CN102199207A - Preparation method for multiple mycoplasma monoclonal antibodies and multi-linked immunoassay reagent - Google Patents
Preparation method for multiple mycoplasma monoclonal antibodies and multi-linked immunoassay reagent Download PDFInfo
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Abstract
The invention relates to a preparation method for multiple mycoplasma monoclonal antibodies and a multi-linked immunoassay reagent. The preparation method is characterized by separately immunizing mice with mycoplasma pneumoniae, mycoplasma hominis and ureaplasma urealyticum for cell fusion and cloning to obtain three monoclonal antibodies with high specificity and high potency. A mycoplasma multi-linked immunoassay reagent is prepared by mixing the three monoclonal antibodies according to a certain ratio, is used for detecting the diseases infected by the three mycoplasmas, and can quickly and accurately detect mycoplasma diseases; and sampling without phlebotomizing does not bring pain to patients, thus providing patients with timely and effective treatment. The multi-linked immunoassay reagent does not generate harmful waste gas or waste water during preparation and use processes, and also does not bring any injury to patients and operators, thereby being an environment-friendly, safe, accurate and sensitive biological preparation.
Description
Technical field
The present invention relates to multiple MONOCLONAL ANTIBODIES SPECIFIC FOR method and application.
Background technology
Mycoplasma pneumoniae is the important pathogenic agent of human especially childrens respiratory tract and pulmonary infection, accounts for respiratory tract and pulmonary infection crowd 30%～40%.Molten urea urea substance (Ureaplasma urealyticum) and mycoplasma hominis are the important pathogenic agent of urogenital infections, account for urogenital infections crowd's 35%～45%.Mycoplasma is a kind of prokaryotic microorganism of volume minimum of acellular wall.Because the acellular wall of mycoplasma, there is significant difference in the treatment of sick medication treatment of mycoplasma infection and bacterium, virus infection disease, has only early stage accurately differential diagnosis to treat timely and effectively this disease.
China has the detection method of mycoplasma infection disease at present, gold mark method, culture method, PCR (polymerase chain reaction) method, present domestic popular gold mark method is to go to seek the method for the mycoplasma antibody in the blood samples of patients with antigen, the advantage of this method is easy and simple to handle, quick, and can go out examining report the same day.But this method has fatal defective, and the first can not be done early diagnosis, just the antibody that can examine can be arranged in the blood samples of patients because need behind the mycoplasma infection about 10 days.It two is to detect positive findings can not confirm as and work as subinfection, because after all previous infection cured, the antibody that produces in the body still can retain for a long time.Culture method is the characteristic of life according to mycoplasma, adopts conditioned medium that mycoplasma is cultivated and detects.The advantage of this method is easy and simple to handle, accurately.Its shortcoming is generally to need 24 hours just can go out the result, gives the timely medication of doctor, and patient in time treatment makes troubles.The PCR method is a kind of employing multiplex PCR, detects the method for mycoplasma DNA, and this method advantage is quick, sensitive, but also exists not enough.The one, need expensive equipment (PCR instrument), the 2nd, need to cultivate the specialized personnel who is always or usually as specified, otherwise just occur false negative and false positive easily.So health ministry had once been issued official document, forbid doing general clinical diagnosis with the PCR method.
Immunodetection is the antigenic method that the monoclonal antibody of employing mycoplasma removes to seek infection site secretory product, this method is simple and efficient to handle, the result is accurate, whole process does not need expensive device, it is a kind of comparatively ideal detection method, but through the new patent searching retrieval, commodity and the patent report with the mycoplasma immunologic function test reagent of mycoplasma Monoclonal Antibody do not seen by China so far.
Summary of the invention
The objective of the invention is to adopt hybridoma technology, filter out and to produce high specificity, the hybridoma cell strain of the high monoclonal antibody of tiring, produce multiple mycoplasma monoclonal antibody through amplification cultivation, with certain proportion they are mixed, be prepared into the multi-joint immunologic function test reagent of mycoplasma, can fast accurately detect various mycoplasmosiss, for the timely and effective treatment of mycoplasmosis is given security.
