CN1715920A - Serum or plasma sample diluting liquid and application thereof - Google Patents
Serum or plasma sample diluting liquid and application thereof Download PDFInfo
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- CN1715920A CN1715920A CN 200410062222 CN200410062222A CN1715920A CN 1715920 A CN1715920 A CN 1715920A CN 200410062222 CN200410062222 CN 200410062222 CN 200410062222 A CN200410062222 A CN 200410062222A CN 1715920 A CN1715920 A CN 1715920A
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Abstract
The present invention discloses a kind of serum or plasma sample diluting liquid, be used in sandwich ELISA and detect dilute serum (blood plasma) sample in people's lipocyte complement related protein (Acrp30), it comprises a kind of reductive agent and buffer components, specifically can be made up of the Tris damping fluid of 25mM DTT, 100mM pH8.0, EDTA and the 200mM NaCl of 2mM pH8.0.Use dilution of the present invention that sample preparation, dilution, last sample are once finished, and make diluted sample to be checked front and back need not boil processing, easy and simple to handle, and by changing reaction conditions, shortened the reaction time, realized human serum (blood plasma) Acrp30 albumen fast quantification is detected.
Description
Technical field:
The present invention relates in the enzyme linked immunosorbent assay (ELISA) (ELISA), the processing of serum or plasma sample, dilution, last sample are applied to the content that sandwich ELISA detects lipocyte complement related protein 30 (Acrp30) in the human serum (blood plasma) especially.
Background technology:
At present, the bag in the most of ELISA system by, enzyme labelled antibody be with recombinant expression protein as antigen, immune animal produces.If the recombinant protein great expression, form the inclusion body precipitation, must use denaturant with its complete sex change dissolving after immune animal again.And the conformation of metaprotein and native protein has certain difference, and the antibody that its immune animal produces may be difficult to combine with native protein, sets up thereby influence ELISA.
Acrp30 is the plasma proteins of adipocyte-specific secretion, and nineteen ninety-five, Scherer etc. have at first reported existence and the cDNA sequence thereof of Acrp30.People Acrp30 gene is positioned at 3q27, its encoding proteins contains 247 amino acid, monomer molecule is made up of 4 domains, holds the C end to be followed successively by signal peptide sequence, non-homogeneous sequence, collagen spline structure territory, globular domain from N, and C holds spherical district (110～247 amino acid) to account for major part.After the Acrp30 translation glycosylation can take place, be a kind of glycoprotein, and its spherical district tripolymer structure and TNF (TNF) family have extremely strong homology.Acrp30 has two kinds of forms in blood plasma, i.e. low-molecular-weight homotrimer (LMW) and high molecular complex (HMW).The Acrp30 oligomer connects by the disulfide bond on 39 halfcystines (Cys-39), and the Cys-39 sudden change causes collagenous region albumen to shear and forms tripolymer.Nearest Tsao etc. studies show that most of Acrp30 (＞80%) exists with the HMW form in the blood circulation, and small part is six aggressiveness (＜10%) and tripolymer (＜10%).
The physiologic function of Acrp30 comprises regulates sugar, lipid metabolism, improves insulin sensitivity, antiatherosclerosis etc.Recently studies show that the effect that Acrp30 has anti-inflammatory to protect the liver, might become a kind of new liver disease medicine.At present, the assay of lipocyte complement related protein 30 (Acrp30) in the human serum (blood plasma), the ELISA reagent of use (Linco Research, Inc. etc.) only uses for laboratory study, and sense cycle is long, and operation is complicated; Domestic still do not have a reagent set.
Making the bag quilt with mouse-anti people Acrp30 monoclonal antibody, the anti-people Acrp30 of rabbit how mark by the anti-enzyme of doing, the recombinant protein A crp30 that is used for immune animal is with the inclusion body formal representation, before immunity, make in the experimental design of its sex change dissolving with 8M urea, adopt the diluted sample formula of liquid in the existing ELISA reagent, all can not detect the Acrp30 in the human blood, thereby be necessary to seek a kind of suitable sample diluting liquid.
Summary of the invention
The object of the present invention is to provide a kind of dilution that is used for enzyme linked immunosorbent assay (ELISA) at serum or plasma sample.
Sample diluting liquid provided by the invention is used for enzyme linked immunosorbent assay (ELISA), and it comprises a kind of reductive agent and buffer components.
