CN106526169A - Kit used for determination of triiodothyronine - Google Patents
Kit used for determination of triiodothyronine Download PDFInfo
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- CN106526169A CN106526169A CN201611110779.8A CN201611110779A CN106526169A CN 106526169 A CN106526169 A CN 106526169A CN 201611110779 A CN201611110779 A CN 201611110779A CN 106526169 A CN106526169 A CN 106526169A
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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
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Abstract
The invention provides a kit used for determination of triiodothyronine. The invention aims to overcome the problems of poor specificity and poor sensitivity of conventional T3 immunoassay reagents. The kit used for determination of triiodothyronine comprises magnetic beads coated by a goat monoclonal antibody, an ANS dissociation agent and an enzyme operation fluid, wherein the magnetic beads coated by the goat monoclonal antibody is labeled with biotin. The kit provided by the invention employs the magnetic beads coated by the goat monoclonal antibody; magnetic particles (the magnetic beads) are coated by anti-biotin antibody which is the goat monoclonal antibody; so the kit has higher specificity and sensitivity compared with the conventional T3 immunoassay reagents.
Description
Technical field
The present invention relates to a kind of kit, more particularly to a kind of kit for determining trilute.
Background technology
Trilute (T3) belongs to small molecule project, generally many grams of the antibody of existing T3 immunoassay reagents
Grand antibody or the monoclonal antibody of mouse, have all been short of in terms of specificity and sensitivity.Prior art does not dilute to be marked
, to fixed concentration, inconvenient operation during mark micro antibody, while differences between batches are big, the reagent kit function of different batches is poor for antibody
Away from larger, cause stability relatively low.
The content of the invention
The invention provides a kind of kit for determining trilute, it is intended to solve T3 in prior art
All poor problem in terms of the specificity of immunoassay reagent and sensitivity.
In order to solve above technical problem, the present invention is achieved through the following technical solutions：It is a kind of to be used to determine triiodo first shape
The kit of gland original ammonia acid, including the coated magnetic bead of sheep monoclonal antibody, ANS dissociation agent and enzyme working solution；On the coated magnetic bead of sheep monoclonal antibody
It is marked with biotin.ANS dissociation agent is the dissociation agent (ANS containing ANS：8- aniline -1-naphthalene sulfonic aicd 8-Anilino-1-
naphthalenesulfonic acid)。
Further, the preparation method of the coated magnetic bead of the sheep monoclonal antibody is：Sheep monoclonal antibody is diluted to into the concentration of setting, to dilute
Sheep monoclonal antibody after releasing carries out biotin labeling and obtains biotin antibody, and biotin antibody is coated on acquisition sheep monoclonal antibody on Magnetic particles
Coated magnetic bead；The concentration for setting is as fixed value.
Before to sheep monoclonal antibody mark, sheep monoclonal antibody is diluted to into specific concentration, the stability of kit can be improved, no
Kit function with batch is close.
Further, the concentration of setting value in 0.1mg/mL～0.6mg/mL.
When sheep monoclonal antibody is diluted to specific concentration within the range, the potency of kit is higher, and good stability.
Further, the preparation method of the coated magnetic bead of the sheep monoclonal antibody is：
A, using PBS to sheep monoclonal antibody dialyse, take out dialysis finish after sheep monoclonal antibody；Take out after dialysis is finished
Sheep monoclonal antibody is diluted to the concentration of setting；
B, BNHS is dissolved in DMSO, is configured to BNHS solution；Wherein BNHS (N-hydroxy-succinamide biotin esters；
EZ-Link NHS-LC-Biotin, 50mg, producer：Thermo Prod#21336)；
C, the sheep monoclonal antibody and BNHS solution after dilution is mixed and reacted, after reaction setting time, add Tris buffer solutions
Terminating reaction, obtains biotin antibody；
E, biotin antibody is dialysed using PBS；
Biotin antibody after F, taking-up dialysis, biotin antibody coating Magnetic particles, obtains the coated magnetic bead of sheep monoclonal antibody.
