CN102707048B - A kind of insolubilized antibody preparation method for immunoassay - Google Patents
A kind of insolubilized antibody preparation method for immunoassay Download PDFInfo
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- CN102707048B CN102707048B CN201210153804.6A CN201210153804A CN102707048B CN 102707048 B CN102707048 B CN 102707048B CN 201210153804 A CN201210153804 A CN 201210153804A CN 102707048 B CN102707048 B CN 102707048B
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Abstract
The present invention is a kind of insolubilized antibody preparation method for immunoassay, specifically, provides a kind of antibody and is fixed on polystyrene solid phase material after treatment and the method keeping insolubilized antibody activity stabilized by protection liquid.Preparation method provided by the invention comprises: (1) coated antibody concentration adjusts; (2) activate: in coated antibody, add activator, mixing, room temperature leaves standstill and activates; (3) bag quilt: the coated antibody of activation and bag are buffered liquid and mix and wrap by solid phase carrier; (4) close: dry the coating buffer on solid phase carrier, confining liquid is added solid phase carrier and close room and protection antibody activity; (5) dry: dry the confining liquid on solid phase carrier, removal moisture drying, encapsulates for subsequent use.
Description
Technical field
The present invention be a kind of biomolecule bag by the method for solid phase carrier, specifically, provide a kind of antibody and be fixed on polystyrene solid phase material after treatment and keep the activity stabilized method of insolubilized antibody by protection liquid.
Background technology
Labelling immunoassay is the technology of a series of detection target substances multiple labelling technique being combined with immunological technique and set up.Nineteen fifty-nine Yalow and Berson application of radiation nucleic
125the principle of I labelled antigen and determined antigen and limitation antibody competition establishes the first sensitive, quantitative radiommunoassay (Radioimmunoassay, RIA), and this is homogeneous immunoassay.Developed again afterwards solid matrix antibody-antigen-
125the heterogeneous immunoradiometric assay (Immuno-radiometric assay, IRMA) of I labelled antibody.The immunoassay of nonradioactive isotope mark develops rapidly on this basis, and mostly be the heterogeneous immunoassay adopting solid phase separation means, representational have 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 must have solid phase carrier, being required component, is again simple and easy effective separation means.Material as solid phase carrier mainly contains cellulose, gel, magnetic particle, nylon, polystyrene etc., two large classes can be divided by support, first surface is hydrophilic with chemical functional group, another kind of is that surface is hydrophobic without chemical functional group, the former is combined with antigen or antibody with covalent bond, Hou Zhetong
Cross physisorption and be coated in carrier surface.In multiple solid phase carrier, polystyrene support is combined with antibody that method is simple, quick, economical, is applicable to large-scale production operation, thus conventional, as microwell plate, ball, test tube etc. that polystyrene material is made.The solid phases such as antibody p-poly-phenyl ethene have stronger absorption affinity, are combined by physisorption with polystyrene solid phase carrier, lean on be hydrophobic grouping on molecular structure and surface of solid phase carriers hydrophobic grouping between acting force.There is researcher by polystyrene board, pearl or effective glutaraldehyde process and r radiation exposure etc. to improve the function be combined with antibody; the present invention from another approach improve antibody and polystyrene support in conjunction with effect; improve detection sensitivity; simultaneously by the application of protection liquid; the activity of protection insolubilized antibody, meets the requirement that the immunoassay kits shelf life of nonradioactive isotope mark is long.
Summary of the invention
The object of this invention is to provide a kind of insolubilized antibody preparation method for immunoassay.
Preparation method provided by the invention, comprising:
(1) concentration adjustment: coated antibody 0.02mol/L pH7.4 phosphate buffer carries out concentration adjustment;
(2) activate: in coated antibody, add activator, mixing, room temperature leaves standstill and activates for 3 minutes, and wherein said activator is 0.05mol/L pH2.4 glycocoll-HCL damping fluid;
(3) bag quilt: the coated antibody of activation and bag are buffered liquid and are mixed into coating buffer, gained coating buffer is added solid phase carrier, 2-8 DEG C of placement is placed 2 hours for 16-24 hour or 37 DEG C, and it is 0.02mol/L pH7.4 phosphate buffer or 0.01mol/L pH9.5 carbonate buffer solution that described bag is buffered liquid;
(4) close: dry the coating buffer on solid phase carrier, confining liquid is added solid phase carrier, place 2 hours or 2-8 DEG C of placement 16-24 hour for 37 DEG C, wherein said confining liquid is the 0.02mol/L pH7.4 phosphate buffer containing 8% sorbierite, 0.5%BSA, 0.1%Proclin300;
(5) dry: dry the confining liquid on solid phase carrier, room temperature dehumidifies more than 24 hours, the solid phase carrier being coated with antibody of drying is loaded in aluminium foil bag to vacuumize encapsulation for subsequent use.
