CN102707048A - Method for preparing solid phase antibody for immunoassay - Google Patents
Method for preparing solid phase antibody for immunoassay Download PDFInfo
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- CN102707048A CN102707048A CN2012101538046A CN201210153804A CN102707048A CN 102707048 A CN102707048 A CN 102707048A CN 2012101538046 A CN2012101538046 A CN 2012101538046A CN 201210153804 A CN201210153804 A CN 201210153804A CN 102707048 A CN102707048 A CN 102707048A
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Abstract
The invention discloses a method for preparing a solid phase antibody for immunoassay and specifically provides a method that after treatment, an antibody is fixed on a polystyrene solid phase material and a solid phase antibody activity is maintained to be stable by a protective liquid. The method includes that step one, adjusting concentration of a coated antibody; step two, activating, adding an activating agent into the coated antibody, uniformly mixing, and performing an activation by still standing at the room temperature; step three, coating, mixing the activated coated antibody and a coated buffer solution to coat a solid phase carrier; step four, sealing, drying a coating liquid on the solid phase carrier in a swing mode, and adding a confining liquid into the solid phase carrier, sealing vacancy and protecting antibody activity; and step five, drying, drying the confining liquid on the solid phase carrier in a swing mode, dehumidifying, drying, and sealing for a reservation.
Description
Technical field
The present invention is the method that a kind of biomolecule encapsulates solid phase carrier, specifically, provides a kind of antibody to be fixed in the polystyrene solid phase material after treatment and passed through protection liquid to keep the activity stabilized method of insolubilized antibody.
Background technology
The labelled immune analysis is that multiple labelling technique is combined with immunological technique and the technology of a series of detection target substances of setting up.Nineteen fifty-nine Yalow and Berson application of radiation property nucleic
125The principle of I labelled antigen and determined antigen and the antibody competition of limiting the quantity of has been set up first kind of sensitivity, (Radioimmunoassay, RIA), this is a homogeneous immunoassay in quantitative radiommunoassay.Developed again afterwards solid matrix antibody-antigen-
125The heterogeneous immunoradiometric assay of I labelled antibody (Immuno-radiometric assay, IRMA).The immunoassay of nonradioactive isotope mark develops rapidly on this basis; And be mostly to adopt the heterogeneous immunoassay of solid phase separation means; Representational have an Enzyme Linked Immunoadsorbent Assay (Enzyme-Linked Immunosorbent assay; ELISA), time resolved fluoro-immunoassay (time-resolued fluoroimmunoassay; TRFIA), chemiluminescence enzyme immunoassay (chemiluminescence enzyme immunoassay, CLEIA), chemiluminescence immune assay (chemiluminescence immunoassay, CLIA), immuno-gold labeling technology (Immunogold labelling techique) etc.
In heterogeneous immunoassay, all solid phase carrier must be arranged, be essential component, is again simple and easy effective separation means.Material as solid phase carrier mainly contains cellulose, gel, magnetic particle, nylon, polystyrene etc.; Can divide two big types by carrier character; It is hydrophilic that first surface has chemical functional group; Another kind of is that the no chemical functional group in surface is hydrophobic, and the former is with covalent bond and antigen or antibodies, and the latter is logical
Crossing physisorption encapsulates in carrier surface.In multiple solid phase carrier, method is simple for polystyrene support and antibodies, quick, economical, is fit to the large-scale production operation, thereby the most commonly used, the microwell plate of processing like polystyrene material, ball, test tube etc.Solid phases such as antibody p-poly-phenyl ethene have stronger absorption affinity, combine through physisorption with the polystyrene solid phase carrier, and what lean on is the acting force between the hydrophobic grouping of hydrophobic grouping and surface of solid phase carriers on the molecular structure.The researcher is arranged with function such as XPS, pearl or processing of effective glutaraldehyde and r radiation exposure etc. with raising and antibodies; The present invention improves the effect that combines of antibody and polystyrene support from another approach; Improve detection sensitivity; Through the application of protection liquid, the activity of protection insolubilized antibody satisfies the long requirement of immunoassay kits shelf life of nonradioactive isotope mark simultaneously.
Summary of the invention
The purpose of this invention is to provide a kind of insolubilized antibody preparation method who is used for immunoassay.
