CN110483644A - Positive serum substitute of mycoplasma pneumoniae IgM antibody and preparation method thereof - Google Patents
Positive serum substitute of mycoplasma pneumoniae IgM antibody and preparation method thereof Download PDFInfo
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- CN110483644A CN110483644A CN201910853071.9A CN201910853071A CN110483644A CN 110483644 A CN110483644 A CN 110483644A CN 201910853071 A CN201910853071 A CN 201910853071A CN 110483644 A CN110483644 A CN 110483644A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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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
- C07K16/1253—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
- C07K16/461—Igs containing Ig-regions, -domains or -residues form different species
Abstract
The present invention provides the positive serum substitutes of mycoplasma pneumoniae IgM antibody, it is characterized by comprising rabbit-anti mycoplasma pneumoniae IgM antibodies and Normal human immunoglobulin M, rabbit-anti mycoplasma pneumoniae IgM antibody and Normal human immunoglobulin M form MP-IgM antibody-people's IgM albumen cross-linking agent by cross-linking reaction.The preparation method of the positive serum substitute of mycoplasma pneumoniae IgM antibody as described above, it is characterised in that: the following steps are included: S1, preparation rabbit-anti mycoplasma pneumoniae IgM antibody；S2, rabbit-anti mycoplasma pneumoniae IgM antibody and Normal human immunoglobulin M are crosslinked.With the above mentioned technical proposal, compared with prior art, the invention has the advantages that not only having realized the IgM antibody performance reacted with MP, but also realize the antigen performance reacted with human IgM antibody, it is important to eliminate the risk of the contagious pathogenic factor that may be present in human serum.
Description
Technical field
The present invention relates to immunological technique fields.
Specifically, being to be related to positive serum substitute of mycoplasma pneumoniae IgM antibody and preparation method thereof.
Background technique
The rapid development of external diagnosis reagent field, each side's science of law, each Testing index become increasingly abundant, refer to the clinic of the mankind
Lead provide convenience, fast, accurately diagnose direction.With the large-scale production and application of mycoplasma pneumoniae IgM antibody, pneumonia
The Quality Control of mycoplasma IgM antibody positive serum, clinical control demand are gradually increased, but its potential infection risk, can not be ignored.
Although positive serum used at present, there are stringent inactivation process and sterilization treatment mode, its infection wind that may be present
Danger is still without clear conclusion.In addition, country strictly supervises human serum, qualification requirement height is bought or collects, source is few, price
Height, this improves threshold to the use unit of positive serum, increases difficulty.
Summary of the invention
It is an object of the invention to overcome the shortcoming of above-mentioned traditional technology, a kind of mycoplasma pneumoniae of high-titer is provided
Positive serum substitute of IgM antibody and preparation method thereof had not only realized the IgM antibody performance reacted with MP, but realize with
The antigen performance of human IgM antibody reaction, it is important to eliminate the risk of the contagious pathogenic factor that may be present in human serum.
The purpose of the present invention is what is reached by following technical measures:
The positive serum substitute of mycoplasma pneumoniae IgM antibody, it is characterised in that: including rabbit-anti mycoplasma pneumoniae IgM antibody and
Normal human immunoglobulin M, rabbit-anti mycoplasma pneumoniae IgM antibody and Normal human immunoglobulin M are formed by cross-linking reaction
MP-IgM antibody-people's IgM albumen cross-linking agent.
The preparation method of the positive serum substitute of mycoplasma pneumoniae IgM antibody as described above, it is characterised in that: including
Following steps:
S1, preparation rabbit-anti mycoplasma pneumoniae IgM antibody；
S2, rabbit-anti mycoplasma pneumoniae IgM antibody and Normal human immunoglobulin M are crosslinked.
