CN110187132A - A kind of analysis method detecting thyrotropic hormone - Google Patents
A kind of analysis method detecting thyrotropic hormone Download PDFInfo
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- CN110187132A CN110187132A CN201910398648.1A CN201910398648A CN110187132A CN 110187132 A CN110187132 A CN 110187132A CN 201910398648 A CN201910398648 A CN 201910398648A CN 110187132 A CN110187132 A CN 110187132A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/583—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with non-fluorescent dye label
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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
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
Abstract
The invention discloses a kind of analysis methods for detecting thyrotropic hormone, it is coupled using the processed antithyrotropic hormone specific IgY antibody of surfactant as capture antibody and with magnetic particle, it regard the IgG antibody coupling of acridinium ester and antithyrotropic hormone pairing as tracer, double antibody sandwich method is established by design and detects thyrotropic hormone.The present invention can effectively exclude the influence of endogenous disturbing factor, and IgY antibody, after the processing of specific surfactant, coupling efficiency and luminous value greatly improve, to keep testing result more accurate.
Description
Technical field
The present invention relates to technical field of immune assay, more particularly to a kind of detection thyrotropic hormone
Analysis method.
Background technique
Thyrotropic hormone (Thyroid-stimulating hormone, TSH) is a kind of sugar secreted by anterior pituitary
Protein hormones are made of two different subunit α, β.Its major physiological effect is to adjust the synthesis of thyroid hormone and divide
It secretes, when thyroid function changes, the fluctuation of serum thyroid stimulating hormone is more rapidly and significant compared with thyroid hormone, therefore, clinical
Upper measurement serum TSH is to diagnosis hyperthyroidism (hyperthyroidism) and declines (first subtracts), the primary identification with secondary hypothyroidism, prison
Curative effect, the diagnosis of subclinical hyperthyroidism, the newborn's first that survey hyperthyroidism and first subtract low screening and the laboratory diagnosis of TSH of pituitary tumor etc.
It is of great significance.
Method currently used for detecting thyrotropic hormone (TSH) mainly has immunochromatographic method, time-resolved fluoroimmunoassay
Analytic approach, enzyme-linked immunosorbent assay, electrochemistry and chemoluminescence method etc..Match used in traditional double antibody sandwich method detection TSH
It is IgG antibody to antibody, since there are more endogenous disturbing factors, such as heterophil antibody, class wind in blood sample
The wet factor, autoantibody, complement etc., testing result are often subject to interfere and can not obtain accurate result.Such as patent of invention
CN101949943A (2011.01) discloses a kind of Thyrotropic hormone quantitative detection kit and preparation method thereof, and invention is special
Sharp CN102426249A (2012.04) discloses Thyrotropic hormone quantitative assay kit and its detection method, these patents
Used in a pair of antibody be IgG antibody, theoretically nevertheless suffer from the impression of endogenous disturbing factor, analysis result can not
It leans on.
Therefore, how providing one kind can accurately detect that thyrotropic hormone and detection sensitivity are high, detection range is wide
Analysis method the problem of being those skilled in the art's urgent need to resolve.
