CN109738642A - The kit and its detection method of dual-antigen sandwich method detection immunoglobulin M - Google Patents
The kit and its detection method of dual-antigen sandwich method detection immunoglobulin M Download PDFInfo
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Abstract
The present invention relates to the kits and its detection method of a kind of dual-antigen sandwich method detection immunoglobulin M.Using IgM pentamer structure, an IgM molecule can be detected with multiple antigen bindings using double antigens sandwich spatial neighbor chemiluminescence technique；Kit includes: enzyme marker, luminous marker, adjuvant, triggering agent and calibration object；Enzyme marker includes the antigen of peroxidase labelling；Luminous marker includes the antigen and Tris buffer of acridan label；Adjuvant includes shine adjuvant and citrate buffer.The homogeneous chemistry luminescence technology of dual-antigen sandwich method detection MP-IgM of the present invention, using double antigens sandwich spatial neighbor luminesceence analysis detection technique, without carrier, it is not coated with, washing process, reagent constituents are few, the detection sensitivity and specificity for substantially increasing MP-IgM, so that testing result is more genuine and believable；Reaction time and reaction step are optimized simultaneously, keeps operation simpler.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of reagent of dual-antigen sandwich method detection immunoglobulin M
Box and its detection method, the technology utilize IgM pentamer structure, IgM molecule can with multiple antigen bindings, and using dual anti-
Former sandwich spatial neighbor chemiluminescence technique detection.
Background technique
IgM is the abbreviation of Immunoglobulin M, i.e. immunoglobulin M.According to the different by immunoglobulin of structure
It is divided into five kinds of IgA, IgG, IgE, IgD and IgM；Wherein, IgM is pentamer, is Ig middle-molecular-weihydroxyethyl the maximum, and molecular structure is in ring
Shape.IgM is primarily present in blood, accounts for the 5%~10% of serum immune globulin total amount, serum-concentration about 1mg/mL.IgM is
The antibody generated at first in humoral immune response after antibody that ontogeny synthesizes earliest and antigenic stimulus, is the anti-sense of body
The vanguard of dye, serum IgM level increases in course of infection, illustrates recent infection.Therefore clinically by detection serum IgM
Early diagnosis of the level for infection.
Respiratory tract infection is relatively conventional one of the disease of children, because to account for children dead for death of child caused by respiratory tract infection
Die 20% or so of rate.However in recent years, being widely used due to antibiotic, the bacterium infection of respiratory tract show decline and become
Gesture, and respiratory tract infection caused by virus, mycoplasma pneumoniae, chlamydia pneumoniae etc. gradually increases, and accounts for childrens respiratory tract infection
80%, huge harm, therefore the diagnosis of fast explicit pathogenic infection are constituted to the health of children, clinical rational is referred to
Determine therapeutic scheme, reduction abuse of antibiotics, the reduction unnecessary economic loss of patient to be of great significance.By detecting pathogen
IgM antibody in serum carries out etiological diagnosis for childrens respiratory tract infection, has obtained satisfied effect.
Mycoplasma pneumoniae (MP) infection can occur at any age, especially common with 5~20 years old.The past foreign data is also shown
Show, school-ager and youth are more common in MP infection.The disease incidence of Eaton agent pneumonia can account for all pneumonia disease incidence 20%~
30%, in epidemic peak, its disease incidence can reach the 70%~80% of pneumonia total incidence.After MP infection, go out earliest in serum
Existing is specific IgM antibodies, therefore the detection of MP-IgM class antibody is that early diagnosis MP related disease is most important and most main
The early detection of the means wanted, MP has very important significance to treatment disease as caused by MP.
Currently used dual-antigen sandwich method detects Ig, is to add an Antigen adsorption in solid phase surface or magnetic bead surfaces
The antigen binding for entering special Ig and absorption adds the antigen of enzyme label in conjunction with Ig, adds substrate and shine, to detect
The content of Ig.Whole process needs to be coated with and wash, and reaction principle is as shown in Figure 1.