Preparation method provided by the invention comprises:
(1) concentration adjustment: coated antibody carries out the concentration adjustment with 0.02mol/L pH7.4 phosphate buffer;
(2) activation: in coated antibody, add active agent, mixing, room temperature leaves standstill carried out activation in 3 minutes, and wherein said active agent is 0.05mol/L pH2.4 glycocoll-HCL damping fluid;
(3) encapsulate: with the coated antibody of activation with encapsulate damping fluid and be mixed into coating buffer; The gained coating buffer is added solid phase carrier; 2-8 ℃ of placement placed 2 hours in 16-24 hour or 37 ℃, and the said damping fluid that encapsulates is 0.02mol/L pH7.4 phosphate buffer or 0.01mol/L pH9.5 carbonate buffer solution;
(4) sealing: dry the coating buffer on the solid phase carrier; Confining liquid is added solid phase carrier; 37 ℃ of placements were placed 16-24 hour in 2 hours or 2-8 ℃, and wherein said confining liquid is the 0.02mol/L pH7.4 phosphate buffer that contains 8% sorbierite, 0.5%BSA, 0.1%Proclin300;
(5) drying: dry the confining liquid on the solid phase carrier, room temperature dehumidify more than 24 hours, with the solid phase carrier that is coated with antibody of drying pack into vacuumize in the aluminium foil bag encapsulate subsequent use.
In the preparation method of the invention described above, solid phase carrier is a polystyrene material, can be transparent microwell plate, white microwell plate, black microwell plate, test tube, ball etc.The said preferred 0.02M pH7.4 of the damping fluid phosphate buffer that encapsulates, preferred 2-8 of the time that encapsulates ℃ 16-24 hour.2 hours preferred 37 ℃ of said off-periods.Antibody can be monoclonal antibody, polyclonal antibody, and the antibody type is IgG.
In the preparation method of the invention described above,, the ratio and the action time of control active agent and coated antibody, outside making antibody more multiple binding sites being exposed to, obviously improve detection sensitivity through adjustment coated antibody concentration.Room point after the absorption of confining liquid sealing surface of solid phase carriers antibody molecule; Reduce non-specific responding in the subsequent detection process; Simultaneously confining liquid forms layer protecting film at surface of solid phase carriers, has effectively protected the activity of insolubilized antibody, can tolerate 50 ℃ and reach more than 10 days activity and do not fall.
Description of drawings
Accompanying drawing 1 has shown that three groups encapsulate and detect 0.5ng/ml calibration object point OD value variation tendency after 50 ℃ of plates are placed different number of days.
Accompanying drawing 2 has shown that three groups encapsulate and detect 1ng/ml calibration object point OD value variation tendency after 50 ℃ of plates are placed different number of days.
Accompanying drawing 3 has shown that three groups encapsulate and detect 5ng/ml calibration object point OD value variation tendency after 50 ℃ of plates are placed different number of days.
Accompanying drawing 4 has shown that three groups encapsulate and detect 50ng/ml calibration object point OD value variation tendency after 50 ℃ of plates are placed different number of days.
The constant insolubilized antibody activity that shows of OD value remains unchanged, and the OD value descends and shows the active decline of insolubilized antibody indirectly.
Embodiment
Among the preparation method of the present invention (1), the coated antibody protein concentration is adjusted to 1mg/ml with 0.02mol/L pH7.4 phosphate buffer.
Among the preparation method of the present invention (2), the volume that concentration is calculated required coated antibody that encapsulates according to selected takes out respective volume antibody, adds 2 times of volume 0.05mol/L pH2.4 glycocoll-HCL damping fluids, mixing, and room temperature left standstill 3 minutes.
Among the preparation method of the present invention (3), the coated antibody of activation is diluted with encapsulating damping fluid, join in the microwell plate micropore, addition is 100 μ l/ holes, with plate change over to 2-8 ℃ encapsulate spend the night (16-24 hour) or 37 ℃ encapsulated 2 hours.The wherein said damping fluid that encapsulates is 0.02mol/L pH7.4 phosphate buffer or 0.01mol/L pH9.5 carbonate buffer solution.
Among the preparation method of the present invention (4); Get rid of coating buffer; On thieving paper, buckle and do; Every hole adds confining liquid 130 μ l, and be 37 ℃ 2 hours or 2-8 ℃ spend the night (16-24 hours) off-period, and wherein said confining liquid is the 0.02mol/L pH7.4 phosphate buffer that contains 8% sorbierite, 0.5%BSA, 0.1%Proclin300.
Among the preparation method of the present invention (5), get rid of deblocking liquid, on thieving paper, buckle and do, room temperature dehumidifies more than 24 hours.Pack the plate that encapsulates in advance of drying into vacuumize encapsulation in the aluminium foil bag subsequent use.