Wherein, described reductive agent is a dithiothreitol (DTT), code name DTT, and the concentration of DTT to 500mM, is preferably 20mM to 100mM at 5mM, is preferably 25mM.
Wherein, described buffer components is the combination of three (methylol) aminomethane (Tris) damping fluid, ethylenediamine tetraacetic acid (EDTA) and NaCl.Wherein to 200mM, to 5mM, the concentration of NaCl arrives 400mM at 100mM to the EDTA concentration of pH8.0 to the Tris buffer concentration of pH8.0 at 1mM at 50mM.
Diluted sample formula of liquid of the present invention is preferably the Tris damping fluid of 25mM DTT, 100mM pH8.0, EDTA and the 200mM NaCl of 2mMpH8.0.
Another object of the present invention is to provide a kind of sandwich method ELISA to detect the method for human serum or blood plasma lipocyte complement related protein 30 (Acrp30) content.
A kind of sandwich method ELISA provided by the invention detects the method for human serum or blood plasma lipocyte complement related protein 30 (Acrp30) content, is to use foregoing any serum or plasma sample diluting liquid to dilute testing sample.
These method concrete steps comprise:
Every hole adds the mouse-anti people Acrp30 monoclonal antibody of 100 microlitres with the dilution in 1: 2000 of 0.05M pH9.6 carbonate buffer solution, and incubated at room is spent the night; Discard liquid then, wash plate 2 times, pat dry plank with the 0.01M PBST of pH7.4; Every hole adds the confining liquid that 150 microlitres are formed with 1.5% bovine serum albumin(BSA), 4% saturated ammonium sulfate, 5% sucrose, 5%EDTA, 0.01M PBS and 0.2 ‰ Sodium Mercurothiolates, incubated at room 1 hour; Discard liquid then, pat dry plank;
As the standard items dilution, after the dilution in 1: 50 of standard items do, remake doubling dilution to 1: 1600 with the sample diluting liquid that does not add DTT; In centrifuge tube, add 100 microlitre sample diluting liquids, add 25 microlitre human serums or blood plasma slurry sample again, the supernatant of this sample for getting after centrifugal 1 minute through 5000g; Every hole adds 100 microlitres and dilutes good standard items, people's blood specimen, and 37 ℃ jolt and hatched 40 minutes; Discard liquid, wash plate 5 times, pat dry plank with the 0.01MPBST of pH7.4; Every hole adds the enzyme labelled antibody of 100 microlitres with the dilution in 1: 300 of enzyme mark dilution, 37 ℃ jolt and hatch 30 minutes, wherein this enzyme mark dilution is made up of 20% calf serum, 15% glycerine, 4% Macrogol 6000,0.75% saturated ammonium sulfate, 0.01M PBS and 0.1% Tween-20, and this enzyme labelled antibody is the anti-people Acrp30 of the rabbit polyclonal antibody with horseradish peroxidase on the sodium periodate method mark; Discard liquid, wash plate 5 times, pat dry plank with 0.01M PBST (pH7.4); Every then hole adds each one of A, B colour developing liquid, 37 ℃ jolt and hatch 15 minutes, wherein A colour developing liquid is that disodium hydrogen phosphate 18.41g/L, citric acid 7.65g/L and 30% hydrogen peroxide 0.24ml/L form, and B colour developing liquid is that tetramethyl benzidine 0.4g/L, dimethyl formamide 40ml/L, EDTA0.525g/L and citric acid 4.2g/L form; Every then hole adds 50 microlitre 1M sulfuric acid stop buffers, and OD450nm detects;
Step 3. according to typical curve, obtain human blood Acrp30 content in the test sample.
According to technique scheme, the present invention makes diluted sample to be checked front and back need not boil processing because of proposing to contain the sample diluting liquid of reductive agent, easy and simple to handle, and, shortened the reaction time by changing reaction conditions, realized human serum (blood plasma) Acrp30 albumen fast quantification is detected.
Description of drawings
Fig. 1 produces the dilution synoptic diagram of Acrp30 standard items for bacterium among the present invention;
Fig. 2 detects the canonical plotting that bacterium produces Acrp30 for ELISA among the present invention.
Embodiment
The present invention relates in the enzyme linked immunosorbent assay (ELISA) (ELISA) processing of serum or plasma sample, dilution, last sample.