Summary of the invention
For the defects in the prior art, it is an object of that present invention to provide a kind of dual-antigen sandwich methods to detect immunoglobulin
The kit and its detection method of M.The present invention utilizes IgM pentamer structure, IgM molecule can with multiple antigen bindings, and
It is detected using double antigens sandwich spatial neighbor chemiluminescence technique, is a kind of homogeneous chemistry luminescence technology truly, nothing
Carrier is needed, is not coated with, washing process, reagent constituents are few, substantially increase the detection sensitivity of mycoplasma pneumoniae (MP) IgM
And specificity, so that testing result is more genuine and believable；Reaction time and reaction step are optimized simultaneously, keeps operation simpler.
To achieve the above object, technical solution provided by the invention are as follows:
In a first aspect, the present invention provides the detection kit of IgM a kind of, kit utilizes IgM pentamer structure, one
IgM molecule and multiple antigen bindings, and detected using double antigens sandwich spatial neighbor chemiluminescence technique；Preferably, kit
It is horizontal using dual-antigen sandwich method detection IgM, it specifically includes: enzyme marker, luminous marker, adjuvant, triggering agent and calibration
Product；Wherein, the raw material components of enzyme marker include the antigen of peroxidase labelling, and the raw material components of luminous marker include 9,
The antigen of 10- acridan label.
Preferably, the raw material components of enzyme marker further include 0.05M phosphate buffer；It is highly preferred that preparation method packet
It includes: weighing 5mg HRP and be dissolved in 1mL distilled water, the 0.1M sodium periodate solution that 0.2mL newly matches is added later, keeps away at room temperature
Light stirs 20min, and above-mentioned solution is fitted into bag filter, dialyses to the sodium-acetate buffer of 1mM pH 4.4, and 4 DEG C overnight；Add
20 μ L 0.2M pH, 9.5 carbonate buffer solution, addition 0.1~1.0mg antigen room temperature, which is protected from light, immediately after is gently mixed 2h, adds
The 4mg/mL sodium borohydride liquid newly matched is mixed, in 4 DEG C of placement 2h, above-mentioned liquid is fitted into bag filter, to 0.15M pH
7.4PBS dialysis, 4 DEG C overnight；Dialysate is taken out, adds qs glycerin to set -20 DEG C of refrigerators and saves.
Preferably, the raw material components of luminous marker further include 0.05M Tris buffer；It is highly preferred that preparation method packet
Include: the luminous substrate Acridan DMF of 500 μ L dissolves；The Acridan41.3 μ L of dissolution is drawn, it is slow that 0.05M Boratex is added
708.7 μ L of fliud flushing adds 50~250 μ g antigens, and overturning mixes 4~5 times, stands 30min at room temperature；Reaction tube will be marked
It is placed on shaking table, is mixed at 2~8 DEG C overnight, take out addition qs glycerin and set -20 DEG C of refrigerators preservations.
Preferably, the component of adjuvant includes 6.0 citrate buffer of adjuvant and pH that shines；It is highly preferred that preparation
Method includes: to weigh citric acid 1.82g, and sodium citrate 10.45g adds pure water to dissolve and is settled to 1000mL, slow in citrate
Suitable luminous adjuvant is added in fliud flushing, is dispensed after mixing, every bottle of 10mL is placed in 4 DEG C of refrigerators and saves backup.
Preferably, 0.05M pH 8.0Tris-HCl buffer is selected in triggering agent；It is highly preferred that preparation method includes: to claim
Tris 6.06g, sodium chloride 9g are taken, appropriate pure water is added to dissolve, dense HCl 2.1mL is added and mixes.It is added and spits in above-mentioned solution
- 20 2mL of temperature are settled to 1000mL after mixing, and dispense after mixing, every bottle of 200mL is placed in 4 DEG C of refrigerators and saves backup.
Preferably, antigen selects mycoplasma pneumoniae antigen.
Preferably, calibration object includes MP-IgM calibration object and 0.1M phosphate buffer；It is highly preferred that preparation method packet
It includes: preparing calibration object dilution: weighing potassium dihydrogen phosphate 14.1g, NaH2PO4·2H2O 3.0g, adds ultrapure water to dissolve,
0.5~1mL of Proclin-300 after mixing, adds ultrapure water to be settled to 1000mL, obtains calibration object dilution, 2~8 DEG C of storages
It is spare；It prepares calibration object: aimed concn being diluted to for MP-IgM is purified using calibration object dilution, 2~8 DEG C store for future use.
