CN108776232A - A kind of detection human growth hormone (HGH) chemical luminescence immune assay determination reagent kit and preparation method thereof - Google Patents
A kind of detection human growth hormone (HGH) chemical luminescence immune assay determination reagent kit and preparation method thereof Download PDFInfo
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- 239000000854 Human Growth Hormone Substances 0.000 title claims abstract description 45
- 238000001514 detection method Methods 0.000 title claims abstract description 41
- 238000002360 preparation method Methods 0.000 title claims abstract description 40
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 39
- 239000000126 substance Substances 0.000 title claims abstract description 24
- 238000004020 luminiscence type Methods 0.000 title claims abstract description 21
- 238000003556 assay Methods 0.000 title claims abstract description 20
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- 238000002372 labelling Methods 0.000 claims abstract description 19
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- 238000005119 centrifugation Methods 0.000 claims description 10
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- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 6
- 239000004472 Lysine Substances 0.000 claims description 6
- -1 acridine ester Chemical class 0.000 claims description 6
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- 238000004140 cleaning Methods 0.000 claims description 4
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- 102000018997 Growth Hormone Human genes 0.000 description 10
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- 108090000445 Parathyroid hormone Proteins 0.000 description 5
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- 229930192334 Auxin Natural products 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid group Chemical class C(CC(O)(C(=O)O)CC(=O)O)(=O)O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 1
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- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 1
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Classifications
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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
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- Urology & Nephrology (AREA)
- Hematology (AREA)
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- Endocrinology (AREA)
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Abstract
A kind of detection human growth hormone (HGH) chemical luminescence immune assay determination reagent kit of present invention offer and preparation method thereof, belongs to technical field of immunoassay.The kit includes：Be coated with the bead suspension of Streptavidin, the GH antibody of acridinium ester label and biotin labeling GH antibody.The present invention also provides a kind of preparation methods of detection human growth hormone (HGH) chemical luminescence immune assay determination reagent kit.The kit is using the GH in sandwich method principle quantitative determination serum or blood plasma, biotin-GH antibody complexes are added in Streptavidin-magnetic particle suspension, pass through the compatible reaction of biotin and Streptavidin, form magnetic microsphere-Avidin-Biotin-GH compounds, pass through antigen-antibody reaction, it forms antigen antibody complex and forms magnetic particle-biotin-GH antibody-GH-GH antibody-acridinium ester, the detection method has the advantages that high sensitivity, high specificity, reproducible, detection range is wide, advantage of lower cost, can be widely used for clinical detection.
Description
Technical field
The invention belongs to technical field of immunoassay, are related to a kind of detection human growth hormone (HGH) chemiluminscence immunoassay
Kit and preparation method thereof.
Background technology
Human growth hormone (HGH) (human Growth Hormone) is the single peptide secreted by anteriorpituitary acidophic cell
Chain protein hormone is made of 191 amino acid, and there is extensive physiological function, the growth hormone of the mankind mainly to pass through stimulation
Hepatic secretion insulin-like growth factor (IGF-I), acts on bone and cartilage, causes the growth of height, while can promote people
Body protein matter synthesizes and lipolysis, adjusts internal water salt balance.
1. the clinical manifestation of auxin exception
Growth hormone is broadly divided into extremely：1) growth hormone is excessive；2) growth hormone deficiency.Growth hormone excessively would generally
There is gigantism and acromegalia.Gigantism is a kind of abnormal longitudinal growth, the reason is that hGH and IGF-1 over effects, add
Upper epiphyseal growth plate childhood open cause stature excessively high.Acromegalia is also a kind of disease caused by hGH and IGF-1 excess
Disease occurs after manhood growth plate cartilage fusion.The diverse clinical manifestations of acromegalia, existing slight sign, such as
Acra hyperplasia and facial characteristics roughening, apparent metabolism, angiocarpy and the respiratory tract for also thering are morbidity and mortality to be risen
The form of expression.Children growth anhormonia can make longitudinal growth more slow compared to the stone age, and if being grown up has serious growth to swash
Element, which lacks, will appear muscular strength decline, and bone amount is reduced, and insulin sensitivity declines, and abdominal obesity and cardiovascular risk factors increase
(i.e. dyslipidemia is horizontal, atherosclerosis).Kidney, bone and the intestinal cell pair of the adult of progressive growth anhormonia
Parathormone (PTH) is insensitive, and then generates slight PTH and resist, and with the raising of serum PTH levels.With end-organ
Official's sensitivity decline is consistent, is similarly postponed to the blood calcium reaction of PTH.
