CN104931685A - Luminescence immune detection method based on recombinant antigen carrying His tag - Google Patents
Luminescence immune detection method based on recombinant antigen carrying His tag Download PDFInfo
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Abstract
The invention provides a luminescence immune detection method based on a recombinant antigen carrying a His tag. His-tag-carried recombinant protein serving as a known antigen, His antibody coating resistant luminescent microspheres, biotin labeled second antibodies and streptavidin labeled light sensing microspheres jointly form a detection reagent. According to the method, the recombinant protein carrying the His tag is taken as the known antigen, the method has the most important characteristic that the known antigen is arranged in liquid-phase three-dimensional space, which is essentially different from an immobilized antigen in the prior art. Due to the change, the technical problems caused by the immobilization process in a traditional antibody detection method are effectively solved. Besides, a homogeneous-phase luminescence immunoassay system based on active oxygen energy transmission is adopted and has higher analysis sensitivity and precision.
Description
Technical field
The invention belongs to the antibody test technical field of Medical Laboratory Specialty, mainly comprise anti-allergen antibodies, pathogen antigen and autoantibody, especially relating to a kind of electrochemiluminescent immunoassay detection method based on carrying His label recombinant antigen.
Background technology
Immunochemistry (antigen-antibody reaction) technology is the important method of detection specificity antibody, namely adopts known antigens to detect unknown antibody format.Enzyme linked immunosorbent assay (ELISA) indirect method is usually used in detecting above-mentioned antibody, also enzyme can be adopted to join dot blot assays (dot-ELISA) to be wrapped by multiple related antigen with " linear mode ", to realize detecting Multiple Antibodies (as antinuclear antibodies spectrum, HIV validation test etc.) simultaneously simultaneously.The ultimate principle of above-mentioned two kinds of methods is as follows: with known antigens bag by solid phase carrier (polystyrene micropore plate or nitrocellulose filter); Add serum temperature to be checked bath, antibody to be checked is combined with known antigens and forms immune complex (or antibody to be checked is caught by solid phase antigen); Add enzymic-labelled antibody again and temperature bath, labelled antibody is combined with antibody to be checked, and non-binding antibody is removed by " washing "; Finally add substrate colour developing, read absorbance (A) by microplate reader after stopping enzymatic reaction or directly observe film pimple result of determination.
Find in practical application that said method exists the defect of some technical elements, and these defective effect testing result accuracys or to commercially produce bring some difficulty, main manifestations in the following areas:
It is that isolation technics is commonly used in non-homogeneous enzyme immunoassay analysis that one, immobilization problem solid phase adsorption are separated, its principle is at solid phase material surface (polystyrene micropore or nitrocellulose membrane) by antigen solid phase, the antigen formed with antibody to be checked and enzyme labelled antibody-antibody-hrp-antibody complex to be checked is present in solid phase material surface, and free label is distributed in liquid phase.Topple over liquid solution and easily can remove interference measurement, free enzymic-labelled antibody by " washing ".But after known antigens is connected with solid phase material (bag quilt), be distributed in the antigen molecule on solid phase material surface, its structure no longer has the native conformation in liquid phase.The change of this molecular conformation will affect known antigens and be combined with antibody to be checked; Meanwhile, solid phase antigen is positioned at solid phase material surface, is combined with antibody to be checked because space steric effect can have a strong impact on.In addition, when envelope antigen is the peptide molecule of molecular weight, often can be lost its biologically active because of bag by process, or binding antibody ability significantly declines.
Two, envelope antigen purity and plurality of antigens combination problem solid-phase coating be by antigen coated in solid phase plane, the bag of microwell plate or nitrocellulose membrane is by limited area, antigen for wrapping quilt requires higher purity, and antigen purification process can increase the production cost of detection kit.Meanwhile, no matter anaphylactogen sIgE antibody, or pathogen antigen or autoantibody, generally need multiple protein as known antigens, and above-mentioned solid phase bag is not suitable for multiple antigenic carries out bag quilt simultaneously by mode.Multiple antigenic bag is related to ratio and the sterically hindered mutual interference caused between component.
Three, preparation process and use procedure complexity no matter enzyme linked immunosorbent assay or enzyme connection dot blot assays, antigen immobilization process is the key link that diagnostic reagent is produced, the processes such as antigen coated-to close-drying are more complicated, and realizing standardization needs standard operating procedure (SOP) and strict implement.From user perspective, the use also more complicated of above-mentioned two kinds of detection reagent, clinical samples need dilution, twice incubation and repeatedly washing process needs for a long time, and labelled antibody (enzyme) is by various factors etc., is also unfavorable for the use of operator.