As an improvement: the preparation of rabbit-anti mycoplasma pneumoniae IgM antibody the following steps are included:
S11, it is immunized；
First day, Freund's complete adjuvant 0.8-1.2ml, mycoplasma pneumoniae recombinant protein 0.8-1.2mg, neck, Medial Thigh Skin, back
The injection of 10 points of portion；
4th day, incomplete Freund's adjuvant 0.8-1.2ml, mycoplasma pneumoniae recombinant protein 0.8-1.2mg, neck, Medial Thigh Skin,
The injection of 10 points of back；
7th day, incomplete Freund's adjuvant 0.8-1.2ml, mycoplasma pneumoniae recombinant protein 0.8-1.2mg, neck, Medial Thigh Skin,
The injection of 10 points of back；
Tenth day, incomplete Freund's adjuvant 0.8-1.2ml, mycoplasma pneumoniae recombinant protein 0.8-1.2mg, neck, Medial Thigh Skin,
The injection of 10 points of back；
12nd day, blood is taken, centrifuging and taking supernatant detects antiserum titre, such as reaches requirement, can start to take within every 4-6 days a blood
Clearly；0.45 μm of filtering with microporous membrane.
As an improvement: centrifuging and taking supernatant detects antiserum titre, does not reach requirement such as, then every 3 days booster immunizations one
It is secondary, take serum to detect after 2 days, until detection potency is qualified.
As an improvement: preparing for rabbit-anti mycoplasma pneumoniae IgM antibody is further comprising the steps of:
S12, purifying；
A1, take that 20 parts by volume of antiserum is separately added into 15-25 parts by volume PBS buffer solution and 8-12 parts by volume saturated ammonium sulfate is molten
Liquid mixes well；
A2,7000-9000r/min are centrifuged 18-22min, abandon supernatant, remove albumin, are precipitated as the immunoglobulin slightly mentioned；
A3, precipitating plus 15-25 parts by volume PBS buffer solution, add saturated ammonium sulfate solution 8-12 parts by volume, mix well；
A4,7000-9000r/min are centrifuged 18-22min, abandon supernatant；
A5, precipitating plus 8-12 parts by volume PBS buffer solution, are packed into bag filter, are put in 4 DEG C of dialysis 24-48h in PBS buffer solution, until
Without SO42-Or NH4+Until appearance；
After A6, dialysis, 2500-3500r/min is centrifuged 18-22min, and 0.22 μm of filtering with microporous membrane of supernatant is as pure
The MP-IgM antibody of change.
As an improvement: further comprising the steps of:
A7, MP-IgM antibody sample is taken to dilute 100 times, ultraviolet-uisible spectrophotometer measures it at wavelength 260nm, 280nm
Light absorption value, then calculate to obtain protein concentration.
As an improvement: further comprising the steps of:
A8, it takes MP-IgM antibody sample by SDS-PAGE electrophoresis, completes electrophoresis and test purity.
As an improvement: the configuration of the saturated ammonium sulfate solution the following steps are included: claim 400-425 grams of ammonium sulfate,
It is dissolved with 50-80 DEG C of distilled water 500mL, concentrated ammonia liquor tune pH to 7.3-7.5.
As an improvement: rabbit-anti mycoplasma pneumoniae IgM antibody and Normal human immunoglobulin M crosslinking include following step
It is rapid:
S21,2 mass parts people IgM are dissolved in 450000-500000 parts by volume combination buffer MES, are sufficiently dissolved；
S22,10 mass parts EDC are dissolved in 800-1200 parts by volume water；
S23, the product of step S21 is added in the product of step S22, mass parts containing 1.8-2.2 is slowly added into mixed liquor
The 180-200 parts by volume combination buffer of rabbit-anti MP-IgM antibody；
S24, dialysis separate product to get the cross-linking products of rabbit-anti MP-IgM antibody and human immunoglobulin M.
By adopting the above-described technical solution, compared with prior art, being reacted the invention has the advantages that both realizing with MP
IgM antibody performance, and realize the antigen performance reacted with human IgM antibody, it is important to eliminate in human serum there may be
The contagious pathogenic factor risk.
