CN108535491B - A kind of latex enhancing immune of Troponin I is than turbid detection kit - Google Patents
A kind of latex enhancing immune of Troponin I is than turbid detection kit Download PDFInfo
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N2800/00—Detection or diagnosis of diseases
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Abstract
This application involves a kind of latex enhancing immunes of Troponin I than turbid detection kit.Specifically, kit includes the first reagent and the second reagent；First reagent includes buffer, blocking agent, surfactant, electrolyte, stabilizer, polyethylene glycol, discrete dose, preservative；Second reagent is coated with latex particle, buffer, surfactant, stabilizer, the preservative of anti-human Troponin I monoclonal antibody.The kit of the application shows better anti-interference ability.
Description
Technical field
This Gong Kai Wataru and medicine, immune and in-vitro diagnosis field, Ju Ti Wataru and a kind of Immunity transmission turbidity detection flesh
The kit of calcium protein I.
Background technique
Cardiac muscle troponin I (cTn I) is one of three kinds of subunits of cardiac troponin (cTn), in addition two kinds of subunits point
It Wei not serum cardiac troponin T (cTnT) and troponin C (TnC).CTnI is about made of 209 amino acid residues, molecular weight
23.9kD, isoelectric point 9.87 are a kind of protein rich in α spiral.Only 3-6% is present in cytoplasm to cTnI in a free form
In, the overwhelming majority is present on actin filament in the form of compound.After myocardial damage, cardiac troponin complex is discharged into
In blood, after 4-6 hours, start to increase in blood, raised Troponin I can be kept in blood 6-10 days.
In these protein related with Muscle cell contract, Troponin I has height Cardiac-specific and sensitivity,
It is unique Troponin I different from being found in skeletal muscle.It is thus extensively studied as the sensitive and special of myocardial damage
Different marker.
The detection method of cardiac muscle troponin I is more at present, such as colloidal gold method, immunofluorescence technique, enzyme-linked immunization, chemistry
Luminescence method, Electrochemiluminescince, latex enhancing immune turbidimetry etc..The shortcomings that colloidal gold method, is accurately to be quantified,
And differences between batches are larger, can not continue the level for monitoring patient's Troponin I；The immunofluorescence technique reaction time is longer, to environment
It is more demanding, be easy to be influenced by dirt in air, and mostly 96 person-portions/box, using the disadvantages of rare earth marker but also this is immune
Method can not large-scale use；Enzyme-linked immunization is easy to operate but sensitivity is low；Chemoluminescence method is to instrument requirements height and reagent
At high cost, detection speed is relatively slow, and popularity rate is low；The application of latex enhancing immune turbidimetry reagent is convenient, at low cost, automation
By force, facilitate clinical application, but this reagent mostly uses how anti-preparation, sensitivity is low, and zero negative value and differences between batches are big etc. asks
Topic, also brings very big puzzlement to clinical application.
Rheumatoid factor existing for patient's body (RF) can interfere many immunological detection methods.IgM type RF and IgG type RF
In conjunction with the Fc of capture antibody and label secondary antibody in detection reagent, cause to detect false positive results.RF is to different detection methods
Annoyance level it is different, influence degree is not directly proportional to RF concentration.Currently, the detection reagent used in clinic does not have mostly
Take well prevent RF interfere measure (progress that rheumatoid factor interferes immunoassays, Medical review 2015
Vol 21,19:3495).Currently, the strategy for preventing RF from interfering includes: for capturing antibody, with F (ab ')2Segment substitution is complete
IgG；Sample to be tested is pre-processed (Euroimmune company) with the solid-phase adsorbent for being connected with thermal denaturation IgG, and 4 DEG C overnight
It is detected again after centrifugation；It when detecting antigen, can be added to 2 mercapto ethanol in Sample dilution or sample, drop IgM type RF
Solution.
For example, disclosing a kind of method and its detection kit for detecting cardiac muscle troponin I in CN102539784A.
