CN112834736A - Pathological detection kit for detecting multiple antigens and preparation method and application thereof - Google Patents
Pathological detection kit for detecting multiple antigens and preparation method and application thereof Download PDFInfo
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- CN112834736A CN112834736A CN202011455212.0A CN202011455212A CN112834736A CN 112834736 A CN112834736 A CN 112834736A CN 202011455212 A CN202011455212 A CN 202011455212A CN 112834736 A CN112834736 A CN 112834736A
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Abstract
The invention relates to a kit for simultaneously detecting N antigens, belonging to the technical field of biological detection and the field of immunology, wherein the kit comprises: a first antibody composition consisting of N first antibodies capable of specifically binding to said N antigens; a second antibody composition consisting of N second antibodies against the N first antibodies, the N second antibodies being conjugated to N artificial polypeptides, respectively; and the third antibody or the antigen binding part thereof is respectively coupled with the polymer, and N color developing agents are coupled on the polymer coupled with the N third antibodies. Different color developing agents are utilized, the kit can be used for simultaneously detecting N antigens, and has the advantages of good specificity, high sensitivity and wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biological detection and the field of immunology, and particularly relates to a pathological detection kit for simultaneously detecting multiple antigens, and a preparation method and application thereof.
Background
Immunohistochemistry (IHC) refers to an immunological detection technique for qualitatively, quantitatively or locally measuring the corresponding antigen in pathological tissues by using specific antibodies with color reagent marks in situ in tissue cells through antigen-antibody reaction and histochemical color generation reaction. The immunohistochemical detection principle is as follows: after the tissue slice is subjected to antigen heat repairing treatment, incubating the tissue slice with a primary antibody reagent to form an antigen-antibody compound of primary antibody and target antigen in situ; the primary antibody molecule in the antigen-antibody compound is then combined with the enzyme-labeled polymer secondary antibody through incubation, and the antigen-antibody-secondary antibody polymer compound is further formed in situ; finally, a coloured deposit is formed at the antigenic site by the enzyme-catalysed substrate. And (4) observing the brown part under an optical microscope to determine whether the target antigen exists and the expression condition of the target antigen. Determining the type and morphology of pathological tissue cells, identifying the source of tissue cell products, and determining the degree of differentiation of tissue cells by immunohistochemical staining; especially, the application in clinical pathology is the most important, such as identifying pathological nature, finding micro-focus, discussing tumor origin or differentiation phenotype, determining tumor stage, and guiding treatment and prognosis; aid in disease diagnosis and classification, search for infection causes, and the like.
At present, the companies supplying the secondary antibody products of the antibody-enzyme labeling-polymer detection system in the domestic market mainly comprise: (1) the Envision secondary antibody of Dako uses chain glucan as a polymer carrier, a plurality of enzyme molecules and secondary antibody molecules are connected on the carrier, so that a detection signal is amplified, but the molecular weight of the chain glucan is higher, the whole enzyme-antibody polymer generates larger steric hindrance when being combined with tissue protein, and the detection sensitivity of the protein expressed in low abundance is not high; (2) CN105566499A discloses a pathology enhanced secondary antibody principle, which is an enzyme-labeled secondary antibody prepared by using a grape cluster polymer as a carrier, so that a plurality of enzymes are tightly connected to a ballThe preparation of one-step secondary antibody belongs to the field of multi-branch carrier molecule. (3) AmpliStain, invented by Baesweiler, SDT GmbH, GermanyTMdetection systems used serpentine linker crop backbones to prepare enzyme-vector-secondary antibody complexes. (4) The secondary antibody developed by Roche diagnostics is characterized in that naturally-occurring small molecule hapten (such as pesticide, veterinary drug, antibiotic, quantum dot and the like) is coupled with an antibody, and the naturally-occurring small molecule hapten is combined with a corresponding enzyme polymer anti-small molecule hapten antibody, so that immunohistochemical detection of tissue protein is realized; however, the method needs natural hapten micromolecules, needs more toxic and harmful chemical reagents in the cross-linking process, and has complex coupling process, so the production cost is higher.
At present, the prior art has no great improvement on the polymer structure design and preparation process, and is specifically represented by: the number of enzymes and antibodies in unit volume is not obviously increased; the steric hindrance of the polymer is not obviously reduced, so that the sensitivity is not improved, and a pathology detection enhanced secondary antibody with better effect is urgently needed.