2. the detection method of growth hormone
The method of currently used detection growth hormone has radioimmunoassay technique (RIA), ELISA technology
(ELISA), but both methods there are many deficiencies, such as RIA there are radioactive pollution, marker half-life short, to operator
Have the shortcomings that radioactive damage, cumbersome, detection time is long；ELISA have sensitivity is low, detection range is narrow, detection when
Between long, poor repeatability the shortcomings of.
Invention content
The purpose of the present invention is to solve it is existing detection growth hormone method detection time it is long, sensitivity is low, again
The problem of renaturation difference, and a kind of detection human growth hormone (HGH) chemical luminescence immune assay determination reagent kit is provided and preparation method thereof.
Present invention firstly provides a kind of detection human growth hormone (HGH) chemical luminescence immune assay determination reagent kit, the kit packets
It includes：
It is coated with the bead suspension of Streptavidin
The GH antibody of acridinium ester label
The GH antibody of biotin labeling
Preferably, described to be coated in the bead suspension of Streptavidin, it is coated with the magnetic bead of Streptavidin
Grain size be 1-3 μm.
Preferably, in the GH antibody of the acridinium ester label, the molar ratio of acridinium ester and GH antibody is (3-15)：1.
Preferably, in the GH antibody of the biotin labeling, the molar ratio of biotin and GH antibody is (3-15)：1.
Preferably, the kit further includes calibration object and quality-control product.
The invention also includes a kind of preparation method of detection human growth hormone (HGH) chemical luminescence immune assay determination reagent kit, packets
It includes：
Step 1：It is coated with the preparation of the bead suspension of Streptavidin
After Streptavidin magnetic particle solution and TBST solution mixings, be placed on magnetic separator, until supernatant without
Muddiness abandons supernatant, leaves and takes magnetic particle, is made into solid-phase reagent R1 after cleaning in buffer solution；
Step 2：The preparation of the GH antibody of acridinium ester label
GH antibody is put into centrifuge tube and is centrifuged, carbonic acid buffer is then added, the centrifugation of acridine ester solution is added after mixing,
It is protected from light being put into after the centrifugation seal of tube in magazine, magazine is then put into mixing in gas bath constant temperature oscillator, confining liquid is added, puts
Enter mixing in gas bath constant temperature oscillator, by the antibody closed by purifying, collection, be then placed in buffer solution and dilute, preserve,
Obtain R2 reagents；
Step 3：The preparation of the GH antibody of biotin labeling
GH antibody is taken, ultrafiltration centrifugation is carried out, GH antibodies buffers is replaced into PBS, it is abundant that biotin solution is then added
The biotin labelled antibodies being collected into after being placed at room temperature for 2-4 hours, are selected buffer solution dilution, obtain R3 reagents by mixing；
Preferably, the buffer solution of the step 1 be 50mM MES, 0.05% tween, 0.05%Proclin300,
pH6.0。
Preferably, in the step 2 GH antibody quality (μ g)：The volume (μ l) of acridine ester solution is 100：1.
Preferably, the confining liquid of the step 2 is preferably lysine, mass fraction 20%.
Preferably, the step 2 buffer solution be 50mM MES, 0.05% Tween-20,0.05%Proclin300,
pH6.0。
Preferably, the buffer solution of the step three is to contain 1%BSA, the 100mM citrate buffer solutions that PH is 6.0.