The solution compound method that relates among the preparation method of the present invention:
0.05mol/L pH2.4 glycocoll-HCL damping fluid: 375.25 milligrams of glycocoll are used deionized water dissolving, add 1.62 milliliters of 2N HCL, are settled to 100 milliliters with deionized water.
0.02mol/L pH7.4 phosphate buffer: Na
2HPO
412H
2O 5.802 grams, NaH
2PO
42H
2O 0.593 gram is with being settled to 1000 milliliters behind the deionized water dissolving.
0.01mol/L pH9.5 carbonate buffer solution: Na
2CO
30.318 gram, NaHCO
30.588 gram is with being settled to 1000 milliliters behind the deionized water dissolving.
Confining liquid: sorbierite 80 grams, BSA 5 grams with being settled to 1000 milliliters after the dissolving of 0.02mol/L pH7.4 phosphate buffer, add 1 milliliter of Proclin300.
Pass through embodiment further explain the present invention below, but the present invention is not limited to this.
Coated antibody concentration is 1mg/ml, and the selected concentration that encapsulates is 4 μ g/ml, desire preparation 500ml coating buffer.Get coated antibody 2ml, add 4ml 0.05mol/L pH2.4 glycocoll-HCL damping fluid, mixing, room temperature left standstill 3 minutes, was added in the 494ml 0.02mol/L pH7.4 phosphate buffer and was mixed into coating buffer.In the microwell plate micropore, add coating buffer, 100 μ l/ holes change plate over to 2-8 ℃ and encapsulate spend the night (16-24 hour).Get rid of coating buffer, on thieving paper, buckle and do, every hole adds confining liquid 130 μ l, off-period be 37 ℃ 2 hours.Get rid of deblocking liquid, on thieving paper, buckle and do, room temperature dehumidifies more than 24 hours.Pack the plate that encapsulates in advance of drying into vacuumize encapsulation in the aluminium foil bag subsequent use.
Prepare coating buffer by embodiment 1, polystyrene bead is added in the coating buffer, addition there was not pearl to be as the criterion with coating buffer, changed 2-8 ℃ over to and encapsulated spend the night (16-24 hour).Discard coating buffer, pour confining liquid into, the confining liquid addition was not as the criterion there not to be pearl, and 37 ℃ were sealed 2 hours.Discard confining liquid, the bead surface confining liquid is done in control, and room temperature dehumidifies more than 24 hours.It is subsequent use in the air-tight bottle that the pearl of drying is packed into.
Divided by the polystyrene test tube is solid phase carrier, coating buffer 200 μ l/ pipe, and outside the confining liquid 300 μ l/ pipe, all the other all identical with embodiment 1 method preparation solid phases encapsulate pipe in advance.
Encapsulate plate in advance by embodiment 1 preparation, different is the time of encapsulating be 37 ℃ 2 hours.
Encapsulate plate in advance by embodiment 1 preparation, different is is 2-8 ℃ off-period and spends the night (16-24 hour).
Encapsulate plate in advance by embodiment 1 preparation, different is with 0.01mol/L pH9.5 carbonate buffer solution preparation coating buffer.
1. material and method
Polystyrene chemiluminescence white microwell plate, anti-C-peptide monoclonal antibody, C-peptide detection by quantitative kit (chemoluminescence method), variable concentrations C-peptide calibration object (0ng/ml, 0.2ng/ml, 0.5ng/ml, 1ng/ml, 2ng/ml, 5ng/ml, 10ng/ml)
To resist C-peptide monoclonal antibody to encapsulate the chemiluminescence plate in advance by embodiment 1 preparation, this is plate A.Encapsulate chemiluminescence plate B in advance by embodiment 1 preparation simultaneously, different is to omit " activation " that step.Detect 0ng/ml simultaneously with plate A and plate B, 0.2ng/ml, 0.5ng/ml, 1ng/ml, 2ng/mll, 5ng/ml, 10ng/mlC-peptide calibration object is pressed the operation of C-peptide detection by quantitative kit (chemoluminescence method) instructions.Detect concrete steps: in microwell plate, add each concentration C of 20 μ l-peptide calibration point respectively, wherein 0ng/ml adds 20 holes, adds 100 μ l enzyme conjugates again; The rearmounted 37 ℃ of reactions of concussion mixing 1.5 hours are cleaned 5 times with cleansing solution, and button is done; Add luminous substrate liquid A 50 μ l, luminous substrate liquid B 50 μ l, mixing; Room temperature was placed 10 minutes, measured each hole luminous value with Chemiluminescence Apparatus.Get the calibration curve regression equation with the match of Log-Log mathematical model; 20 holes " 0 " calibration object luminous value mean values (X) add twice standard deviation (SD), and the concentration value when utilizing the dose-response curve regression equation calculation to go out luminous value for X+2SD is the sensitivity of kit.