Disclosure of Invention
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the first aspect of the present invention provides an artificial polypeptide composition comprising at least one of an artificial polypeptide PL, an artificial polypeptide HM and an artificial polypeptide LH, wherein,
the amino acid sequence of the artificial polypeptide PL comprises AAP (AADAAD) nAAPAAA, wherein n is 1-10, namely the amino acid sequence has an amino acid sequence shown in SEQ ID NO. 1-10. In some embodiments of the invention, to facilitate coupling of the protein, 1 cysteine (C) is added at the N-terminus or C-terminus of the amino acid.
The amino acid sequence of the artificial polypeptide HM comprises GQA (T) nAQ, wherein n is 1-10, namely the amino acid sequence has an amino acid sequence shown in SEQ ID NO. 11-20. In some embodiments of the invention, to facilitate coupling of the protein, 1 cysteine (C) is added at the N-terminus or C-terminus of the amino acid.
The amino acid sequence of the artificial polypeptide LH comprises TTT (PTT) n, wherein n is 1-10, namely the amino acid sequence has an amino acid sequence shown in SEQ ID NO. 21-30. In some embodiments of the invention, to facilitate coupling of the protein, 1 cysteine (C) is added at the N-terminus or C-terminus of the amino acid.
In the present invention, the artificial polypeptide is also referred to as a non-natural polypeptide, a synthetic polypeptide or an artificial polypeptide. It does not occur naturally in mammals, especially humans, and thus cross-reactivity can be avoided.
In a second aspect, the present invention provides a monoclonal antibody composition comprising at least one monoclonal antibody against the artificial polypeptide PL, the artificial polypeptide HM and the artificial polypeptide LH of the first aspect of the present invention, respectively. Wherein, the monoclonal antibody is also called monoclonal antibody.
In some embodiments of the invention, the heavy chain variable region CDR-H1, CDR-H2, CDR-H3 and the light chain variable region CDR-L1, CDR-L2 and CDR-L3 of the monoclonal antibody against the artificial polypeptide PL have the amino acid sequences shown in SEQ ID NO:31-36, respectively. The antibody is a rabbit-derived antibody and can be specifically bound with artificial polypeptide PL, and EC50 is 9.94 ng/mL.
In some embodiments of the invention, the heavy chain variable region CDR-H1, CDR-H2, CDR-H3 and the light chain variable region CDR-L1, CDR-L2 and CDR-L3 of the monoclonal antibody against the artificial polypeptide HM have amino acid sequences shown in SEQ ID NO:37-42, respectively. The antibody is a rabbit-derived antibody, can be specifically bound with artificial polypeptide HM, and has EC50 of 11.6 ng/mL.
In some embodiments of the invention, the heavy chain variable region CDR-H1, CDR-H2, CDR-H3 and the light chain variable region CDR-L1, CDR-L2 and CDR-L3 of the monoclonal antibody against the artificial polypeptide LH have the amino acid sequences shown in SEQ ID NO 43-48, respectively. The antibody is a rabbit-derived antibody, can be specifically combined with artificial polypeptide LH, and has EC50 of 1.78 ng/mL.
In a third aspect, the present invention provides an artificial polypeptide-antibody conjugate composition comprising at least one of an artificial polypeptide PL-antibody conjugate formed by the artificial polypeptide PL according to the first aspect of the present invention and an antibody, an artificial polypeptide HM-antibody conjugate formed by the artificial polypeptide HM according to the first aspect of the present invention and an antibody, and an artificial polypeptide LH-antibody conjugate formed by the artificial polypeptide LH according to the first aspect of the present invention and an antibody, wherein the antibody is a polyclonal antibody.
In some embodiments of the invention, the polyclonal antibody is a goat anti-mouse polyclonal antibody or a goat anti-rabbit polyclonal antibody.
In a fourth aspect, the present invention provides a polymer composition comprising at least one of a polymer to which an antigen-binding portion of a mab against artificial polypeptide PL according to the second aspect of the present invention is coupled, a polymer to which an antigen-binding portion of a mab against artificial polypeptide HM according to the second aspect of the present invention is coupled, and a polymer to which an antigen-binding portion of a mab against artificial polypeptide LH according to the second aspect of the present invention is coupled, wherein the polymer is further coupled with a color-developing agent. Thus, the anti-human polypeptide PL monoclonal antibody-color reagent marker-polymer, the anti-human polypeptide HM monoclonal antibody-color reagent marker-polymer or the anti-human polypeptide LH monoclonal antibody-color reagent marker-polymer is formed.
In some embodiments of the invention, the chromogenic agent is a fluorescein, an enzyme, a metal ion, a quantum dot, or an isotope. In some embodiments of the invention, the enzyme is horseradish peroxidase, alkaline phosphatase, or a beta-glucosidase.
