CN109870570A - A kind of enzyme linked immunological kit detecting monkey IL-18 - Google Patents
A kind of enzyme linked immunological kit detecting monkey IL-18 Download PDFInfo
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Abstract
A kind of double antibodies sandwich enzyme linked immunological kit detecting monkey IL-18, including by the coated ELISA Plate of capture antibody, detection antibody, dilution, concentrated cleaning solution, conjugate, developing solution, terminate liquid and standard items；Wherein, the monoclonal antibody that capture antibody is IL-18；Detect the IL-18 polyclonal antibody that antibody is biotin labeling；Dilution is the pH7.4 phosphate buffer of 0.5~4% casein sodium；Capture antibody is coated on ELISA Plate, and antibody closing is carried out using the pH7.4 phosphate buffer of 1~5% bovine serum albumin(BSA) of mass fraction as confining liquid；Standard items are IL-18 albumen.For the present invention using the level of the IL-18 of the test sample of sandwich sxemiquantitative ELISA, detection method is easy to operate, and result precision is high, is able to detect large batch of sample.Suitable for the specific detection for monkey IL-18 of the non-clinical diagnostic purpose of scientific research field, there is very big application value.
Description
Technical field
The present invention relates in vitro diagnostic techniques fields, more particularly, to a kind of enzyme linked immunological for detecting monkey IL-18
Kit.
Background technique
IL-18 is generated by macrophage and other cells, belongs to IL-1 ligand family, structure and IL-1 protein family phase
Seemingly.IL-18cDNA encodes 193 amino acid, and cysteine aspartase is hydrolyzed to mature IL-18 in N-terminal, plays its life
Object activity.It is an anticusp inflammation factor, and various kinds of cell development and cytokine secretion is adjusted.Studies have shown that it is only
Special dependent cells factor ambient enviroment and the cell factor for stimulating Thl and Th2 cell effect.It can promote peripheral blood mononuclear cells
The cell factors such as IFN-γ, IL-2 and granulocyte macrophage colony stimulating factor are generated, the thin of NK cell and Thl cell is enhanced
Cytotoxicity promotes the proliferation of T cell, and has promotion and adjustment effect in Thl cell differentiation and immune response.It is adjusted immune
It plays an important role in section, anti-infective, antitumor and chronic inflammation disease pathogenic process.In scientific research field, non-clinical diagnosis
The IL-18 detection of purpose has very big market prospects.
But currently on the market still suitable for the specificity inspection for monkey IL-18 of the non-clinical diagnostic purpose of scientific research field
Test agent box lacks the enzyme linked immunological kit of monkey IL-18 easy to operate, detection sensitivity is high, accuracy is strong a kind of.
Summary of the invention
The purpose of the invention is to overcome the prior art lack it is a kind of it is easy to operate, detection sensitivity is high, accuracy
The enzyme linked immunological kit of strong monkey IL-18 is insufficient, provides a kind of monkey IL- easy to operate, detection sensitivity is high, accuracy is strong
18 enzyme linked immunological kit.
To achieve the goals above, the present invention is achieved by the following technical programs:
The technical scheme is that capture antibody is coated in ELISA Plate, and after detection antibody and IL-18 albumen is added, shape
At capture antibody, antigen and the compound for detecting antibody.After conjugate and developing solution to be added, terminates and react and read sample
Light absorption value.Content by the IL-18 being calculated compared with standard curve in sample to be tested.
A kind of double antibodies sandwich enzyme linked immunological kit detecting monkey IL-18, which is characterized in that including being coated with by capture antibody
ELISA Plate, detection antibody, dilution, concentrated cleaning solution, conjugate, developing solution, terminate liquid and standard items；
Wherein, the monoclonal antibody that capture antibody is IL-18；
Detect the IL-18 polyclonal antibody that antibody is biotin labeling；
Dilution is the pH7.4 phosphate buffer of 0.5~4% casein sodium；
Capture antibody is coated on ELISA Plate, with the pH7.4 phosphate buffer of 1~5% bovine serum albumin(BSA) of mass fraction
Antibody closing is carried out as confining liquid；
Standard items are IL-18 albumen.
