CN106771139A - A kind of rheumatoid factor IgM chemiluminescence immune detection reagent kits and preparation method thereof - Google Patents
A kind of rheumatoid factor IgM chemiluminescence immune detection reagent kits and preparation method thereof Download PDFInfo
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- CN106771139A CN106771139A CN201611018764.9A CN201611018764A CN106771139A CN 106771139 A CN106771139 A CN 106771139A CN 201611018764 A CN201611018764 A CN 201611018764A CN 106771139 A CN106771139 A CN 106771139A
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- rheumatoid factor
- igm
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- acridinium ester
- factor igm
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The invention discloses a kind of rheumatoid factor IgM chemiluminescence immune detection reagent kits, the kit includes：The acridinium ester of the anti-human IgM marks of the coated magnetic particle of rheumatoid factor IgM monoclonal antibody, mouse, Sample dilution, rheumatoid factor IgM calibrations product, preexciting liquid, exciting liquid.In addition the invention also discloses a kind of preparation method of rheumatoid factor IgM chemiluminescence immune detection reagent kits.Kit of the present invention is easy to operate compared with available reagent box, and sensitivity is high, the advantages of detection range is wide.
Description
Technical field
The present invention relates to in-vitro diagnosis field of immunodetection, specifically, the invention provides a kind of inspection of chemiluminescence immunoassay
Survey rheumatoid factor IgM kits and preparation method thereof.
Background technology
Rheumatoid factor（Rheumatoid factor, RF）Detection is clinically the more commonly used detection project, mainly
Used in the diagnosis to autoimmune disease, particularly to the diagnosis of rheumatoid arthritis.Rheumatoid factor（RF）Be it is a kind of with
Denaturation IgG is the autoantibody of target antigen, and the predominantly IgM of 19S also shows the IgG and IgA of 7S.It is due to hemolytic hammer
Caused by the infectants such as metabolite, the slow virus of bacterium or other bacteriums.RF is initially by Rose and Waller（1984
Year）In rheumatoid arthritis（RA）Found in patients serum.It can be natural with autologous, allosome or xenogenesis or denaturation
IgG is combined, and has IgG, IgM, IgA, IgE and IgD Multiple Antibodies type RF, is primarily present in patient with rheumatoid arthritis
In serum and joint fluid.
It is generally believed that RF has two kinds of sources, one kind is abiogenous RF, and another kind is pathologic RF.It is abiogenous
RF play its regulation organism immune response, activating complement accelerate remove microorganism infection and remove immune complex make body from
The physiological actions such as the damage of circulating complexe.Pathologic RF then has pathogenic effects, makes the activity of RA conditions of patients, joint sclerotin
Destroy, necrotizing angitis and rheumatoid nodules etc. occur.The incidence of disease women of rheumatoid arthritis is higher than male, Nv Xingshi
2~3 times of male；The incidence of disease of American-European countries is apparently higher than compatriots.
Biological agent to IgM type rheumatoid factor in recent years has been had gained some understanding, and these biological agents include:
1st, vivo immunization reaction is adjusted；
2nd, activating complement, accelerates to remove microorganism infection；
3rd, removing immune complex makes body from the damage of circulating complexe.
Claim rheumatoid factor positive when only the amount of rheumatoid factor exceedes certain titre.Due to IgM types rheumatoid because
Son is the main Types of rheumatoid factor, and the characteristics of with aggegation high, it is easy to precipitated, therefore clinically main determines IgM types
Rheumatoid factor.Rheumatoid factor determines the rheumatoid factor concentration that reagent is qualitatively judged in blood of human body.Rheumatoid factor
The positive is also shown in virus infection as comment, tumour, chronic infection such as pulmonary tuberculosis, Asia are anxious in addition to rheumatoid arthritis is seen
Property bacterial endocarditis and other autoimmune diseases.