Summary of the invention
In view of this, surfactant is handled the present invention provides a kind of analysis method for detecting thyrotropic hormone
The antithyrotropic hormone specific IgY antibody crossed is coupled as capture antibody and with magnetic particle, by acridinium ester and anti-rush first shape
The IgG antibody coupling of glandular hormone pairing is used as tracer, establishes double antibody sandwich method by two-step method design and detects thyroid
Hormone.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of analysis method detecting thyrotropic hormone, specifically includes the following steps:
(1) egg obtained through the immune processing of thyrotropic hormone antigen is subjected to grease removal and purifying, obtains anti-rush first shape
Glandular hormone IgY antibody, -20 DEG C of preservations；
(2) by antithyrotropic hormone IgY antibody obtained in step (1) and surfactant hybrid reaction, then plus
Enter buffer ultrafiltration cleaning, then antithyrotropic hormone IgY antibody is sucked out, obtains through the processed anti-rush first of surfactant
Shape glandular hormone IgY antibody；
(3) coupling reagent is added into magnetic particle after pretreatment and carries out first time incubation at room temperature, step is then added
(2) second is carried out through the processed antithyrotropic hormone IgY antibody of surfactant obtained in be incubated at room temperature, and removes supernatant
Liquid, washing obtain magnetic particle suspension, and 2-8 DEG C saves backup；
(4) antithyrotropic hormone IgG antibody is added into label buffer to mix, it is anti-adds acridinium ester concussion mixing
It answering, the lysine reaction 15min that concentration is 10%, which is added, to be made to mark reaction terminating, and hyperfiltration treatment is transferred in new centrifuge tube,
Acridinium ester label is obtained, 2-8 DEG C saves backup；
(5) thyrotropic hormone calibration object is prepared, then magnetic particle obtained in calibration object, step (3) is suspended respectively
Liquid sequentially adds in reaction tube, and oscillation mixes, and is incubated for for the first time, separates, then acridine obtained in step (4) is added in cleaning
Ester marker, second of incubation, separates, and cleaning is put into and carries out detecting its luminous intensity in light-emitting appearance, establishes luminous intensity and school
The standard curve of quasi- product concentration；
(6) sample to be tested is detected into its luminous intensity according to the method in step (5), can be calculated according to standard curve
The content of thyrotropic hormone in sample to be tested.
The principle of present invention detection thyrotropic hormone is the two-step method in double-antibody method: first by sample to be tested and idol
It is associated with the magnetic particle mixing of the IgY antibody handled through surfactant, the thyrotropic hormone in sample to be tested is captured, after reaction
First step cleaning is carried out, the substance being not bound with is removed；The pairing IgG tracer antibody of coupling acridinium ester, reaction is added in second step
After the completion, second step cleaning is carried out；Photon survey is carried out after exciting liquid is added, sample to be tested can be calculated according to standard curve
The content of middle thyrotropic hormone.
The beneficial effects of the present invention are:
The present invention is using yolk antibody IgY as capture antibody, and the design feature of IgY is it is possible to prevente effectively from endogenous interferes
The influence of factor, if but directly apply IgY, it is not high with the joint efficiency of target, sandwich method immunoassay can not be applied to
In；The present invention is creatively carried out the IgY antibody of surfactant processing and magnetic particle even by prolonged tortuous research
Connection, IgY antibody can keep its stereochemical structure after handling through surfactant, thus be easier to effectively be combined with target,
Coupling efficiency and luminous value can be greatly improved in immunoluminescence analysis, and IgY after treatment is analyzed in immunoluminescence
In sensitivity, detection range improves a lot.
Further, in above-mentioned steps (2), surfactant can be cationic surfactant cetyl trimethyl chlorine
Change ammonium (CTAC), cetyl trimethylammonium bromide (CTAB), amphoteric surfactant N- dodecyl-N, N- dimethylamino
Propane sulfonic acid salt (DDAPS), ten alkyl dimethylamine oxides ammoniums (DDAO), nonionic surfactant Triton X-100 or anion
Surfactant SDS (SDS), preferably amphoteric surfactant, more preferably N- dodecyl-N, N- bis-
Methylamino propane sulfonic acid salt (DDAPS) or ten alkyl dimethylamine oxides ammoniums (DDAO), more preferably N- dodecyl-N, N- diformazan
Base aminopropanesulfonic acid salt (DDAPS)；Concentration is 0.01%-0.5%, preferably 0.1%.
It is using above-mentioned further beneficial effect, being added after surfactant is handled can be such that anti-thyroid swashs
Plain IgY antibody keeps its stereochemical structure, to be easier to effectively be combined with target.
Further, in above-mentioned steps (2), the mass ratio of antithyrotropic hormone IgY antibody and surfactant is 0.02-
1.5:1.