The kit includes reagent 1, reagent 2, and wherein reagent 1 is buffer, reagent 2 by coupled antibody composition polystyrene latex
Particle, buffer composition.The buffer is PBS buffer solution, Tris buffer, glycine buffer, borate buffer solution, vinegar
Phthalate buffer, citrate phosphate buffer, carbonate bicarbonate buffer, 2-morpholine ethane sulfonic acid buffer, ammonium chloride
One or more of buffer.The composition that at least three kinds of above antibody are used in the kit, meets high-precision as much as possible
Condition needed for spending cTnI detection antibody, balanced sensibility of the antibody to various determination influences factors, can be minimized it is negative or
Positive interference, increases the accuracy of measuring method；Meanwhile also cTnI antibody being coated with to latex particle using orientation coupling method
On, antibody coupling position is Fc segment, and antigen binding site is made to be directed toward mobile phase, and the feelings of antigen binding capacity loss will not occur
Condition；In addition to this, due to during chemical coupling Fc segment the change in structure has occurred, reduce RF and heterophil antibody
Interference.
There is still a need for the cardiac muscle troponin I kits of a kind of high sensitivity, good in anti-interference performance for this field.
Summary of the invention
According to some embodiments, a kind of detection kit of Troponin I is provided, it includes the first reagent and second
Reagent.
In some embodiments, the first reagent includes:
In some embodiments, second reagent includes:
In some embodiments, buffer is selected from: glycine buffer, Tris buffer, MOPS buffer, MES are slow
Fliud flushing, Hepes buffer.In some embodiments, the buffer in first reagent and second reagent is identical
Or it is different.In some embodiments, the buffer in first reagent and second reagent is 50mM to 100mM
Glycine buffer.
In some embodiments, the electrolyte is selected from: sodium chloride, potassium chloride, magnesium chloride, magnesium sulfate, zinc chloride, chlorine
Change calcium.
In some embodiments, the stabilizer is selected from the following a kind of or combination: mannose, glucose, chitosan,
Sorbierite, bovine serum albumin(BSA), trehalose, fructose, sucrose.
In some embodiments, the surfactant is selected from: Triton, Tween20, Tween80, NP40,
thesit,BrijL2；Preferably Tween80.
In some embodiments, it is selected from for described discrete dose: potassium rhodanide, choline chloride.
In some embodiments, the preservative is selected from: Sodium azide, phenol, P-hydroxybenzoic acid, PC300.
In some embodiments, the monoclonal antibody is originated from: mouse, rabbit or fowl.
In some embodiments, the blocking agent is selected from: mouse IgG, Mouse IgM, mice serum.
In some specific embodiments, the blocking agent is Mouse IgM.
In some specific embodiments, the blocking agent is mouse anti human IgM monoclonal antibody.
Without wishing to be held to specific theoretical limitation, it is however to be understood that present inventor it was unexpectedly found that
Non-specific antibody (such as mouse IgG, Mouse IgM, mice serum) is affinity to RF, so that RF is blocked, to avoid false sun
Property.
In some specific embodiments, the surfactant is 10g/L to 20g/LTween 80.
In some specific embodiments, the latex particle is polystyrene latex particles, and surface modification group is
Carboxyl.
In some specific embodiments, the concentration of the blocking agent is 12g/L to 18g/L, preferably 15g/L.
In other specific embodiments, anti-human Troponin I monoclonal antibody is anti-comprising two different source of mouse
Human troponin I monoclonal antibody identifies different epitopes (epitope aa 60-90 and the aa 20- of human troponin I respectively
40).In some specific embodiments, the ratio of the amount of the substance of two different anti-human Troponin I monoclonal antibodies is
2:1.
In some specific embodiments, the detection kit of the application also includes calibration object or quality-control product.Calibration object
Or quality-control product can voluntarily prepare be also possible to it is commercially available.
According to some embodiments, a kind of detection kit of Troponin I is provided, it includes the first reagent and second
Reagent；
Wherein, first reagent includes:
Second reagent includes:
The blocking agent is the mouse anti human IgM monoclonal antibody of purifying.
According to some embodiments, purposes of the blocking agent in anti-interference is additionally provided.The blocking agent is selected from the following
A kind of or combination: mouse IgG, Mouse IgM, mice serum.
In some embodiments, the mouse anti human IgM monoclonal antibody for providing purifying is improving Troponin I detection
The purposes of middle anti-interference ability.Preferably, interference refers to the interference of rheumatoid factor；More specifically, interference refers to due to class wind
Troponin I testing result false positive caused by the interference of the wet factor.