2. the result sees the following form 1:
Two kinds of chemiluminescences of table 1 encapsulate plate sensitivity testing result
Under the same conditions, each calibration object luminous value of plate A is higher than plate B, and S1/S0 is 4.834, and sensitivity is 0.027ng/ml, and the S1/S0 of plate B is 2.771, and sensitivity is 0.046ng/ml.The antibody of plate A shows better more active than plate B, and sensitivity proves that apparently higher than plate B the present invention can significantly improve detection sensitivity.
1. material and method
The polystyrene elisa plate, anti--the HBs monoclonal antibody, the HBsAg enzyme linked immunological kit, variable concentrations HBsAg calibration object (0ng/ml, 0.5ng/ml, 1ng/ml, 2ng/ml)
To resist-the HBs monoclonal antibody encapsulates elisa plate in advance by embodiment 1 preparation, and this is plate C.Encapsulate elisa plate D in advance by embodiment 1 preparation simultaneously, different is to omit " activation " that step.Detect 0ng/ml simultaneously with plate C and plate D, 0.5ng/ml, 1ng/ml, 2ng/ml HBsAg calibration object is pressed the operation of HBsAg enzyme linked immunological kit instructions.Detect concrete steps: in microwell plate, add each concentration HBsAg calibration point of 50 μ l respectively, add 50 μ l enzyme conjugates again, the rearmounted 37 ℃ of reactions of concussion mixing 1 hour; Clean 4 times with cleansing solution, button is done, and adds TMB developer A 50 μ l; Developer B 50 μ l, mixing, 37 ℃ were developed the color 10 minutes; Add 50 μ l 2M sulfuric acid cessation reactions, read to remember the OD value in the 450nm place with ELIASA.
2. the result sees the following form 2:
Two kinds of enzymes couplet of table 2 encapsulate plate sensitivity testing result
The OD value is mean value in these table data.According to HBsAg enzyme linked immunological kit instructions, boundary value CO=0.105, S/CO >=1 o'clock is judged to be surface antigen positive, and S/CO＜1 o'clock is judged as the surface antigen feminine gender.Plate C detects 0.5ng/ml, and plate D can only detect 1ng/ml, proves that the present invention can significantly improve detection sensitivity.
The insolubilized antibody study on the stability of embodiment 9 the present invention preparation
1. material and method
The polystyrene elisa plate, anti--the HBs monoclonal antibody, the HBsAg enzyme linked immunological kit; Variable concentrations HBsAg calibration object (0ng/ml, 0.5ng/ml, 1ng/ml; 5ng/ml, 50ng/ml), U.S. HY technology company is exclusively used in the product Supercoat that protection is adsorbed in protein on the microwell plate.
To resist-the HBs monoclonal antibody encapsulates elisa plate in advance by embodiment 1 preparation, and different is to replace confining liquid according to the invention with the product Supercoat of U.S. HY technology company, and this is plate I; To resist-the HBs monoclonal antibody encapsulates elisa plate in advance by embodiment 1 preparation, and this is plate II; To resist-the HBs monoclonal antibody encapsulates elisa plate in advance by embodiment 1 preparation, and different is to replace confining liquid according to the invention with 1%BSA, and this is plate III.
These three groups of laths are put into 50 ℃ of drying boxes; Respectively at detecting 0ng/ml, 0.5ng/ml, 1ng/ml in 0,3,5,7,10,13 day simultaneously; 5ng/ml; 50ng/ml HBsAg calibration object is pressed the operation of HBsAg enzyme linked immunological kit instructions, changes to follow the tracks of according to each calibration object OD value and detects its activity change.Detect concrete steps with embodiment 8.
2. the result sees the following form 3:
Table 3 encapsulates the active testing result of following the tracks of of plate for three groups
The OD value is mean value in these table data.I group, II group lath stability are all fine, very nearly the same, and 50 ℃ of activity in 10 days remain unchanged basically.Supercoat it be unclear that its composition; The III group only has the effect of albumen, less stable, 50 ℃ of 5 days loss of activity nearly 75%; Prove that confining liquid according to the invention has good protection to be adsorbed in the effect of antibody on the microwell plate, can match in excellence or beauty with the Supercoat of HY technology company.