The main method of clinical detection rheumatoid factor IgM be enzyme linked immunosorbent assay, but the method exist it is following
Weak point：
（1）Using 12 × 8 types, 6 × 8 types, 8 × 12 types or the hole Special micro porous plate of complete plate 96 as antigen coat apparatus
And reaction vessel, 12 batches, 6 batches, 8 batches or whole plate first use can only be divided into when in use, it is impossible to carry out independence
, the detection of single part；
（2）Quantitative determination reagent type used is more, and each detection reagent will be contained with reagent bottle, and often be made
It is required for changing imbibition nozzle during with a kind of reagent being filled into the micropore of microwell plate respectively, not only reagent bottle species is more, filling
The operation of reagent is also extremely cumbersome；
（3）Lack the corresponding mark to detection information, can only just will appreciate that or know by checking the mark of kit external packing box
The product batch number and term of validity information of detection reagent are known, and the information known is uncontrolled in detection process, with very big
Randomness；
（4）Detection reagent, in open space, easily causes the cross pollution between various reagents and shadow in detection process
Ring the accuracy of testing result；
（5）Using manual operations more than detection process, the dosage of reagent or sample is not bery accurate, and operating process is extremely cumbersome and multiple
It is miscellaneous, bust is susceptible to, the degree of accuracy of testing result and precision are poor；
（6）In the quantity configuration of detection project reagent set and using item number × 48/96 person-portion is above, if necessary to examine
10 projects are surveyed, then the configuration of reagent and the use of number must be 10 × 48/96 person-portions, if only a sample needs detection 10
Individual different project, it is also desirable to configure the reagent of 10 × 48/96 person-portions, haves the shortcomings that inadequate economical rationality.
The content of the invention
Current rheumatoid factor IgM detection techniques have the following disadvantages：Testing cost is high, detection sensitivity is low, detection line
Property narrow range, reappearance is low, can not quantify, complex operation etc..
The present invention discloses that a kind of testing cost is low, sensitivity is high, detection is linear precisely in order to overcome the above shortcoming
Scope is wide, reappearance is high, can quantify, rheumatoid factor IgM kits simple to operate and preparation method thereof.The present invention is first
Chemical luminescence immune analysis reagent box is prepared, is mainly included：The coated magnetic particle of rheumatoid factor IgM monoclonal antibody, mouse resist
The acridinium ester and rheumatoid factor IgM calibration product of people IgM marks；Then using Full-automatic chemiluminescence immunoassay analysis meter to fixed
Mark product are detected that drafting standard curve is built in computer software, tests actual sample, and sample is calculated according to sample luminous value
Concentration；Performance finally is carried out to rheumatoid factor IgM automatic chemiluminescence immunoassays system（Sensitivity, linear, precision
Degree, interference）Evaluation.
It is of the invention compared with current technology, with advantages below：
1st, present invention selection acridinium ester is used as marker material, and is applied to chemiluminescence immunoassay system, and the luminescence system is
Direct chemiluminescence, compared with traditional enzyme-catalyzed chemical luminescence, the reaction does not need the participation of enzyme, more cost-effective；
2nd, the acridinium ester chemiluminescent immunoassay system detection sensitivity that the present invention is selected is high, can reach 0.2 IU/mL, phase
10 times are at least improve than the sensitivity of other rheumatoid factor IgM detection methods；
3rd, the acridinium ester chemiluminescent immunoassay system range of linearity that the present invention is selected is wide, can reach 5-240 IU/mL, other
Rheumatoid factor IgM chemistry hair detection method the inspection range of linearity be 10-200 IU/mL；
4th, the acridinium ester chemiluminescent immunoassay system repeatability that the present invention is selected is high, in batch and difference between batch is within 5%,
This is that other chemiluminescence immunoassay systems are unapproachable；
5th, chemiluminescence immunoassay system of the invention has realized quantifying for sample, soft to testing by built-in standard curve
Part, only needs test sample to directly obtain the concentration value of sample；
6th, chemiluminescence immunoassay system of the invention has realized full-automation, and the addition of reagent and sample has instrument complete entirely
Into operation is easier, reduces artificial error.