The invention will be further described for specific embodiment below.
Specific embodiment
Embodiment 1: the positive serum substitute of mycoplasma pneumoniae IgM antibody, including rabbit-anti mycoplasma pneumoniae IgM antibody and
Normal human immunoglobulin M, rabbit-anti mycoplasma pneumoniae IgM antibody and Normal human immunoglobulin M are formed by cross-linking reaction
MP-IgM antibody-people's IgM albumen cross-linking agent.
Embodiment 2: the preparation method of the positive serum substitute of mycoplasma pneumoniae IgM antibody as described in Example 1, packet
Include following steps:
S1, preparation rabbit-anti mycoplasma pneumoniae IgM antibody.
Select it is excellent, the preparation of rabbit-anti mycoplasma pneumoniae IgM antibody the following steps are included:
S11, it is immunized.
First day, Freund's complete adjuvant 0.8ml, mycoplasma pneumoniae recombinant protein 0.8mg, neck, Medial Thigh Skin, back 10
Point injection；
4th day, incomplete Freund's adjuvant 0.8ml, mycoplasma pneumoniae recombinant protein 0.8mg, neck, Medial Thigh Skin, 10 points of back
Injection；
7th day, incomplete Freund's adjuvant 0.8ml, mycoplasma pneumoniae recombinant protein 0.8mg, neck, Medial Thigh Skin, 10 points of back
Injection；
Tenth day, incomplete Freund's adjuvant 0.8ml, mycoplasma pneumoniae recombinant protein 0.8mg, neck, Medial Thigh Skin, 10 points of back
Injection；
12nd day, take blood 10ml, 3500r/min, centrifugation 12min takes supernatant to detect antiserum titre, such as reach requirement (1:
2000 or more), can start to take a serum in every 4 days.0.45 μm of filtering with microporous membrane, filtering packing is into sterile tube, and -20 DEG C
It saves.
It does not reach requirement such as, then every 3 days booster immunizations are primary, take serum to detect after 2 days, until detection potency is qualified.
S12, purifying.
A1,20 parts by volume of antiserum is taken to be separately added into 15 parts by volume PBS buffer solution and 8 parts by volume saturated ammonium sulfate solution,
It is stirring while adding, it mixes well, stands 30min.
The configuration of the saturated ammonium sulfate solution is the following steps are included: claim 400 grams of ammonium sulfate, with 50 DEG C of distilled water
500mL dissolution, concentrated ammonia liquor tune pH to 7.3.
A2,7000r/min are centrifuged 18min, abandon supernatant, remove albumin, are precipitated as the immunoglobulin slightly mentioned.
A3, precipitating plus 15 parts by volume PBS buffer solution, make it completely dissolved, add 8 parts by volume of saturated ammonium sulfate solution,
It mixes well, 4 DEG C of standing 30min；.
A4,7000r/min are centrifuged 18min, abandon supernatant.
Repetition step A3, A4,2 times.
A5, precipitating plus 8 parts by volume PBS buffer solution after being completely dissolved, are packed into bag filter, and desalination of dialysing is put in PBS buffering
4 DEG C of dialysis for 24 hours, are changed liquid 5 times, with 1%BaCl in liquid2Check the SO4 in dialyzate2-Or NH4 is checked with nessler reagent+(take 3-
4ml dialyzate, reagent adding 1-2 drop occur brick-red being to think there is NH4+In the presence of), up to no SO42-Or NH4+Until appearance.
After A6, dialysis, 2500r/min is centrifuged 18min, and 0.22 μm of filtering with microporous membrane of supernatant as purifies
MP-IgM antibody.
A7, take MP-IgM antibody sample dilute 100 times, ultraviolet-uisible spectrophotometer measure its wavelength 260nm,
Then light absorption value at 280nm calculates to obtain protein concentration.
Protein concentration calculation formula are as follows: protein concentration (mg/ml)=1.45xOD280-0.74xOD260 detectable concentration.