For reaching above purpose, the present invention takes following technical scheme to be achieved:
Multiple mycoplasma MONOCLONAL ANTIBODIES SPECIFIC FOR method is characterized in that, comprises the steps:
(1) after three kinds of reference cultures difference amplification cultivation with mycoplasma pneumoniae, mycoplasma hominis and molten urea urea substance, gets three mycoplasma species immunity original bacteria liquid: mycoplasma pneumoniae original bacteria liquid, mycoplasma hominis original bacteria liquid, molten urea urea substance original bacteria liquid;
(2) every kind of original bacteria liquid concentration is transferred to 10
5/ ml, deactivation in 50 ℃ ± 5 ℃ thermostat containers, and mix with the equivalent Freund's complete adjuvant, with the amount of every 0.2ml, the abdominal injection immune mouse, supplementary immunization was once respectively in the 7th, 14,28 day in immunity back for the first time;
Got immune mouse spleen cell in (3) the 31st days and make splenocyte suspension, concentration is transferred to 1 * 10
8/ ml; Get the sp20 murine myeloma cell again, make myeloma cell's suspension, concentration is transferred to 2～3 * 10
7/ ml carries out cytogamy with above two kinds of cell suspensions mixing;
(4) will be added to through the cell suspension of cytogamy then and auxilliary have in 96 well culture plates of feeder cell, every hole 0.1ml puts 37 ℃ ± 1 ℃ with culture plate, contains volumetric concentration 5%CO
2Incubator in cultivate, detect positive hole with the enzyme linked immunoassay method, the hybridoma that is defined as the male hole is carried out cloning with limiting dilution assay;
(5) join in the positive hybridoma cell after the cloning with serum-free medium and carry out amplification cultivation, concentration is adjusted into 1 * 10
6/ ml is inoculated into the Balb/C mouse with the amount abdominal injection of every 0.5ml, and mouse is put to death in inoculation back 7～14 days, collect ascites, centrifugal, get the ascitic type monoclonal antibody that supernatant is this mycoplasma species;
(6) adopt the enzyme linked immunoassay method, respectively to mycoplasma pneumoniae, mycoplasma hominis and molten urea urea substance ascitic type monoclonal antibody tire, hypotype and cross matching, filtering out tires is 1 * 10
-5～1 * 10
-7, high specificity mycoplasma pneumoniae monoclonal antibody, mycoplasma hominis monoclonal antibody and molten urea urea substance monoclonal antibody, wherein, described high specificity is meant except that self mycoplasma not and other mycoplasma generation cross reaction.
In the aforesaid method, the described cytogamy of step (3) comprises the steps
1. draw respectively and contain 1 * 10
8Individual splenocyte suspension, contain 2～3 * 10
7Individual myeloma cell's suspension adds in the centrifuge tube, adds incomplete nutrient solution to 30ml, fully shakes up;
2. 1000rpm is centrifugal 7 minutes, supernatant discarded;
3. add volumetric concentration and be 50% fusogen PEG solution 0.7ml;
4. add the incomplete nutrient solution of 25ml, make the PEG solution dilution;
5. 800rpm is centrifugal 7 minutes, supernatant discarded;
6. the HAT nutrient solution that adds 20ml, the pressure-vaccum sedimentation cell overhangs it and mixing gently.
A kind of multi-joint immunologic function test reagent is characterized in that, tiring of three mycoplasma species monoclonal antibodies adjusted to 1 * 10 respectively
-4, mix with 1: 1: 1 volume ratio, be that first antibody is prepared into multi-joint immunologic function test reagent with this mixing monoclonal antibody.
Advantage of the present invention is, the prepared multi-joint immunologic function test reagent that goes out can specificly detect mycoplasma pneumoniae, mycoplasma hominis and molten urea urea substance disease.Easy and simple to handle, result accurately, can go out the result same day.Whole process need not drawn blood, and avoids bringing misery to the patient; Do not need expensive device, easy to utilize.This multi-joint immunologic function test reagent can carry out suitability for industrialized production, production and use do not produce harmful exhaust waste water, patient and operator are not produced infringement, be a kind of environmental protection, safety, accurate, sensitive in-vitro diagnosis biological products, it carried out industrialization development will produce huge society and economic benefit.