Preferably, the monoclonal antibody that capture antibody is anti-human IL-18.
Preferably, detection antibody is the IL-18 Mouse Polyclonal Antibody of biotin labeling.
IL-18 albumen is NCBI Reference Sequence:NP_001028006.1.
IL-18 albumen is the IL-18 of Bacillus coli expression.
Preferably, standard items are IL-18 albumen dried frozen aquatic products.
Preferably, capture antibody is coated on ELISA Plate, comprising the following steps:
S11. antibody dilution will be captured with pH9.4 phosphate buffer, ELISA Plate is added, it is pre-coated；
S12. S1 ELISA Plate is washed with the PBS of the pH7.4 containing 0.01~0.2% polysorbas20 of volume fraction；
S13. it pats dry, confining liquid, closing is added；
S14. it pats dry, it is dry, it is vacuum-packed, saves.
Preferably, in step S11, coated condition is 1~10 DEG C, no less than 12 hours.
Preferably, in step S12, after washing S1 coating with the PBS of the pH7.4 containing 0.05% polysorbas20 of volume fraction
ELISA ELISA Plate
Preferably, in step S13, closed condition is 25~37 DEG C, 2~8 hours.
It is highly preferred that closed condition is 25 DEG C, 4 hours in step S13.
Preferably, in step S14, dry condition be 25~37 DEG C, 4~12 hours, 10%~60% humidity.
It is highly preferred that in step S14, dry condition is 25 DEG C, 6 hours, 30% humidity.
Preferably, in step S14, the condition of preservation is 4~25 DEG C.
It is highly preferred that in step S14,4 DEG C of the condition of preservation.
Preferably, the peridium concentration for capturing antibody is 0.5~4 μ g/ml, and package amount is 50~300 μ L.
Preferably, the peridium concentration for capturing antibody is 1 μ g/ml, and package amount is 100 μ L.
Preferably, the use concentration for detecting antibody is 0.25~2 μ g/ml, and usage amount is 50~300 μ L.
Preferably, the use concentration for detecting antibody is 0.5 μ g/ml, and usage amount is 50 μ L.
Preferably, concentrated cleaning solution is the phosphate-buffered of the pH7.4 of the tween containing volume fraction 0.01~0.2%
Liquid, working concentration need to dilute 20 times.
It is highly preferred that concentrated cleaning solution is the phosphate buffer of the pH7.4 of the tween containing volume fraction 0.05%, work
Need to dilute 20 times as concentration.
Preferably, conjugate is the Streptavidin of horseradish peroxidase label, and working concentration is 0.1~1. μ g/ml.
It is highly preferred that the working concentration of conjugate is 0.25 μ g/ml.
Preferably, it is 0.01~0.05% tetramethyl benzidine that developing solution, which is mass fraction,.
It is highly preferred that it is 0.02% tetramethyl benzidine that developing solution, which is mass fraction,.
Preferably, terminate liquid is 1~4mol/L sulfuric acid.
It is highly preferred that terminate liquid is 2mol/L sulfuric acid.
The application method of above-described double antibodies sandwich enzyme linked immunological kit, comprising the following steps:
S21. into ELISA Plate, detection antibody is added；
S22. the standard items and sample to be tested that will be diluted with liquid gradient dilution are added in corresponding hole, and oscillation mixes, room temperature
It is incubated for；
S23. ELISA Plate is washed with cleaning solution；
S24. conjugate is added, oscillation mixes, incubation at room temperature；
S25. ELISA Plate is washed with cleaning solution；
S26. developing solution is added, oscillation mixes, and room temperature is protected from light colour developing；
S27. terminate liquid is added, terminates reaction；
S28. the OD value for detecting 450nm and 620nm respectively, calculates its difference；
S29. with OD450With OD620Difference be ordinate, using the concentration of standard items as abscissa, obtain standard curve and
Linear regression equations；
S210. by the OD of sample to be tested450With OD620Difference bring linear regression equations into, calculate its concentration.