A8, it takes MP-IgM antibody sample by SDS-PAGE electrophoresis, completes electrophoresis and test purity.
S2, rabbit-anti mycoplasma pneumoniae IgM antibody and Normal human immunoglobulin M are crosslinked.
S21,2 mass parts people IgM are dissolved in 450000 parts by volume combination buffer MES(2-(N- morpholinoes) ethanesulfonic acid)
In, sufficiently dissolve.
S22, by 10 mass parts EDC(1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride) equilibrate to room temperature,
It is dissolved in 800 parts by volume water.
S23, the product of step S21 is added in the product of step S22, is slowly added into mixed liquor containing 1.8 mass parts
180 parts by volume combination buffers of rabbit-anti MP-IgM antibody, react 2h at room temperature.
S24, dialysis separation product, change a dialyzate every 5h, change liquid 5 times.
Up to the cross-linking products of rabbit-anti MP-IgM antibody and human immunoglobulin M, -20 DEG C of preservations.
Embodiment 3: the preparation method of the positive serum substitute of mycoplasma pneumoniae IgM antibody as described in Example 1, packet
Include following steps:
S1, preparation rabbit-anti mycoplasma pneumoniae IgM antibody.
Select it is excellent, the preparation of rabbit-anti mycoplasma pneumoniae IgM antibody the following steps are included:
S11, it is immunized.
First day, Freund's complete adjuvant 1ml, mycoplasma pneumoniae recombinant protein 1mg, neck, Medial Thigh Skin, 10 points of back note
It penetrates；
4th day, incomplete Freund's adjuvant 1ml, mycoplasma pneumoniae recombinant protein 1mg, neck, Medial Thigh Skin, 10 points of back note
It penetrates；
7th day, incomplete Freund's adjuvant 1ml, mycoplasma pneumoniae recombinant protein 1mg, neck, Medial Thigh Skin, 10 points of back note
It penetrates；
Tenth day, incomplete Freund's adjuvant 1ml, mycoplasma pneumoniae recombinant protein 1mg, neck, Medial Thigh Skin, 10 points of back note
It penetrates；
12nd day, take blood 10ml, 4000r/min, centrifugation 15min takes supernatant to detect antiserum titre, such as reach requirement (1:
2000 or more), can start to take within every 4-6 days a serum.0.45 μm of filtering with microporous membrane, filtering packing is into sterile tube, and -20
DEG C save.
It does not reach requirement such as, then every 3 days booster immunizations are primary, take serum to detect after 2 days, until detection potency is qualified.
S12, purifying.
A1,20 parts by volume of antiserum is taken to be separately added into 20 parts by volume PBS buffer solution and 10 parts by volume saturated ammonium sulfate solution,
It is stirring while adding, it mixes well, stands 30min.
The configuration of the saturated ammonium sulfate solution is the following steps are included: claim 410 grams of ammonium sulfate, with 65 DEG C of distilled water
500mL dissolution, concentrated ammonia liquor tune pH to 7.4.
A2,8000r/min are centrifuged 20min, abandon supernatant, remove albumin, are precipitated as the immunoglobulin slightly mentioned.
A3, precipitating plus 20 parts by volume PBS buffer solution, make it completely dissolved, add 10 parts by volume of saturated ammonium sulfate solution,
It mixes well, 4 DEG C of standing 30min；.
A4,8000r/min are centrifuged 20min, abandon supernatant.
Repetition step A3, A4,2 times.
A5, precipitating plus 10 parts by volume PBS buffer solution after being completely dissolved, are packed into bag filter, and desalination of dialysing is put in PBS buffering
4 DEG C of dialysis 36h in liquid, are changed liquid 5 times, with 1%BaCl2Check the SO4 in dialyzate2-Or NH4 is checked with nessler reagent+(take 3-
4ml dialyzate, reagent adding 1-2 drop occur brick-red being to think there is NH4+In the presence of), up to no SO42-Or NH4+Until appearance.