Embodiment
The present invention is described in further detail below in conjunction with embodiment.
Bacterial classification:
Mycoplasma pneumoniae, mycoplasma hominis, molten urea urea substance are provided by U.S. ATCC company, and above bacterial strain produces monoclonal antibody as immunogen.Mycoplasma genitalium, penetrate mycoplasma, mainly as the monoclonal antibody cross matching also available from U.S. ATCC company.Cell strain: the strain of sp20 murine myeloma cell is provided by The Fourth Military Medical University cell engineering research centre, makes cytogamy and uses.
The preparation method:
The preparation and the deactivation of immunogen mycoplasma bacterial strain: with mycoplasma pneumoniae, mycoplasma hominis, molten each lml of urea urea substance liquid nutrient medium is added to corresponding mycoplasma pneumoniae, mycoplasma hominis, in the molten urea urea substance reference culture lyophilized powder, after the dissolving, get mycoplasma pneumoniae respectively, mycoplasma hominis, the bacterium liquid of molten urea urea substance, prepare 10 bottles of mycoplasma pneumoniae liquid nutrient mediums, 10 bottles of mycoplasma hominis liquid nutrient mediums, 10 bottles of molten urea urea substance liquid nutrient mediums, draw 100 μ l mycoplasma pneumoniae bacterium liquid respectively with pipettor, 100 μ l mycoplasma hominis bacterium liquid, the molten urea urea of 100 μ l substance bacterium liquid is added in the corresponding mycoplasma liquid nutrient medium, after cultivating into positive reaction, each mycoplasma liquid nutrient medium will be wherein 9 bottles is put-25 ℃ of refrigerators and is made seed and preserve, one bottle as the secondary amplification cultivation, and increase into 10 bottles, the back results are positive, preserve in 4 ℃ of refrigerators, in order to being used as immune original bacteria liquid.
The immunity of Balb/C mouse:
Get 30 of the Balb/C mouse in 8 ages in week, be divided into 3 groups, 10 every group, with every kind of concentration (mycoplasma number/ml) be transferred to 10 of making immunogenic mycoplasma original bacteria liquid
5/ ml, after putting deactivation in 30 minutes of 50 ℃ ± 5 ℃ thermostat containers and equivalent Freund's complete adjuvant and mixing, with the amount of every 0.2ml, abdominal injection is immune Balb/C mouse respectively.In immune back the 7th, 14,28 day for the first time difference supplementary immunization once, extracting spleen cell carried out cytogamy in the 31st day.
Cytogamy and cultivation:
(1) preparation of immune spleen cell:
1. get the immune mouse spleen, put into the plate that fills the incomplete nutrient solution of 5ml (RPMI-1640 that does not add calf serum), place on the 200 purpose stainless (steel) wires.Grind spleen with plunger, and wash stainless (steel) wire gently, splenocyte all is expressed in the solution by mesh with incomplete nutrient solution in the plate.
2. splenocyte suspension is changed in the 50ml centrifuge tube, add incomplete nutrient solution to 30ml, behind the mixing, through 1000rpm centrifugal 5 minutes, supernatant discarded.
3. the full nutrient solution that toos many or too much for use is centrifugal again with sedimentation cell, washing once (repeating step 2.), and sedimentary cell is overhang to 10ml, and mixing is with cell counting version counting.Total cell count is adjusted to 1 * 10
8
(2) preparation of sp20 murine myeloma cell suspension
1. the sp20 murine myeloma cell is carried out amplification cultivation.
2. the myeloma cell with 4 bottles of amplification cultivation collects in the centrifuge tube of 50ml.
3. 1000rpm is centrifugal 5 minutes, supernatant discarded.