Further, in above-mentioned steps (2), mixed reaction time 0.5-1h, preferably 0.5h.
It is that hybrid reaction makes surfactant and antithyrotropic hormone IgY using above-mentioned further beneficial effect
Antibody sufficiently acts on.
Further, in above-mentioned steps (2), buffer is MES buffer, and the concentration of MES buffer is 0.05-0.2mol/
L, preferably 0.1mol/L；The pH of MES buffer be 5.0-6.5, preferably 5.0.
It is using above-mentioned further beneficial effect, the MES buffer under this concentration and pH value range can be preferably
Wash off remaining surfactant.
Further, in above-mentioned steps (3), coupling reagent is 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide
(EDC)。
It is using above-mentioned further beneficial effect, EDC can be living with functional group's carboxyl on activated carboxyl magnetic particle surface
It can specific antibodies in succession after change.
Further, in above-mentioned steps (3), the mass ratio of magnetic particle and coupling reagent is 1-10:1, preferably 1:1；Coupling
The mass ratio of reagent and antithyrotropic hormone IgY antibody is 10-30:1.
Further, in above-mentioned steps (3), being incubated at room temperature the time for the first time is 15-60min, preferably 30min；Second
Greenhouse incubation time is 1-4h, preferably 2h.
It is using above-mentioned further beneficial effect, the effect of incubation at room temperature is the carboxylic made on carboxyl magnetic particle for the first time
Base sufficiently activates, and incubation time is too long to reduce coupling efficiency instead；The effect of second of incubation at room temperature is to have antibody
Effect is adequately bonded on the carboxyl magnetic particle after activation.
Further, in above-mentioned steps (3), after carrying out second of greenhouse incubation, it is (dense that the Tris buffer containing 1%BSA is added
Degree is 50mmol/L) it is closed, time 1h；Supernatant is removed, cleaning buffer solution (TBS+0.05%Tween-20) is added and washes
It washs 3 times, the magnetic particle suspension prepared is placed in and is saved in liquid (Tris-HCl), 2-8 DEG C of preservation.
It is using above-mentioned further beneficial effect, Tris buffer is added and is used to blockade combination vacant on magnetic particle
Site.
Further, in above-mentioned steps (4), label buffer is Na2CO3-NaHCO3Solution, concentration 0.5-0.05M are excellent
It is selected as 0.1M；PH is 8-10, preferably 8.5.
Further, in above-mentioned steps (4), it is slow that 500-2000 μ l label is added in the antithyrotropic hormone IgG antibody of every 1mg
Fliud flushing；The molar ratio of acridinium ester and antithyrotropic hormone IgG antibody is 5-40:1, preferably 10:1；The anti-rush first shape of every 1mg
The lysine solution that 10-200 μ L concentration is 10% is added in glandular hormone IgG antibody.
It is using above-mentioned further beneficial effect, finds what acridinium ester was reacted with antithyrotropic hormone IgG antibody
Optimum buffer environment and concentration.
Further, in above-mentioned steps (4), reaction is mixed to react 0.5-3h, preferably 2h at room temperature.
It is using above-mentioned further beneficial effect, reacts acridinium ester sufficiently with antithyrotropic hormone IgG antibody.
Further, in above-mentioned steps (5), the dosage of calibration object is 10-200 μ L, and the dosage of magnetic particle suspension is 10-
100 μ L, the dosage of acridinium ester label are 10-200 μ L.
Further, in above-mentioned steps (5), first time incubation temperature is 37 DEG C, time 5-10min, preferably 9min；The
Secondary incubation temperature is 37 DEG C, time 10-20min, preferably 10min.
It is using above-mentioned further beneficial effect, be incubated for makes resisting in antibody and calibration object on magnetic particle for the first time
Former sufficiently combine forms conjugate；It is sufficiently anti-that second of incubation makes antibody and first time on acridinium ester be incubated for the conjugate formed
It answers.