After A6, dialysis, 3000r/min is centrifuged 20min, and 0.22 μm of filtering with microporous membrane of supernatant as purifies
MP-IgM antibody.
A7, take MP-IgM antibody sample dilute 100 times, ultraviolet-uisible spectrophotometer measure its wavelength 260nm,
Then light absorption value at 280nm calculates to obtain protein concentration.
Protein concentration calculation formula are as follows: protein concentration (mg/ml)=1.45xOD280-0.74xOD260 detectable concentration.
A8, it takes MP-IgM antibody sample by SDS-PAGE electrophoresis, completes electrophoresis and test purity.
S2, rabbit-anti mycoplasma pneumoniae IgM antibody and Normal human immunoglobulin M are crosslinked.
S21,2 mass parts people IgM are dissolved in 470000 parts by volume combination buffer MES(2-(N- morpholinoes) ethanesulfonic acid)
In, sufficiently dissolve.
S22, by 10 mass parts EDC(1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride) equilibrate to room temperature,
It is dissolved in 1000 parts by volume water.
S23, the product of step S21 is added in the product of step S22, is slowly added into mixed liquor containing 2 mass parts rabbits
190 parts by volume combination buffers of anti-MP-IgM antibody, react 2h at room temperature.
S24, dialysis separation product, change a dialyzate every 5h, change liquid 5 times.
Up to the cross-linking products of rabbit-anti MP-IgM antibody and human immunoglobulin M, -20 DEG C of preservations.
Embodiment 4: the preparation method of the positive serum substitute of mycoplasma pneumoniae IgM antibody as described in Example 1, packet
Include following steps:
S1, preparation rabbit-anti mycoplasma pneumoniae IgM antibody.
Select it is excellent, the preparation of rabbit-anti mycoplasma pneumoniae IgM antibody the following steps are included:
S11, it is immunized.
First day, Freund's complete adjuvant 1.2ml, mycoplasma pneumoniae recombinant protein 1.2mg, neck, Medial Thigh Skin, back 10
Point injection；
4th day, incomplete Freund's adjuvant 1.2ml, mycoplasma pneumoniae recombinant protein 1.2mg, neck, Medial Thigh Skin, 10 points of back
Injection；
7th day, incomplete Freund's adjuvant 1.2ml, mycoplasma pneumoniae recombinant protein 1.2mg, neck, Medial Thigh Skin, 10 points of back
Injection；
Tenth day, incomplete Freund's adjuvant 1.2ml, mycoplasma pneumoniae recombinant protein 1.2mg, neck, Medial Thigh Skin, 10 points of back
Injection；
12nd day, blood 10ml, 4500r/min are taken, centrifugation 12-18min takes supernatant to detect antiserum titre, such as reaches requirement
(1:2000 or more), can start to take a serum in every 6 days.0.45 μm of filtering with microporous membrane, filtering are dispensed into sterile tube ,-
20 DEG C of preservations.
It does not reach requirement such as, then every 3 days booster immunizations are primary, take serum to detect after 2 days, until detection potency is qualified.
S12, purifying.
A1,20 parts by volume of antiserum is taken to be separately added into 25 parts by volume PBS buffer solution and 12 parts by volume saturated ammonium sulfate solution,
It is stirring while adding, it mixes well, stands 30min.
The configuration of the saturated ammonium sulfate solution is the following steps are included: claim 25 grams of ammonium sulfate, with 80 DEG C of distilled water 500mL
Dissolution, concentrated ammonia liquor tune pH to 7.5.
A2,9000r/min are centrifuged 22min, abandon supernatant, remove albumin, are precipitated as the immunoglobulin slightly mentioned.
A3, precipitating plus 25 parts by volume PBS buffer solution, make it completely dissolved, add 12 parts by volume of saturated ammonium sulfate solution,
It mixes well, 4 DEG C of standing 30min；.
A4,9000r/min are centrifuged 22min, abandon supernatant.