Further, the preparation method of the coated magnetic bead of the sheep monoclonal antibody is：
A, using 0.1～0.3mol/L PBS pair the dialysis of sheep monoclonal antibody, the temperature of dialysis is 2～8 DEG C；Take out
The sheep monoclonal antibody after finishing is analysed in dialysis, and sheep monoclonal antibody is diluted to the concentration of setting；
B, BNHS is dissolved in DMSO, is configured to the BNHS solution of 8～12mmol/L；
C, by step A parse after sheep monoclonal antibody and step B prepare BNHS solution mix and react 28～33 minutes；
D, the reaction for terminating step C with the Tris buffer solutions of 0.8～1.2mol/L；Obtain biotin antibody；
E, adopt concentration and biotin antibody is dialysed for the PBS of 0.1～0.3mol/L, the temperature of dialysis is
2～8 DEG C；
F, glycerine is added to the biotin antibody after dialysis, biotin antibody and glycerine volume ratio are 4:5～5:4；
G, biotin antibody coating Magnetic particles, obtain the coated magnetic bead of sheep monoclonal antibody；With the buffer solution of 0.08～0.12%BSA
Setting concentration is diluted to the coated magnetic bead of sheep monoclonal antibody.
Further, mixed solution of the enzyme working solution for the T3 derivatives of the buffer solution and ALP mark of BSA.The T3 of ALP marks
Derivative is the T3 derivatives of alkali phosphatase enzyme mark, connects alkaline phosphatase, both had on a kind of molecule of similar T3 antigens
There are the high catalytic property of enzyme, and the specificity with T3 antigens.
Compared with prior art it is an advantage of the invention that：The present invention includes the coated magnetic bead of sheep monoclonal antibody, magnetic particle (magnetic in adopting
Pearl) on it is coated be biotin antibody, antibody for sheep monoclonal antibody, kit specificity and sensitivity all than before
T3 immunoassay reagents it is high.
Specific embodiment
Embodiment one：
A kind of kit for determining trilute, including the coated magnetic bead of sheep monoclonal antibody, ANS dissociation agent and
Enzyme working solution, is marked with biotin on the coated magnetic bead of sheep monoclonal antibody.
The preparation method of the coated magnetic bead of the sheep monoclonal antibody is：
A, 0.25mg sheep monoclonal antibodies are taken, 2～8 DEG C of dialysed overnights are carried out with 0.2mmol/L PBS (PH7.4) buffer solution 5L.
The sheep monoclonal antibody that B, taking-up have been dialysed accurately measures volume and records, sheep monoclonal antibody is diluted in appropriate containers
0.5mg/mL is standby.
C, the BNHS for weighing 1.0mg, are fully dissolved with 217 μ LDMSO, are configured to the BNHS solution of 10mmol/L.
D, by the ratio of the BNHS for preparing and sheep monoclonal antibody be 20：1 amount is added in the sheep monoclonal antibody after dialysis, fully mixed
Even rear room temperature reaction 30min.That is the antibody of 1.0mg adds 13.3 μ L of 10mmol/LBNHS.0.25mg adds what is configured
10mmol/LBNHS 3.3μL。
E, take 1mol/LTris buffer solutions and carry out terminating reaction, add 1mol/LTris buffer solutions in reactant liquor.Add
The body fluid of Tris is one of percentage of above-mentioned reactant liquor final volume, is fully mixed, room temperature reaction 10Min.
Reactant liquor after F, taking-up terminate loads bag filter, carries out 2～8 with 0.2mol/L PBS (PH7.4) buffer solution 5L
DEG C dialysed overnight.
G, the biotin antibody dialysed is taken out, and accurately measure volume, add isopyknic glycerine, labeling, and
Record concentration, preserves in -20 DEG C, standby.
H, by biotin labeling good biotin antibody coating Magnetic particles, package amount is 1 μ g/mg, obtains sheep monoclonal antibody coating
Magnetic bead.It is after with the buffer B uffer dilution of 1%BSA, standby.
Embodiment two：
A kind of kit for determining trilute, including the coated magnetic bead of sheep monoclonal antibody, ANS dissociation agent and
Enzyme working solution, is marked with biotin on the coated magnetic bead of sheep monoclonal antibody.
The preparation method of the coated magnetic bead of the sheep monoclonal antibody is：
A, 0.2mg sheep monoclonal antibodies are taken, 2～8 DEG C of dialysed overnights are carried out with 0.12mol/L PBS (PH7.4) buffer solution 4.5L.
The sheep monoclonal antibody that B, taking-up have been dialysed accurately measures volume and records, sheep monoclonal antibody is diluted in appropriate containers
0.2mg/mL is standby.
C, BNHS is dissolved in DMSO, is configured to the BNHS solution of 8mmol/L.
D, by the ratio of the BNHS for preparing and sheep monoclonal antibody be 18：1 amount is added in the sheep monoclonal antibody after dialysis, fully mixed
Even rear room temperature reaction 28min.
E, the Tris buffer solutions for taking 1mol/L carry out terminating reaction, add 1mol/LTris buffer solutions in reactant liquor.Plus
The body fluid for entering Tris is one of percentage of above-mentioned reactant liquor final volume, is fully mixed, room temperature reaction 11Min.