Preferably, in step S21, the amount that detection antibody is added is 50~300 holes μ L/.
It is highly preferred that the amount that detection antibody is added is 50 holes μ L/ in step S21.
Preferably, in step S22, the amount that antigen or sample to be tested is added is 50~300 holes μ L/.
It is highly preferred that the amount that antigen or sample to be tested is added is 50 holes μ L/ in step S22.
Preferably, in step S22, incubation time is 1~3h.
It is highly preferred that in step S22, incubation time 2h.
Preferably, in step S23, washing ELISA Plate is 200~400 hole μ L/ of cleaning solution, washs 2~6 times, every time 20~
It 60 seconds, pats dry.
It is highly preferred that washing ELISA Plate is 300 hole μ L/ of cleaning solution in step S23, washs 3 times, 30 seconds every time, pat dry.
Preferably, in step S24,50~200 hole μ L/ of amount of conjugate is added.
It is highly preferred that 100 hole μ L/ of amount of conjugate is added in step S24.
Preferably, in step S24, incubation time is 15~60min.
It is highly preferred that in step S24, incubation time 30min.
Preferably, in step S25, washing ELISA ELISA Plate be 200~400 hole μ L/ of cleaning solution, wash 2~6 times, every time
It 20~60 seconds, pats dry.
It is highly preferred that in step S25, washing ELISA ELISA Plate is 300 hole μ L/ of cleaning solution, washs 5 times, 30 seconds every time,
It pats dry.
Preferably, in step S26,50~200 hole μ L/ of amount of developing solution is added.
It is highly preferred that 100 hole μ L/ of amount of developing solution is added in step S26.
Preferably, in step S26, developing time is 15~60min.
It is highly preferred that in step S26, developing time 15min.
Preferably, in step S27,25~100 hole μ L/ of amount of terminate liquid is added.
It is highly preferred that 50 hole μ L/ of amount of terminate liquid is added in step S27.
Compared with prior art, the invention has the following beneficial effects:
Level of the present invention using the IL-18 of the test sample of sandwich sxemiquantitative ELISA, detection method operation letter
Single, result precision is high, is able to detect large batch of sample.Monkey IL- is directed to suitable for the non-clinical diagnostic purpose of scientific research field
18 specific detection has very big application value.
Detailed description of the invention
Fig. 1 is monkey IL-18 standard items examination criteria curve in embodiment 1.
Fig. 2 is the monkey IL-18 standard items standard curve that selection one-step method obtains in embodiment 3.
Fig. 3 is the monkey IL-18 standard items standard curve that selection two step method obtains in embodiment 3.
Fig. 4 is the monkey IL-18 standard for selecting the pH7.4 phosphate buffer of 2%BSA to obtain as confining liquid in embodiment 4
Product standard curve.
Fig. 5 is the monkey IL-18 standard for selecting the pH7.4 phosphate buffer of 3%BSA to obtain as confining liquid in embodiment 4
Product standard curve.
Fig. 6 is the monkey IL-18 for selecting the pH7.4 phosphate buffer of 1% casein sodium to obtain as dilution in embodiment 5
Standard items examination criteria curve.
Fig. 7 is the monkey IL-18 standard for selecting the pH7.4 phosphate buffer of 1%BSA to obtain as dilution in embodiment 5
Product examine mark directrix curve.
Specific embodiment
The present invention is made with specific embodiment with reference to the accompanying drawings of the specification and further being elaborated, the embodiment
It is served only for explaining the present invention, be not intended to limit the scope of the present invention.Test method as used in the following examples is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
Following embodiment is with MBL (MEDICAL&BIOLOGICAL LABORATORIES CO., LTD), article No. D044-3
Anti-human IL-18 monoclonal antibody as capture antibody, MBL (MEDICAL&BIOLOGICAL LABORATORIES CO.,
LTD) company's article No. be D045-6 Mouse Polyclonal Antibody as detection antibody, for illustrate technical solution of the present invention,
It is not the selection of uniquely suitable antibody of the invention.