In some embodiments of the invention, the antigen binding portion is selected from the group consisting of a Fab fragment, a Fab 'fragment, a F (ab')2 fragment, a Fv fragment, a scFv fragment, a Fd fragment, and a single domain antibody. In some embodiments of the invention, the antigen binding portion is a Fab' fragment.
The fifth aspect of the present invention provides the use of the artificial polypeptide composition of the first aspect of the present invention or the combination thereof with the monoclonal antibody composition of the second aspect of the present invention in the preparation of a kit for pathological detection.
In a sixth aspect, the invention provides a kit for use in pathological detection comprising an artificial polypeptide-antibody conjugate composition according to the third aspect of the invention and a polymer composition according to the fourth aspect of the invention.
In some embodiments of the invention, the kit further comprises a primary antibody. The polyclonal antibody in the artificial polypeptide-antibody conjugate can be combined with the monoclonal antibody.
In some embodiments of the invention, the kit further comprises a staining agent and a counterstaining agent. Further, the kit also includes sample processing reagents.
In some embodiments of the invention, the complex comprises a biological sample comprising two antigens, a first primary antibody that binds to a first antigen and a second primary antibody that binds to a second antigen, two of the artificial polypeptide-antibody conjugate compositions; in two of the polymer compositions, the antigen-binding portion of the monoclonal antibody on the polymer binds to a corresponding artificial polypeptide, and the polyclonal antibody on the artificial polypeptide-antibody conjugate binds to a first primary antibody and a second primary antibody, respectively, which bind to a first antigen and a second antigen on the biological sample, respectively.
In some embodiments of the invention, the complex comprises a biological sample comprising two antigens, a first primary antibody that binds to the first antigen and a second primary antibody that binds to the second antigen, an artificial polypeptide PL-antibody conjugate and an artificial polypeptide HM-antibody conjugate; anti-artificial polypeptide PL monoclonal antibody-color reagent label-polymer and anti-artificial polypeptide HM monoclonal antibody-color reagent label-polymer. The first antibody is a rabbit monoclonal antibody, the second antibody is a mouse monoclonal antibody, the artificial polypeptide PL-antibody conjugate is an artificial polypeptide PL-goat anti-rabbit polyclonal antibody conjugate, and the artificial polypeptide HM-antibody conjugate is an artificial polypeptide HM-goat anti-mouse polyclonal antibody conjugate. Wherein the anti-artificial polypeptide PL monoclonal antibody is combined with the artificial polypeptide PL, and the anti-artificial polypeptide HM monoclonal antibody is combined with the artificial polypeptide HM; the goat anti-rabbit polyclonal antibody is combined with the rabbit monoclonal antibody, and the goat anti-mouse polyclonal antibody is combined with the mouse monoclonal antibody; the rabbit monoclonal antibody binds to the first antigen and the mouse monoclonal antibody binds to the second antigen. Thereby forming the complex.
In other embodiments of the invention, the complex comprises a biological sample comprising two antigens, a first primary antibody that binds to the first antigen and a second primary antibody that binds to the second antigen, an artificial polypeptide PL-antibody conjugate and an artificial polypeptide LH-antibody conjugate; anti-artificial polypeptide PL monoclonal antibody-color-developing agent label-polymer and anti-artificial polypeptide LH monoclonal antibody-color-developing agent label-polymer. The first primary antibody is a rabbit monoclonal antibody, the second primary antibody is a mouse monoclonal antibody, the artificial polypeptide PL-antibody conjugate is an artificial polypeptide PL-goat anti-rabbit polyclonal antibody conjugate, and the artificial polypeptide LH-antibody conjugate is an artificial polypeptide LH-goat anti-mouse polyclonal antibody conjugate. Wherein the anti-artificial polypeptide PL monoclonal antibody is combined with the artificial polypeptide PL, and the anti-artificial polypeptide LH monoclonal antibody is combined with the artificial polypeptide LH; the goat anti-rabbit polyclonal antibody is combined with the rabbit monoclonal antibody, and the goat anti-mouse polyclonal antibody is combined with the mouse monoclonal antibody; the rabbit monoclonal antibody binds to the first antigen and the mouse monoclonal antibody binds to the second antigen. Thereby forming the complex.