Further, in above-mentioned steps (5), the preparation of calibration object and the method for building up of standard curve are as follows:
With the PBS buffer solution containing 0.5%BSA by thyrotropic hormone sterling be configured to mark concentration be 0 μ IU/mL,
0.25μIU/mL、1.25μIU/mL、2.5μIU/mL、5μIU/mL、6.25μIU/mL、12.5μIU/mL、25μIU/mL、50μIU/
A series of calibration objects of mL, 75 μ IU/mL, 150 μ IU/mL, respectively by 50 μ L of calibration object, coupling 150 μ L of magnetic particle suspension according to
In secondary addition reaction tube, oscillation is mixed, 37 DEG C of incubation 9min, is separated, cleaning；100 μ are continuously added to the reaction vessel after washing
L acridinium ester label continues to be incubated for 10min, separates, cleaning；It is put into and carries out detecting its luminous intensity in light-emitting appearance, tied to surveying
Fruit uses double-log linear fit, establishes the standard curve and normal equation of luminous intensity and calibration object concentration.
The normal equation of standard curve are as follows: y=1.004x+1.937.
It can be seen via above technical scheme that compared with prior art, the present disclosure provides a kind of detections to promote first shape
The analysis method of glandular hormone, by surfactant, treated that antithyrotropic hormone IgY antibody can keep antibody form,
It is easier in conjunction with target, coupling efficiency and luminous value can be greatly improved in immunoluminescence analysis, and resist after treatment
Thyrotropic hormone IgY antibody immunoluminescence analysis in sensitivity, detection range improve a lot, in the linear range
With good linear relationship, R2It can reach 0.99 or more.
Detailed description of the invention
Fig. 1 is that it shines after different surfaces activating agent processing antithyrotropic hormone IgY antibody in the embodiment of the present invention 5
Value, sensitivity and linear relationship chart；
Fig. 2 is that the DDAO surfactant of various concentration in the embodiment of the present invention 5 handles antithyrotropic hormone IgY antibody
Its luminous value, sensitivity and linear relationship chart afterwards；
Fig. 3 is that the DDAPS surfactant processing antithyrotropic hormone IgY of various concentration in the embodiment of the present invention 5 is anti-
Its luminous value, sensitivity and linear relationship chart after body；
Fig. 4 is the luminous intensity of 5 alignment product of the embodiment of the present invention and the canonical plotting of calibration object concentration.
Specific embodiment
The following is a clear and complete description of the technical scheme in the embodiments of the invention, it is clear that described embodiment
Only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
Art personnel every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
The preparation of 1 antithyrotropic hormone IgY antibody of embodiment
1. pair chicken carries out initial immunity
After being emulsified with Freund's complete adjuvant, using chest muscle and the subcutaneous multi-point injection method of neck, with the thyroid prepared
Hormone antigen injects 4 points in chest muscle, and subcutaneous 1 point of neck, every 0.2mL is primary every six weeks booster immunizations.
The extraction of 2.IgY antibody
First immunisation collected egg after 2 weeks, passed through grease removal, purification IgY antibody.
(1) grease removal
Using the albumen in Yolk separator removal egg, thyrotropic hormone yolk is inhaled in rolling on filter paper later
Dry egg white, with sterile distilled water cleaning down yolk, until egg white is rushed it is cleared after puncture vitellinae membrana, collect yolk；Then with 9
The distilled water of times volume dilutes yolk, stirs 10min, is fully dissolved out water-soluble IgY, then adjusts pH with the HCl of 1mol/L
To 5.2, freeze thawing 2 times (- 20 DEG C of storage 20h, taking-up put 37 DEG C of baking ovens melt), 12000r/min is centrifuged 30min at 4 DEG C, then will
Supernatant filters grease removal with Suction filtration device.