A kind of enzyme linked immunological kit for detecting monkey IL-18 of embodiment 1
One, it forms
It is coated with the ELISA Plate of capture antibody: the monoclonal antibody that capture antibody is anti-human IL-18；
Detect the Mouse Polyclonal Antibody that antibody is biotin labeling；
Concentrated cleaning solution: the phosphate buffer of the pH7.4 of the tween containing volume fraction 0.5%, working concentration need
20 times of dilution；
Conjugate: the Streptavidin of horseradish peroxidase label, working concentration dilution 2000 × it is 0.25 μ g/ml；
Developing solution: mass fraction is 0.02% tetramethyl benzidine；
Terminate liquid: 2mol/L sulfuric acid；
Dilution: the pH7.4 phosphate buffer of 1% casein sodium of mass fraction；
Standard items: standard items are lyophilized in recombination monkey IL-18 albumen.
Wherein, the ELISA ELISA Plate for being coated with capture antibody is prepared in accordance with the following methods:
1, it is coated with
It is diluted to 1 μ g/mL by antibody is captured with coating buffer (pH9.4 phosphate buffer), 100 holes μ L/, 4 DEG C were coated with
Night.
2, it closes
Topple over liquid in plate, is washed 2 times with the PBS containing 0.05% polysorbas20,300 holes μ L/.It pats dry.Confining liquid is added
(the pH7.4 phosphate buffer containing 3% bovine serum albumin(BSA)), 300 holes μ L/, room temperature are closed 4 hours.Topple over liquid in plate, claps
It is dry.
3, dry
25 DEG C, 30% humidity is 6 hours dry, is packed with vacuum packing machine, 4 DEG C save backup.
Two, application method
1, it is loaded
50 hole μ L/ of biotin labelled antibodies is added (detection antibody concentration is 0.5 μ g/mL).Then, it will be diluted with liquid dilution
Good 6 concentration gradient 20ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, the standard items of 0.625ng/mL,
(dilution is then added in blank well) and sample to be tested are added in corresponding experimental port, and 50 μ L are added in every hole.Sealing plate film is covered, gently
Oscillation mixes, and room temperature (referring both to 18 DEG C~30 DEG C below) is incubated for 2h.
2, it washs
Liquid in pouring aperture, 300 hole μ L/ of cleaning solution wash 3 times, 30 seconds every time, wash pat dry on blotting paper every time.Such as
Fruit is machine-washed with board-washing and is washed, and washing times increase primary.
3, conjugate
Conjugate is added in 100 holes μ L/, covers sealing plate film, and gently oscillation mixes, and is incubated at room temperature 30min.
4, it washs
Liquid in pouring aperture, 300 hole μ L/ of cleaning solution wash 5 times, 30 seconds every time, wash pat dry on blotting paper every time.Such as
Fruit is machine-washed with board-washing and is washed, and washing times increase primary.
5, it develops the color
Developing solution is added in 100 holes μ L/.Sealing plate film is covered, gently oscillation mixes, and room temperature is protected from light colour developing 15min.
6, it terminates
Take out ELISA ELISA Plate, terminate liquid is added in the direct hole 50 μ L/, terminates reaction (blue switchs to yellow immediately at this time).
7, it detects
After termination, detected with microplate reader in dominant wavelength 450nm and 620~650nm of reference wavelength, OD450Subtract
OD620Read result (OD620 is subtracted with OD450 below and calculates testing result).Detection is completed in 15 minutes after terminating reaction.
8, the foundation of standard curve
The concentration of monkey IL-18 standard items is abscissa, and corresponding OD value (refers both to OD below450Subtract OD620) it is ordinate, it does
Corresponding standard curve (Fig. 1) is obtained, the linear regression equations (ignoring zero standard's product when calculating) of standard curve are calculated.
The OD value of sample is substituted into equation and calculates its IL-18 concentration.