Brief description of the drawings
Fig. 1 is the rheumatoid factor IgM canonical plottings that embodiment 3 is obtained.
Specific embodiment
Embodiment 1：Rheumatoid factor IgM chemiluminescence immune detection reagent kit preparation methods
（1）It is prepared by the coated nanometer magnetic bead of rheumatoid factor IgM monoclonal antibody：
Take the magnetic particle of 50mg carboxylated（Particle diameter is 0.05-1um）Suspension, Magneto separate removes supernatant, and it is 5.5 to use 0.02 M, pH
MES buffer solutions are resuspended, add the EDC aqueous solution of 10 mg/mL of the new configurations of 0.5-2mL, activated magnetic beads surface carboxyl groups to add 3-5
Mg rheumatoid factor IgM monoclonal antibodies, suspension 2-10 h, Magneto separate, remove supernatant, with the 0.1M containing 2% BSA at room temperature
PH is that 8.0 Tris buffer solutions are resuspended to 1mg/mL, obtains the coated magnetic particle of rheumatoid factor IgM monoclonal antibody, every bottle
5mL packing be stored in 4 DEG C it is standby.
（2）The preparation of the acridinium ester of the anti-human IgM marks of mouse：
The anti-human IgM of mouse of 50 uL 25mg/mL is taken, the carbonate buffer solution of 150 uL 0.1-0.2 M pH 9.0-9.5 is added,
Mix, the acridinium ester for being subsequently adding the mg/mL of 1-2 uL 5 is mixed, at room temperature lucifuge reaction, is taken out after 1-2 h, with 2 mL's
Zeba is centrifuged desalting column desalting processing, is processed with pure water and TBS buffer solutions respectively first in desalination processes, is eventually adding
The acridine ester solution of the anti-human IgM marks of mouse for obtaining, collects liquid to the preservation in centrifuge tube and is in control the anti-human IgM marks of mouse
Acridinium ester, every bottle of 5 mL packing be stored in 4 DEG C it is standby.
（3）Rheumatoid factor IgM calibrates the preparation of product：
Use standard items buffer solution（40 mM Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0）By rheumatoid factor IgM configurations
Into concentration be 0.0 IU/mL, 4.9 IU/mL, 20.5 IU/mL, 50.7 IU/mL, 100.8 IU/mL, 299.1 IU/mL, often
Bottle 0.5 mL packing, 4 DEG C save backup.
(4) preparation of chemiluminescence preexciting liquid：
1.0 liters of purified waters are measured, the hydrogen peroxide (H that 80uL mass fractions are 20% is sequentially added2O2), 1.0 grams of sodium azide, 1.5
Gram polysorbas20, shakes up rear lucifuge storage.
(5) preparation of chemiluminescence exciting liquid：
1.0 liters of purified waters are measured, 0.6 gram of NaOH, 0.5 gram of PC300,0.5g sodium azide, 1.5 grams of Triton are sequentially added
405, shake up rear lucifuge storage.
Embodiment 2：Rheumatoid factor IgM chemical luminous immune detection methods：
The present invention is detection instrument with Full-automatic chemiluminescence immunoassay analysis meter, and method of the present invention pattern is indirect method, i.e.,
Instrument sequentially adds the sample of 10 uL（1:20 dilutions）, 50 uL the coated magnetic particle of rheumatoid factor IgM monoclonal antibody
After reacting 10 min, cleaning adds the acridinium ester of the anti-human IgM marks of mouse of 100 uL, after 10 min of reaction, carries out Magneto separate,
Reactant mixture is sent into darkroom by instrument, and sequentially adding 50uL chemiluminescence preexcitings liquid, 50uL chemiluminescences exciting liquid is carried out
Luminescence-producing reaction, finally records luminous intensity, and the rheumatoid factor IgM contents of sample are calculated from standard curve.