In the research that the present invention relates to, make the bag quilt with mouse-anti people Acrp30 monoclonal antibody, the anti-people Acrp30 of rabbit resists more is made the enzyme mark, and the recombinant protein A crp30 that is used for immune animal is with the inclusion body formal representation, makes its sex change dissolving with 8M urea before immunity.In this experimental design, need to detect the Acrp30 content in the human blood, find that sample diluting liquid wherein can influence the detection of Acrp30.
For this reason, grope and test, the present invention proposes a kind of serum or plasma sample diluting liquid through arduous.
The principal feature of the dilution that the present invention proposes is to contain reductive agent, and experimental result shows that reductive agent is that dithiothreitol (DTT) (DTT) is best, and effect was better when working concentration arrived 100mM at 20mM, and optium concentration is about 25mM.
Also comprising the damping fluid composition in the sample diluting liquid of the present invention, is the combination of three (methylol) aminomethane (Tris) damping fluid, ethylenediamine tetraacetic acid (EDTA) and NaCl.Wherein, the concentration of Tris damping fluid (pH8.0) arrives 200mM about 50mM greatly, and optium concentration is about 100mM; The concentration of EDTA (pH8.0) arrives 5mM about 1mM greatly, and optium concentration is about 2mM; The concentration of NaCl arrives 400mM about 100mM greatly, and optium concentration is about 200mM.
Embodiment one: the preparation of serum of the present invention (blood plasma) sample diluting liquid
The most preferred serum of the present invention (blood plasma) sample diluting liquid is for 25mM DTT, 100mM Tris damping fluid (pH8.0), 2mM EDTA (pH8.0), 200mM NaCl form, referring to table 1.
Table 1: sample diluting liquid composition
| Component | Concentration | The source |
| DTT Tris damping fluid (pH8.0) EDTA (pH8.0) NaCl | 25mM 100mM 2mM 200mM | Sigma Promega Beijing Beijing Yili Fine Chemicals Co., Ltd. of chemical reagents corporation |
Listed component in the table 1, prepare stand-by by following mode respectively:
Tris: claim 24.22g, adding distil water is to 100ml, and compound concentration is 2M;
HCl: get the HCl 172.4ml of 11.6N, adding distil water is to 1000ml, and compound concentration is 2M;
Tris damping fluid (pH8.0): 2M Tris 50ml adds 2M HCl 29.2ml, and adding distil water is to 100ml; Compound concentration is 1M;
EDTA (pH8.0): in 80ml distilled water, add 186.1g two water disodium ethylene diamine tetraacetate (EDTA-Na2H
2O) vigorous stirring on magnetic stirring apparatus transfers pH value of solution to 8.0 (needing the 20gNaOH particle approximately) to be settled to 1L then with NaOH, and autoclaving is standby after the packing, and compound concentration is 0.5M;
NaCl: claim 5.844g, adding distil water is to 100ml, and compound concentration is 1M;
DTT: claim 1.545g, adding distil water is put-20 ℃ and is deposited to 10ml, and compound concentration is 1M.
Above-mentioned each component is prepared with the listed step of table 2, forms 100ml sample diluting liquid of the present invention:
The compound method of table 2 sample diluting liquid
| | Volume | |
| 1. adding 1M Tris damping fluid (pH8.0) 2. adds 0.5M EDTA (pH8.0) and 3. adds 1M NaCl 4. and add 1M DTT 5. adding distil waters | 10ml 0.4ml 20ml 2.5ml 67.1ml |
Embodiment two to four: use the method identical with embodiment one, prepare respectively table 3 listed each form and the sample diluting liquid of content.
Table 3: embodiment two to four each components and concentration of sample diluting liquid
Embodiment five: the ELISA detection by quantitative
One, the dilution of standard items: operation is referring to Fig. 1, and concrete steps are:
1. the standard items dilution for preparing is added in six centrifuge tubes, first pipe adds 500 μ l, and other pipe adds 250 μ l;
2. the bacterium product Acrp30 that in first pipe, adds concentration known (1.4mg/ml), to final concentration be 28 μ g/ml.
3. make doubling dilution, the concentration that makes Acrp30 in second to six pipe is 14 μ g/ml, 7 μ g/ml, 3.5 μ g/ml, 1.75 μ g/ml, 0.875 μ g/ml.
Two, the ELISA of standard model detects:
1. every hole adds the Acrp30 standard items of 100 μ l step 1 preparation, and blooming is put 37 ℃ and jolted 40 minutes.