Beneficial effects of the present invention
A kind of detection human growth hormone (HGH) chemical luminescence immune assay determination reagent kit of present invention offer and preparation method thereof, should
Kit is added using the GH in sandwich method principle quantitative determination serum or blood plasma in Streptavidin-magnetic particle suspension
Biotin-GH antibody complexes form magnetic microsphere-Avidin-biology by the compatible reaction of biotin and Streptavidin
Element-GH compounds form antigen antibody complex and form magnetic particle-biotin-GH antibody-GH-GH by antigen-antibody reaction
Compound is adsorbed on reaction cup bottom by antibody-acridinium ester with magnetic field, washes off free ingredient, and soda acid exciting liquid is added, according to
The content of GH in luminous intensity quantitative measurment sample.The present invention combines chemiluminescence and Enzyme Immunoassay,
The detection method has the advantages that high sensitivity, high specificity, reproducible, detection range is wide, advantage of lower cost, can be extensive
For clinical detection.
Description of the drawings
Fig. 1 is the preparation flow design of the preparation method of the present inventor's growth hormone chemiluminescence immune detection reagent kit
Figure.
Fig. 2 is to survey relative light unit using the detection calibration product examine of the present inventor's growth hormone chemiluminescence quantification kit
Standard curve.
Specific implementation mode
Present invention firstly provides a kind of detection human growth hormone (HGH) chemical luminescence immune assay determination reagent kit, the kit packets
It includes：
It is coated with the bead suspension of Streptavidin
The GH antibody of acridinium ester label
The GH antibody of biotin labeling
According to the present invention, described is coated in the bead suspension of Streptavidin, is coated with the magnetic of Streptavidin
The grain size of pearl is preferably 1-3 μm, when being coated with the grain size of magnetic bead of Streptavidin less than 1 μm, antigen or antibody and magnetic
Pearl Percentage bound is low, and whole light quantity subnumber may be caused relatively low；When being coated with the grain size of magnetic bead of Streptavidin higher than 3 μm,
Non-specific binding is with obvious effects, may lead to kit poor sensitivity etc..
According to the present invention, in the GH antibody of the acridinium ester label, the molar ratio of acridinium ester and GH antibody is preferably (3-
15)：1.
According to the present invention, in the GH antibody of the biotin labeling, the molar ratio of biotin and GH antibody is preferred (3-
15)：1.
According to the present invention, the kit further includes calibration object and quality-control product.The system of the calibration object and quality-control product
It is standby identical, it preferably includes：
GH is diluted to calibration object with the 100mM PBS buffer solution containing 20% calf serum, be distributed into 0.03ng/ml,
0.1ng/ml、0.5ng/ml、1ng/ml、2ng/ml、5ng/ml、10ng/ml、25ng/ml、50ng/ml。
The invention also includes a kind of preparation method of detection human growth hormone (HGH) chemical luminescence immune assay determination reagent kit, tools
Body flow is as shown in Figure 1, include：
Step 1：It is coated with the preparation of the bead suspension of Streptavidin
After Streptavidin magnetic particle solution and TBST solution mixings, be placed on magnetic separator, until supernatant without
Muddiness abandons supernatant, leaves and takes magnetic particle, is made into solid-phase reagent R1 after cleaning in buffer solution；The Streptavidin magnetic particle
The concentration of solution is preferably 100mg/ml；Streptavidin magnetic particle solution and the volume ratio of TBST solution are preferably 0.5：10；
Buffer solution is 50mM MES, 0.05% tween, 0.05%Proclin300, pH6.0；The solid-phase reagent coating strepto- is affine
The magnetic bead concentration of element is preferably 0.05%-0.1%；
Step 2：The preparation of the GH antibody of acridinium ester label
GH antibody is put into centrifuge tube and is centrifuged, preferably centrifuges 10s~30s at room temperature, ensures that antibody is located at centrifuge tube
Then bottom position is added carbonic acid buffer, the centrifugation of acridine ester solution is added after mixing, the centrifuging temperature is preferably room