A kind of preparation for the enzyme linked immunological kit for detecting monkey IL-18 of embodiment 2
One, chessboard method gropes the optimum antibody of detection kit to concentration
1, experimental method
It is 4 μ g/mL, 2 μ g/mL, 1 μ g/mL and 0.5 μ g/mL, anti-IL-18 that the capture antibody of anti-IL-18, which is diluted to concentration,
Detection antibody be diluted to concentration be 2 μ g/mL, 1 μ g/mL, 0.5 μ g/mL and 0.25 μ g/mL, recombination IL-18 albumen be diluted to
40ng/mL, 20ng/mL and 10ng/mL are detected according to embodiment 1.
2, experimental result
Comprehensive analysis blank absorbency and response guarantee that blank value is lower than 0.1, and the lower response the better.Select antibody
Peridium concentration be 1 μ g/mL, detection antibody concentration be 0.5 μ g/mL.
Two, the preparation of ELISA Plate
1, it is coated with
It is diluted to 1 μ g/mL by antibody is captured with coating buffer (pH9.4 phosphate buffer), 100 holes μ L/, 4 DEG C were coated with
Night.
2, it closes
Topple over liquid in plate, is washed 2 times with the PBS containing 0.05% polysorbas20,300 holes μ L/.It pats dry.Confining liquid is added
(the pH7.4 phosphate buffer containing 3% bovine serum albumin(BSA)), 300 holes μ L/, room temperature are closed 4 hours.Topple over liquid in plate, claps
It is dry.
3, dry
25 DEG C, 30% humidity is 6 hours dry, is packed with vacuum packing machine, 4 DEG C save backup.
The selection of 3 detection method of embodiment optimizes
1, experimental method
The detection method in embodiment 1 is replaced with two step method, is carried out using the enzyme-linked immunization kit that embodiment 1 constructs
Detection.It is compared with the detection method in embodiment 1.Respectively by standard items diluted to 0.625,1.25,2.5,
5,10 and 20ng/ml is detected.
The specific detecting step of two step method is as follows:
(1), antigen is added
By above-mentioned dilution good 6 concentration gradients 20ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL and
The standard items of 0.625ng/mL are added in corresponding experimental port, and 100 μ L are added in every hole.Sealing plate film is covered, room temperature (refers both to 18 below
DEG C~30 DEG C) it is incubated for 2h.
(2), it washs
Liquid in pouring aperture, 300 hole μ L/ of cleaning solution wash 3 times, 30 seconds every time, wash pat dry on blotting paper every time.Such as
Fruit is machine-washed with board-washing and is washed, and washing times increase primary.
(3), detection antibody is added
Addition concentration is 0.5 μ g/mL biotin labelled antibodies, 100 hole μ L/.Sealing plate film is covered, room temperature (refers both to 18 DEG C below
~30 DEG C) it is incubated for 1h.
(4), it washs
Liquid in pouring aperture, 300 hole μ L/ of cleaning solution wash 3 times, 30 seconds every time, wash pat dry on blotting paper every time.Such as
Fruit is machine-washed with board-washing and is washed, and washing times increase primary.
(5), conjugate
Conjugate is added in 100 holes μ L/, covers sealing plate film, and gently oscillation mixes, and is incubated at room temperature 30min.
(6), it washs
Liquid in pouring aperture, 300 hole μ L/ of cleaning solution wash 5 times, 30 seconds every time, wash pat dry on blotting paper every time.Such as
Fruit is machine-washed with board-washing and is washed, and washing times increase primary.
(7), it develops the color
Developing solution is added in 100 holes μ L/.Sealing plate film is covered, gently oscillation mixes, and room temperature is protected from light colour developing 15min.
(8), it terminates
Take out ELISA Plate, terminate liquid is added in the direct hole 50 μ L/, terminates reaction (blue switchs to yellow immediately at this time).
(9), it detects
After termination, detected with microplate reader in dominant wavelength 450nm and 620~650nm of reference wavelength, OD450 subtracts
OD620 reads result (subtract OD620 below with OD450 and calculate testing result).Detection is completed in 15 minutes after terminating reaction.