Repetition step A3, A4,3 times.
A5, precipitating plus 12 parts by volume PBS buffer solution after being completely dissolved, are packed into bag filter, and desalination of dialysing is put in PBS buffering
4 DEG C of dialysis 48h in liquid, are changed liquid 6 times, with 1%BaCl2Check the SO4 in dialyzate2-Or NH4 is checked with nessler reagent+(take 3-
4ml dialyzate, reagent adding 1-2 drop occur brick-red being to think there is NH4+In the presence of), up to no SO42-Or NH4+Until appearance.
After A6, dialysis, 3500r/min is centrifuged 22min, and 0.22 μm of filtering with microporous membrane of supernatant as purifies
MP-IgM antibody.
A7, take MP-IgM antibody sample dilute 100 times, ultraviolet-uisible spectrophotometer measure its wavelength 260nm,
Then light absorption value at 280nm calculates to obtain protein concentration.
Protein concentration calculation formula are as follows: protein concentration (mg/ml)=1.45xOD280-0.74xOD260 detectable concentration.
A8, it takes MP-IgM antibody sample by SDS-PAGE electrophoresis, completes electrophoresis and test purity.
S2, rabbit-anti mycoplasma pneumoniae IgM antibody and Normal human immunoglobulin M are crosslinked.
S21,2 mass parts people IgM are dissolved in 500000 parts by volume combination buffer MES(2-(N- morpholinoes) ethanesulfonic acid)
In, sufficiently dissolve.
S22, by 10 mass parts EDC(1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride) equilibrate to room temperature,
It is dissolved in 1200 mass parts by volume water.
S23, the product of step S21 is added in the product of step S22, is slowly added into mixed liquor containing 2.2 mass parts
200 mass parts by volume combination buffers of rabbit-anti MP-IgM antibody, react 2h at room temperature.
S24, dialysis separation product, change a dialyzate every 5h, change liquid 6 times.
Up to the cross-linking products of rabbit-anti MP-IgM antibody and human immunoglobulin M, -20 DEG C of preservations.
Several embodiments of the invention are described in detail above, but the content is only preferable implementation of the invention
Example, should not be considered as limiting the scope of the invention.It is all according to all the changes and improvements made by the present patent application range
Deng should all fall within the scope of the patent of the present invention.
Claims (9)
1. the positive serum substitute of mycoplasma pneumoniae IgM antibody, it is characterised in that: including rabbit-anti mycoplasma pneumoniae IgM antibody
With Normal human immunoglobulin M, rabbit-anti mycoplasma pneumoniae IgM antibody and Normal human immunoglobulin M are formed by cross-linking reaction
MP-IgM antibody-people's IgM albumen cross-linking agent.
2. the preparation method of the positive serum substitute of mycoplasma pneumoniae IgM antibody as described in claim 1, feature exist
In: the following steps are included:
S1, preparation rabbit-anti mycoplasma pneumoniae IgM antibody；
S2, rabbit-anti mycoplasma pneumoniae IgM antibody and Normal human immunoglobulin M are crosslinked.
3. the preparation method of the positive serum substitute of mycoplasma pneumoniae IgM antibody according to claim 2, feature exist
In: rabbit-anti mycoplasma pneumoniae IgM antibody preparation the following steps are included:
S11, it is immunized；
First day, Freund's complete adjuvant 0.8-1.2ml, mycoplasma pneumoniae recombinant protein 0.8-1.2mg, neck, Medial Thigh Skin, back
The injection of 10 points of portion；
4th day, incomplete Freund's adjuvant 0.8-1.2ml, mycoplasma pneumoniae recombinant protein 0.8-1.2mg, neck, Medial Thigh Skin,
The injection of 10 points of back；
7th day, incomplete Freund's adjuvant 0.8-1.2ml, mycoplasma pneumoniae recombinant protein 0.8-1.2mg, neck, Medial Thigh Skin,
The injection of 10 points of back；
Tenth day, incomplete Freund's adjuvant 0.8-1.2ml, mycoplasma pneumoniae recombinant protein 0.8-1.2mg, neck, Medial Thigh Skin,
The injection of 10 points of back；
12nd day, blood is taken, centrifuging and taking supernatant detects antiserum titre, such as reaches requirement, can start to take within every 4-6 days a blood
Clearly；
0.45 μm of filtering with microporous membrane.