Temperature, centrifugation time are preferably 0.5min~3min；It is protected from light being put into after the centrifugation seal of tube in magazine, magazine is then put into gas bath
Mixing in constant temperature oscillator (25 DEG C), the mixing time is preferably 2-4h, and confining liquid is added, is put into gas bath constant temperature oscillator
The closing of middle mixing, the off-period is preferably 1-2h, by 250 column purification of sephadex G on the antibody closed, uses PB
Buffer solution elutes, and collects, is then placed in buffer solution and dilutes, and preserves, obtains R2 reagents；
The quality (μ g) of the GH antibody：The volume (μ l) of acridine ester solution is preferably 100：1；The change acridinium ester
The concentration of solution is preferably 2mg/mL；The confining liquid is preferably lysine, and mass fraction is preferably 20%；The buffering
Liquid is 50mM MES, 0.05% Tween-20,0.05%Proclin300, pH6.0；The GH of the R2 reagent acridinium ester labels
Antibody concentration is preferably 1-3ug/mL；
Step 3：The preparation of the GH antibody of biotin labeling
GH monoclonal antibodies are added in 50KDa ultra-filtration centrifuge tubes, PBS buffer solution is added, centrifugation is repeated 3 times, by antibody
Interior buffer exchange is biotin labeling buffer solution, collects solution after displacement, in molar ratio antibody：Biotin is 1:3-1:15 add
Enter biotin, stands 2-4h.The full-automatic protein purification instrument of GE company AKTA is selected to purify biotin labelled antibodies solution,
Purified solution is collected, purified antibodies are formulated as R3 reagents, 4 DEG C of preservations with containing citrate buffer solution；
The quality (μ g) of the GH monoclonal antibodies：The volume (μ l) of PBS buffer solution is preferably 3：1；The PBS is slow
The concentration of fliud flushing is preferably 50mM-200mM；The centrifugal rotational speed is preferably 8000-12000rpm, and centrifugation time is preferably 5-
15min；The GH antibody concentrations of the R3 reagent biotin labelings are preferably 1-3ug/mL；The citrate buffer solution be containing
There are 1%BSA, the 100mM citrate buffer solutions that PH is 6.0.
The detecting step of kit of the present invention：Sample to be tested and R2 reagents are incubated 5min, R3 and R1 reagents are added and are incubated
Exciting liquid record relative luminous intensity (RLU) is added in 5min, washing.In a certain range, the content and relative luminous intensity of GH
It is directly proportional.
The kit of the present invention is using the GH in sandwich method principle quantitative determination serum or blood plasma, in Streptavidin-magnetic
Biotin-GH antibody complexes are added in microparticle suspending liquid, by the compatible reaction of biotin and Streptavidin, it is micro- to form magnetic
Ball-Avidin-Biotin-GH compounds forms antigen antibody complex and forms magnetic particle-biology by antigen-antibody reaction
Element-GH antibody-GH-GH antibody-acridinium ester washes off free ingredient by magnetic separator separating immune complexes.Pass through soda acid
Exciting liquid excites acridinium ester to shine, record relative luminous intensity (RLU), in the range of linearity, in sample the content of GH with it is opposite
Luminous intensity is directly proportional.
Further detailed description is done to the present invention with reference to specific embodiment.
Embodiment 1 prepares the GH chemiluminescence detection kits of the present invention
One, the preparation of calibration object
GH is diluted to calibration object with the 100mM PBS buffer solution containing 20% calf serum, be distributed into 0.03ng/ml,
0.1ng/ml、0.5ng/ml、1ng/ml、2ng/ml、5ng/ml、10ng/ml、25ng/ml、50ng/ml。
Two, the R1 solution of coating Streptavidin MagneSphere is prepared
The Streptavidin magnetic particle solution 0.5 milliliter (50mg) of a concentration of 100mg/ml is taken, 10 milliliters of TBST is added
Solution mixes well after ten minutes, is positioned on magnetic separator, until supernatant without muddiness, abandons supernatant, leaves and takes magnetic particle.It repeats clear
It is a concentration of in 50mM MES, 0.05% tween, 0.05%Proclin300, the buffer solution of pH6.0 it to be made into magnetic bead after washing 3 times
0.05% solid-phase reagent, 2~8 DEG C of preservations.