For the defect of above-mentioned technical elements, this technological invention relates to a kind of known recombinant antigen based on carrying His label, active oxygen ability is adopted to transmit homogeneous luminescent immunoassay principle, set up a kind of novel light-emitting immunoassay system, detect for specific IgE, pathogen antigen and autoantibody.
Summary of the invention
In view of this, the present invention is intended to propose a kind of electrochemiluminescent immunoassay detection method based on carrying His label recombinant antigen, adopt active oxygen energy transferring homogeneous luminescent immunoassay principle, known recombinant antigen is placed in liquid phase three dimensions, essential distinction is there is with conventional art immobilization antigen, the present invention sets up a kind of novel light-emitting immunoassay system, detect for specific IgE, pathogen antigen and autoantibody, there is higher analysis susceptibility and precision, and preparation technology is simple, easy to operate.
For achieving the above object, the technical scheme of the invention is achieved in that
Based on an electrochemiluminescent immunoassay detection method of carrying His label recombinant antigen, comprise the steps,
1) His-recombinant protein is obtained;
2) the luminous microspheres solution of serum to be checked, His-recombinant protein solution, His antibody bag quilt is fully mixed, and in 37 DEG C of temperature bath 30 ~ 45min;
3), after temperature bath, through high speed centrifugation 10 ~ 15 minutes, supernatant is abandoned;
4) add the photosensitive microspheres solution of biotin labeled second antibody solution and Streptavidin bag quilt, fully mix, 37 DEG C of temperature bath 15 ~ 30min;
5) by light stimulated luminescence detector sensed light signal under 615nm.
Described His-recombinant protein, His label is carried in upstream (left end), downstream be antibody to be checked for recombinant protein (known antigens), recombinant protein is with different epitope (if diagram is containing 3 kinds of epitopes), and this recombinant protein can catch antibody to be checked.
Described biotin labeled second antibody, is marked with biotin molecule, because antibody to be checked is human immunoglobulin(HIg), can use rabbit anti human IgE (for detecting IgE antibody-like) or rabbit anti-human igg's (for detecting IgG antibody-like).
Described Streptavidin bag is by photosensitive microballoon, and photosensitive microballoon is combined with biotin, carries photoactive substance simultaneously, can produce active oxygen species when irradiating with laser.
The luminous microballoon of described anti-His albumen bag quilt, luminous microballoon and His protein combination, accept active oxygen energy simultaneously, can produce by induction light signal.
Preferably, described step 1) in, His-recombinant protein is obtained by following steps, according to antigen primary structure synthesis target DNA, and through Protocols in Molecular Biology, genes of interest and the expression plasmid carrying His label are recombinated, transfection suitable host carries out the expression of destination protein, then obtains His-recombinant protein through nickel affinity chromatography post preliminary purification, this material as known antigens for detecting antibody.
Preferably, described step 2) in, after three kinds of materials mix, temperature bath 30 ~ 45min at 37 DEG C.
Preferably, described step 2) in, the volume ratio of the luminous microballoon of serum to be checked, His-recombinant protein mixed solution, His antibody bag quilt is 1:1:1; And serum Tirs-HCl solution to be checked is that dilution process is carried out in 1:10 ~ 20 according to volume ratio, preferably, 1:10.
The dilution ratio of the luminous microspheres solution of described His-recombinant protein mixed solution, His antibody bag quilt is determined according to the type of test antibodies.
Preferably, described step 3) in, high speed centrifugation speed is 10 × 10
3r/min, and the ultracentrifugal time be 10min.
Preferably, described step 4) in, biotin labeled second antibody, the photosensitive microballoon of Streptavidin bag quilt and the volume ratio of serum to be checked are 2:4:1.
Preferably, described step 4) in, described biotin labeled second antibody is the one in rabbit anti human IgE or rabbit anti-human igg.
Present invention also offers a kind of as above based on the application of electrochemiluminescent immunoassay detection method in specific IgE detects of carrying His label recombinant antigen.
Present invention provides a kind of as above based on the application of electrochemiluminescent immunoassay detection method in pathogen IgG or IgM detects of carrying His label recombinant antigen.
Invention also provides a kind of as above based on the application of electrochemiluminescent immunoassay detection method in autoantibody IgG detects of carrying His label recombinant antigen.
Present invention also offers a kind of for the kit of electrochemiluminescent immunoassay detection method based on carrying His label recombinant antigen as above, wherein kit comprises biotin labeled second antibody, the photosensitive microballoon of Streptavidin bag quilt, biotin labeling second antibody, carries the recombinant protein of His label.