Kit of the present invention detects the content of special IgM in human serum (slurry) using dual-antigen sandwich method.By sample, horseradish
The MP antigen of peroxidase (HRP) label, the MP antigen one of 9,10- acridan (Acridan) label react, and two anti-
Former marker in conjunction with same IgM molecule, is able to them spatially close to each other simultaneously, and triggering agent adjuvant and touching is added
Agent is sent out, flash type chemiluminescence is generated；And unbonded free HRP labelled antigen and Acridan labelled antigen does not shine then.
MP-IgM content is higher in sample, and the luminous value (RLU) of measurement is bigger.So passing through the luminous value of calibration object and the hair of sample
Light value calculates COI, and result is used for clinical diagnosis.
Second aspect, application of the kit provided by the invention in dual-antigen sandwich method detection mycoplasma pneumoniae IgM.
The third aspect, above-mentioned detection kit provided by the invention is when dual-antigen sandwich method detects mycoplasma pneumoniae IgM
Method, comprising steps of S1: anti-respectively at the MP that 25 μ L standard items/sample, 25 μ L peroxidase labellings are added in reaction tube
Former and 25 μ L luminous markers, concussion mix；S2: in 37 DEG C of constant-temperature incubation 15min；S3: 5 μ L adjuvants are separately added into reaction
Pipe, concussion mix, and stand 1~2min；It is separately added into triggering 75 μ L of agent later, concussion is mixed, detected immediately, reads signal value；
S4: COI is calculated according to calibration object and sample luminous value；Wherein, COI>=1 is the positive, and COI<1 is feminine gender.
Technical solution provided by the invention, have it is following the utility model has the advantages that
(1) IgG, IgE, IgD, serotype IgA are the basic structure being made of a pair of of L chain and a pair of H chain, i.e. monomer；And
IgM is the pentamer there are five monomer composition, has 10 antigen binding sites, structural schematic diagram is as shown in Figure 2.The present invention makes
With spatial neighbor chemiluminescence, when being marked with the antigen of HRP and be marked with the antigen of 9,10- acridan (Acridan)
It when in conjunction with same IgM molecule, is able to them spatially close to each other, adjuvant and triggering agent is added, generates flash type
Chemiluminescence.Whole process is homogeneous reaction, is also not required to be coated with and wash.And traditional IgM detection method, usually with anti-human
IgM antibody (antiantibody) captures IgM, adds antigenic label and substrate shines, disturbing factor is more.In other words, of the invention
According to the pentamer structure of IgM, dexterously it is combined with that the sandwich method of same IgM molecule detects using double antigenic labels
IgM, sensitive special, disturbing factor is few.
(2) the homogeneous chemistry luminescence technology of dual-antigen sandwich method detection IgM of the present invention comprising any not synantigen；This hair
It is bright also entirely different with common spatial neighbor chemiluminescence (double antibody sandwich method).
(3) it is needed with traditional chemiluminescence using microwell plate or magnetic particle as carrier coated antibody or antigen phase
Than, carrier is not necessarily in kit provided by the invention and detection method, is not coated with, washing process, be it is a kind of truly
Homogeneous chemistry luminescence technology.
(4) traditional enzyme-catalyzed chemical luminescence technology is in the test analyte successively detection antibody with capture antibody and enzyme label
It must be cleaned after combining 2~3 times, to remove the unstable non-specific substance of be not associated with or combination；And the present invention is
After the combination of test analyte and two specific antigens, the interference that shines is eliminated by adjuvant effect, whole process is without washing
It washs.
(5) traditional enzyme-catalyzed chemical luminescence technology needs substrate for enzymatic activity, and 5~10min or so detects luminous value；And this hair
Bright is flash type chemiluminescence, generates luminous signal immediately after triggering agent is added.
