CN111647080B - C-line antibody for immune colloidal gold mixed labeling and application thereof - Google Patents
C-line antibody for immune colloidal gold mixed labeling and application thereof Download PDFInfo
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Abstract
The invention discloses a C-line antibody for immune colloidal gold mixed labeling and application thereof, belonging to the technical field of immune colloidal gold. The C line antibody is bovine serum albumin antibody; the amino acid sequence of the heavy chain of the C-line antibody comprises the amino acid sequence shown as SEQ ID NO: 1; the amino acid sequence of the light chain of the C-line antibody comprises the amino acid sequence shown as SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof. The C-line antibody can be prepared into a sample mixed solution with a sample to be marked for immune colloidal gold marking, and the pH value of the marking is 7.8-8.4. The invention can realize the combination with real C line goat anti-mouse by mixing the bovine serum albumin antibody with the sample to be marked, thereby increasing the length-reduction transformation of the strength of the T line and the C line, improving the sensitivity of the lateral chromatography detection and avoiding the occurrence of false positive.
Description
Technical Field
The invention relates to the technical field of immune colloidal gold, in particular to a C-line antibody for immune colloidal gold mixed labeling and application thereof.
Background
The Immune colloidal gold technology (Immune colloidal gold technology) is an Immune labeling technology which applies colloidal gold as a tracer marker to antigen-antibody detection, and is abbreviated in English as follows: and (4) GICT. The colloidal gold is prepared from chloroauric acid (HAuCl)4) Under the action of reducing agent such as ascorbic acid and sodium citrate, the nano gold particles are reductively polymerized into nano gold particles with specific size, and become a stable colloidal state due to electrostatic action, and the nano gold particles are called colloidal gold. The colloidal gold particles are negatively charged and can form firm combination with the positive charge groups of protein molecules, and the combination is electrostatic combination, so that the biological characteristics of the protein are not influenced. Colloidal gold, in addition to binding to proteins, can also bind to many other biomacromolecules, such as SPA, PHA, ConA, and the like. According to some physical properties of colloidal gold, e.g. high electron densityParticle size, shape and color reactions, coupled with the immunological and biological properties of the conjugate, have led to the widespread use of colloidal gold in the fields of immunology, histology, pathology and cell biology.
The lateral chromatography technology is a novel in vitro diagnosis technology developed on the basis of monoclonal antibody technology, colloidal gold immunochromatography technology and new material technology in the 90 th century, has the advantages of rapidness, simplicity, single-person detection and economy, and is widely applied to the fields of medical detection, food quality monitoring, environment monitoring, agriculture and animal husbandry, entry and exit inspection and quarantine, forensic case setting and the like.
The lateral chromatography technology takes a nitrocellulose membrane (NC membrane) as a carrier, fixes a specific antigen or antibody on the NC membrane, when a sample to be detected is added on a sample pad at one end of a test strip, moves laterally through capillary action, generates specific immunoreaction with a reagent marked by colloidal gold or microspheres on a binding pad, moves on the NC membrane, is captured by an antigen or antibody detection line (T line) fixed on the surface of the NC membrane, and is gathered on a detection strip, and an intuitive color development result is obtained by visually observing a light reflection signal of a marker (colloidal gold or latex particles) on the surface of the nitrocellulose membrane. The other unbound label crosses the detection zone and is captured by the control line (C-line) antibody (usually goat anti-mouse). And realizing qualitative or semi-quantitative detection of the object to be detected through the existence and the darkness of the T line and the C line.
The antibody is a main marker marked by immune colloidal gold, and the spatial structure of the antibody is Y-shaped and consists of two light chains and two heavy chains. The amino terminal of the heavy chain and the light chain form a Fab segment combined with antigen, and the carboxyl terminals of the two heavy chains are connected by disulfide bond to form a conserved Fc segment.
In the immune colloidal gold labeling process, the binding configuration of the antibody and the colloidal gold particles has directionality in space. To describe the directionality of this spatial binding configuration, one defined the Fab fragment of the antibody as the Head (Head) and the Fc fragment as the Foot (Foot). Thus, the spatial binding configuration of the antibody molecule and the colloidal gold particles has three states: 1, binding state of Fab segment and colloidal gold particle, Head-down binding state, Head-on; 2, the Fc segment is combined with the colloidal gold particles, and the feet face down, Foot-on; side-on, Side-lying position. In the production labeling, it is found that only Foot-on binding state exists after some antibodies are labeled, or Foot-on and Side-on binding mixed state does not exist, and Head-on binding state does not exist. The visual performance of the test result of the lateral chromatography test strip is judged to be that the T line has good color development strength and good inhibition rate, but the antibody C line has very weak color development or no color development.