4. add the incomplete nutrient solution of 30ml, repeating step 3., centrifuge washing once overhangs cell to 10ml then again, mixing, the counting.Total cell count is adjusted to 2～3 * 10
7
(3) cytogamy
1. draw respectively and contain 1 * 10
8Individual splenocyte suspension and 2～3 * 10
7Individual myeloma cell's suspension adds in the 50ml centrifuge tube, adds incomplete nutrient solution to 30ml, fully shakes up.
2. 1000rpm is centrifugal 7 minutes, supernatant discarded.
3. fusogen polyoxyethylene glycol (PEG) the solution 0.7ml that adds volumetric concentration 50%.
4. add the incomplete nutrient solution of 25ml, make PEG dilution and lose the short effect of melting.
5. 800rpm is centrifugal 7 minutes, supernatant discarded.
6. the HAT nutrient solution (the selectivity nutrient solution that contains xanthoglobulin, aminopterin, thymus pyrimidine) that adds 20ml, the pressure-vaccum sedimentation cell overhangs it and mixing gently.
(4) cell suspension is added auxilliaryly have in 96 well culture plates of feeder cell, every hole 0.1ml, containing volumetric concentration at 37 ℃ is 5%CO
2Incubator in cultivate.
Antibody positive hole detection-indirect elisa method
(1) with known mycoplasma antigen bacterium liquid bag quilt
(2) be added with culture supernatant 100 μ l in the hole of clonal growth.
(3) the anti-mouse IgG of enzyme-added mark antibody 100 μ l.
(4) add substrate solution 100 μ l, room temperature lucifuge colour developing 10～20 minutes.
(5) result of determination: under enzyme-linked immunosorbent assay instrument 492 wavelength, measure the OD value, the blank zeroing.If treat observing and controlling OD value more than or equal to 2.1 times of negative control hole OD values, the positive hole of decidable then, growing in the hole has positive colony.
Cloning-limiting dilution assay
The hole mark that culture supernatant in 96 orifice plates is detected as antibody positive comes out.With sample injector the hybridoma in the hole is blown and beaten even back counting repeatedly, carry out stepwise dilution with limiting dilution assay then, be added to 96 orifice plates again and cultivate.Observe clonal growth, record clone hole, and in time carry out positive detection.Forward the cell of positive colony to 24 orifice plates and cultivate, treat that it is bred after some amount to change enlarged culturing in the cell bottle again over to.
Hybridoma frozen
The hybridoma that will be logarithmic growth is collected in centrifuge tube, centrifugal 10 minutes of 1000rpm, and supernatant discarded adds 1ml frozen storing liquid mixing, is added to frozen pipe then, changes the liquid nitrogen container prolonged preservation over to after-70 ℃ of cryogenic refrigerators spend the night.
The ascitic type Monoclonal Antibody
With frozen hybridoma recovery amplification cultivation, the hybridoma of amplification cultivation is blown and beaten, centrifugal 10 minutes of 1000rpm, it is standby to collect supernatant.Add serum-free medium in sedimentary cell, adjusting cell count is 1 * 10
6/ ml, every Balb/C mouse peritoneal injection 0.5ml, behind the inoculation hybridoma 7～14 days, draw the neck dislocation to put to death mouse, it is centrifugal to draw ascites, and collect supernatant and be the ascitic type monoclonal antibody, packing postposition-20 ℃ refrigerator, frozen standby.
The evaluation of monoclonal antibody
The mycoplasma pneumoniae monoclonal antibody screens the hybridoma cell strain of 5 strains energy stably excreting antibody, called after 1G6,1H7,2F5,2B3,2E4 respectively.Identify that through hypotype wherein the monoclonal antibody of 1G6,2F5,2B3 generation is IgG3; The monoclonal antibody that 1H7,2E4 produce is IgG1.Through titration: tiring of above 5 kinds of ascitic type monoclonal antibodies is 1 * 10
-5～1 * 10
-7Through cross reaction measure have monoclonal antibody that two strain 1G6 and 2F5 hybridoma cell strain produce except that mycoplasma pneumoniae not with other mycoplasma generation cross reaction.
