is many
Clonal antibody.
Background technology
The drug resistance of bacterial antibiotic constantly changes with new and broad-spectrum antibiotic widely using, wherein
Gram positive bacteria (Gram-positive bacteria, G+) drug resistance problems are especially prominent, with staphylococcus, streptococcus and intestines
The drug resistance of coccus is most commonly seen and serious.Peptide glycan is Gram-positive cell wall main component.And people, animal and plant are thin
In born of the same parents and such composition is not contained.Therefore, peptide glycan can as diagnosis whether be gram-positive bacterial infections target.Hospital
Interior diagnosis gram positive bacteria, generally by the method for microculture.This is required to obtain the biological tissue at sufferer,
In many cases, this can not.Such as staphylococcal pneumonia is common in neonate and child, in this case,
The means of tissue cultures are infeasible.On the other hand, it is also possible to pathogen is circulated, the weaker disease of infection resistance
People.Therefore, it is the life for being not only related to gram-positive bacterial infections patient to develop non-culture, the diagnostic method of Non-Invasive,
It is related to the safety of other patients in hospital.Wherein, ELISA method is with the advantages of its is easy to operate, sensitivity is high, extensive use
In clinical detection and scientific research.
At present, the gram positive bacteria quick diagnosis reagent kit that not can apply to clinic is gone up at home.Country of China
Bureau of Drugs Supervision is also not given to any producer field admission to market and licensing.Therefore, the domestic research in the field is empty
In vain.In the international market, only the production of WAKO a companys of Japan can be used for detecting gram positive bacteria quick diagnosis reagent kit
SLP, but only supply scientific research.Therefore, peptide glycan detection kit of the research and development with independent intellectual property right is significant.And
The key of ELISA method is to obtain suitable antibody, and double-antibodies sandwich ELISA is one kind height that the applicant prepares research and development
Effect detection method, the method be bound to antagonist requirement it is very high, it is necessary to can preferably be matched between antibody.The applicant has obtained
One plant of peptide glycan monoclonal antibody 3F8, Patent No. CN102675457A that there is broad spectrum activity to detect gram positive bacteria is taken.Only
The indirect competitive ELISA method of detection gram-positive bacteria is set up using this plant of monoclonal antibody, sensitivity is 1ug/ml, for
The detection of human serum sample has certain limitation, but does not obtain expected results.Based on the highly sensitive of double-antibody sandwich elisa
The advantage of degree, is more high-sensitivity detection human serum sample, therefore, another antibody of preparation is most important, therefore prepares and be directed to peptide
The antibody of glycan antigen turns into the key for developing the antigen detection kit.
The advantage of polyclonal antibody can be the multiple epitopes for recognizing same antigen.Therefore in immune detection, can recognize that more
Many antigen, is influenceed less by antigen conformation change.Therefore, using polyclonal antibody coated elisa plate, can capture more
Antigen, then combined with the monoclonal antibody of high specificity, both improve the sensitivity of method, also increase the special of method
Property.
The content of the invention
It is an object of the invention to overcome the deficiencies in the prior art, and provide a kind of many of aureus peptide glycan
Clonal antibody, is that the realization of double-antibodies sandwich ELISA lays the first stone, for the gram-positive bacteria for being applied to clinic is quickly examined
The listing early of disconnected kit provides safeguard.
The present invention realizes that the technical scheme of purpose is as follows：
A kind of aureus peptide glycan polyclonal antibody, the peptide glycan polyclonal antibody takes by the following method
：
First, animal is immunized
(1) the aureus peptide glycan using immunogene identical with monoclonal antibody 3F8 is immunized new as immunogene
Western orchid rabbit；Specific method is：
1. new zealand rabbit, screening conditions are：Male, substance is between 2.5kg-3.0kg；
2. first immunisation dosage be 500ug sugar/only, adjuvant is Freund's complete adjuvant, with secondary immunity interval time be 3
Week；
3. secondary immunity immunizing dose be 250ug sugar/only, adjuvant is incomplete Freund's adjuvant, during with three immunization intervals
Between be 2 weeks；
4. three times and the interval time between immunizing dose, approach, adjuvant are immunized with second immunization ways, twice later
It is 2 weeks, it is immune to the 6th time；
2nd, the acquisition of polyclonal antibody
(1) titration：In immunologic process, after exempting from from the 3rd, immune blood sampling in latter 7 days every time determines potency, to the 6th
Exempt to determine serum titer stabilization；
(2) antiserum is separated：Six exempt from rear serum titer reaches 1：More than 10000, serum sample is collected in femoral artery bloodletting, point
- 20 DEG C of preservations after dress；
(3) saturated ammonium sulfate salting out method carries out preliminary purification
1. after taking quantitative rabbit anteserum and the isometric mixing of PBS, 2 times of saturations of rabbit anteserum volume are slowly added to
Ammonium sulfate, and carry out 4 DEG C overnight；
2. next day centrifugation, takes precipitation and is dissolved with PBS, adds the saturation sulfuric acid isometric with PBS
Ammonium salt solution, 4 DEG C of dialysis, obtains preliminary purification product；
(4) affinity chromatography is further purified and obtains polyclonal antibody
Affinitive layer purification is carried out using GE ProteinA pillars, polyclonal antibody is obtained after being further purified.