The present invention is based on the recombinant protein carrying His label and jointly formed detection reagent as known antigens, anti-His antibody bag by luminous microballoon, biotin labeling second antibody (antibody for antibody isotype epi-position to be checked), the photosensitive microballoon of marked by streptavidin.
Under there is situation in antibody to be checked, antibody to be checked and recombinant protein form immune complex, this compound is combined by the luminous microballoon of His and the His antibody bag quilt of upstream again, be combined with biotin labeling second antibody by antibody to be checked, and be combined with the photosensitive microballoon of marked by streptavidin through biotin.By above-mentioned course of reaction, photosensitive microballoon and luminous microballoon close to each other, distance is less than 200 nanometers, meet active oxygen and transmit light-induced chemiluminescent condition, system luminescence (615nm) when irradiating with laser, prove that antibody to be checked exists, and according to luminous intensity, quantitative test is carried out to antibody to be checked.As there is not antibody to be checked in sample to be checked, in conjunction with restructuring known antigens, also just can not can not induce chain reaction, the spacing of photosensitive microballoon and luminous microballoon is greater than 200nm, can not meet active oxygen and transmit light-induced chemiluminescent condition, when irradiating with laser, system can not detect light signal.
Relative to prior art, a kind of electrochemiluminescent immunoassay detection method based on carrying His label recombinant antigen of the present invention, has following advantage:
1) Solid Free bag is by process, and in liquid phase, antigen can keep native conformation, can not to the activity generation significant impact of known antigens in conjunction with antibody to be checked;
2) known antigens is placed in liquid phase, be different from bag by rear solid phase surface antigen, liquid phase provides " three-dimensional space ", and solid phase provides " two dimensional surface space ".Effect that three-dimensional space is deposited hardly " sterically hindered ", and effectively can reduce Ag-Ab and reach balance required time.
3) system detecting antibody of polycomponent as known antigens is applicable to.Food allergen sIgE detects, and because any single diet contains multiple allergic protein, this antibody-like (sIgE) is a kind of potpourri.Pathogen antigen is also like this.This type of antibody test needs multiple related antigen, is not too applicable to solid-phase coating, and adopts " liquid phase reactor " pattern designed by this technological invention, can provide sufficient space for multiple antigenic antibody response.
4) preparation technology is simple.Anti-His antibody bag in the detection system of this technological invention is general detection reagent by luminous microballoon, biotin labeling second antibody, the photosensitive microballoon of marked by streptavidin, is applicable to Testing index.The design of recombinant protein also adopts identical His label, for protein purification creates conditions.Meanwhile, because not needing solid-phase coating, system is less demanding to restructuring antigen purity, simple purification.
Accompanying drawing explanation
The accompanying drawing forming a part of the present invention is used to provide a further understanding of the present invention, and schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 is a kind of principle schematic of electrochemiluminescent immunoassay detection method based on carrying His label recombinant antigen of the present invention;
Description of reference numerals:
1-His-recombinant protein, 2-antibody to be checked, the biotin labeled second antibody of 3-, 4-Streptavidin bag by photosensitive microballoon, the luminous microballoon of 5-anti-His albumen bag quilt.
Embodiment
Embodiment one
To detect milk allergy component, " " antibody illustrates the specific implementation process of this technological invention to beta lactoglobulin below.
Detect reagent:
(1) recombinant beta lactoglobulin, His-beta lactoglobulin
(2) biotin labeled rabbit antihuman IgE antibody
(3) the photosensitive microballoon of Streptavidin bag quilt
(4) the luminous microballoon of anti-His albumen bag quilt
Wherein the photosensitive microballoon of biotin labeled rabbit antihuman IgE antibody, Streptavidin bag quilt, the luminous microballoon of anti-His albumen bag quilt are general detection reagent.
Serum sample:
The patients serum of doubtful milk allergy
Detection method:
(1) in detection system, add the luminous microspheres solution (luminous microballoon concentration is 20 mcg/ml) of 25 μ L His antibody bag quilts respectively, 25 μ L are with the recombinant antigen solution (protein concentration is 10 mcg/ml) of His label, 25 μ L serum to be checked (Tris-Hcl solution 1:10 dilutes), fully mixes;
(2) 37 DEG C of temperature bath 30min; Through high speed centrifugation (10 × 10 after incubation
3r/min) 10 minutes, supernatant is abandoned;
(3) add the biotin labeled second antibody solution of 50 μ L, the photosensitive microspheres solution (concentration of photosensitive microballoon is 20 mcg/ml) of 100 μ l Streptavidin bag quilts, fully mixes;
(4) 37 DEG C of temperature bath 30min;
(5) with light stimulated luminescence detector sensed light signal (615nm).