Embodiment 3：Rheumatoid factor IgM chemiluminescence immune detection reagent kit performance evaluations
Detection curve is shown in accompanying drawing 1.
The detection of sensitivity：
With reference to CLSI EP17-A file recommendation experimental programs, rheumatoid factor IgM chemiluminescence immunoassays chromatography kit is calculated
Sensitivity, the sensitivity tried to achieve be 0.2 IU/ mL.
Linear detection：
It is that 4.9 IU/mL, 20.5 IU/mL, 50.7 IU/mL, 100.8 IU/mL, 299.1 IU/mL standard items make line to concentration
Property analysis, calculate linearly dependent coefficient, r=0.9996, in addition, linear model of the kit to rheumatoid factor IgM sample detections
It is 5-240 IU/mL to enclose.
Precision is determined：
Concentration is taken for two rheumatoid factor IgM samples of 53.7 IU/mL and 206.9 IU/mL, each sample each concentration is respectively done
3 parallel, is detected with three batches of kits, and calculating kit criticizes interior and difference between batch, as a result shows in the kit batch and criticizes
Between difference be respectively less than 5%.
Interference is tested：
Taking pooled serum and adding chaff interference respectively includes：It is combined with bilirubin, unconjugated bilirubin, hemoglobin, ascorbic acid, sweet
Grease, adding proportion is according to 1:20 are carried out, and are determined pooled serum respectively and be with the addition of the survey of pooled serum after various chaff interferences
Value, calculates deviation therebetween, with ± 10% for tolerance interval.Result shows that interference reaches the file of NCCLS
Standard, can be used for the accurate evaluation of clinical trial rheumatoid factor IgM situations.
Embodiment 4:The Sensitivity comparison experiment of rheumatoid factor IgM chemiluminescence immune detection reagent kits
It is respectively the calibration object or sample of 0 IU/mL to concentration with chemical luminescence detection method and traditional enzyme linked immunosorbent assay
Dilution is detected that replication 20 times draws 20 RLU values of measurement result for sample（Relative light unit）, calculate it
Average value（M）And standard deviation（SD）, M+2SD is drawn, luminous value substitution calibration curve is calculated corresponding concentration value.Adopt
The concentration value obtained with chemical luminescence detection method is 0.2 IU/mL, relative to traditional enzyme linked immunosorbent assay lowest detection
2.5 IU/mL are limited, about 12 times are improve.
Claims (10)
1. a kind of rheumatoid factor IgM chemiluminescence immune detection reagent kits, the kit includes：Rheumatoid factor IgM is mono-
Clonal antibody is coated-nanometer magnetic microsphere, chemiluminescent labels, Chemoluminescent substrate, rheumatoid factor IgM calibration product.
2. kit according to claim 1, it is characterised in that the rheumatoid factor IgM monoclonal antibody is coated
Solid phase carrier is magnetic particle.
3. kit according to claim 1, it is characterised in that the rheumatoid factor IgM monoclonal antibody is coated
Solid phase carrier is 0.05-1um magnetic particles for the particle diameter of carboxylated.
4. kit according to claim 1, it is characterised in that the chemiluminescent labels are acridinium ester, acridinium ester
Sulfonamide, acridinium ester toluenesulfonamide, acridinium ester are to methylsulfonamides, acridinium ester trimethyl fluoride sulfonyl amine.
5. kit according to claim 1, it is characterised in that the preferred acridinium ester of chemiluminescent labels.
6. kit according to claim 1, it is characterised in that the Chemoluminescent substrate is excited including chemiluminescence
Liquid, chemiluminescence preexciting liquid.
7. kit according to claim 1, it is characterised in that the chemiluminescence preexciting liquid is mass fraction
0.005% ~ 0.5% hydrogen peroxide (H2O2) solution, exciting liquid is the NaOH of 0.005mol/L ~ 0.025mol/L
(NaOH) solution.