Reactant liquor after F, taking-up terminate loads bag filter, carries out 2～8 with PBS (PH7.4) the buffer solution 5L of 0.2mol/L
DEG C dialysed overnight.
G, the biotin antibody dialysed is taken out, and accurately measure volume, third is added to the biotin antibody after dialysis
Triol, biotin antibody are 4 with glycerine volume ratio:5, labeling, and concentration is recorded, and preserve in -20 DEG C, it is standby.
H, by biotin labeling good biotin antibody coating Magnetic particles, package amount is 0.12 μ g/mg, obtains Yang Dankangbao
The magnetic bead of quilt.It is after with the buffer B uffer dilution of 1%BSA, standby.
Embodiment three：
A kind of kit for determining trilute, including the coated magnetic bead of sheep monoclonal antibody, ANS dissociation agent and
Enzyme working solution, is marked with biotin on the coated magnetic bead of sheep monoclonal antibody.
The preparation method of the coated magnetic bead of the sheep monoclonal antibody is：
A, 0.3mg sheep monoclonal antibodies are taken, 2～8 DEG C of dialysed overnights are carried out with 0.12mol/LPBS (PH7.4) buffer solution 6L.
The sheep monoclonal antibody that B, taking-up have been dialysed accurately measures volume and records, sheep monoclonal antibody is diluted in appropriate containers
0.1mg/mL is standby.
C, BNHS is dissolved in DMSO, is configured to the BNHS solution of 11mmol/L.
D, by the ratio of the BNHS for preparing and sheep monoclonal antibody be 21：1 amount is added in the sheep monoclonal antibody after dialysis, fully mixed
Even rear room temperature reaction 31min.
E, the Tris buffer solutions for taking 1.1mol/L carry out terminating reaction, add 1.1mol/LTris buffer solutions in reactant liquor
In.The body fluid for adding Tris is one of percentage of above-mentioned reactant liquor final volume, is fully mixed, room temperature reaction 9Min.
Reactant liquor after F, taking-up terminate loads bag filter, carries out 2 with PBS (PH7.5) the buffer solution 5.5L of 0.3mol/L
～8 DEG C of dialysed overnights.
G, the biotin antibody dialysed is taken out, and accurately measure volume, third is added to the biotin antibody after dialysis
Triol, biotin antibody are 5 with glycerine volume ratio:4, labeling, and concentration is recorded, and preserve in -20 DEG C, it is standby.
H, by biotin labeling good biotin antibody coating Magnetic particles, package amount is 0.09 μ g/mg, obtains Yang Dankangbao
The magnetic bead of quilt.It is after with the buffer B uffer dilution of 0.11%BSA, standby.
Beneficial effects of the present invention are further illustrated with reference to experiment：
Experiment one：, under square one, kit sensitivity compares real with specificity for anti-, mouse monoclonal antibody and sheep monoclonal antibody more than rabbit
Test.
Experiment material：BNHS, 0.2mol/L PBS (PH7.4) buffer solution, 1mmol/LTris buffer solutions, glycerine,
3 kinds of DMSO, T3 antibody (rabbit resists more, mouse monoclonal antibody, sheep monoclonal antibody), buffer B uffer containing 1%BSA, the T3 of ALP marks derive
Thing, ANS dissociation agent, Magnetic particles, T3 calibration object cal-1, cal-5 (T3 sterlings are diluted to into buffer B uffer containing 5%BSA,
The concentration of Cal-1 to Cal-5 is 0,0.3,1,3,10ng/mL), high concentration T4, substrate solution, cleaning fluid, shore pine photon are semi-automatic
Chemical luminescence detector.
Using the method for embodiment one, resisting above-mentioned rabbit more, mouse monoclonal antibody and the coated magnetic bead of sheep monoclonal antibody respectively with dissociation agent,
Enzyme working solution collectively constitutes kit, determines T3 calibration object cal-1 to cal-5, and the T4 of high concentration, observes the survey of different antibodies
Determine curve and specificity.
Reaction pattern is：30 μ L of calibration object, 50 μ L of magnetic bead are initially charged, after 50 μ L37 DEG C of dissociation agent are incubated 15min jointly, then
Add 37 DEG C of incubation 15Min of enzyme working solution, addition cleaning fluid to clean in magnetic field after separating three times, add 37 DEG C of incubations of substrate solution
After 5Min, detect on semi-automatic chemical luminescence detector.