2. discard liquid, wash plate five times, pat dry with 0.01M pH7.4 PBST.
3. every hole adds 100 μ l enzyme conjugates, and blooming is put 37 ℃ and jolted 30 minutes.
4. discard liquid, wash plate five times, pat dry with 0.01M pH7.4 PBST.
5. every hole adds colour developing liquid A, each 50 μ l of B, and blooming is put 37 ℃ and jolted 10 minutes.Wherein A colour developing liquid is that disodium hydrogen phosphate 18.41g/L, citric acid 7.65g/L and 30% hydrogen peroxide 0.24ml/L form, and B colour developing liquid is that tetramethyl benzidine 0.4g/L, dimethyl formamide 40ml/L, EDTA0.525g/L and citric acid 4.2g/L form;
6. every hole adds 50 μ l 1M H
2SO
4Cessation reaction, OD450nm detects.
7. testing result is depicted as typical curve, referring to Fig. 2.
Three, the content of Acrp30 in the ELISA test sample:
1. 1 volume serum is joined in the 4 volume sample dilutions and mix, get 100 μ l samples and place the hole, blooming is put 37 ℃ and is jolted 40 minutes.
2.～6. with the step 1 in the step 2～6.
7. reference standard curve obtains the Acrp30 content of specimen.
Experiment one: different diluent ELISA detects the Acrp30 control experiment
To standard model and sample to be checked, dilute with different dilutions, detect the content of Acrp30 then according to the method ELISA of embodiment five step 2, the result is as shown in table 4.Wherein, to be LincoResearch in the same old way, the dilution of Inc., this sample are the dilution of the embodiment of the invention one preparation.
Table 4: different diluent ELISA detects the Acrp30 control experiment
| Sample preparation | Sample to be checked (people's blood specimen) |
| Handle with this sample diluting liquid | 0.9-20μg/ml |
| Handle with the control sample dilution | Not-(not detecting) |
From this experiment as can be seen, use dilution of the present invention, can more accurately detect the content of Acrp30, and use, then can't detect Acrp30 dilution in the same old way.
Experiment two: detect the degree of accuracy experiment
One) processing of standard items dilution and blood serum sample: standard items dilute with embodiment five; Sample preparation: 1 volume serum joined in the 3 volume sample dilutions mix.
Two) ELISA detects:
1. degree of accuracy in the plate: a duplicate samples repeats to add 12 holes.
2. degree of accuracy between plate: a duplicate samples is divided ten secondary detection.
3. operation is with embodiment one
Three) result: see Table 5.CV is 5% in the check-out console as can be seen, CV is 8% between plate from table 5 data, and degree of accuracy is good.
Table 5:ELISA detects degree of accuracy
| Acrp30 concentration (X) | Standard deviation (S) | The coefficient of variation (CV) | |
| Between the plate inner panel | 3.2 6.8 | 0.16 0.544 | 5％ 8％ |
Experiment three: ELISA detection accuracy experiment
One) processing of standard items dilution and blood serum sample
1. the standard items dilution is with embodiment five.
2. add the standard items of high, medium and low three kinds of concentration known in a serum, diluted sample is with experiment two.
Two) ELISA detects: operation is with embodiment five.
Three) result: see Table 6.The accuracy that detects as can be seen from table 6 data is at 88-108%, and accuracy is good.
Table 6.ELISA detection accuracy
| Measured value (O) | Desired value (E) | O/E(％) |
| 1.48 4.2 9.6 15.5 | - 4.78 8.9 15.0 | 88 108 103 |
The linear relationship experiment that test four: ELISA detects
One) processing of standard items dilution and blood serum sample
1. the standard items dilution is with embodiment five.
2. diluted sample is with experiment two.The serum that dilution is good is done 2 times, 4 times, 8 times dilutions with sample liquid.
Two) ELISA detects: operation is with embodiment five.
Three) result: see Table 7.The detection line sexual intercourse is at 80-102% as can be seen from table 7 data, and linear relationship is good.
The linear relationship that table 7:ELISA detects
| Extension rate | Measured value (O) | Desired value (E) | O/E(％) |
| 1∶2 1∶4 1∶8 | 13.54 6.83 2.70 1.73 | - 6.77 3.385 1.6925 | 101 80 102 |
Claims (10)
1. serum or plasma sample diluting liquid are used for enzyme linked immunosorbent assay (ELISA), it is characterized in that it comprises a kind of reductive agent and buffer components.