(2) it purifies: ammonium sulfate salting-out process
Supernatant is placed in a beaker, in the ammonium sulfate powder for being slowly added to 20% on blender while stirring, stands 1h
12000r/min is centrifuged 30min at 4 DEG C afterwards, and extracting waste precipitating, it is that 0.01mol/L, pH are that white precipitate, which is then dissolved in concentration,
In 7.4 phosphate buffer PB, dialysis, filtering obtain extract, as antithyrotropic hormone IgY antibody after degerming, and -20
DEG C save.
(3) protein content detects
Using protein content in 260nm and 280nm dual wavelength measurement extract.
(4) titration
Use agar double immunodiffusion method: egg yolk liquid is made doubling dilution with physiological saline and is directly measured.
Start within two weeks after first immunisation to collect egg, carry out number, detects an antibody titer every two weeks, make every two weeks
It is standby primary.
Embodiment 2 handles antithyrotropic hormone IgY antibody with different surfactants
Taking 500 μ L concentration is respectively 0.01%, 0.05%, 0.1%, 0.3%, 0.5% a variety of different ion surfaces
Activating agent (cationic surfactant: hexadecyltrimethylammonium chloride (CTAC), cetyl trimethylammonium bromide
(CTAB), amphoteric surfactant: N- dodecyl-N, N- dimethylamino propane sulfonic acid salt (DDAPS), ten alkyl dimethyl oxygen
Change ammonium (DDAO), nonionic surfactant: Triton X-100, anionic surfactant: lauryl sodium sulfate
(SDS)) it is mixed with 12 μ L (5mg/mL, 60 μ g) antithyrotropic hormone IgY antibody, after 30min, 800 μ L concentration, which are added, is
The MES buffer that 0.1mol/L, pH are 5.0, with ultra-filtration centrifuge tube by antithyrotropic hormone IgY antibody ultrafiltration 3-4 times, then plus
Entering 400 μ L concentration is the MES buffer that 0.1mol/L, pH are 5.0, antithyrotropic hormone IgY antibody is sucked out, as surface
The antithyrotropic hormone IgY antibody of activating agent processing, it is spare.
Embodiment 3 is coupled the system of the magnetic particle suspension for the antithyrotropic hormone IgY antibody for having surfactant to handle
It is standby
(1) take 3mg carboxyl magnetic particle in 0.5mL centrifuge tube, it is 5.0 that 300 μ L concentration of addition, which are 0.1mol/L, pH,
MES buffer is vortexed and mixes, is placed on magnetic frame, and standing 5min separates magnetic particle with liquid, discards supernatant liquid, washing 3
Secondary, adding 300 μ L concentration is the MES buffer that 0.1mol/L, pH are 5.0, is vortexed；
(2) coupling reagent 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide that 50 μ L concentration are 20mg/mL is added
(EDC), in being vortexed on rotatable reactor, it is incubated at room temperature 30min；
(3) the antithyrotropic hormone IgY antibody handled in 0.1mg embodiment 2 through surfactant is added, it is anti-in rotation
It answers and is vortexed on device, be incubated at room temperature 2h；
(4) it is that 50mmol/L is closed containing the Tris buffer of 1% (mass fraction) BSA that 1mL concentration, which is added, and the time is
1h；
(5) supernatant is removed, 300 μ L cleaning buffer solutions (TBS+0.05%Tween-20) are added, is washed 3 times；
(6) the above-mentioned magnetic particle suspension prepared is placed in 1.5mL to save in liquid (Tris-HCl), 2-8 DEG C of preservation.
Wherein, liquid -- 25mM TBS (pH7.2) 0.15M NaCl 0.1%PC300 is saved
Tris (tri methylol amino methane) 0.605g and NaCl 1.755g is weighed, distilled water is added to dissolve, 0.1g is added
Tween20 and 1g BSA, 0.2gPC300, then plus distilled water to 200mL, it is 7.2 that dense HCl to pH is added dropwise under magnetic stirring.