In the preparation method of the invention described above, solid phase carrier is polystyrene material, can be clear microplate, white microwell plate, black microwell plate, test tube, ball etc.Described bag is buffered liquid preferred 0.02M pH7.4 phosphate buffer, wraps by time preferred 2-8 DEG C 16-24 hour.Described off-period preferably 37 DEG C 2 hours.Antibody can be monoclonal antibody, polyclonal antibody, and Antibody types is IgG.
In the preparation method of the invention described above, by adjustment coated antibody concentration, control ratio and the action time of activator and coated antibody, make antibody more multiple binding sites be exposed to the outside, significantly improve detection sensitivity.Confining liquid closes the room point after the absorption of surface of solid phase carriers antibody molecule; reduce non-specific responding in subsequent detection process; simultaneously confining liquid forms layer protecting film at surface of solid phase carriers, the available protecting activity of insolubilized antibody, can tolerate 50 DEG C and reach more than 10 days activity and do not fall.
Accompanying drawing explanation
Figure 1 shows after three groups of bags are placed different number of days by plate 50 DEG C and detect 0.5ng/ml calibration object point OD value variation tendency.
Figure 2 shows after three groups of bags are placed different number of days by plate 50 DEG C and detect 1ng/ml calibration object point OD value variation tendency.
Figure 3 shows after three groups of bags are placed different number of days by plate 50 DEG C and detect 5ng/ml calibration object point OD value variation tendency.
Figure 4 shows after three groups of bags are placed different number of days by plate 50 DEG C and detect 50ng/ml calibration object point OD value variation tendency.
OD value is constant shows that insolubilized antibody activity remains unchanged, and OD value declines and indirectly shows insolubilized antibody activity decrease.
Embodiment
In preparation method of the present invention (1), with 0.02mol/L pH7.4 phosphate buffer, coated antibody protein concentration is adjusted to 1mg/ml.
In preparation method of the present invention (2), calculated the volume of required coated antibody according to selected bag by concentration, take out respective volume antibody, add 2 times of volume 0.05mol/L pH2.4 glycocoll-HCL damping fluids, mixing, room temperature leaves standstill 3 minutes.
In preparation method of the present invention (3), the coated antibody bag of activation is buffered liquid dilute, join in microwell plate micropore, addition is 100 μ l/ holes, plate is proceeded to 2-8 DEG C bag spent the night (16-24 hour) or 37 DEG C bag by 2 hours.It is 0.02mol/L pH7.4 phosphate buffer or 0.01mol/L pH9.5 carbonate buffer solution that wherein said bag is buffered liquid.
In preparation method of the present invention (4), get rid of coating buffer, thieving paper is buckled dry, every hole adds confining liquid 130 μ l, off-period is 37 DEG C 2 hours or 2-8 DEG C spend the night (16-24 hour), and wherein said confining liquid is the 0.02mol/L pH7.4 phosphate buffer containing 8% sorbierite, 0.5%BSA, 0.1%Proclin300.
In preparation method of the present invention (5), get rid of deblocking liquid, thieving paper is buckled dry, room temperature dehumidifies more than 24 hours.For subsequent use by vacuumizing encapsulation in the pre-coated plate loading aluminium foil bag of drying.
The solution preparation method related in preparation method of the present invention:
0.05mol/L pH2.4 glycocoll-HCL damping fluid: 375.25 milligrams of glycocoll deionized water dissolvings, add 1.62 milliliters of 2N HCL, be 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, with being settled to 1000 milliliters after deionized water dissolving.
0.01mol/L pH9.5 carbonate buffer solution: Na
2cO
30.318 gram, NaHCO
30.588 gram, with being settled to 1000 milliliters after deionized water dissolving.
Confining liquid: sorbierite 80 grams, BSA 5 grams, is settled to 1000 milliliters after dissolving, adds 1 milliliter of Proclin300 with 0.02mol/L pH7.4 phosphate buffer.
Further describe the present invention below by embodiment, but the present invention is not limited to this.