And, in the method for obtaining peptide glycan polyclonal antibody, immune in the step (1) is specifically included：Back
Hypodermic injection is immune, intraperitoneal injection is immune and intravenous immune.
And, in the method for obtaining peptide glycan polyclonal antibody, the immune new zealand rabbit of selection in the step (1)
Substituted by the mouse of artificial feeding, horse, ox and sheep, immunizing dose determines according to specific immune animal species, body weight.
Advantages of the present invention and effect are：
1st, invention provides a kind of polyclonal antibody of anti-Staphylococcus aureus peptide glycan antigen, is with many
Clonal antibody sensitivity advantage high, the good advantage of monoclonal antibody specificity is matched two kinds of antibody, sets up detection leather
The double-antibodies sandwich ELISA of Lan Shi positive bacterias lays the foundation.Can not only make up in hospital at microculture sufferer
Biological tissue is difficult the deficiency for obtaining.And detection time can be shortened, and testing result is obtained with most fast speed, it is easy to patient
Treat early.
2nd, anti-peptide glycan polyclonal antibody of the invention not only has more efficient valency, and is recognizing other Gram-positives
Nonrecognition Gram-negative bacteria peptide glycan while bacterium peptide glycan, with relatively strong specificity.
Brief description of the drawings：
Fig. 1 is rabbit polyclonal antibody SDS-PAGE figures after purification；
Fig. 2 is the titration result of rabbit polyclonal antibody；
Fig. 3 is polyclonal antibody to staphylococcus aureus, lactobacillus acidophilus, Lactobacillus casei, Lactobacillus sake and plant
The peptide glycan cross reacting rate figure in thing lactobacillus source；
Fig. 4 is polyclonal antibody to aureus peptide glycan, saccharomycete 1,3- calloses, Escherichia coli peptide
The cross reacting rate figure of glycan and its lipopolysaccharides.
Specific embodiment
With reference to specific embodiment, the present invention is further described, and its specific embodiment is construed as only lifting
Example explanation, is not limited, it is impossible to limit protection scope of the present invention with following illustrations.
A kind of aureus peptide glycan polyclonal antibody, the peptide glycan polyclonal antibody takes by the following method
：
First, animal is immunized
(1) the aureus peptide glycan using immunogene identical with monoclonal antibody 3F8 is immunized new as immunogene
Western orchid rabbit；Specific method is：
1. new zealand rabbit, screening conditions are：Male, substance is between 2.5kg-3.0kg；
2. first immunisation dosage be 500ug sugar/only, adjuvant is Freund's complete adjuvant, with secondary immunity interval time be 3
Week；
3. secondary immunity immunizing dose be 250ug sugar/only, adjuvant is incomplete Freund's adjuvant, during with three immunization intervals
Between be 2 weeks；
4. three times and the interval time between immunizing dose, approach, adjuvant are immunized with second immunization ways, twice later
It is 2 weeks, it is immune to the 6th time；
Wherein, described being immunized specifically includes：Dorsal sc injection is immune, intraperitoneal injection is immune and intravenous immune.
In specific implementation of the invention, the immune new zealand rabbit of selection is replaced by the mouse of artificial feeding, horse, ox and sheep
Generation, immunizing dose specifically determines according to immune animal species, body weight.