(6) reagent constituents provided by the invention are few, and production process is simple, and production cost reduces, and are easy to amplify production；And
Detection process is convenient, it is easy to accomplish full-automatic.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Detailed description of the invention
Fig. 1 is the reaction principle figure that currently used dual-antigen sandwich method detects Ig in background of invention；
Fig. 2 is the schematic arrangement of IgM of the present invention.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical solution of the present invention is clearly and completely described.Implement below
Example is only used for clearly illustrating technical solution of the present invention, therefore is intended only as example, and cannot be used as a limitation and limit this hair
Bright protection scope.
Experimental method in following embodiments is unless otherwise specified conventional method.Examination as used in the following examples
Material is tested, is to be commercially available from routine biochemistry reagent shop unless otherwise specified.Quantitative test in following embodiment,
Three repeated experiments are respectively provided with, data are the average value or mean+SD of three repeated experiments.
The present invention provides the detection kit of IgM a kind of, which utilizes IgM pentamer structure, IgM molecule and more
A antigen binding, and detected using double antigens sandwich spatial neighbor chemiluminescence technique；Preferably, kit is using dual anti-former folder
Heart method detects IgM level, specifically includes: enzyme marker, luminous marker, adjuvant, triggering agent and calibration object；Wherein, enzyme mark
The component of note object includes the antigen and 0.05M phosphate buffer of peroxidase labelling；The component of luminous marker includes 9,
The antigen and 0.05M Tris buffer of 10- acridan label；The component of adjuvant includes 6.0 Chinese holly of adjuvant and pH that shines
Same regimen acid salt buffer；It triggers agent and selects 0.05M pH 8.0Tris-HCl buffer；Calibration object include MP-IgM calibration object and
0.1M phosphate buffer.
One, the preparation of calibration object
(1) it prepares calibration object dilution: weighing potassium dihydrogen phosphate 14.1g, sodium dihydrogen phosphate (2H2O) 3.0g adds ultrapure water
Dissolution, 0.5~1mL of Proclin-300 after mixing, add ultrapure water to be settled to 1000mL, obtain phosphate buffer, that is, calibrate
Product dilution, 2~8 DEG C store for future use.
(2) it prepares calibration object: suitable concentration being diluted to for MP-IgM is purified using phosphate buffer, 2~8 DEG C of storages are standby
With.
Two, the preparation of enzyme marker
(1) it weighs 5mg HRP to be dissolved in 1mL distilled water, it is molten that the 0.1M sodium periodate that 0.2mL newly matches is added in upper liquid
Liquid is protected from light stirring 20min at room temperature, above-mentioned solution is fitted into bag filter, dialyses to the sodium-acetate buffer of 1mM pH 4.4,
4 DEG C overnight.
(2) add 20 μ L 0.2M pH, 9.5 carbonate buffer solution, 0.1~1.0mg antigen room temperature is added immediately after and is protected from light
It is gently mixed 2h, adds the 4mg/mL sodium borohydride liquid newly matched, mixes, in 4 DEG C of placement 2h, above-mentioned liquid is fitted into bag filter, it is right
0.15M pH 7.4PBS dialysis, 4 DEG C overnight.
(3) dialysate is taken out, adds qs glycerin to set -20 DEG C of refrigerators and saves.
Three, the preparation of luminous marker
(1) the luminous substrate Acridan DMF of 500 μ L is dissolved.
(2) the 41.3 μ L of Acridan for drawing dissolution, is added 708.7 μ L of 0.05M sodium borate buffer liquid, add 50~
250 μ g antigens, overturning mix 4~5 times, stand 30min at room temperature.
(3) label reaction tube is placed on shaking table, is mixed at 2~8 DEG C overnight, taken out addition qs glycerin and set -20 DEG C
Refrigerator saves.
Four, the preparation of adjuvant
Citric acid 1.82g, sodium citrate 10.45g are weighed, pure water is added to dissolve and is settled to 1000mL, it is slow in citrate
The adjuvant that shines is added in fliud flushing, is dispensed after mixing, is placed in 4 DEG C of refrigerators and saves backup；Wherein, more preferably per bottled 10mL.