Three, the preparation of the GH monoclonal antibody R2 solution of acridinium ester label
500ug antibody is put into centrifuge tube, ensures that antibody is located at centrifuge tube bottom position (centrifuge room temperature centrifuges 20s)
After carbonate buffer solution is added, mix well, the DMF solution of 5 μ l 2mg/mL acridinium esters be added after mixing, with centrifuge room temperature
Under the conditions of centrifuge 0.5 minute.It will centrifuge to be put into after effective sealed membrane seals and be protected from light in magazine, magazine is put into gas bath constant temperature later
Oscillator (25 DEG C), mixing 4 hours.20% lysine confining liquids of 1mL are added, are put into gas bath constant temperature oscillator (25 DEG C), middling speed
Mixing, off-period 1h.It by 250 column purification of sephadex G on the antibody closed, is eluted with PB buffer solutions, branch receives
Collection.The antibody-solutions gathered are positioned over 2~8 DEG C of preservations.When use by after purification GH antibody concentrated solutions 50mM MES,
The buffer solution of 0.05% tween, 0.05%Proclin300, pH6.0 are diluted to final concentration of 0.1ug/ml, 2~8 DEG C of preservations.
Four, the preparation of the GH monoclonal antibody R3 solution of biotin labeling
GH monoclonal antibodies 300ug is added in 50KDa ultra-filtration centrifuge tubes, the 100mMPBS buffer solutions of 500ul are added,
10000rpm centrifuges 10min, is repeated 3 times, and is biotin labeling buffer solution by buffer exchange in antibody.It collects molten after replacing
Liquid, by antibody：Biotin=1:Biotin is added in 15 molar ratios, stands 2h.Select the full-automatic protein purification instrument of GE company AKTA
Biotin labelled antibodies solution is purified, purified solution is collected, with containing 1%BSA, the 100mM citric acids that PH is 6.0
Purified antibodies are formulated as the R3 solution of 0.5ug/ml, 4 DEG C of preservations by buffer solution.
Embodiment 2 prepares the GH chemiluminescence detection kits of the present invention
One, the preparation of calibration object
GH is diluted to calibration object with the 100mM PBS buffer solution containing 20% calf serum, be distributed into 0.03ng/ml,
0.1ng/ml、0.5ng/ml、1ng/ml、2ng/ml、5ng/ml、10ng/ml、25ng/ml、50ng/ml。
Two, the R1 solution of coating Streptavidin MagneSphere is prepared
The Streptavidin magnetic particle solution 0.5 milliliter (50mg) of a concentration of 100mg/ml is taken, 10 milliliters of TBST is added
Solution mixes well after ten minutes, is positioned on magnetic separator, until supernatant without muddiness, abandons supernatant, leaves and takes magnetic particle.It repeats clear
It is a concentration of in 50mM MES, 0.05% tween, 0.05%Proclin300, the buffer solution of pH6.0 it to be made into magnetic bead after washing 3 times
0.03% solid-phase reagent, 2~8 DEG C of preservations.
Three, the preparation of the GH monoclonal antibody R2 solution of acridinium ester label
500ug antibody is put into centrifuge tube, ensures that antibody is located at centrifuge tube bottom position (centrifuge room temperature centrifuges 20s)
After carbonate buffer solution is added, mix well, the DMF solution of 5 μ l 2mg/mL acridinium esters be added after mixing, with centrifuge room temperature
Under the conditions of centrifuge 10s.It will centrifuge to be put into after effective sealed membrane seals and be protected from light in magazine, magazine is put into gas bath constant temperature oscillation later
Device (37 DEG C), mixing 3 hours.20% lysine confining liquids of 1mL are added, are put into gas bath constant temperature oscillator (37 DEG C), middling speed is mixed
It is even, off-period 45min.It by 250 column purification of sephadex G on the antibody closed, is eluted with PB buffer solutions, branch receives
Collection.The antibody-solutions gathered are positioned over 2~8 DEG C of preservations.When use by after purification GH antibody concentrated solutions 50mM MES,
The buffer solution of 0.05% tween, 0.05%Proclin300, pH6.0 are diluted to final concentration of 0.05ug/ml, 2~8 DEG C of preservations.