2. serum according to claim 1 or plasma sample diluting liquid is characterized in that, described reductive agent is a dithiothreitol (DTT), code name DTT.
3. serum according to claim 2 or plasma sample diluting liquid is characterized in that, the concentration of DTT arrives 500mM at 5mM in the described dilution.
4. serum according to claim 3 or plasma sample diluting liquid is characterized in that the concentration of DTT is preferably 20mM to 100mM in the described dilution.
5. serum according to claim 3 or plasma sample diluting liquid is characterized in that the concentration of DTT is preferably 25mM in the described dilution.
6. serum according to claim 1 or plasma sample diluting liquid, described buffer components are the combination of three (methylol) aminomethane (Tris) damping fluid, ethylenediamine tetraacetic acid (EDTA) and NaCl.
7. serum according to claim 6 or plasma sample diluting liquid is characterized in that, to 200mM, to 5mM, the concentration of NaCl arrives 400mM at 100mM to the EDTA concentration of pH8.0 to the Tris buffer concentration of pH8.0 at 1mM at 50mM in the described dilution.
8. serum according to claim 1 or plasma sample diluting liquid is characterized in that, comprise the Tris damping fluid of 25mMDTT, 100mM pH8.0, EDTA and the 200mM NaCl of 2mM pH8.0.
9. a sandwich method ELISA detects the method for human serum or blood plasma lipocyte complement related protein 30 (Acrp30) content, it is characterized in that, uses arbitrary described serum of claim 1 to 8 or plasma sample diluting liquid to dilute testing sample.
10. sandwich method ELISA according to claim 9 detects the method for human serum or blood plasma lipocyte complement related protein 30 (Acrp30) content, it is characterized in that concrete steps comprise:
Step 1. wrap by elisa plate:
Every hole adds the mouse-anti people Acrp30 monoclonal antibody of 100 microlitres with the dilution in 1: 2000 of 0.05M pH9.6 carbonate buffer solution, and incubated at room is spent the night; Discard liquid then, wash plate 2 times, pat dry plank with the 0.01M PBST of pH7.4; Every hole adds the confining liquid that 150 microlitres are formed with 1.5% bovine serum albumin(BSA), 4% saturated ammonium sulfate, 5% sucrose, 5%EDTA, 0.01M PBS and 0.2 ‰ Sodium Mercurothiolates, incubated at room 1 hour; Discard liquid then, pat dry plank;
Step 2. detect step:
As the standard items dilution, after the dilution in 1: 50 of standard items do, remake doubling dilution to 1: 1600 with the sample diluting liquid that does not add DTT; In centrifuge tube, add 100 microlitre sample diluting liquids, add 25 microlitre human serums or blood plasma slurry sample again, the supernatant of this sample for getting after centrifugal 1 minute through 5000g; Every hole adds 100 microlitres and dilutes good standard items, people's blood specimen, and 37 ℃ jolt and hatched 40 minutes; Discard liquid, wash plate 5 times, pat dry plank with the 0.01M PBST of pH7.4; Every hole adds the enzyme labelled antibody of 100 microlitres with the dilution in 1: 300 of enzyme mark dilution, 37 ℃ jolt and hatch 30 minutes, wherein this enzyme mark dilution is made up of 20% calf serum, 15% glycerine, 4% Macrogol 6000,0.75% saturated ammonium sulfate, 0.01M PBS and 0.1% Tween-20, and this enzyme labelled antibody is the anti-people Acrp30 of the rabbit polyclonal antibody with horseradish peroxidase on the sodium periodate method mark; Discard liquid, wash plate 5 times, pat dry plank with 0.01M PBST (pH7.4); Every then hole adds each one of A, B colour developing liquid, 37 ℃ jolt and hatch 15 minutes, wherein A colour developing liquid is that disodium hydrogen phosphate 18.41g/L, citric acid 7.65g/L and 30% hydrogen peroxide 0.24ml/L form, and B colour developing liquid is that tetramethyl benzidine 0.4g/L, dimethyl formamide 40ml/L, EDTA0.525g/L and citric acid 4.2g/L form; Every then hole adds 50 microlitre 1M sulfuric acid stop buffers, and OD450nm detects;
Step 3. according to typical curve, obtain human blood Acrp30 content in the test sample.
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