8. kit according to claim 1, it is characterised in that the rheumatoid factor IgM calibrations product are to use standard items
Buffer solution by rheumatoid factor IgM be configured to concentration for 0.0 IU/mL, 4.9 IU/mL, 20.5 IU/mL, 50.7 IU/mL,
100.8 IU/mL, 299.1 IU/mL, 4 DEG C save backup.
9. kit according to claim 1, it is characterised in that the preparation method of the kit, it is characterised in that bag
Include preparation, preparation, the chemistry of the acridinium ester of the anti-human IgM marks of mouse of the coated magnetic particle of rheumatoid factor IgM monoclonal antibody
The preparation of product is calibrated in the preparation of luminous substrate liquid, rheumatoid factor IgM.
10. according to claim 1 and claim 9 kit preparation method, it is characterised in that comprise the following steps：
1）The preparation of the coated magnetic particle of rheumatoid factor IgM monoclonal antibody：
The nanometer magnetic bead suspension of carboxylated is taken, Magneto separate removes supernatant, and MES buffer solutions are resuspended, add the EDC aqueous solution, activate magnetic
Bead surface carboxyl, adds rheumatoid factor IgM monoclonal antibody, at room temperature suspension 2-10 h, and Magneto separate removes supernatant, Tris
Buffer solution is resuspended, obtains the coated magnetic particle of rheumatoid factor IgM monoclonal antibody；Optionally, carboxylated nanometer magnetic bead diameter
It is 0.1 μm ~ 2.0 μm；MES buffer concentrations are 10mM ~ 100mM, pH 5.5 ~ 8.5；
2）The preparation of the acridinium ester of the anti-human IgM marks of mouse：
The anti-human IgM of mouse is taken, carbonate buffer solution is added, mixed, be subsequently adding acridinium ester mixing, at room temperature lucifuge reaction, 1-2 h
After take out, be centrifuged desalting column desalting processing, processed with pure water and TBS buffer solutions respectively first in desalination processes, finally
The acridine ester solution of the anti-human IgM marks of mouse that addition is obtained, collects liquid to the preservation in centrifuge tube and is in control the anti-human IgM marks of mouse
The acridinium ester of note；
3）Rheumatoid factor IgM calibrates the preparation of product：
With standard items buffer solution by rheumatoid factor IgM be configured to concentration for 0.0 IU/mL, 4.9 IU/mL, 20.5 IU/mL,
50.7 IU/mL, 100.8 IU/mL, 299.1 IU/mL, 4 DEG C save backup；
4）The preparation of chemiluminescence preexciting liquid：
1.0 liters of purified waters are measured, the hydrogen peroxide (H that 0.5 ~ 100uL mass fractions are 20% is sequentially added2O2), 0.5 ~ 5 gram prevent
Rotten agent, 0.5 ~ 5 gram of surfactant, shake up rear lucifuge storage；Optionally, preservative be commercialization sodium azide, PC300,
Surfactant is polysorbas20, Tween 80, Triton X100, Triton 405；
5）The preparation of chemiluminescence exciting liquid：
1.0 liters of purified waters are measured, 0.2 ~ 1 gram of NaOH, 0.5 ~ 5 gram of preservative, 0.5 ~ 5 gram of surface is sequentially added and is lived
Property agent, shake up the storage of rear lucifuge；Optionally, preservative is commercialization sodium azide, PC300, and surfactant is polysorbas20, tells
Temperature 80, Triton X100, Triton 405.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201620686118 | 2016-06-30 | ||
| CN2016206861189 | 2016-06-30 |
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| CN106771139A true CN106771139A (en) | 2017-05-31 |
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Citations (7)
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|---|---|---|---|---|
| CN103185788A (en) * | 2011-12-30 | 2013-07-03 | 深圳市亚辉龙生物科技有限公司 | Reagent device for detecting rheumatoid factor and method thereof |
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2016
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| Title |
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