Result interpretation:
| Sequence number | Optical signal value | Result |
| Negative control | 40 | |
| Positive control | 682 | |
| Sample 1# | 205 | Positive |
| Sample 2# | 421 | Moderate positive |
| Sample 3# | 846 | Strong positive |
| Sample 4# | 468 | Moderate positive |
| Sample 5# | 168 | Positive |
| Sample 6# | 398 | Moderate positive |
The foregoing is only the preferred embodiment of the invention; not in order to limit the invention; within all spirit in the invention and principle, any amendment done, equivalent replacement, improvement etc., within the protection domain that all should be included in the invention.
Claims (10)
1., based on an electrochemiluminescent immunoassay detection method of carrying His label recombinant antigen, it is characterized in that: comprise the steps,
1) His-recombinant protein is obtained;
2) the luminous microspheres solution of serum to be checked, His-recombinant protein solution, His antibody bag quilt is fully mixed, and in 37 DEG C of temperature bath 30 ~ 45min;
3), after temperature bath, through high speed centrifugation 10 ~ 15 minutes, supernatant is abandoned;
4) add the photosensitive microspheres solution of biotin labeled second antibody solution and Streptavidin bag quilt, fully mix, 37 DEG C of temperature bath 15 ~ 30min;
5) by light stimulated luminescence detector sensed light signal under 615nm.
2. the electrochemiluminescent immunoassay detection method based on carrying His label recombinant antigen according to claim 1, it is characterized in that: described step 1) in, His-recombinant protein is obtained by following steps, according to antigen primary structure synthesis target DNA, and through Protocols in Molecular Biology, genes of interest and the expression plasmid carrying His label are recombinated, transfection suitable host carries out the expression of destination protein, obtain His-recombinant protein through nickel affinity chromatography post preliminary purification again, this material is used for unknown detection antibody as known antigens.
3. the electrochemiluminescent immunoassay detection method based on carrying His label recombinant antigen according to claim 1, is characterized in that: described step 2) in, after three kinds of materials mix, temperature bath 30 ~ 45min at 37 DEG C.
4. described according to claim 1 or 3 based on the electrochemiluminescent immunoassay detection method of carrying His label recombinant antigen, it is characterized in that: described step 2) in, the volume ratio of the luminous microspheres solution of serum to be checked, His-recombinant protein solution, His antibody bag quilt is 1:1:1; And serum Tirs-HCl solution to be checked is that dilution process is carried out in 1:10 ~ 20 according to volume ratio, preferably, 1:10.
5. the electrochemiluminescent immunoassay detection method based on carrying His label recombinant antigen according to claim 1, is characterized in that: described step 3) in, high speed centrifugation speed is 10 × 10
3r/min, and the ultracentrifugal time be 10min.
6. the electrochemiluminescent immunoassay detection method based on carrying His label recombinant antigen according to claim 1, it is characterized in that: described step 4) in, biotin labeled second antibody solution, the photosensitive microspheres solution of Streptavidin bag quilt and the volume ratio of serum to be checked are 2:4:1.
7. according to claim 1 or 6 based on the electrochemiluminescent immunoassay detection method of carrying His label recombinant antigen, it is characterized in that: described step 4) in, described biotin labeled second antibody is the one in rabbit anti human IgE or rabbit anti-human igg.
8. according to any one of claim 1 ~ 7 based on the application of the electrochemiluminescent immunoassay detection method of carrying His label recombinant antigen in specific IgE or autoantibody IgG detect.
9. according to any one of claim 1 ~ 7 based on the application of the electrochemiluminescent immunoassay detection method of carrying His label recombinant antigen in pathogen IgG or IgM detects.
10. one kind for as described in any one of claim 1 ~ 7 based on the kit of electrochemiluminescent immunoassay detection method carrying His label recombinant antigen, it is characterized in that, comprise biotin labeled second antibody, the photosensitive microballoon of Streptavidin bag quilt, biotin labeling second antibody, carry the recombinant protein of His label.
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| CN108351351A (en) * | 2015-11-09 | 2018-07-31 | 生物辐射实验室股份有限公司 | Use the experiment of Avidin and biotin |
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| CN115839945A (en) * | 2023-02-13 | 2023-03-24 | 上海索昕生物科技有限公司 | Photosensitive microsphere for light-activated chemiluminescence detection |
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