The mycoplasma hominis monoclonal antibody screens the hybridoma cell strain of six strains energy stably excreting monoclonal antibody, called after 1E3,1F8,1B2,2G8,2F7,2H5 respectively.Identify that through hypotype wherein the monoclonal antibody of 1E3,1B2,2H5 generation is IgG3; The monoclonal antibody that 1F8,2G8,2F7 produce is IgG2a.Through titration: tiring of above 6 kinds of ascitic type monoclonal antibodies is 1 * 10
-5～1 * 10
-7Through cross reaction measure monoclonal antibody that 1E3,1F8,2H5 hybridoma cell strain produce except that mycoplasma hominis not with other mycoplasma generation cross reaction.
Molten urea urea substance monoclonal antibody screens the hybridoma cell strain of five strains energy stably excreting monoclonal antibody, called after 1D2,1F5,1A6,2A3,2D3 respectively.Identify that through hypotype wherein the monoclonal antibody of 1D2,1A6,2A3 generation is IgG1; The monoclonal antibody that 1F5,2D3 produce is IgG3.Through titration: the tiring of ascitic type monoclonal antibody that above 5 strain of hybridoma produce is 1 * 10
-5～1 * 10
-7Through cross reaction measure monoclonal antibody that 1D2,2A3, hybridoma cell strain produce remove molten urea urea former external not with other mycoplasma generation cross reaction.
The multi-joint immunologic function test reagent finished product preparation of mycoplasma
With tiring respectively of anti-mycoplasma pneumoniae 1G6 monoclonal antibody, anti-mycoplasma hominis 1E3 monoclonal antibody, anti-molten urea urea substance 2A2 monoclonal antibody from original 1 * 10
-7Adjust to 1 * 10
-4, mix by 1: 1: 1 volume ratio then, be that first antibody is prepared into the multi-joint ELISA immunologic function test reagent of mycoplasma with this mixing monoclonal antibody.
The multi-joint immunologic function test reagent of this mycoplasma can detect mycoplasma pneumoniae, mycoplasma hominis and molten urea urea pathogen infection disease simultaneously.With known mycoplasma reference culture this mycoplasma immunologic function test reagent and the detection of mycoplasma substratum that obtains the registration certification are compared test, its identical rate is 100%.It is to operate more simple and efficient, reaction times weak point than the advantage of culture method, just can go out to be reported as mycoplasmosis diagnosis in time and effective the treatment same day and give security.
Claims (3)
1. many kinds of mycoplasma MONOCLONAL ANTIBODIES SPECIFIC FOR methods is characterized in that, comprise the steps:
(1) after three kinds of reference cultures difference amplification cultivation with mycoplasma pneumoniae, mycoplasma hominis and molten urea urea substance, gets three mycoplasma species immunity original bacteria liquid: mycoplasma pneumoniae original bacteria liquid, mycoplasma hominis original bacteria liquid, molten urea urea substance original bacteria liquid;
(2) every kind of original bacteria liquid concentration is transferred to 10
5/ ml, deactivation in 50 ℃ ± 5 ℃ thermostat containers, and mix with the equivalent Freund's complete adjuvant, with the amount of every 0.2ml, the abdominal injection immune mouse, supplementary immunization was once respectively in the 7th, 14,28 day in immunity back for the first time;
Got immune mouse spleen cell in (3) the 31st days and make splenocyte suspension, concentration is transferred to 1 * 10
8/ ml; Get the sp20 murine myeloma cell again, make myeloma cell's suspension, concentration is transferred to 2～3 * 10
7/ ml carries out cytogamy with above two kinds of cell suspensions mixing;
(4) will be added to through the cell suspension of cytogamy then and auxilliary have in 96 well culture plates of feeder cell, every hole 0.1ml puts 37 ℃ ± 1 ℃ with culture plate, contains volumetric concentration 5%CO
2Incubator in cultivate, detect positive hole with the enzyme linked immunoassay method, the hybridoma that is defined as the male hole is carried out cloning with limiting dilution assay;
(5) join in the positive hybridoma cell after the cloning with serum-free medium and carry out amplification cultivation, concentration is adjusted into 1 * 10
6/ ml is inoculated into the Balb/C mouse with the amount abdominal injection of every 0.5ml, and mouse is put to death in inoculation back 7～14 days, collect ascites, centrifugal, get the ascitic type monoclonal antibody that supernatant is this mycoplasma species;
(6) adopt the enzyme linked immunoassay method, respectively to mycoplasma pneumoniae, mycoplasma hominis and molten urea urea substance ascitic type monoclonal antibody tire, hypotype and cross matching, filtering out tires is 1 * 10
-5～1 * 10
-7, high specificity mycoplasma pneumoniae monoclonal antibody, mycoplasma hominis monoclonal antibody and molten urea urea substance monoclonal antibody, wherein, described high specificity is meant except that self mycoplasma not and other mycoplasma generation cross reaction.