According to some embodiments, the mouse anti human IgM monoclonal antibody for additionally providing purifying is improving Troponin I inspection
Purposes in the false positive results of survey.
According to some embodiments, a kind of method for improving anti-interference ability in Troponin I detection is additionally provided, including
Step: blocking agent is introduced in Troponin I detection reagent, the blocking agent is selected from the following a kind of or combines: mouse IgG,
Mouse IgM, mice serum.
According to some embodiments, a kind of method for improving false positive in Troponin I detection, including step are additionally provided
It is rapid: to introduce blocking agent in Troponin I detection reagent, the blocking agent is selected from the following a kind of or combination: mouse IgG, small
Mouse IgM, mice serum.
In some embodiments, Troponin I detection reagent refers to latex enhancing immune than turbid detection reagent.
According to some embodiments, the combination for additionally providing blocking agent and surfactant is improving Troponin I detection
The purposes of middle anti-interference ability.The blocking agent is selected from the following a kind of or combines: mouse IgG, Mouse IgM, mice serum；And
The surfactant is Tween 80.In some embodiments, anti-interference ability is selected from the following a kind of or combines: anti-RF
Interference, the interference of anti-binding bilirubin, antivitamin C interference, the interference of anti-Intralipid fat emulsion, antiheparin sodium interfere, are anti-
Hemoglobin interference.In some embodiments, the mass ratio of blocking agent and surfactant in same reagent is 3:2.
Detailed description of the invention
Fig. 1: disclosure Troponin I kit standard curve.
Specific embodiment
Embodiment 1: the preparation of the kit of the application
1. the first reagent preparation process is as follows:
First reagent includes
Under room temperature, according to the preparation of above-mentioned the first reagent of carry out, the blocking agent is that the mouse anti human IgM of purifying is mono-
It is anti-(to be purchased from California, USA Scantibodies Laboratory Inc.9336Abraham Way, Santee, 92071, article No.
3KC542；95%) purity is greater than.
2. the second reagent preparation process is as follows:
4.5ml 0.05M MES is added in the polystyrene colloidal milk solution (concentration 10%) of 0.5ml diameter 300nm
(PH6.0) and 100 μ l EDC (carbodiimides, concentration 5mg/mL), 1 hour is stood in 37 DEG C；
Troponin I monoclonal antibody is added to above-mentioned immediately after being diluted with 5ml 0.05M Tris buffer (PH7.5)
In buffer, reacted 2 hours at 37 DEG C；
1ml 0.1M glycine buffer (PH7.0) is added and stirs the reaction of termination in 1 hour；
It is washed with the glycine buffer (PH7.0) of 20ml 100mM, supernatant is removed in centrifugation, washs 3 times；
It is allowed to be dispersed into white latex suspension with buffer resuspension latex microsphere is saved；
The coated polystyrene latex particles concentration of Troponin I monoclonal antibody is 0.25% (% table in second reagent
Show g/100ml)；Concentration of the antibody in the second reagent is 0.08g/L.
Wherein, the composition for saving buffer:
The example more preferred as one: monoclonal antibody in the second reagent can with more than one monoclonal antibody,
Such as two or more monoclonal antibodies, the different loci (coating of different monoclonal antibodies of Troponin I is identified respectively
Method also presses above-mentioned steps progress).In an example, the mass ratio (or the mass ratio of the material) of two kinds of monoclonal antibodies is 2:1, is known respectively
Epitope aa 60-90 and the aa 20-40 of other Troponin I.
Embodiment 2: the preparation of contrast agents box
The method that the preparation method is the same as that of Example 1, difference, which is only that, is substituted for polyclonal antibody for monoclonal antibody.
The comparison of 3. surfactant of embodiment
Reagent preparation box according to the method for embodiment 1, difference are only that: the Tween 80 in the first reagent is substituted for down
Each surfactant of various various concentrations in table；And the standard items of various concentration are tested respectively, it is as a result as follows.
The comparison of 1. surfactant of table
The data of S1-S6 are calibration reaction absorbances, and reaction gradient (i.e. slope) is bigger, and blank reacts absorbance (i.e. S1
React absorbance) it is more lower better.From the results, it was seen that effect is best when Tween 80 is 10g/L, when 20g/L Tween80
Take second place.