Claims (7)
1. insolubilized antibody preparation method who is used for immunoassay is characterized in that may further comprise the steps:
(1) concentration adjustment: coated antibody carries out the concentration adjustment with 0.02mol/L pH7.4 phosphate buffer;
(2) activation: in coated antibody, add active agent, mixing, room temperature leaves standstill carried out activation in 3 minutes, and wherein said active agent is 0.05mol/L pH2.4 glycocoll-HCL damping fluid;
(3) encapsulate: with the coated antibody of activation with encapsulate damping fluid and be mixed into coating buffer; The gained coating buffer is added solid phase carrier; 2-8 ℃ of placement placed 2 hours in 16-24 hour or 37 ℃, and the said damping fluid that encapsulates is 0.02mol/L pH7.4 phosphate buffer or 0.01mol/L pH9.5 carbonate buffer solution;
(4) sealing: dry the coating buffer on the solid phase carrier; Confining liquid is added solid phase carrier; 37 ℃ of placements were placed 16-24 hour in 2 hours or 2-8 ℃, and wherein said confining liquid is the 0.02mol/L pH7.4 phosphate buffer that contains 8% sorbierite, 0.5%BSA, 0.1%Proclin300;
(5) drying: dry the confining liquid on the solid phase carrier, room temperature dehumidify more than 24 hours, with the solid phase carrier that is coated with antibody of drying pack into vacuumize in the aluminium foil bag encapsulate subsequent use.
2. preparation method as claimed in claim 1 is characterized in that, said solid phase carrier is a polystyrene material, can be transparent microwell plate, white microwell plate, black microwell plate, test tube, ball etc.
3. preparation method as claimed in claim 1 is characterized in that, said antibody can be monoclonal antibody, polyclonal antibody, and the antibody type is IgG.
4. preparation method as claimed in claim 1 is characterized in that, with 0.02mol/L pH7.4 phosphate buffer the coated antibody protein concentration is adjusted to 1mg/ml.
5. preparation method as claimed in claim 1 is characterized in that, antibody sandwich is preceding through 0.05mol/L pH2.4 glycocoll-HCL damping fluid activation processing.
6. preparation method as claimed in claim 1; Wherein said step (2) comprises following steps again: according to the selected volume that concentration is calculated required coated antibody that encapsulates; Take out respective volume antibody; Add 2 times of volume 0.05mol/L pH2.4 glycocoll-HCL damping fluids, mixing, room temperature left standstill 3 minutes.
7. preparation method as claimed in claim 1 is characterized in that, the 0.02mol/L pH7.4 phosphate buffer that contains 8% sorbierite, 0.5%BSA, 0.1%Proclin300 is as confining liquid.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106053785A (en) * | 2016-05-18 | 2016-10-26 | 北京北方生物技术研究所有限公司 | Solid antibody pre-coating method for competitive chemiluminescent immunoreactions |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0820448B2 (en) * | 1989-01-18 | 1996-03-04 | 帝人株式会社 | Method for producing fixed antibody |
| US20120045778A1 (en) * | 2010-08-23 | 2012-02-23 | The Ohio State University Research Foundation | Elisa for haptoglobin-matrix metalloproteinase 9 complex as a diagnostic test for conditions including acute inflammation |
-
2012
- 2012-05-18 CN CN201210153804.6A patent/CN102707048B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0820448B2 (en) * | 1989-01-18 | 1996-03-04 | 帝人株式会社 | Method for producing fixed antibody |
| US20120045778A1 (en) * | 2010-08-23 | 2012-02-23 | The Ohio State University Research Foundation | Elisa for haptoglobin-matrix metalloproteinase 9 complex as a diagnostic test for conditions including acute inflammation |
Non-Patent Citations (2)
| Title |
|---|
| EIJI ISHIKAWA 等: "An improved preparation of antibody-coated polystyrene beads for sandwich enzyme immunoassay", 《JOURNAL OF IMMUNOASSAY》, vol. 1, no. 3, 31 December 1980 (1980-12-31) * |
| ELISABETH PAUS 等: "TD-11 workshop report: characterization of monoclonal antibodies to S100 proteins", 《TUMOR BIOLOGY》, vol. 32, no. 1, 24 July 2010 (2010-07-24) * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106053785A (en) * | 2016-05-18 | 2016-10-26 | 北京北方生物技术研究所有限公司 | Solid antibody pre-coating method for competitive chemiluminescent immunoreactions |
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Address after: 100076 Beijing city Fengtai District pan Temple No. 20 Patentee after: BEIJING NORTH INSTITUTE OF BIOLOGICAL TECHNOLOGY Address before: 100076 Beijing city Fengtai District pan Temple No. 20 Patentee before: Beijing North Institute of Biological Technology |