4 acridinium ester of embodiment and antithyrotropic hormone IgG antibody are coupled
(1) Na of 160 μ L is added in 2mL centrifuge tube2CO3-NaHCO3Buffer is marked, it is anti-to add 0.2mg (40 μ L)
Thyrotropic hormone IgG antibody mixes；
(2) the NSP-SA-NHS DMF solution that 2.1 μ L concentration are 6.5mmol/L is added to be protected from light, acridinium ester is made
Molar ratio with antithyrotropic hormone IgG antibody is 10:1, and concussion mixes, and reacts 2h at room temperature；
(3) lysine that 10 μ L concentration are 10% is added, reaction 15min makes to mark reaction terminating, through hyperfiltration treatment, transfer
Into new centrifuge tube, acridinium ester label is obtained, 2-8 DEG C saves backup.
Wherein, buffer -- the Na that concentration 0.1M, pH is 8.5 is marked2CO3-NaHCO3Solution
A liquid: the Na of 1.06g is weighed2CO31L is settled to distilled water；
B liquid: the NaHCO of 7.56g is weighed3It is settled to 100mL with distilled water, adjusts pH to 8.5.
5 different surfaces activating agent of embodiment handles influence and standard curve of the antithyrotropic hormone IgY antibody to experiment
Foundation
(1) 0.1% surfactant (cationic surfactant: hexadecyltrimethylammonium chloride (CTAC), ten is added
Six alkyl trimethyl ammonium bromides (CTAB), amphoteric surfactant: N- dodecyl-N, N- dimethylamino propane sulfonic acid salt
(DDAPS), ten alkyl dimethylamine oxides ammonium (DDAO), nonionic surfactant: Triton X-100, anion surface active
Agent: lauryl sodium sulfate (SDS)) processing antithyrotropic hormone IgY antibody after, be coupled respectively with magnetic particle, it
Reaction test is carried out according to above-mentioned experimental procedure afterwards, test result is as shown in Figure 1, wherein abscissa is the logarithm of calibration object concentration
Value, ordinate by survey luminous value logarithm.
As shown in Figure 1, after different surfactant processing antithyrotropic hormone IgY antibody is added, luminous value, spirit
Sensitivity and linear relationship situation are as follows: DDAO (amphoteric surfactant), DDAPS (amphoteric surfactant) > TritonX-
100 (nonionic surfactant) > without, SDS (anionic surfactant), CTAC (cationic surfactant), CTAB
(cationic surfactant).
The above test result explanation, it is living that addition amphoteric surfactant DDAPS and DDAO are better than addition non-ionic surface
Property agent TritonX-100 and be not added surfactant processing antithyrotropic hormone IgY antibody, thus select DDAO and
Two kinds of surfactants of DDAPS, and be further optimized.
(2) optimize the concentration of amphoteric surfactant DDAO
Anti- rush first is handled with the DDAO surfactant that concentration is respectively 0.01%, 0.05%, 0.1%, 0.3%, 0.5%
Shape glandular hormone IgY antibody, carries out reaction test later, and test result is as shown in Fig. 2, wherein abscissa is pair of calibration object concentration
Numerical value, ordinate by survey luminous value logarithm.
As shown in Figure 2, with the increase of surfactant concentration, the luminous value of product is also being gradually increased, when surface is living
Property agent DDAO concentration when being 0.1%, luminous value reaches maximum；Continuing growing with surfactant concentration later,
Curve starts overhead kick phenomenon occur.
The above test result explanation, the optimal use concentration of surfactant D DAO are 0.1%.
(3) optimize the concentration of amphoteric surfactant DDAPS
Anti- rush is handled with the DDAPS surfactant that concentration is respectively 0.01%, 0.03%, 0.05%, 0.1%, 0.3%
Thyroid hormone IgY antibody, carries out reaction test later, and test result is as shown in figure 3, wherein abscissa is calibration object concentration
Logarithm, ordinate by survey luminous value logarithm.