(10), the foundation of standard curve
The concentration of monkey IL-18 standard items is abscissa, and corresponding OD value (refer both to OD450 below and subtract OD620) is ordinate,
It is made corresponding standard curve, calculates the linear regression equations (ignoring zero standard's product when calculating) of standard curve.It will
The OD value of sample substitutes into equation and calculates its IL-18 concentration.
2, experimental result
Testing result is shown in Table 1 and Fig. 2~3, and the regression equation of the standard curve of detection method provided by the invention is y=
0.1023x+0.0271(R2=0.9992)；The regression equation for the standard curve for selecting two step method to obtain is y=0.0647x+
0.0853(R2=0.9993).Detection method provided by the invention is high relative to traditional two step method OD value, and can save original
The dosage of material.
Table 1:
The selection of 4 confining liquid of embodiment optimizes
1, experimental method
Confining liquid selects the pH7.4 phosphate buffer containing 2% and 3%BSA respectively, and rest part is same as Example 1.Structure
The enzyme linked immunological kit of detection monkey IL-18 is built, according to the method for embodiment 1 examination criteria product (recombination monkey IL-18 albumen).Point
Standard items are not detected with diluted to 0,5,10 and 20ng/ml.
2, experimental result
It the results are shown in Table the mark for selecting the pH7.4 phosphate buffer of 2%BSA to obtain shown in 2 and Fig. 4, Fig. 5 as confining liquid
Directrix curve are as follows: y=0.1181x+0.0372 (R2=0.9867)；Select the pH7.4 phosphate buffer of 3%BSA as confining liquid
Obtained standard curve are as follows: y=0.1117x+0.0251 (R2=0.9971).It follows that the pH7.4 phosphoric acid of selection 3%BSA
The linear equation R that buffer is obtained as confining liquid2> 0.99, and the detected value of blank sample is lower, and detection method is more quasi-
Really.
Table 2:
The selection of 5 dilution of embodiment optimizes
1, experimental method
Dilution selects the phosphate buffer of 1% casein sodium and the PBS buffer solution of 1%BSA, rest part and reality respectively
It is identical to apply example 1.The enzyme linked immunological kit of building detection monkey IL-18, examination criteria product (recombinate monkey according to the method for embodiment 1
IL-18 albumen).Standard items are detected with diluted to 0,5,10 and 20ng/ml respectively.
2, experimental result
It the results are shown in Table shown in 3, table 4 and Fig. 6, Fig. 7, the standard curve for selecting the phosphate buffer of 1% casein sodium to obtain
Regression equation be y=0.1002x-0.0226 (R2=1)；The standard curve that the PBS buffer solution of selection 1%BSA obtains returns
Returning equation is y=0.1098x+0.2125 (R2=0.9638).It follows that the phosphate buffer of 1% casein sodium of selection is made
The linear equation R obtained for dilution2Bigger, detection method is more accurate.
Table 3: the PBS buffer solution of 1% casein sodium of selection does dilution
Table 4: the PBS buffer solution of 1%BSA is selected to do dilution
The measurement of 6 kit major parameter of embodiment
One, the range of linearity of kit
1, experimental method
IL-18 freeze-drying standard items are diluted to 20ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL with 1% casein sodium,
1.25ng/mL, 0.625ng/mL and 0ng/mL, using the enzyme linked immunological kit in embodiment 1, according to the side in embodiment 1
Method is detected.
2, experimental result
When the concentration of IL-18 freeze-drying standard items is between 0.625~20ng/mL, its line of the equation of linear regression of acquisition
Property related coefficient be 0.99 or more, i.e., the range of linearity of the kit be 0.625~20ng/mL.
Two, the specificity of kit
1, experimental method
By non-human primates CX3CL1, CXCL10 of recombination, CCL17, CXCL13, PD-1, TWEAK, TRAIL, CXCL12,
CXCL7 is diluted to 2 μ g/mL, using the enzyme linked immunological kit in embodiment 1, is detected according to the method in embodiment 1.