Experimental result is as shown in table 1：
Table 1
Experiment conclusion：Poor using the kit sensitivity and specificity that resist rabbit, the kit using mouse monoclonal antibody is bent more
Line substantially meets use, and sensitivity is enough, but specificity is poor.Had very using the kit sensitivity and specificity of sheep monoclonal antibody
Big raising.
Experiment two：The sheep monoclonal antibody of variable concentrations, kit sensitivity and specific comparative experiments.
There are three batches of sheep monoclonal antibodies, concentration difference 5mg/mL, 2.5mg/mL, 1mg/mL, after marking biotin respectively, according to experiment
One method is measured to T3 calibration objects, observes difference between batch.
Experiment material：BNHS, 0.2mol/L PBS (PH7.4) buffer solution, the Tris buffer solutions of 1mol/L, glycerine,
DMSO, T3 antibody (is sheep monoclonal antibody, concentration is respectively 5mg/mL, 2.5mg/mL, 1mg/mL), the buffer solution containing 1%BSA
The T3 derivatives of Buffer, ALP mark, ANS dissociation agent, T3 sterlings (are diluted to by Magnetic particles, T3 calibration object cal-1, cal-5
Buffer B uffer containing 5%BSA, the concentration of Cal-1 to Cal-5 is 0,0.3,1,3,10ng/mL), substrate solution, cleaning fluid,
The semi-automatic chemical luminescence detector of shore pine photon.
Experimental technique is with reference to experiment one；Remarks：After the antibody of three kinds of concentration is dialysed, recovery volume is basically unchanged,
Concentration after then dialysing is still consistent with original content.
Experimental result such as table 2：
Table 2
Experiment conclusion：Sheep monoclonal antibody is all, but concentration is different during due to marking biotin, as prepared by identical labeling method
Kit difference between batch it is larger, and AC is bigger, and the potency of kit is lower.
Experiment three：Fixed T3 ACs, kit sensitivity and specific comparative experiments.
According to two conclusions that drawn of experiment, sheep monoclonal antibody is diluted to into identical concentration, then carries out the mark of biotin,
Impact of the observation to difference between batch and potency.
Experiment material：BNHS, 0.2mol/LPBS (PH7.4) buffer solution, 1mol/LTris buffer solutions, glycerine, DMSO,
T3 antibody (is sheep monoclonal antibody, concentration is respectively 5mg/mL, 2.5mg/mL, 1mg/mL), buffer B uffer containing 1%BSA,
The T3 derivatives of ALP marks, ANS dissociation agent, Magnetic particles, T3 calibration object cal-1, cal-5 (are diluted to T3 sterlings containing 5%BSA
Buffer B uffer, the concentration of Cal-1 to Cal-5 is 0,0.3,1,3,10ng/mL), substrate solution, cleaning fluid, shore pine photon
Semi-automatic chemical luminescence detector.
Experimental technique " will take out the sheep monoclonal antibody dialysed in appropriate containers, standard in one step B of embodiment with reference to experiment one
Really measure volume and record, sheep monoclonal antibody is diluted to into 0.5mg/mL standby." replace with：" the sheep monoclonal antibody that taking-up has been dialysed is in appropriate
Container, accurately measures volume and records, and the antibody of three kinds of variable concentrations is diluted to 0.5mg/mL standby.”
Experimental result such as table 3：
Table 3
Experiment conclusion：The sheep monoclonal antibody of three kinds of variable concentrations is diluted to after 0.5mg/mL and marks biotin, be then coated with
The potency of three kinds of T3 kits that magnetic bead is constituted is basically identical, and curve is essentially identical, and after dilution, antibody labeling biotin can
The difference between batch of effective control product.
Experiment four：The sheep monoclonal antibody of variable concentrations carries out biotin labeling, kit sensitivity and specific comparative experiments.
Experiment material：2.5mg/mL and 2.5mg/mL is diluted to into 0.5mg/mL carries out biology in experiment two and experiment three
Coated two kinds of magnetic beads after element mark, enzyme working solution, agent of dissociating, T3 calibration objects, substrate solution, cleaning fluid, shore pine photon are semi-automatic
Chemical luminescence detector, constant incubator.
Experimental technique：Prepare two kinds of magnetic beads, every kind of equal-volume are divided into two parts, a to preserve with 2-8 DEG C, Yi Fenyu
37 DEG C of preservations in constant incubator, took out after 6 days.Four parts of magnetic beads are coordinated respectively at dissociation agent and enzyme working solution and determines T3 calibrations
Product.The luminous signal of observation calibration object, calculates signal retention rate, verifies the stability of magnetic bead.