Five, the preparation of agent is triggered
Tris 6.06g, sodium chloride 9g are weighed, appropriate pure water is added to dissolve, dense HCl 2.1mL is added and mixes；It is added later
Tween-20 2mL is settled to 1000mL after mixing, packing is placed in 4 DEG C of refrigerators and saves backup；Wherein, more preferably per bottled
200mL。
Use IgM detection kit when dual-antigen sandwich method detects mycoplasma pneumoniae IgM the present invention also provides a kind of
Method, comprising steps of
S1: respectively at the MP antigen and 25 μ L that 25 μ L standard items/sample, 25 μ L peroxidase labellings are added in reaction tube
Luminous marker, concussion mix.
S2: in 37 DEG C of constant-temperature incubation 15min.
S3: being separately added into 5 μ L adjuvants to chemiluminescence reaction pipe, and concussion mixes, and stands 1~2min；Add respectively later
Enter to trigger 75 μ L of agent, concussion is mixed, detected immediately, reads signal value.
S4: critical value index number COI is calculated according to calibration object and sample luminous value；Wherein, COI>=1 is the positive, COI<1
It is as negative.
Technical solution provided by the invention is described further combined with specific embodiments below.
Embodiment one
The present embodiment provides the detection kits of MP-IgM a kind of, comprising: enzyme marker, luminous marker, adjuvant, touching
Send out agent and calibration object；Wherein, calibration object includes MP-IgM calibration object and 0.1M phosphate buffer；Enzyme marker includes peroxidating
The MP antigen and 0.05M phosphate buffer of object enzyme label；Luminous marker include acridan label MP antigen and
0.05M Tris buffer；The component of adjuvant includes 6.0 citrate buffer of adjuvant and pH that shines；Agent is triggered to select
0.05M pH 8.0Tris-HCl buffer.
One, the preparation of calibration object
(1) it prepares phosphate buffer: weighing potassium dihydrogen phosphate 14.1g, sodium dihydrogen phosphate (2H2O) 3.0g adds ultrapure water
Dissolution, Proclin-300 0.8mL after mixing, add ultrapure water to be settled to 1000mL, obtain phosphate buffer, and 4 DEG C of storages are standby
With.
(2) it prepares calibration object: suitable concentration being diluted to for MP-IgM is purified using phosphate buffer, 4 DEG C store for future use.
Two, the preparation of enzyme marker
(1) it weighs 5mg HRP to be dissolved in 1mL distilled water, it is molten that the 0.1M sodium periodate that 0.2mL newly matches is added in upper liquid
Liquid is protected from light stirring 20min at room temperature, above-mentioned solution is fitted into bag filter, dialyses to the sodium-acetate buffer of 1mM pH 4.4,
4 DEG C overnight；
(2) add 20 μ L 0.2M pH, 9.5 carbonate buffer solution, 0.1mg MP antigen room temperature is added immediately after and is protected from light gently
Light stirring 2h, adds the 4mg/mL sodium borohydride liquid newly matched, mixes, in 4 DEG C of placement 2h, above-mentioned liquid is fitted into bag filter, right
0.15M pH 7.4PBS dialysis, 4 DEG C overnight；
(3) dialysate is taken out, adds qs glycerin to set -20 DEG C of refrigerators and saves.
Three, the preparation of luminous marker
(1) the luminous substrate Acridan DMF of 500 μ L is dissolved；
(2) the 41.3 μ L of Acridan for drawing dissolution, is added 708.7 μ L of 0.05M sodium borate buffer liquid, adds 100 μ g
MP antigen, overturning mix 5 times, stand 30min at room temperature；
(3) label reaction tube is placed on shaking table, is mixed at 4 DEG C overnight, taken out addition qs glycerin and set -20 DEG C of refrigerators
It saves.
Four, the preparation of adjuvant
Citric acid 1.82g, sodium citrate 10.45g are weighed, pure water is added to dissolve and is settled to 1000mL, it is slow in citrate
The adjuvant that shines is added in fliud flushing, is dispensed after mixing, every bottle of 10mL is placed in 4 DEG C of refrigerators and saves backup.
Five, the preparation of agent is triggered
Tris 6.06g, sodium chloride 9g are weighed, appropriate pure water is added to dissolve, dense HCl 2.1mL is added and mixes；It is added later
Tween-20 2mL is settled to 1000mL after mixing, packing, every bottle of 200mL is placed in 4 DEG C of refrigerators and saves backup.