2, experimental result
The results show that the kit and non-human primates CX3CL1, CXCL10, CCL17, CXCL13, PD-1, TWEAK,
TRAIL, CXCL12, CXCL7 do not have visible cross reaction.
Three, the sensitivity of kit
1, experimental method
Using the enzyme linked immunological kit in embodiment 1, the average absorbance value of 16 blank samples is detected plus twice of mark
Quasi- deviation calculates the content of IL-18.
2, experimental result
The results show that it is 99.7pg/mL that lowest detection, which is limited to sensitivity,.Illustrate that this kit has higher detection sensitive
Degree.
Four, kit precision
(I) precision in testing
1, experimental method
Have chosen 4 groups of samples, every group containing there are two various concentration (10ng/mL and 2.5ng/mL), each concentration has 10
It repeats, calculates separately the coefficient of variation (C.V) of testing result.
2, experimental result
It the results are shown in Table 5, average coefficient of variation 2.56%.
Table 5:
(II) measurement of test bay precision
1, experimental method
Two various concentrations (10ng/mL and 2.5ng/mL) is had chosen, each concentration there are 10 repetitions, has carried out 3 realities
It tests, calculates the coefficient of variation (C.V) of 3 batch testing results.
2, experimental result
It the results are shown in Table 6, average coefficient of variation 7.96%.
Table 6:
Five, the kit rate of recovery
1, experimental method
The recombinant protein IL-18 of 3 various concentrations (20ng/mL, 10ng/mL and 5ng/mL) is respectively added to machin
Using the enzyme linked immunological kit in embodiment 1 in 1640 culture medium of serum and RPMI, carried out according to the method in embodiment 1
Detection, calculates the rate of recovery of each concentration.
2, experimental result
In the average recovery rate that the average recovery rate in machin serum matrix is in 31.8%, RPMI, 1640 culture medium
It is 158.33%.
Claims (10)
1. a kind of double antibodies sandwich enzyme linked immunological kit for detecting monkey IL-18, which is characterized in that including coated by capture antibody
ELISA Plate, detection antibody, dilution, concentrated cleaning solution, conjugate, developing solution, terminate liquid and standard items；
Wherein, the monoclonal antibody that capture antibody is IL-18；
Detect the IL-18 polyclonal antibody that antibody is biotin labeling；
Dilution is the pH7.4 phosphate buffer of 0.5~4% casein sodium；
Capture antibody is coated on ELISA Plate, using the pH7.4 phosphate buffer of 1~5% bovine serum albumin(BSA) of mass fraction as envelope
It closes liquid and carries out antibody closing；
Standard items are IL-18 albumen.
2. double antibodies sandwich enzyme linked immunological kit according to claim 1, which is characterized in that capture antibody is coated on enzyme mark
On plate, comprising the following steps:
S11. antibody dilution will be captured with pH9.4 phosphate buffer, ELISA Plate is added, it is pre-coated；
S12. the ELISA enzyme mark after washing S11 coating with the PBS of the pH7.4 containing 0.01~0.2% polysorbas20 of volume fraction
Plate；
S13. it pats dry, confining liquid, closing is added；
S14. it pats dry, it is dry, it is vacuum-packed, saves.
3. double antibodies sandwich enzyme linked immunological kit according to claim 1, which is characterized in that capture the peridium concentration of antibody
For 0. 5~4 μ g/ml, package amount is 50~300 μ L.
4. double antibodies sandwich enzyme linked immunological kit according to claim 3, which is characterized in that capture the peridium concentration of antibody
For 1g/ml, package amount is 100 μ L.
5. double antibodies sandwich enzyme linked immunological kit according to claim 1, which is characterized in that detect the use concentration of antibody
For 0.25~2 μ g/ml, usage amount is 50~300 μ L.
6. double antibodies sandwich enzyme linked immunological kit according to claim 5, which is characterized in that detect the use concentration of antibody
For 0.5 μ g/ml, usage amount is 50 μ L.