Influence of 4. surfactant of embodiment to anti-interference ability
Reagent preparation box according to the method for embodiment 1, step difference are only that following: Tween80 is substituted for following table institute
The different surfaces activating agent of the various various concentrations shown.
Contained with each kit test of preparation or ((in conjunction with bilirubin, also referred to as direct gallbladder is red by DB without chaff interferent
Element), Vc, Intralipid fat emulsion, heparin sodium, hemoglobin) sample.As a result as follows:
Influence of 2. surfactant of table to anti-interference ability
From the results, it was seen that when reagent addition 80 anti-jamming effectiveness of 10g/L Tween is more preferable；The Tween 80 of 8g/L
Take second place.
The comparison of the different blocking agents of embodiment 5.
Reagent preparation box according to the method for embodiment 1, step difference are only that without blocking agent.
The sample contained or without RF is tested with each kit of preparation.As a result as follows:
The comparison of the different blocking agents of table 3.
Obviously there is when as can be seen that containing blocking agent anti-RF interference effect from data, and specific absorbance is influenced
It is relatively small.
Embodiment 6. determines the concentration of blocking agent
Reagent preparation box according to the method for embodiment 1, step difference are only that blocking agent concentration is different (see the table below).With system
Standby each kit test contains or the sample without RF.As a result as follows:
The comparison of the different blocking agent concentration of table 4.
From the results, it was seen that the optimal dosage of blocking agent is 15g/L, 18g/L takes second place.
Influence of 7. blocking agent of embodiment to calibration
Reagent preparation box according to the method for embodiment 1, step difference are only that uninterrupted dose.With each kit of preparation
Test the standard items of various concentration.As a result as follows:
Whether there is or not the comparisons of blocking agent for table 5.
The degree of fitting of calibration curve is smaller, and illustrative graph fitting is better.
8. blocking agent of embodiment improves false positive
It prepares the kit for containing or not contain blocking agent respectively according to the method for embodiment 7, and is distinguished with each kit
Test false positive sample, and conventional sample (i.e. true positives and true negative sample).
Siemens's measured value is the negative or lower sample of measured value, if the application reagent does not add blocking agent, a other style
Originally it will appear extremely high value, as shown in the table, false positive is significantly improved after blocking agent is added.
Whether there is or not the comparisons of blocking agent for table 6.
Whether there is or not the comparisons of blocking agent for table 7.
The result shows that blocking agent, which is added, corrects for false positive issue, to the measured values of true positives and true negative sample without statistics
Significantly affect.
9. difference between batch of embodiment compares
Experimental method: according to the form below experiment parameter table is tested, and embodiment 1 (when being related to two kinds of monoclonal antibodies) takes 3 batches of reagents, real
It applies example 2 and takes 2 batches of reagents (only 2 batches of antibody at present), calibrate and detect 10 samples.
8. experiment parameter table of table
| R1:R2:S | 150:50:15 | Pair/dominant wavelength | Without/600 |
| Read point | 19-34 | Calibrate type | spline |
| The Direction of Reaction | It is incremented by | Point (point)/skip point (span point) | 6/3 |
10. embodiment of table, 1 kit the calibration results
11. embodiment of table, 2 kit the calibration results
The comparison of 12.10 pattern detection results of table
It can be, it is evident that embodiment 1 (disclosure) can improve how anti-flesh calcium from the calibration results and pattern detection result
Protein I latex enhancing immune is than difference between batch problem existing for turbid reagent, and 2 the calibration results of embodiment and pattern detection result are all deposited
In notable difference.
10. repeatability of embodiment
Experimental method: with kit (when being related to using two kinds of monoclonal antibodies) detection low value sample and height of the embodiment of the present application 1
It is worth sample, retest 10 times, calculates mean value and the coefficient of variation.
13. repeated experiment result of table
| Sequencing | Low concentration sample | High concentration sample |
| 1 | 1.147 | 4.794 |
| 2 | 1.149 | 4.71 |
| 3 | 1.193 | 4.722 |
| 4 | 1.147 | 4.811 |
| 5 | 1.186 | 4.789 |
| 6 | 1.201 | 4.784 |
| 7 | 1.134 | 4.461 |
| 8 | 1.128 | 4.756 |
| 9 | 1.195 | 4.732 |
| 10 | 1.095 | 4.747 |
| Mean value | 1.1575 | 4.7306 |
| SD | 0.035 | 0.100 |
| CV | 3.02% | 2.12% |
The CV of upper table show lower concentration sample and high concentration sample is respectively 3.02% and 2.12%, the results showed that the disclosure
Measured value repeatability is good.