Monoclonal antibodies of many antibiotics, pesticides and drugs are difficult to screen, and receptor proteins are used for detection, do not have Fc sections and cannot be combined with the antibodies.
The current solution is to scribe a false C line on the NC membrane using Bovine Serum Albumin (BSA) blocking colloidal gold particles mixed with a chemical dye. The color intensity of the false C line is constant, and no attenuation transformation between the intensity of the T line and the intensity of the C line exists, so that part of sensitivity is lost. In addition, the false C line is irrelevant to the activity of the colloidal gold marker and is asynchronous with the activity state of the immune gold, so that the activity state of the immune gold cannot be indicated, and false positive is easily caused in quality control.
Disclosure of Invention
The present invention is directed to a C-line antibody for immunocolloidal gold mixed labeling, which solves the problems of the background art mentioned above.
In order to achieve the above purpose, the embodiments of the present invention provide the following technical solutions:
a C line antibody for immune colloidal gold mixed labeling is disclosed, wherein the C line antibody is bovine serum albumin antibody; the amino acid sequence of the heavy chain of the C-line antibody comprises the amino acid sequence shown as SEQ ID NO: 1; the amino acid sequence of the light chain of the C-line antibody comprises the amino acid sequence shown as SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.
As a preferable embodiment of the present invention, the C-line antibody is a mouse monoclonal antibody obtained by screening bovine serum albumin as an immunogen, and is named CAB 01; the cell strain of the mouse monoclonal antibody is preserved in China general microbiological culture collection center in 1 month and 8 days of 2020, and the preservation number is CGMCC No. 19284.
Another objective of the embodiments of the present invention is to provide an application of the above-mentioned C-line antibody in immune colloidal gold mixed labeling.
As another preferable aspect of the embodiment of the present invention, the application includes the steps of: and preparing a sample mixed solution from the C-line antibody and a sample to be marked for immune colloidal gold marking.
In another preferred embodiment of the present invention, the concentration of the C-line antibody in the sample mixture is 0.8 to 1.5. mu.g/mL.
As another preferable scheme of the embodiment of the invention, the concentration of the sample to be marked in the sample mixed solution is 4-6 mug/mL.
In another preferable embodiment of the invention, in the step, the pH of the immune colloidal gold label is 7.8-8.4.
Compared with the prior art, the embodiment of the invention has the beneficial effects that: the embodiment of the invention provides a C-line antibody for immune colloidal gold mixed labeling, which is a bovine serum albumin antibody, can be mixed with a sample to be labeled, and can realize the combination with a real C-line goat-anti-mouse, thereby increasing the length-reduction transformation of the strength of a T line and a C line, improving the sensitivity of lateral chromatography detection and avoiding the occurrence of false positive.
Drawings
FIG. 1 is a graph showing the relationship between the pH value of a colloidal gold solution and the color intensity of line C.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In addition, the apparatus and reagents used in the following examples are commercially available ones unless otherwise specified.
Example 1
The embodiment provides a C line antibody for immune colloidal gold mixed labeling, wherein the C line antibody is a bovine serum albumin antibody, and the preparation and screening method comprises the following steps:
(1) selecting bovine serum albumin as immunogen; the content abundance of blocking proteins such as bovine serum albumin and the like in a natural sample is large, the blocking proteins are easy to obtain, the specificity is good, cross reaction with other detection objects basically does not exist, and the blocking proteins have the advantages of easiness in obtaining, low cost and the like.
(2) Injecting bovine serum albumin into a mouse body, and performing primary immunization, secondary immunization, tertiary immunization and four-time boosting immunization; the specific immunization program is as follows:
firstly, the method avoids: d0 days, mixing bovine serum albumin with Freund's complete adjuvant, and injecting into mouse abdominal cavity with dosage of 50 mg;
and (2) avoiding: d21 days, mixing bovine serum albumin with Freund's incomplete adjuvant, and injecting into mice subcutaneously in an amount of 30 mg;
and (3) three-step (I): d35 days, mixing bovine serum albumin with Freund's complete adjuvant, and injecting into mice subcutaneously in an amount of 30 mg;
strengthening and avoiding: d42 days, bovine serum albumin was injected into the abdominal cavity in a 50mg dose in admixture with Freund's incomplete adjuvant.