4. the preparation method of the positive serum substitute of mycoplasma pneumoniae IgM antibody according to claim 3, feature exist
In: centrifuging and taking supernatant detects antiserum titre, does not reach requirement such as, then every 3 days booster immunizations are primary, takes serum to detect after 2 days,
Until detection potency is qualified.
5. the preparation method of the positive serum substitute of mycoplasma pneumoniae IgM antibody according to claim 2, feature exist
In: preparing for rabbit-anti mycoplasma pneumoniae IgM antibody is further comprising the steps of:
S12, purifying；
A1, take that 20 parts by volume of antiserum is separately added into 15-25 parts by volume PBS buffer solution and 8-12 parts by volume saturated ammonium sulfate is molten
Liquid mixes well；
A2,7000-9000r/min are centrifuged 18-22min, abandon supernatant, remove albumin, are precipitated as the immunoglobulin slightly mentioned；
A3, precipitating plus 15-25 parts by volume PBS buffer solution, add saturated ammonium sulfate solution 8-12 parts by volume, mix well；
A4,7000-9000r/min are centrifuged 18-22min, abandon supernatant；
A5, precipitating plus 8-12 parts by volume PBS buffer solution, are packed into bag filter, are put in 4 DEG C of dialysis 24-48h in PBS buffer solution, until
Without SO42-Or NH4+Until appearance；
After A6, dialysis, 2500-3500r/min is centrifuged 18-22min, and 0.22 μm of filtering with microporous membrane of supernatant is as pure
The MP-IgM antibody of change.
6. the preparation method of the positive serum substitute of mycoplasma pneumoniae IgM antibody according to claim 5, feature exist
In: further comprising the steps of:
A7, MP-IgM antibody sample is taken to dilute 100 times, ultraviolet-uisible spectrophotometer measures it at wavelength 260nm, 280nm
Light absorption value, then calculate to obtain protein concentration.
7. the preparation method of the positive serum substitute of mycoplasma pneumoniae IgM antibody according to claim 5, feature exist
In: further comprising the steps of:
A8, it takes MP-IgM antibody sample by SDS-PAGE electrophoresis, completes electrophoresis and test purity.
8. the preparation method of the positive serum substitute of mycoplasma pneumoniae IgM antibody according to claim 5, feature exist
In: the configuration of the saturated ammonium sulfate solution is the following steps are included: claim 400-425 grams of ammonium sulfate, with 50-80 DEG C of distilled water
500mL dissolution, concentrated ammonia liquor tune pH to 7.3-7.5.
9. the preparation method of the positive serum substitute of mycoplasma pneumoniae IgM antibody according to claim 2, feature exist
Be crosslinked in: rabbit-anti mycoplasma pneumoniae IgM antibody and Normal human immunoglobulin M the following steps are included:
S21,2 mass parts people IgM are dissolved in 450000-500000 parts by volume combination buffer MES, are sufficiently dissolved；
S22,10 mass parts EDC are dissolved in 800-1200 parts by volume water；
S23, the product of step S21 is added in the product of step S22, mass parts containing 1.8-2.2 is slowly added into mixed liquor
The 180-200 parts by volume combination buffer of rabbit-anti MP-IgM antibody；
S24, dialysis separate product to get the cross-linking products of rabbit-anti MP-IgM antibody and human immunoglobulin M.
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| 刘威 等: "猪肺炎支原体脂蛋白LppT的多克隆抗体制备及鉴定", 《江苏农业科学》 * |
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