From the figure 3, it may be seen that with the increase of surfactant concentration, the luminous value of product is also being gradually increased, when surface is living
Property agent DDAPS concentration when being 0.1%, luminous value reaches maximum；Continuing growing with surfactant concentration later,
Curve starts overhead kick phenomenon occur.
The above test result explanation, the optimal use concentration of surfactant D DAPS are 0.1%.
(4) compare 0.1%DDAO and 0.1%DDAPS, find out the optium concentration and optimum value of surfactant
From the point of view of the test result of (2) and (3), after handling antithyrotropic hormone IgY antibody with 0.1%DDAPS, hair
Light value is higher than the processed antithyrotropic hormone IgY antibody of 0.1%DDAO, and is 0.25-150 μ IU/mL in the range of linearity
In range, there is good linear relationship, R2It can reach 0.99 or more, therefore final choice handles anti-rush with 0.1%DDAPS
Thyroid hormone IgY antibody.And magnetic particle suspension is made by the operating procedure of embodiment (3).
(5) foundation of the preparation of calibration object and standard curve
With the PBS buffer solution containing 0.5%BSA by thyrotropic hormone sterling be configured to mark concentration be 0 μ IU/mL,
0.25μIU/mL、1.25μIU/mL、2.5μIU/mL、5μIU/mL、6.25μIU/mL、12.5μIU/mL、25μIU/mL、50μIU/
A series of calibration objects of mL, 75 μ IU/mL, 150 μ IU/mL, respectively by 50 μ L of calibration object, coupling 150 μ L of magnetic particle suspension according to
In secondary addition reaction tube, oscillation is mixed, 37 DEG C of incubation 9min, is separated, cleaning；100 μ are continuously added to the reaction vessel after washing
L acridinium ester label continues to be incubated for 10min, separates, cleaning；It is put into and carries out detecting its luminous intensity in light-emitting appearance, tied to surveying
Fruit uses double-log linear fit, establishes the standard curve and normal equation of luminous intensity and calibration object concentration.
Show that as shown in Figure 4 (knot of 0.1%DDAPS processing antithyrotropic hormone IgY antibody is added in its standard curve
Fruit), wherein abscissa be calibration object concentration logarithm, ordinate by survey luminous value logarithm.Normal equation are as follows: y=
1.004x+1.937。
The detection of thyrotropic hormone in 6 sample to be tested of embodiment
50 μ L of sample to be tested, coupling 150 μ L of magnetic particle suspension are sequentially added in reaction tube, oscillation mixes, and 37 DEG C incubate
9min is educated, is separated, cleaning；100 μ L acridinium ester labels are continuously added to the reaction vessel after washing to continue to be incubated for 10min, point
From cleaning；It is put into and carries out detecting its luminous intensity in light-emitting appearance, rush first shape in sample to be tested can be calculated according to standard curve
The content of glandular hormone.
7 performance indicator testing result of embodiment
Accuracy
It is coupled with the processed antithyrotropic hormone IgY antibody of above-mentioned 0.1% DDAPS surfactant and magnetic particle
Kit obtained is tested afterwards, and the caliberator of 0.7,2.9,75 μ IU/mL is separately added into the serum of known concentration, is surveyed
Determine the accuracy that the rate of recovery carrys out detection reagent, calculation formula is as follows:
The gained rate of recovery, which is calculated, according to formula is respectively as follows: 96.5%, 102.1% and 106.3%.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest scope of cause.