(3) Performing Enzyme linked immunosorbent assay (ELISA) on the four-time immunized mice, and if the serum titer is more than 10 ten thousand, aseptically taking spleen cells and myeloma cells (SP 20) of the immunized mice and performing fusion treatment; then, culturing and screening the cells subjected to fusion treatment to obtain the mouse monoclonal antibody of the bovine serum albumin, namely the bovine serum albumin antibody, wherein the clone number of the antibody is as follows: 10G12, named: CAB01, wherein the amino acid sequence of the heavy chain of the antibody comprises the amino acid sequence shown as SEQ ID NO: 1, and the amino acid sequence of the light chain comprises the amino acid sequence shown as SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof. In addition, the cell line of the mouse monoclonal antibody was deposited in the China general microbiological culture Collection center on 1 month and 8 days 2020 at the address: china Beijing, the preservation number is CGMCC No. 19284.
Wherein, the screening method comprises the following steps:
1. the plate was coated with bovine serum albumin at a concentration of 5. mu.g/mL in an amount of 100. mu.L/well.
2. Preparing bovine serum albumin monoclonal antibody, diluting culture supernatant by 1000 times, and then OD450nmThe value should be greater than 2.0.
3. Purifying the bovine serum albumin monoclonal antibody, adjusting the concentration to be 5 mug/mL, directly marking by immune colloidal gold, and setting the marking pH to be 7.8-8.4.
4. NC membrane, 0.5mg/mL bovine serum albumin for T line, 0.5mg/mL goat anti-mouse antibody for C line, 0.05M PBS for membrane scribing, and drying at 40 deg.C for 24 hr.
5. Screening conditions are as follows: the ELISA detection has strong affinity with bovine serum albumin, and the serum titer is more than 10 ten thousand; no cross reaction with other blocking proteins; the 5 mug/mL bovine serum albumin monoclonal antibody is directly marked, the immune colloidal gold T line does not develop color, and the color development intensity of the C line is more than 1200.
Example 2
This embodiment provides a sample mixture comprising a sample to be labeled and the bovine serum albumin antibody provided in embodiment 1 above; the concentration of the bovine serum albumin antibody is 0.8 mug/mL. The concentration of the sample to be labeled is 4. mu.g/mL. The solvent of the sample mixed solution is water. Wherein, the sample to be marked is a sample which needs to be marked actually; bovine serum albumin antibody was prepared in the same manner as in example 1.
Example 3
This embodiment provides a sample mixture comprising a sample to be labeled and the bovine serum albumin antibody provided in embodiment 1 above; the concentration of the bovine serum albumin antibody is 1.5 mug/mL. The concentration of the sample to be labeled is 6. mu.g/mL. The solvent of the sample mixed solution is water. Wherein, the sample to be marked is a sample which needs to be marked actually; bovine serum albumin antibody was prepared in the same manner as in example 1.
Example 4
This embodiment provides a sample mixture comprising a sample to be labeled and the bovine serum albumin antibody provided in embodiment 1 above; the concentration of the bovine serum albumin antibody is 1 mug/mL. The concentration of the sample to be labeled is 5. mu.g/mL. The solvent of the sample mixed solution is water. Wherein, the sample to be marked is a sample which needs to be marked actually; bovine serum albumin antibody was prepared in the same manner as in example 1.
Example 5
The embodiment provides a method for screening the labeling pH of the sample mixed solution in the application of immune colloidal gold mixed labeling, which comprises the following steps:
(1) taking 5 mu g/mL bovine serum albumin solution, coating the enzyme label plate by 100 mu L/well, and reserving for later use.
(2) Detecting the bovine serum albumin antibody obtained in the embodiment 1 by using the existing lateral chromatography test strip, wherein the T line on the NC membrane of the test strip is 0.5mg/mL of bovine serum albumin, the C line is 0.5mg/mL of goat anti-mouse, the membrane-scribing solution is 0.05mol/L of phosphate buffer solution, drying the membrane at 40 ℃, and 24 hours; then, the above bovine serum albumin antibodies were directly labeled with colloidal gold solutions of different pH values, and the C-line color intensities thereof are shown in FIG. 1. As can be seen from the figure, the color intensity of the C line rapidly decreases after the pH value of the label is greater than 8.4, and the C line does not develop color after the pH value of the label is greater than 8.6. Therefore, the pH application range of the bovine serum albumin antibody (CAB 01) marked by the immune gold is 7.8-8.4.
Example 6
This embodiment provides a method for hybrid labeling of Beta lactam receptor WG001 with immune colloidal gold, which comprises the following steps:
(1) 2.4mL of the colloidal gold solution was added to a 5mL centrifuge tube, and then 0.1mol/L potassium carbonate solution was added to the centrifuge tube to mix by vortexing for 2 cycles, thereby adjusting the pH of the colloidal gold solution to 7.8.