Embodiment two
The present embodiment provides it is a kind of using IgM detection kit dual-antigen sandwich method detect mycoplasma pneumoniae IgM when
Method, comprising steps of
S1: respectively at the MP antigen and 25 μ L that 25 μ L standard items/sample, 25 μ L peroxidase labellings are added in reaction tube
Luminous marker, concussion mix.
S2: in 37 DEG C of constant-temperature incubation 15min.
S3: being separately added into 5 μ L adjuvants to chemiluminescence reaction pipe, and concussion mixes, and stands 1~2min；Add respectively later
Enter to trigger 75 μ L of agent, concussion is mixed, detected immediately, reads signal value.
S4: critical value index number COI is calculated according to calibration object and sample luminous value；Wherein, COI>=1 is the positive, COI<1
It is as negative.
The present invention detects MP-IgM using dual-antigen sandwich method, to the mycoplasma pneumoniae early infection patient to clarify a diagnosis,
MP-IgM Positive rate is 100% to 20 healthy population MP-IgM detection negative rates up to 95%, so, MP-IgM
Diagnosing total coincidence rate for mycoplasma pneumoniae infection is 97.5%.
Certainly, the case where being enumerated in addition to embodiment one and embodiment two, other conditions and parameter in preparation process etc.
It is possible.
Applicant has found after creative work: the present invention utilizes IgM pentamer structure, IgM molecule can with it is more
A antigen binding, and detected using double antigens sandwich spatial neighbor chemiluminescence technique, it is a kind of homogeneousization truly
Luminescence technology is learned, carrier is not necessarily to, is not coated with, washing process, reagent constituents are few, substantially increase mycoplasma pneumoniae (MP)
The detection sensitivity and specificity of IgM, so that testing result is more genuine and believable；Reaction time and reaction step are optimized simultaneously,
Keep operation simpler.
It should be noted that unless otherwise indicated, technical term or scientific term used in this application should be this hair
The ordinary meaning that bright one of ordinary skill in the art are understood.Unless specifically stated otherwise, it otherwise illustrates in these embodiments
Component and opposite step, numerical expression and the numerical value of step are not limit the scope of the invention.It is illustrated and described herein
In all examples, unless otherwise prescribed, any occurrence should be construed as merely illustratively, not as limitation, because
This, other examples of exemplary embodiment can have different values.
In the description of the present invention, it is to be understood that, term " first ", " second " are used for description purposes only, and cannot
It is interpreted as indication or suggestion relative importance or implicitly indicates the quantity of indicated technical characteristic.Define as a result, " the
One ", the feature of " second " can explicitly or implicitly include one or more of the features.In the description of the present invention,
The meaning of " plurality " is two or more, unless otherwise specifically defined.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme should all cover in protection scope of the present invention.
Claims (9)
1. a kind of detection kit of IgM, it is characterised in that:
The kit utilizes IgM pentamer structure, an IgM molecule and multiple antigen bindings, and empty using double antigens sandwich
Between neighbouring chemiluminescence technique detect；Preferably, the kit is horizontal using dual-antigen sandwich method detection IgM, specifically includes:
Enzyme marker, luminous marker, adjuvant, triggering agent and calibration object；Wherein, the raw material components of the enzyme marker include peroxide
The antigen of compound enzyme label, the raw material components of the luminous marker include the antigen of 9,10- acridan label.
2. the detection kit of IgM according to claim 1, it is characterised in that:
The raw material components of the enzyme marker further include 0.05M phosphate buffer；
Preferably, preparation method includes:
It weighs 5mg HRP to be dissolved in 1mL distilled water, the 0.1M sodium periodate solution that 0.2mL newly matches is added later, keeps away at room temperature
Light stirs 20min, and above-mentioned solution is fitted into bag filter, dialyses to the sodium-acetate buffer of 1mM pH 4.4, and 4 DEG C overnight；
20 μ L 0.2M pH, 9.5 carbonate buffer solution is added, addition 0.1~1.0mg antigen room temperature, which is protected from light, immediately after gently stirs
2h is mixed, the 4mg/mL sodium borohydride liquid newly matched is added, mixes, in 4 DEG C of placement 2h, above-mentioned liquid is fitted into bag filter, to 0.15M
PH 7.4PBS dialysis, 4 DEG C overnight；
Dialysate is taken out, adds qs glycerin to be placed on -20 DEG C of refrigerators and saves.