Experimental result such as table 4：
Table 4
Experiment conclusion：After the 37 degrees Celsius of storages of the coated magnetic bead of biotin antibody are marked after antibody dilution, signal retention rate is more
Height, stability are more preferable.
To sum up four experiments can draw：Show that T3 determines kit when using sheep monoclonal antibody, sensitivity and specificity are equal
Effectively improve.Before biotin labeling, sheep monoclonal antibody is diluted to into a fixed setting concentration, the steady of kit can be improved
Qualitative, setting concentration is too low can waste of resource.
The specific embodiment of the present invention is the foregoing is only, but the technical characteristic of the present invention is not limited thereto, Ren Heben
The technical staff in field in the field of the invention, all cover among the scope of the claims of the present invention by the change or modification made.
Claims (7)
1. a kind of kit for determining trilute, is characterized in that：Including the coated magnetic bead of sheep monoclonal antibody, ANS
Dissociation agent and enzyme working solution, are marked with biotin on the coated magnetic bead of sheep monoclonal antibody.
2. a kind of kit for determining trilute according to claim 1, is characterized in that：Sheep monoclonal antibody
Coated magnetic bead package amount is 0.08～0.12 μ g/mg.
3. a kind of kit for determining trilute according to claim 1, is characterized in that：The sheep
The coated magnetic bead preparation method of monoclonal antibody is：Sheep monoclonal antibody is diluted to into the concentration of setting, biotin is carried out to the sheep monoclonal antibody after dilution
Mark obtains biotin antibody, biotin antibody is coated on Magnetic particles and obtains the coated magnetic bead of sheep monoclonal antibody；The setting
Concentration is fixed value.
4. a kind of kit for determining trilute according to claim 3, is characterized in that：Setting
Concentration value in 0.1mg/mL～0.6mg/mL.
5. a kind of kit for determining trilute according to claim 1 or 2 or 3 or 4, its feature
It is：The preparation method of the coated magnetic bead of the sheep monoclonal antibody is：
A, using PBS to sheep monoclonal antibody dialyse, take out dialysis finish after sheep monoclonal antibody；The sheep list taken out after dialysis is finished
The anti-concentration for being diluted to setting；
B, BNHS is dissolved in DMSO, is configured to BNHS solution；
C, the sheep monoclonal antibody and BNHS solution after dilution is mixed and reacted, after reaction setting time, add Tris buffer solutions to terminate
Reaction, obtains biotin antibody；
E, biotin antibody is dialysed using PBS；
Biotin antibody after F, taking-up dialysis, biotin antibody coating Magnetic particles, obtains the coated magnetic bead of sheep monoclonal antibody.
6. a kind of kit for determining trilute according to claim 5, is characterized in that：The sheep
The preparation method of the coated magnetic bead of monoclonal antibody is：
A, using 0.1～0.3mol/L PBS pair the dialysis of sheep monoclonal antibody, the temperature of dialysis is 2～8 DEG C；Take out dialysis
The sheep monoclonal antibody after finishing is analysed, sheep monoclonal antibody is diluted to into the concentration of setting；
B, BNHS is dissolved in DMSO, is configured to the BNHS solution of 8～12mmol/L；
C, by step A parse after sheep monoclonal antibody and step B prepare BNHS solution mix and react 28～33 minutes；
D, the reaction for terminating step C with the Tris buffer solutions of 0.8～1.2mol/L；Obtain biotin antibody；
E, adopt concentration and biotin antibody is dialysed for the PBS of 0.1～0.3mol/L, the temperature of dialysis is 2～8
℃；
F, glycerine is added to the biotin antibody after dialysis, biotin antibody and glycerine volume ratio are 4:5～5:4；
G, biotin antibody coating Magnetic particles, obtain the coated magnetic bead of sheep monoclonal antibody；With the buffer solution of 0.08～0.12%BSA to sheep
The coated magnetic bead of monoclonal antibody is diluted to setting concentration.
7. a kind of kit for determining trilute according to claim 1, is characterized in that：The enzyme
Mixed solution of the working solution for the T3 derivatives of the buffer solution and ALP mark of BSA.
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| CN113866429A (en) * | 2021-12-03 | 2021-12-31 | 南京岚轩生物科技有限公司 | Dissociation agent for detecting TT3 and TT4 contents and preparation method thereof |
| CN113866429B (en) * | 2021-12-03 | 2022-02-22 | 南京岚轩生物科技有限公司 | Dissociation agent for detecting TT3 and TT4 contents and preparation method thereof |
| CN114216897A (en) * | 2021-12-22 | 2022-03-22 | 武汉生之源生物科技股份有限公司 | sST2 chemiluminescence detection kit and detection method thereof |
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