2nd, the acquisition of polyclonal antibody
(1) titration：In immunologic process, after exempting from from the 3rd, immune blood sampling in latter 7 days every time determines potency, to the 6th
Exempt from；
(2) antiserum is separated：Six exempt from rear serum titer reaches 1：More than 10000, serum sample is collected in femoral artery bloodletting, point
- 20 DEG C of preservations after dress；
(3) saturated ammonium sulfate salting out method carries out preliminary purification
1. take after quantitative rabbit anteserum sample and PBS mix in equal volume, be slowly added to 2 times of rabbit anteserum volumes
Saturated ammonium sulfate solution, and carry out 4 DEG C overnight；
2. next day, centrifugation, takes precipitation and is dissolved with PBS, adds isometric saturated ammonium sulfate solution, 4 DEG C
Dialysis, obtains preliminary purification product；
(4) affinity chromatography is further purified and obtains polyclonal antibody
Affinitive layer purification is carried out using GE ProteinA pillars, polyclonal antibody is obtained after being further purified.
The detection of antibody
1.SDS-PAGE electroresis appraisals
To the preliminary purification product for obtaining and it is further purified product and carries out SDS-PAGE electrophoresis, Fig. 1 is as a result seen, through preliminary
After purification, the product that is further purified for being obtained using affinity chromatography has two obvious bands at 50KD and 25KD, miscellaneous without other
Band, illustrates that the antibody purification purity for obtaining is higher.
2. indirect elisa method determines potency
The goat anti-rabbit igg of Promega horseradish peroxidase-labeleds is selected as this assay method secondary antibody.Coating peptide gathers
Sugar, the serum sample to gathering carries out different gradient dilutions, and blood sampling serum sample determines serum sample as negative control before being immunized
This highest diluted concentration.Evaluation criterion is to regard as the positive more than 2.1 times of negative serum OD.Can from result figure 2
Go out, the antibody titer is 1：More than 10000.
3. the performance measurement of polyclonal antibody
1. the identification of polyclonal antibody broad spectrum activity
Its light spectrality is carried out using indirect ELISA method to the polyclonal antibody for obtaining to identify.Method is the various leather of coating
The peptide glycan of Lan Shi positive bacterias, is measured by primary antibody of the polyclonal antibody of acquisition.Polyclonal antibody carries out quantitative dilution, meter
Calculate the cross reacting rate of obtained antibody and various bacterial strain peptide glycans.
Y=S/Z*100%
S：Concentration when polyclonal antibody is combined with itself aureus peptide glycan 50%
Z：Concentration when polyclonal antibody is combined with other species gram-positive bacteria peptide glycans 50%.
Result is shown in Fig. 3.The polyclonal antibody is respectively provided with higher the intersection with the gram-positive bacteria peptide glycan of separate sources
Reactivity, more than 85%.Illustrate that there is certain broad spectrum activity.
2. the specific detection of polyclonal antibody
The specific detection of the polyclonal antibody is carried out using indirect competitive ELISA method.Coated with staphylococal
The peptide glycan of extraction, with the polyclonal antibody as primary antibody, respectively with aureus peptide glycan, saccharomycete 1,3- β-D- Portugals
Glycan, Escherichia coli peptide glycan and its lipopolysaccharides carry out specific detection for competition antigen.Calculate cross reacting rate：
Y=S/Z*100%
S：Concentration when aureus peptide glycan is with 50% antibody combination
Z：Concentration when interfering material is with 50% antibody combination.
Result is shown in Fig. 4.The polyclonal antibody is to saccharomycete 1,3- calloses, the cross reaction of e. coli lipopolysaccharide
Rate is less than 0.1%, and the cross reacting rate to Escherichia coli peptide glycan is less than 1%, illustrates that the polyclonal antibody is not only examined with fungi
Index 1 is surveyed, 3- calloses, Gram-negative bacteria Testing index lipopolysaccharides do not have cross reaction, and and Gram-negative
Bacterium peptide glycan is in controlled range, it was demonstrated that the polyclonal antibody has preferably specificity.