3. the detection kit of IgM according to claim 1, it is characterised in that:
The raw material components of the luminous marker further include 0.05M Tris buffer；
Preferably, preparation method includes:
The luminous substrate Acridan DMF of 500 μ L is dissolved；
The 41.3 μ L of Acridan of dissolution is drawn, 708.7 μ L of 0.05M sodium borate buffer liquid is added, it is anti-to add 50~250 μ g
Original, overturning mix 4~5 times, are stored at room temperature 30min；
Label reaction tube is placed on shaking table, is mixed at 2~8 DEG C overnight, addition qs glycerin is taken out and is placed on -20 DEG C of refrigerators
It saves.
4. the detection kit of IgM according to claim 1, it is characterised in that:
The component of the adjuvant includes the citrate buffer of luminous adjuvant and pH 6.0；
Preferably, preparation method includes:
Citric acid 1.82g, sodium citrate 10.45g are weighed, pure water is added to dissolve and is settled to 1000mL, in citrate buffer
It is middle that the adjuvant that shines is added, it is dispensed after mixing, is placed in 4 DEG C of refrigerators and saves backup；Wherein, more preferably per bottled 10mL.
5. the detection kit of IgM according to claim 1, it is characterised in that:
0.05M pH 8.0Tris-HCl buffer is selected in the triggering agent；
Preferably, preparation method includes:
Tris 6.06g, sodium chloride 9g are weighed, appropriate pure water is added to dissolve, dense HCl 2.1mL is added and mixes；It is added and spits later
- 20 2mL of temperature, are settled to 1000mL after mixing, packing is placed in 4 DEG C of refrigerators and saves backup；Wherein, more preferably per bottled 200mL.
6. the detection kit of described in any item IgM according to claim 1~5, it is characterised in that:
The antigen selects mycoplasma pneumoniae antigen.
7. the detection kit of IgM according to claim 6, it is characterised in that:
The calibration object includes MP-IgM calibration object and 0.1M calibration object dilution；
Preferably, preparation method includes:
It prepares calibration object dilution: weighing potassium dihydrogen phosphate 14.1g, NaH2PO4·2H2O 3.0g, adds ultrapure water to dissolve,
0.5~1mL of Proclin-300 after mixing, adds ultrapure water to be settled to 1000mL, obtains calibration object dilution, 2~8 DEG C of storages
It is spare；
It prepares calibration object: aimed concn being diluted to for MP-IgM is purified using the calibration object dilution, 2~8 DEG C store for future use.
8. application of the described in any item kits of claim 1~7 in dual-antigen sandwich method detection mycoplasma pneumoniae IgM.
9. method of the described in any item kits of claim 1~7 when dual-antigen sandwich method detects mycoplasma pneumoniae IgM,
Comprising steps of
S1: it shines respectively at 25 μ L standard items/sample, the MP antigen of 25 μ L peroxidase labellings and 25 μ L are added in reaction tube
Marker, concussion mix；
S2: in 37 DEG C of constant-temperature incubation 15min；
S3: being separately added into 5 μ L adjuvants to reaction tube, and concussion mixes, and stands 1~2min；It is separately added into triggering 75 μ L of agent later,
Concussion mixes, and detects immediately, reads signal value；
S4: critical value index number COI is calculated according to calibration object and sample luminous value；Wherein, COI>=1 is the positive, and COI<1 is
It is negative.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110286236A (en) * | 2019-07-25 | 2019-09-27 | 科尼格沃斯(无锡)医学科技有限公司 | The detection method of mycoplasma pneumoniae detection kit, preparation method and mycoplasma pneumoniae |
| CN113759109A (en) * | 2021-08-20 | 2021-12-07 | 捷和泰（北京）生物科技有限公司 | Chemiluminescence immunoassay kit for detecting anti-SS-B antibody by double-antigen sandwich method |
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