2. multiple mycoplasma MONOCLONAL ANTIBODIES SPECIFIC FOR method as claimed in claim 1 is characterized in that the described cytogamy of step (3) comprises the steps:
1. draw respectively and contain 1 * 10
8Individual splenocyte suspension, contain 2～3 * 10
7Individual myeloma cell's suspension adds in the centrifuge tube, adds incomplete nutrient solution to 30ml, fully shakes up;
2. 1000rpm is centrifugal 7 minutes, supernatant discarded;
3. add volumetric concentration and be 50% fusogen PEG solution 0.7ml;
4. add the incomplete nutrient solution of 25ml, make the PEG solution dilution;
5. 800rpm is centrifugal 7 minutes, supernatant discarded;
6. the HAT nutrient solution that adds 20ml, the pressure-vaccum sedimentation cell overhangs it and mixing gently.
3. a multi-joint immunologic function test reagent is characterized in that, the described mycoplasma pneumoniae monoclonal antibody of claim 1, mycoplasma hominis monoclonal antibody and tiring of molten urea urea substance monoclonal antibody are adjusted to 1 * 10 respectively
-4, mix with 1: 1: 1 volume ratio, be that first antibody is prepared into multi-joint immunologic function test reagent with this mixing monoclonal antibody.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011101077740A CN102199207A (en) | 2011-04-28 | 2011-04-28 | Preparation method for multiple mycoplasma monoclonal antibodies and multi-linked immunoassay reagent |
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|---|---|---|---|
| CN2011101077740A CN102199207A (en) | 2011-04-28 | 2011-04-28 | Preparation method for multiple mycoplasma monoclonal antibodies and multi-linked immunoassay reagent |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104267195A (en) * | 2014-10-08 | 2015-01-07 | 哈德逊（天津）生物技术有限责任公司 | Multi-index inflammation detection reagent and reagent kit |
| CN106544324A (en) * | 2015-11-11 | 2017-03-29 | 北京科兴中维生物技术有限公司 | People's mycoplasma pneumoniae monoclonal antibody and its application |
| CN106814186A (en) * | 2016-12-27 | 2017-06-09 | 深圳金石邦信股权投资基金管理有限公司 | A kind of porcine mycoplasmal section Idiotype monoclonal antibody and its application in clinical detection |
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2011
- 2011-04-28 CN CN2011101077740A patent/CN102199207A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104267195A (en) * | 2014-10-08 | 2015-01-07 | 哈德逊（天津）生物技术有限责任公司 | Multi-index inflammation detection reagent and reagent kit |
| CN104267195B (en) * | 2014-10-08 | 2016-08-24 | 哈德逊（天津）生物技术有限责任公司 | A kind of inflammation multiple determination reagent and test kit |
| CN106544324A (en) * | 2015-11-11 | 2017-03-29 | 北京科兴中维生物技术有限公司 | People's mycoplasma pneumoniae monoclonal antibody and its application |
| CN106544324B (en) * | 2015-11-11 | 2020-03-17 | 北京科兴中维生物技术有限公司 | Monoclonal antibody of mycoplasma hyopneumoniae and application thereof |
| CN106814186A (en) * | 2016-12-27 | 2017-06-09 | 深圳金石邦信股权投资基金管理有限公司 | A kind of porcine mycoplasmal section Idiotype monoclonal antibody and its application in clinical detection |
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Application publication date: 20110928 |