Claims (10)
1. a kind of analysis method for detecting thyrotropic hormone, which is characterized in that specifically includes the following steps:
(1) egg obtained through the immune processing of thyrotropic hormone antigen is subjected to grease removal and purifying, obtains anti-thyroid and swashs
Plain IgY antibody, -20 DEG C of preservations；
(2) it by antithyrotropic hormone IgY antibody obtained in step (1) and surfactant hybrid reaction, is then added slow
Fliud flushing ultrafiltration cleaning, then antithyrotropic hormone IgY antibody is sucked out, it obtains through the processed anti-thyroid of surfactant
Hormone IgY antibody；
(3) coupling reagent is added into magnetic particle after pretreatment and carries out first time incubation at room temperature, be then added in step (2)
What is obtained carries out second through the processed antithyrotropic hormone IgY antibody of surfactant and is incubated at room temperature, discard supernatant liquid,
Washing, obtains magnetic particle suspension, 2-8 DEG C saves backup；
(4) antithyrotropic hormone IgG antibody is added into label buffer to mix, adds acridinium ester concussion and mixes reaction,
Lysine is added to reaction terminating is marked, ultrafiltration obtains acridinium ester label, and 2-8 DEG C saves backup；
(5) prepare thyrotropic hormone calibration object, then respectively by magnetic particle suspension obtained in calibration object, step (3) according to
In secondary addition reaction tube, oscillation is mixed, and is incubated for for the first time, is separated, then acridinium ester mark obtained in step (4) is added in cleaning
Remember object, second of incubation separates, and cleaning is put into and carries out detecting its luminous intensity in light-emitting appearance, establishes luminous intensity and calibration object
The standard curve of concentration；
(6) sample to be tested is detected into its luminous intensity according to the method in step (5), can be calculated according to standard curve to be measured
The content of thyrotropic hormone in sample.
2. a kind of analysis method for detecting thyrotropic hormone according to claim 1, which is characterized in that in step (2),
The surfactant is N- dodecyl-N, N- dimethylamino propane sulfonic acid salt or ten alkyl dimethylamine oxides ammoniums.
3. a kind of analysis method for detecting thyrotropic hormone according to claim 1, which is characterized in that in step (2),
The mass ratio of the antithyrotropic hormone IgY antibody and surfactant is 0.02-1.5:1.
4. a kind of analysis method for detecting thyrotropic hormone according to claim 1, which is characterized in that in step (2),
The mixed reaction time is 0.5-1h.
5. a kind of analysis method for detecting thyrotropic hormone according to claim 1, which is characterized in that in step (3),
The mass ratio of the magnetic particle and coupling reagent is 1-10:1；The matter of the coupling reagent and antithyrotropic hormone IgY antibody
Amount is than being 10-30:1.
6. a kind of analysis method for detecting thyrotropic hormone according to claim 1, which is characterized in that in step (3),
Incubation at room temperature time first time is 15-60min, and second of greenhouse incubation time is 1-4h.
7. a kind of analysis method for detecting thyrotropic hormone according to claim 1, which is characterized in that in step (4),
The antithyrotropic hormone IgG antibody of every 1mg is added 500-2000 μ l and marks buffer；The acridinium ester and anti-thyroid swash
The molar ratio of plain IgG antibody is 5-40:1；It is 10% that 10-200 μ L concentration, which is added, in the antithyrotropic hormone IgG antibody of every 1mg
Lysine solution.
8. a kind of analysis method for detecting thyrotropic hormone according to claim 1, which is characterized in that in step (4),
The mixing reaction is to react 0.5-3h at room temperature.
9. a kind of analysis method for detecting thyrotropic hormone according to claim 1, which is characterized in that in step (5),
The dosage of the calibration object is 10-200 μ L, and the dosage of the magnetic particle suspension is 10-100 μ L, the acridinium ester label
Dosage be 10-200 μ L.
10. a kind of analysis method for detecting thyrotropic hormone according to claim 1, which is characterized in that step (5)
In, the first time incubation temperature is 37 DEG C, time 5-10min；Second of incubation temperature is 37 DEG C, time 10-
20min。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112904025A (en) * | 2021-02-02 | 2021-06-04 | 广东云曌医疗科技有限公司 | Chemiluminescence method-based determination kit and determination method thereof |
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