11. accelerated stability of embodiment
Experimental method: the reagent of the embodiment of the present application 1 is put into 37 DEG C of incubators and carries out accelerating processing 7 days, before comparing acceleration
The variation of reaction signal and sample measured value is calibrated afterwards.
Reaction signal is calibrated in the acceleration of table 14. before and after the processing
Table 15. accelerates the variation of sample measured value before and after the processing
| Catalogue number(Cat.No.) | Before acceleration | After acceleration |
| 1 | 1.65 | 1.65 |
| 2 | 2.17 | 2.19 |
| 3 | 2.22 | 2.17 |
| 4 | 1.61 | 1.60 |
| 5 | 1.55 | 1.59 |
| 6 | 1.97 | 2.06 |
| 7 | 0.85 | 1.09 |
| 8 | 10.36 | 10.58 |
| 9 | 11.37 | 11.05 |
| 10 | 5.00 | 4.47 |
Claims (7)
1. a kind of detection kit of Troponin I, it includes the first reagents and the second reagent；Wherein:
First reagent includes:
Second reagent includes:
The partial size of the latex particle is 150nm to 400nm；
The buffer is selected from the following a kind of or combines: glycine buffer, Tris buffer, MOPS buffer, MES buffering
Liquid, Hepes buffer；
Buffer in first reagent and second reagent is same or different；
The electrolyte is selected from the following a kind of or combines: sodium chloride, potassium chloride, magnesium chloride, magnesium sulfate, zinc chloride, calcium chloride；
The stabilizer is selected from the following a kind of or combination: mannose, glucose, chitosan, sorbierite, bovine serum albumin(BSA),
Trehalose, fructose, sucrose；
Described discrete dose selected from the following a kind of or combination: potassium rhodanide, choline chloride；
The preservative is selected from the following a kind of or combines: Sodium azide, phenol, P-hydroxybenzoic acid, PC300；
The monoclonal antibody is originated from below a kind of or combination: mouse, rabbit or fowl；
The anti-human Troponin I monoclonal antibody includes two different anti-human Troponin I monoclonal antibodies, is identified respectively
The epitope 60-90 amino acids and 20-40 amino acids of human troponin I；
The ratio of the amount of the substance of two different anti-human Troponin I monoclonal antibodies is 2:1；
The blocking agent is mouse anti human IgM monoclonal antibody.
2. the detection kit of Troponin I according to claim 1, wherein the latex particle is polystyrene latex
Particle, surface modification group are carboxyls.
3. the detection kit of Troponin I according to claim 1, wherein the concentration of the blocking agent is 15g/L.
4. the detection kit of Troponin I according to any one of claims 1 to 3 also includes calibration object or quality-control product.
5. a kind of detection kit of Troponin I, it includes the first reagents and the second reagent；
Wherein, first reagent includes:
Second reagent includes:
The partial size of the latex particle is 300nm；
The blocking agent is the mouse anti human IgM monoclonal antibody of purifying；
The anti-human Troponin I monoclonal antibody includes two different anti-human Troponin I monoclonal antibodies, is identified respectively
The epitope 60-90 amino acids and 20-40 amino acids of human troponin I；
The ratio of the amount of the substance of two different anti-human Troponin I monoclonal antibodies is 2:1.
6. the detection kit of Troponin I according to claim 5 also includes calibration object or quality-control product.
7. purposes of the combination of blocking agent and Tween 80 in the anti-interference ability for improving Troponin I detection, in which:
The blocking agent is mouse anti human IgM monoclonal antibody；
Concentration of the blocking agent in detection reagent is 15g/L；
Concentration of the Tween 80 in detection reagent is 10g/L；
The anti-interference combination selected from the following: the interference of the resisting rheumatoid disease factor, anti-binding bilirubin are interfered, antivitamin C is interfered,
Anti- Intralipid fat emulsion interference, the interference of antiheparin sodium, the interference of anti-hemoglobin.
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