7. double antibodies sandwich enzyme linked immunological kit according to claim 1, which is characterized in that concentrated cleaning solution is to contain body
The phosphate buffer of the pH7.4 of the tween of fraction 0.01~0.2%, working concentration need to dilute 20 times.
8. double antibodies sandwich enzyme linked immunological kit according to claim 1, which is characterized in that conjugate is horseradish peroxidating
The Streptavidin of enzyme label；Developing solution is tetramethyl benzidine；Terminate liquid is sulfuric acid.
9. the application method of any double antibodies sandwich enzyme linked immunological kit of claim 1~8, comprising the following steps:
S21. to ELISA Plate, detection antibody is added；
S22. the standard items and sample to be tested that will be diluted with liquid gradient dilution are separately added into corresponding hole, and oscillation mixes, room temperature
It is incubated for；
S23. ELISA Plate is washed with cleaning solution；
S24. conjugate is added, oscillation mixes, incubation at room temperature；
S25. ELISA Plate is washed with cleaning solution；
S26. developing solution is added, oscillation mixes, and room temperature is protected from light colour developing；
S27. terminate liquid is added, terminates reaction；
S28. the OD value for detecting 450nm and 620nm respectively, calculates its difference；
S29. with OD450With OD620Difference be ordinate, using the concentration of standard items as abscissa, obtain standard curve and linear
Regression equation；
S210. by the OD of sample to be tested450With OD620Difference bring linear regression equations into, calculate its concentration.
10. application method according to claim 9, which is characterized in that the amount of standard items or sample to be tested is 50~300 μ
The hole L/.
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| CN201811647624.7A CN109870570A (en) | 2018-12-29 | 2018-12-29 | A kind of enzyme linked immunological kit detecting monkey IL-18 |
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| CN112014340A (en) * | 2020-09-01 | 2020-12-01 | 广西玮美生物科技有限公司 | Method for detecting ghrelin of nonhuman primate |
| CN112858694A (en) * | 2021-02-05 | 2021-05-28 | 湖南泰新医药科技有限公司 | Method for simultaneously determining concentrations of anti-PD-1 antibody and anti-PD-L1 antibody in human serum |
| CN113092392A (en) * | 2021-03-05 | 2021-07-09 | 苏州西山中科药物研究开发有限公司 | Universal method for detecting total concentration of monoclonal antibody drug targeting soluble protein in monkey serum |
| CN113702362A (en) * | 2021-08-27 | 2021-11-26 | 宁波熙宁检测技术有限公司 | Method for quantitatively detecting IFN-gamma concentration by using chemiluminescence method and detection kit thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111610333A (en) * | 2020-05-14 | 2020-09-01 | 武汉康珠生物技术有限公司 | Enzyme-linked immunoassay method based on fingertip blood |
| CN112014340A (en) * | 2020-09-01 | 2020-12-01 | 广西玮美生物科技有限公司 | Method for detecting ghrelin of nonhuman primate |
| CN112858694A (en) * | 2021-02-05 | 2021-05-28 | 湖南泰新医药科技有限公司 | Method for simultaneously determining concentrations of anti-PD-1 antibody and anti-PD-L1 antibody in human serum |
| CN112858694B (en) * | 2021-02-05 | 2024-03-12 | 湖南泰新医药科技有限公司 | Method for simultaneously measuring anti-PD-1 antibody and anti-PD-L1 antibody concentration in human serum |
| CN113092392A (en) * | 2021-03-05 | 2021-07-09 | 苏州西山中科药物研究开发有限公司 | Universal method for detecting total concentration of monoclonal antibody drug targeting soluble protein in monkey serum |
| CN113092392B (en) * | 2021-03-05 | 2022-11-08 | 苏州西山中科药物研究开发有限公司 | Universal method for detecting total concentration of monoclonal antibody drug targeting soluble protein in monkey serum |
| CN113702362A (en) * | 2021-08-27 | 2021-11-26 | 宁波熙宁检测技术有限公司 | Method for quantitatively detecting IFN-gamma concentration by using chemiluminescence method and detection kit thereof |
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