Four, the preparation of the GH monoclonal antibody R3 solution of biotin labeling
GH monoclonal antibodies 300ug is added in 50KDa ultra-filtration centrifuge tubes, the 100mM PBS buffer solution of 500ul is added,
12000rpm centrifuges 8min, is repeated 3 times, and is biotin labeling buffer solution by buffer exchange in antibody.Solution after replacing is collected,
By antibody：Biotin=1:Biotin is added in 10 molar ratios, stands 3h.Select the full-automatic protein purification instrument of GE company AKTA to life
Object element labelled antibody solution is purified, and purified solution is collected, with containing 1%BSA, the 100mM lemon acid bufferings that PH is 6.0
Purified antibodies are formulated as the R3 solution of 1ug/ml, 4 DEG C of preservations by liquid.
Embodiment 3 prepares the GH chemiluminescence detection kits of the present invention
One, the preparation of calibration object
GH is diluted to calibration object with the 100mM PBS buffer solution containing 20% calf serum, be distributed into 0.03ng/ml,
0.1ng/ml、0.5ng/ml、1ng/ml、2ng/ml、5ng/ml、10ng/ml、25ng/ml、50ng/ml。
Two, the R1 solution of coating Streptavidin MagneSphere is prepared
The Streptavidin magnetic particle solution 0.5 milliliter (50mg) of a concentration of 100mg/ml is taken, 10 milliliters of TBST is added
Solution mixes well after ten minutes, is positioned on magnetic separator, until supernatant without muddiness, abandons supernatant, leaves and takes magnetic particle.It repeats clear
It is a concentration of in 50mM MES, 0.05% tween, 0.05%Proclin300, the buffer solution of pH6.0 it to be made into magnetic bead after washing 3 times
0.03% solid-phase reagent, 2~8 DEG C of preservations.
Three, the preparation of the GH monoclonal antibody R2 solution of acridinium ester label
500ug antibody is put into centrifuge tube, ensures that antibody is located at centrifuge tube bottom position (centrifuge room temperature centrifuges 20s)
After carbonate buffer solution is added, mix well, the DMF solution of 5 μ l 2mg/mL acridinium esters be added after mixing, with centrifuge room temperature
Under the conditions of centrifuge 30s.It will centrifuge to be put into after effective sealed membrane seals and be protected from light in magazine, magazine is put into gas bath constant temperature oscillation later
Device (37 DEG C), mixing 2 hours.20% lysine confining liquids of 1mL are added, are put into gas bath constant temperature oscillator (37 DEG C), middling speed is mixed
It is even, off-period 30min.It by 250 column purification of sephadex G on the antibody closed, is eluted with PB buffer solutions, branch receives
Collection.The antibody-solutions gathered are positioned over 2~8 DEG C of preservations.When use by after purification GH antibody concentrated solutions 50mM MES,
The buffer solution of 0.05% tween, 0.05%Proclin300, pH6.0 are diluted to final concentration of 0.07ug/ml, 2~8 DEG C of preservations.
Four, the preparation of the GH monoclonal antibody R3 solution of biotin labeling
GH monoclonal antibodies 300ug is added in 50KDa ultra-filtration centrifuge tubes, the 100mM PBS buffer solution of 500ul is added,
9000rpm centrifuges 10min, is repeated 3 times, and is biotin labeling buffer solution by buffer exchange in antibody.Solution after replacing is collected,
By antibody：Biotin=1:Biotin is added in 3 molar ratios, stands 4h.Select the full-automatic protein purification instrument of GE company AKTA to life
Object element labelled antibody solution is purified, and purified solution is collected, with containing 1%BSA, the 100mM lemon acid bufferings that PH is 6.0
Purified antibodies are formulated as the R3 solution of 1.5ug/ml, 4 DEG C of preservations by liquid.