Claims (3)
1. a kind of aureus peptide glycan polyclonal antibody, it is characterised in that the peptide glycan polyclonal antibody is by as follows
Method is obtained：
First, animal is immunized
(1) the aureus peptide glycan using immunogene identical with monoclonal antibody 3F8 is immunized New Zealand as immunogene
Rabbit；Specific method is：
1. new zealand rabbit, screening conditions are：Male, substance is between 2.5kg-3.0kg；
2. first immunisation dosage be 500ug sugar/only, adjuvant is Freund's complete adjuvant, with secondary immunity at intervals of 3 weeks；
3. secondary immunity immunizing dose is 250ug sugar/only, and adjuvant is incomplete Freund's adjuvant, is 2 weeks with three immunization intervals；
4. three times and the interval time between immunizing dose, approach, adjuvant are immunized with second immunization ways, twice later are 2
It is week, immune to the 6th time；
2nd, the acquisition of polyclonal antibody
(1) titration：In immunologic process, after exempting from from the 3rd, immune blood sampling in latter 7 days every time determines potency, exempts from really to the 6th
Determine serum titer stabilization；
(2) antiserum is separated：Six exempt from rear serum titer reaches 1：More than 10000, serum sample, packing are collected in femoral artery bloodletting
- 20 DEG C of preservations afterwards；
(3) saturated ammonium sulfate salting out method carries out preliminary purification
1. after taking quantitative rabbit anteserum and the isometric mixing of PBS, 2 times of saturation sulfuric acid of rabbit anteserum volume are slowly added to
Ammonium salt solution, and carry out 4 DEG C overnight；
2. next day centrifugation, takes precipitation and is dissolved with PBS, adds the saturated ammonium sulfate isometric with PBS molten
Liquid, 4 DEG C of dialysis, obtains preliminary purification product；
(4) affinity chromatography is further purified and obtains polyclonal antibody
Affinitive layer purification is carried out using GE ProteinA pillars, polyclonal antibody is obtained after being further purified.
2. aureus peptide glycan polyclonal antibody according to claim 1, it is characterised in that gather peptide is obtained
In the method for sugared polyclonal antibody, immune in the step (1) is specifically included：Dorsal sc injection is immune, intraperitoneal injection is exempted from
Epidemic disease and intravenous immune.
3. aureus peptide glycan polyclonal antibody according to claim 1, it is characterised in that gather peptide is obtained
In the method for sugared polyclonal antibody, in the step (1) the immune new zealand rabbit of selection by the mouse of artificial feeding, horse, ox and
Sheep substitutes, and immunizing dose determines according to specific immune animal species, body weight.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2469714A1 (en) * | 2001-12-21 | 2003-07-24 | Biosynexus Incorporated | Multifunctional monoclonal antibodies directed to peptidoglycan of gram-positive bacteria |
| CN102089005A (en) * | 2008-05-12 | 2011-06-08 | 斯特罗克斯生物制药有限责任公司 | Staphylococcus aureus-specific antibody preparations |
| CN102675457A (en) * | 2012-05-24 | 2012-09-19 | 天津市一瑞生物工程有限公司 | Preparation of peptidoglycan monoclonal antibody and products of peptidoglycan monoclonal antibod |
| CN103792366A (en) * | 2014-01-21 | 2014-05-14 | 内蒙古必威安泰生物科技有限公司 | Foot-and-mouth disease vaccine host cell protein double-antibody sandwiched enzyme-linked immunosorbent assay kit as well as using method thereof |
| WO2014132150A1 (en) * | 2013-02-27 | 2014-09-04 | Kimberly-Clark Worldwide, Inc. | Rapid identification of organisms in bodily fluids |
| CN104474531A (en) * | 2011-06-19 | 2015-04-01 | 纽约大学 | Methods of treating and preventing staphylococcus aureus infections and associated conditions |
| CN106413737A (en) * | 2014-05-29 | 2017-02-15 | 株式会社绿十字 | Composition for preventing or treating staphylococcus aureus infection |
-
2017
- 2017-03-14 CN CN201710147906.XA patent/CN106749652A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2469714A1 (en) * | 2001-12-21 | 2003-07-24 | Biosynexus Incorporated | Multifunctional monoclonal antibodies directed to peptidoglycan of gram-positive bacteria |
| CN102089005A (en) * | 2008-05-12 | 2011-06-08 | 斯特罗克斯生物制药有限责任公司 | Staphylococcus aureus-specific antibody preparations |
| CN104474531A (en) * | 2011-06-19 | 2015-04-01 | 纽约大学 | Methods of treating and preventing staphylococcus aureus infections and associated conditions |
| CN102675457A (en) * | 2012-05-24 | 2012-09-19 | 天津市一瑞生物工程有限公司 | Preparation of peptidoglycan monoclonal antibody and products of peptidoglycan monoclonal antibod |
| WO2014132150A1 (en) * | 2013-02-27 | 2014-09-04 | Kimberly-Clark Worldwide, Inc. | Rapid identification of organisms in bodily fluids |
| CN103792366A (en) * | 2014-01-21 | 2014-05-14 | 内蒙古必威安泰生物科技有限公司 | Foot-and-mouth disease vaccine host cell protein double-antibody sandwiched enzyme-linked immunosorbent assay kit as well as using method thereof |
| CN106413737A (en) * | 2014-05-29 | 2017-02-15 | 株式会社绿十字 | Composition for preventing or treating staphylococcus aureus infection |
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Application publication date: 20170531 |