(2) Adding 30 mu L of the sample mixed solution provided by the embodiment 4 (the sample to be marked is Beta lactam receptor WG 001) into the centrifuge tube, covering the centrifuge tube cover, performing vortex mixing for 2 rounds, and standing for 10 min; then, adding 24 μ L of bovine serum albumin solution with the mass percentage concentration of 12%, covering a centrifugal tube cover, uniformly mixing by vortex for 2 rounds, and standing for 5 min; then, 24 μ L of polyethylene glycol solution with the mass percentage concentration of 12% is added, a centrifuge tube cover is covered, after 2 rounds of vortex mixing, the centrifuge tube is placed at 10000rpm for centrifugal treatment for 4min by using a desk type high-speed centrifuge, and the supernatant is discarded to obtain a precipitate.
(3) And removing residual liquid on the surface of the precipitate, adding 300 mu L of redissolution, shaking for resuspension, and uniformly mixing for 2 rounds by vortex to obtain a labeling solution, thus finishing the labeling of the sample to be labeled. The labeling solution can be directly added to a lateral chromatography test strip for test analysis.
Example 7
This embodiment provides a method for hybrid labeling of Beta lactam receptor WG001 with immune colloidal gold, which comprises the following steps:
(1) 3.6mL of the colloidal gold solution was added to a 5mL centrifuge tube, and then 0.3mol/L potassium carbonate solution was added to the centrifuge tube to mix by vortexing for 2 cycles, thereby adjusting the pH of the colloidal gold solution to 8.4.
(2) Adding 30 mu L of the sample mixed solution provided by the embodiment 4 (the sample to be marked is Beta lactam receptor WG 001) into the centrifuge tube, covering the centrifuge tube cover, performing vortex mixing for 2 rounds, and standing for 10 min; then, 36 μ L of bovine serum albumin solution with the mass percentage concentration of 8% is added, a centrifugal tube cover is covered, vortex and uniform mixing is carried out for 2 rounds, and standing is carried out for 5 min; then, 36 μ L of polyethylene glycol solution with a mass percentage concentration of 8% was added, the centrifuge tube cap was covered, after 2 rounds of vortex mixing, the centrifuge tube was centrifuged at 15000rpm for 4min using a desk-top high speed centrifuge, and the supernatant was discarded to obtain a precipitate.
(3) And removing residual liquid on the surface of the precipitate, adding 300 mu L of redissolution, shaking for resuspension, and uniformly mixing for 2 rounds by vortex to obtain a labeling solution, thus finishing the labeling of the sample to be labeled. The labeling solution can be directly added to a lateral chromatography test strip for test analysis.
Example 8
This embodiment provides a method for hybrid labeling of Beta lactam receptor WG001 with immune colloidal gold, which comprises the following steps:
(1) 3mL of the colloidal gold solution was added to a centrifuge tube of a 5mL centrifuge tube, and then 0.2mol/L potassium carbonate solution was added to the centrifuge tube to perform vortex mixing for 2 rounds, thereby adjusting the pH value of the colloidal gold solution to 8.2.
(2) Adding 30 mu L of the sample mixed solution provided by the embodiment 4 (the sample to be marked is Beta lactam receptor WG 001) into the centrifuge tube, covering the centrifuge tube cover, performing vortex mixing for 2 rounds, and standing for 10 min; then, 30 μ L of bovine serum albumin solution with the mass percentage concentration of 10% is added, a centrifugal tube cover is covered, vortex and uniform mixing is carried out for 2 rounds, and standing is carried out for 5 min; then, 30 μ L of polyethylene glycol solution with a mass percentage concentration of 10% was added, the centrifuge tube cap was covered, after 2 rounds of vortex mixing, the centrifuge tube was placed at 12000rpm using a desk-top high speed centrifuge for centrifugation for 4min, and the supernatant was discarded to obtain a precipitate.
(3) And removing residual liquid on the surface of the precipitate, adding 300 mu L of redissolution, shaking for resuspension, and uniformly mixing for 2 rounds by vortex to obtain a labeling solution, thus finishing the labeling of the sample to be labeled. The labeling solution can be directly added to a lateral chromatography test strip for test analysis.
Comparative example 1
This comparative example provides a method of directly labeling Beta lactam receptor WG001 using conventional immunocolloidal gold, which is different from example 8 only in that the sample mixture in step (2) was replaced with a 5. mu.g/mL solution of Beta lactam receptor WG001 (i.e., no bovine serum albumin antibody was added).