Embodiment 4GH chemical luminous immune detection methods
With Full-automatic chemiluminescence immunoassay analysis meter (CM-180) for detection instrument, methodology pattern is double-antibody sandwich
Method, i.e. instrument sequentially add the sample of 20 μ l, the GH monoclonal antibodies of biotin labeling of 50 μ l, 50 μ l chemiluminescent substance
The GH monoclonal antibodies of label after reacting 10min, then add 40 μ l Streptavidin magnetic particles, after reacting 10min, carry out magnetic point
From addition cleaning solution cleans 5 times, and instrument will react compound and be sent into detector, sequentially add preexciting liquid and exciting liquid progress
Luminescence-producing reaction acquires optical signal, records luminous value.
Embodiment 5GH chemiluminescence immune detection reagent kit performance evaluations
GH chemiluminescence detection kits obtained are examined and determine according to professional standard in this field and vertification regulation, are tied
Fruit is as follows：
1. the kit range of linearity measures
By the kit of preparation wherein 1 batch, measure range of linearity sample 0.03ng/ml, 0.1ng/ml, 0.5ng/ml,
1ng/ml, 2ng/ml, 5ng/ml, 10ng/ml, 25ng/ml, 50ng/ml show that the range of linearity is 0.03ng/mL~50ng/
ML, linearly dependent coefficient r >=0.9900.Fig. 2 is the test school using the present inventor's growth hormone chemiluminescence quantification kit
The standard curve of relative light unit is surveyed in quasi- product examine, and abscissa is concentration, and unit ng/ml, ordinate is luminous value.As a result such as table
Shown in 1：
1 calibration object concentration of table and relative luminous intensity
2. the measurement of kit sensitivity
By the kit of preparation wherein 1 batch, 20 zero-dose samples are measured, average value (M) and standard deviation (SD) is calculated, obtains
Go out M+2SD, carrying out 2 regression fits according to the concentration-RLU between zero-dose calibration object and adjacent calibration object obtains first power
Journey brings the RLU of M+2SD into equations, show that respective concentration value is kit sensitivity 0.0011ng/ml.1st luminous value
The results are shown in Table 2：
2 blank sample luminous value of table
First point of mean value M that shines1=52.65, SD1=19.58, M1+2SD1=91.80
2nd luminous value is as shown in table 3：
3 second point of table calibrates luminous value
The luminous mean value M=7302 of second point
First point (0,52.65) and second point (0.2,7302) line matched curve, sensitivity=0.0011ng/ of calculating
ml。
3. kit precision is tested
(1) withinrun precision
By the kit of preparation wherein 1 batch, replication is distinguished 10 times to the sample of two various concentrations of high level and low value,
Show that variation within batch coefficient is 1.66%-3.61%.
4 kit withinrun precision of table measures
(2) betweenrun precision
By the kit of preparation wherein 1 batch, replication is distinguished 10 times to the sample of two various concentrations of high level and low value,
Show that interassay coefficient of variation is 1.66%-3.61%.
5 kit betweenrun precision of table measures
4. kit accuracy determination
By the kit of preparation wherein 1 batch, measured concentration is the standard substance of 0.5ng/ml and 20ng/ml, relative deviation
< 10%, test result is as follows：
6 kit accuracy determination of table
5. kit specificity experiments
GH detectable concentrations find following cross reaction when being 1ng/mL and 10ng/mL：
Table 7 kit specificity
6. stabilization of kit is tested
The kit of preparation is carried out to 4 DEG C and 37 DEG C of 14 days stability experiments respectively, the results showed that kit standard product are sent out
The indexs such as variation, precision, the accuracy of luminous intensity are in normal range (NR), and the kit term of validity was up to 24 months.
By a large amount of repeated experiments, kit index of the invention is as follows：
Detection range：0.02-50ng/ml；
Sensitivity：Minimum detection limit is not higher than 0.0011ng/ml；
Precision：Less than 10%；
Accuracy：Measured value standard substance deviation is less than 10%.