Embodiment 1
Coated antibody concentration is 1mg/ml, and selected bag is 4 μ g/ml by concentration, for preparation 500ml coating buffer.Get coated antibody 2ml, add 4ml 0.05mol/L pH2.4 glycocoll-HCL damping fluid, mixing, room temperature leaves standstill 3 minutes, is added in 494ml 0.02mol/L pH7.4 phosphate buffer and is mixed into coating buffer.In microwell plate micropore, add coating buffer, 100 μ l/ holes, plate is proceeded to 2-8 DEG C of bag and spent the night (16-24 hour).Get rid of coating buffer, thieving paper is buckled dry, every hole adds confining liquid 130 μ l, off-period be 37 DEG C 2 hours.Get rid of deblocking liquid, thieving paper is buckled dry, room temperature dehumidifies more than 24 hours.For subsequent use by vacuumizing encapsulation in the pre-coated plate loading aluminium foil bag of drying.
Embodiment 2
Prepare coating buffer by embodiment 1, added by polystyrene bead in coating buffer, addition there was not pearl to be as the criterion with coating buffer, proceeded to 2-8 DEG C of bag and was spent the night (16-24 hour).Discard coating buffer, pour confining liquid into, confining liquid addition was as the criterion there not to be pearl, closed 2 hours for 37 DEG C.Discard confining liquid, the dry bead surface confining liquid of control, room temperature dehumidifies more than 24 hours.The pearl of drying is loaded in air-tight bottle for subsequent use.
Embodiment 3
Be solid phase carrier divided by polystyrene tube, coating buffer 200 μ l/ manages, outside confining liquid 300 μ l/ pipe, all the other all with the pre-coated pipe of embodiment 1 method identical preparation solid phase.
Embodiment 4
Prepare pre-coated plate by embodiment 1, unlike bag by the time be 37 DEG C 2 hours.
Embodiment 5
Pre-coated plate is prepared, unlike being 2-8 DEG C spend the night (16-24 hour) off-period by embodiment 1.
Embodiment 6
Prepare pre-coated plate by embodiment 1, prepare coating buffer unlike with 0.01mol/L pH9.5 carbonate buffer solution.
Embodiment 7 the present invention improves detection sensitivity effect identification 1
1. materials and methods
Polystyrene chemiluminescence white microwell plate, anti-C-peptide monoclonal antibody, C-peptide immue quantitative detection reagent box (chemoluminescence method), variable concentrations C-peptide calibration object (0ng/ml, 0.2ng/ml, 0.5ng/ml, 1ng/ml, 2ng/ml, 5ng/ml, 10ng/ml)
Anti-C-peptide monoclonal antibody is prepared pre-coated Chemiluminescent plate by embodiment 1, and this is plate A.Prepare pre-coated Chemiluminescent plate B by embodiment 1, unlike omission " activation " that step simultaneously.Detect 0ng/ml with plate A and plate B, 0.2ng/ml, 0.5ng/ml, 1ng/ml, 2ng/mll, 5ng/ml, 10ng/mlC-peptide calibration object simultaneously, operate by C-peptide immue quantitative detection reagent box (chemoluminescence method) instructions.Detect concrete steps: in microwell plate, add 20 μ l each concentration C-peptide calibration points respectively, wherein 0ng/ml adds 20 holes, add 100 μ l enzyme conjugates again, the rearmounted 37 DEG C of reactions of concussion mixing 1.5 hours, clean 5 times with cleansing solution, button is dry, add luminous substrate liquid A 50 μ l, luminous substrate liquid B 50 μ l, mixing, room temperature places 10 minutes, measures each hole luminous value with Chemiluminescence Apparatus.Regressive calibration curve equation is obtained with Log-Log Model fitting, 20 holes " 0 " calibration object luminous value mean value (X) adds twice standard deviation (SD), utilizes dose-response curve regression equation calculation to go out luminous value for concentration value during X+2SD and is the sensitivity of kit.
2. the results are shown in following table 1:
Table 1 two kinds of chemiluminescence bags are by plate sensitivity technique result
Under the same conditions, plate A each calibration object luminous value 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 exhibits of plate A goes out better more active than plate B, and sensitivity, apparently higher than plate B, proves that the present invention can significantly improve detection sensitivity.
Embodiment 8 the present invention improves detection sensitivity effect identification 2
1. materials and methods
Polystyrene elisa plate, Anti-HBs antibody monoclonal antibody, HBsAg enzyme linked immunological kit, variable concentrations HBsAg calibration object (0ng/ml, 0.5ng/ml, 1ng/ml, 2ng/ml)
Anti-HBs antibody monoclonal antibody is prepared pre-coated elisa plate by embodiment 1, and this is plate C.Prepare pre-coated elisa plate D by embodiment 1, unlike omission " activation " that step simultaneously.Detect 0ng/ml with plate C and plate D, 0.5ng/ml, 1ng/ml, 2ng/ml HBsAg calibration object simultaneously, operate by 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 DEG C of reactions of concussion mixing 1 hour, clean 4 times with cleansing solution, button is dry, add TMB developer A 50 μ l, developer B 50 μ l, mixing, 37 DEG C are developed the color 10 minutes, add 50 μ l 2M sulfuric acid cessation reactions, read note OD value by microplate reader in 450nm place.