Comparative example 2
This comparative example provides a method of hybrid labeling of Beta lactam receptor WG001 with immune colloidal gold, which is the only difference from example 8 in that bovine serum albumin antibody concentration in the sample mixture used in step (2) was 0.5. mu.g/mL.
Comparative example 3
This comparative example provides a method of hybrid labeling of Beta lactam receptor WG001 with immune colloidal gold, which is the only difference from example 8 in that the concentration of bovine serum albumin antibody in the sample mixture used in step (2) was 2. mu.g/mL.
Comparative example 4
This comparative example provides a method of hybrid labeling of Beta lactam receptor WG001 with immune colloidal gold, which is the only difference from example 8 in that bovine serum albumin antibody concentration in the sample mixture used in step (2) was 4. mu.g/mL.
The labeling methods provided in example 8 and comparative examples 1 to 4 were respectively matched with Beta-lactam lateral chromatography test strips to test samples with different penicillin addition amounts, and the T value, C value and T/C value obtained by the tests are shown in table 1 below (3 strips were tested in parallel under each condition and the average value was taken). Wherein, the Beta lactam lateral chromatography test strip has the quality control standard that: negative, wherein the T value is 1300-1500, the C value is more than 200, and the T/C value is more than 3.0; positive (1 ppb penicillin) and T/C value of 0.7-0.9. In addition, when the marking methods provided in example 8 and comparative examples 2-4 are used for testing, the C line is a true C line, namely, the goat anti-mouse; when the marking method provided in comparative example 1 was used for the test, the line C was a pseudo line C, which was the bovine serum albumin-sealed colloidal gold particle mixed chemical dye.
TABLE 1
As can be seen from the above table 1, in the embodiment of the invention, the bovine serum albumin antibody is mixed with the sample to be marked, so that the combination with the real C line goat-anti-mouse can be realized, the length-reduction transformation of the strength of the T line and the C line can be increased, the sensitivity of the lateral chromatography detection can be improved by 30% -100%, and the occurrence of false positive can be avoided.
In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
Sequence listing
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Claims (7)
1. A C line antibody for immune colloidal gold mixed labeling is characterized in that the C line antibody is a bovine serum albumin antibody; the amino acid sequence of the heavy chain of the C-line antibody comprises the amino acid sequence shown as SEQ ID NO: 1; the amino acid sequence of the light chain of the C-line antibody comprises the amino acid sequence shown as SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.
2. The immune colloidal gold mixed labeling C-line antibody according to claim 1, wherein the C-line antibody is a mouse monoclonal antibody obtained by screening bovine serum albumin as an immune source and is named CAB 01; the cell strain of the mouse monoclonal antibody is preserved in China general microbiological culture collection center in 1 month and 8 days of 2020, and the preservation number is CGMCC No. 19284.
3. Use of the C-line antibody of claim 1 or 2 for immunocolloidal gold mixed labeling.
4. Use according to claim 3, characterized in that it comprises the following steps: and preparing a sample mixed solution from the C-line antibody and a sample to be marked for immune colloidal gold marking.
5. The use according to claim 4, wherein the concentration of the C-line antibody in the sample mixture is 0.8 to 1.5 μ g/mL.
6. The use according to claim 4, wherein the concentration of the sample to be labeled in the sample mixture is 4-6 μ g/mL.
7. The use according to claim 4, wherein in the step of labeling with immunocolloidal gold, the pH is 7.8 to 8.4.
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Citations (3)
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| EP0131415A2 (en) * | 1983-06-28 | 1985-01-16 | Kyowa Hakko Kogyo Kabushiki Kaisha | Monoclonal antibodies, processes for their preparation and methods for their use |
| CN108341870A (en) * | 2018-01-02 | 2018-07-31 | 北京大学 | A kind of anti-BSA nano antibodies, its production method and application |
| WO2019138898A1 (en) * | 2018-01-09 | 2019-07-18 | 東洋紡株式会社 | Immunochromatography test piece, measurement kit, and measurement method |
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2020
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| EP0131415A2 (en) * | 1983-06-28 | 1985-01-16 | Kyowa Hakko Kogyo Kabushiki Kaisha | Monoclonal antibodies, processes for their preparation and methods for their use |
| CN108341870A (en) * | 2018-01-02 | 2018-07-31 | 北京大学 | A kind of anti-BSA nano antibodies, its production method and application |
| WO2019138898A1 (en) * | 2018-01-09 | 2019-07-18 | 東洋紡株式会社 | Immunochromatography test piece, measurement kit, and measurement method |
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| 牛血清白蛋白单克隆抗体杂交瘤细胞株的建立及鉴定;余健等;《微生物学免疫学进展》;20070320(第01期);第30-34页 * |
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