Specificity：Measured concentration is not less than the prolactin(PRL sample of 2000mIU/L, and measurement result should be not more than 0.5ng/
ml。
Claims (10)
1. a kind of detection human growth hormone (HGH) chemical luminescence immune assay determination reagent kit, which is characterized in that the kit includes：
It is coated with the bead suspension of Streptavidin
The GH antibody of acridinium ester label
The GH antibody of biotin labeling.
2. a kind of detection human growth hormone (HGH) chemical luminescence immune assay determination reagent kit according to claim 1, feature
It is, described is coated in the bead suspension of Streptavidin, and the grain size for being coated with the magnetic bead of Streptavidin is 1-3 μ
m。
3. a kind of detection human growth hormone (HGH) chemical luminescence immune assay determination reagent kit according to claim 1, feature
It is, in the GH antibody of the acridinium ester label, the molar ratio of acridinium ester and GH antibody is (3-15)：1.
4. a kind of detection human growth hormone (HGH) chemical luminescence immune assay determination reagent kit according to claim 1, feature
It is, in the GH antibody of the biotin labeling, the molar ratio of biotin and GH antibody is (3-15)：1.
5. a kind of detection human growth hormone (HGH) chemical luminescence immune assay determination reagent kit according to claim 1, feature
It is, the kit further includes calibration object and quality-control product.
6. a kind of preparation side of detection human growth hormone (HGH) chemical luminescence immune assay determination reagent kit according to claim 1
Method, which is characterized in that including：
Step 1：It is coated with the preparation of the bead suspension of Streptavidin
After Streptavidin magnetic particle solution and TBST solution mixings, it is placed on magnetic separator, until supernatant is without muddiness,
Supernatant is abandoned, magnetic particle is left and taken, is made into solid-phase reagent R1 after cleaning in buffer solution；
Step 2：The preparation of the GH antibody of acridinium ester label
GH antibody is put into centrifuge tube and is centrifuged, is then added carbonic acid buffer, the centrifugation of acridine ester solution is added after mixing, it will be from
It is put into and is protected from light in magazine after the heart seal of tube, magazine is then put into mixing in gas bath constant temperature oscillator, confining liquid is added, is put into gas
Mixing in constant temperature oscillator is bathed, by the antibody closed by purifying, collection, is then placed in buffer solution and dilutes, preserve, obtain
R2 reagents；
Step 3：The preparation of the GH antibody of biotin labeling
GH antibody is taken, ultrafiltration centrifugation is carried out, GH antibodies buffers is replaced into PBS, biotin solution is then added and mixes well,
After being placed at room temperature for 2-4 hours, the biotin labelled antibodies being collected into are selected into buffer solution dilution, obtain R3 reagents.
7. a kind of preparation side of detection human growth hormone (HGH) chemical luminescence immune assay determination reagent kit according to claim 6
Method, which is characterized in that the buffer solution of the step 1 is 50mM MES, 0.05% tween, 0.05%Proclin300, pH6.0.
8. a kind of preparation side of detection human growth hormone (HGH) chemical luminescence immune assay determination reagent kit according to claim 6
Method, which is characterized in that the quality (μ g) of GH antibody in the step 2：The volume (μ l) of acridine ester solution is 100：1.
9. a kind of preparation side of detection human growth hormone (HGH) chemical luminescence immune assay determination reagent kit according to claim 6
Method, which is characterized in that the confining liquid of the step 2 is preferably lysine, mass fraction 20%.
10. a kind of preparation of detection human growth hormone (HGH) chemical luminescence immune assay determination reagent kit according to claim 1
Method, which is characterized in that the step 2 buffer solution be 50mM MES, 0.05% Tween-20,0.05%Proclin300,
pH6.0。
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| CN109470872B (en) * | 2018-12-07 | 2022-02-08 | 迈克生物股份有限公司 | Magnetic particle buffer |
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Application publication date: 20181109 |