2. the results are shown in following table 2:
Table 2 two kinds of enzyme connection bags are by plate sensitivity technique result
In these table data, OD value is mean value.According to HBsAg enzyme linked immunological kit instructions, boundary value CO=0.105, is judged to be surface antigen positive during S/CO >=1, is judged as surface antigen negative during S/CO < 1.Plate C detects 0.5ng/ml, and plate D can only detect 1ng/ml, proves that the present invention can significantly improve detection sensitivity.
Insolubilized antibody study on the stability prepared by embodiment 9 the present invention
1. materials and methods
Polystyrene elisa plate; Anti-HBs antibody monoclonal antibody; 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 microwell plate.
Anti-HBs antibody monoclonal antibody is prepared pre-coated elisa plate by embodiment 1, and replace confining liquid of the present invention unlike with HY technology company of U.S. product Supercoat, this is plate I; Anti-HBs antibody monoclonal antibody is prepared pre-coated elisa plate by embodiment 1, and this is plate II; Anti-HBs antibody monoclonal antibody is prepared pre-coated elisa plate by embodiment 1, replaces confining liquid of the present invention unlike with 1%BSA, this is plate III.
These three groups of laths are put into 50 DEG C of drying boxes, detect 0ng/ml respectively at 0,3,5,7,10,13 day simultaneously, 0.5ng/ml, 1ng/ml, 5ng/ml, 50ng/ml HBsAg calibration object, by the operation of HBsAg enzyme linked immunological kit instructions, according to each its activity change of calibration object OD value change tracing detection.Detect concrete steps with embodiment 8.
2. the results are shown in following table 3:
Table 3 three groups of bags are by the active tracing detection result of plate
In these table data, OD value is mean value.I group, II group lath stability are all fine, very nearly the same, and 50 DEG C of activity in 10 days remain unchanged substantially.Supercoat it be unclear that its composition; Group III only has the effect of albumen, less stable, 50 DEG C of 5 days loss of activity nearly 75%; prove that confining liquid of the present invention has well protection to be adsorbed in the effect of antibody on microwell plate, can match in excellence or beauty with the Supercoat of HY technology company.
Claims (2)
1., for an insolubilized antibody preparation method for immunoassay, it is characterized in that comprising the following steps:
(1) concentration adjustment: coated antibody protein concentration is adjusted to 1mg/ml with 0.02mol/L pH7.4 phosphate buffer;
(2) activate: the volume being calculated required coated antibody according to selected bag by concentration, take out the antibody that respective volume concentration is adjusted to 1mg/ml, add 2 times of volume 0.05mol/L pH2.4 glycocoll-HCL damping fluids, mixing, room temperature leaves standstill 3 minutes;
(3) bag quilt: the coated antibody of activation and bag are buffered liquid and are mixed into coating buffer, gained coating buffer is added microwell plate, 100 μ l/ holes, 2-8 DEG C of placement is placed 2 hours for 16-24 hour or 37 DEG C, and it is 0.02mol/L pH7.4 phosphate buffer or 0.01mol/L pH9.5 carbonate buffer solution that described bag is buffered liquid;
(4) close: dry the coating buffer on microwell plate, confining liquid is added microwell plate, 130 μ l/ holes, place 2 hours or 2-8 DEG C of placement 16-24 hour for 37 DEG C, wherein said confining liquid is the 0.02mol/L pH7.4 phosphate buffer containing 8% sorbierite, 0.5%BSA, 0.1%Proclin300;
(5) dry: dry the confining liquid on microwell plate, room temperature dehumidifies more than 24 hours, the microwell plate being coated with antibody of drying is loaded in aluminium foil bag to vacuumize encapsulation for subsequent use;
(6) wherein said coated antibody is anti-C-peptide monoclonal antibody or Anti-HBsAg antibody monoclonal antibody.
2. preparation method as claimed in claim 1, it is characterized in that, described microwell plate is clear microplate, white microwell plate, black microwell plate.
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| An improved preparation of antibody-coated polystyrene beads for sandwich enzyme immunoassay;Eiji Ishikawa 等;《Journal of Immunoassay》;19801231;第1卷(第3期);第385-398页 * |
| TD-11 workshop report: characterization of monoclonal antibodies to S100 proteins;Elisabeth Paus 等;《Tumor Biology》;20100724;第32卷(第1期);第1-12页 * |
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