In still further embodiments of the invention, the complex comprises a biological sample comprising two antigens, a first primary antibody that binds to the first antigen and a second primary antibody that binds to the second antigen, an artificial polypeptide HM-antibody conjugate and an artificial polypeptide LH-antibody conjugate; anti-human polypeptide HM monoclonal antibody-color-developing agent label-polymer and anti-human polypeptide LH monoclonal antibody-color-developing agent label-polymer. The first antibody is a rabbit monoclonal antibody, the second antibody is a mouse monoclonal antibody, the artificial polypeptide HM-antibody conjugate is an artificial polypeptide HM-goat anti-rabbit polyclonal antibody conjugate, and the artificial polypeptide LH-antibody conjugate is an artificial polypeptide LH-goat anti-mouse polyclonal antibody conjugate. Wherein the anti-artificial polypeptide HM monoclonal antibody is combined with the artificial polypeptide HM, and the anti-artificial polypeptide LH monoclonal antibody is combined with the artificial polypeptide LH; the goat anti-rabbit polyclonal antibody is combined with the rabbit monoclonal antibody, and the goat anti-mouse polyclonal antibody is combined with the mouse monoclonal antibody; the rabbit monoclonal antibody binds to the first antigen and the mouse monoclonal antibody binds to the second antigen. Thereby forming the complex.
In some embodiments of the invention, the complex comprises a biological sample comprising three antigens, a first primary antibody that binds to a first antigen, a second primary antibody that binds to a second antigen, and a third primary antibody that binds to a third antigen, an artificial polypeptide PL-antibody conjugate, an artificial polypeptide HM-antibody conjugate, and an artificial polypeptide LH-antibody conjugate; the artificial polypeptide LH-antibody conjugate comprises an artificial polypeptide LH-monoclonal antibody-color reagent marker-polymer, an artificial polypeptide HM-monoclonal antibody-color reagent marker-polymer and an artificial polypeptide LH-monoclonal antibody-color reagent marker-polymer, wherein the first antibody is a first rabbit monoclonal antibody, the second antibody is a second rabbit monoclonal antibody, the third antibody is a mouse monoclonal antibody, the artificial polypeptide PL-antibody conjugate is an artificial polypeptide PL-goat anti-rabbit polyclonal conjugate, the artificial polypeptide HM-antibody conjugate is an artificial polypeptide HM-goat anti-rabbit polyclonal conjugate, and the artificial polypeptide LH-antibody conjugate is an artificial polypeptide LH-goat anti-mouse polyclonal conjugate. Wherein the anti-artificial polypeptide PL monoclonal antibody is combined with the artificial polypeptide PL, the anti-artificial polypeptide HM monoclonal antibody is combined with the artificial polypeptide HM, and the anti-artificial polypeptide LH monoclonal antibody is combined with the artificial polypeptide LH; the goat anti-rabbit polyclonal antibody on the artificial polypeptide PL-goat anti-rabbit polyclonal antibody conjugate is combined with the first rabbit monoclonal antibody, the goat anti-rabbit polyclonal antibody on the artificial polypeptide HM-goat anti-rabbit polyclonal antibody conjugate is combined with the second rabbit monoclonal antibody, and the goat anti-mouse polyclonal antibody is combined with the mouse monoclonal antibody; the first rabbit mab binds to the first antigen, the second rabbit mab binds to the second antigen, and the murine mab binds to the third antigen. Thereby forming the complex.
Further, since the first antibody and the second antibody are both rabbit monoclonal antibodies, in order to avoid cross reaction, when preparing the complex, the first antibody and the third antibody, the artificial polypeptide PL-goat anti-rabbit conjugate, the artificial polypeptide LH-goat anti-mouse multi-antibody conjugate, the anti-human polypeptide PL monoclonal antibody-color reagent label-polymer, and the anti-human polypeptide LH monoclonal antibody-color reagent label-polymer are required to be respectively subjected to primary antibody incubation, primary antibody post-incubation, and secondary antibody incubation, and then the second antibody, the artificial polypeptide HM-goat anti-rabbit conjugate, the anti-human polypeptide HM monoclonal antibody-color reagent label are required to be subjected to primary antibody incubation, primary antibody post-incubation, and secondary antibody incubation again. The usage amount of the artificial polypeptide PL-goat anti-rabbit conjugate needs to be excessive, so that cross reaction caused by the fact that the first primary antibody is not combined with the polyclonal antibody is avoided.
Based on the above-mentioned principle,
the eighth aspect of the present invention provides a kit for simultaneously detecting N antigens, comprising: a first antibody composition consisting of N first antibodies capable of specifically binding to said N antigens; a second antibody composition consisting of N second antibodies against the N first antibodies, the N second antibodies being conjugated to N artificial polypeptides, respectively; and the third antibody or the antigen binding part thereof is respectively coupled with the polymer, and N color developing agents are coupled on the polymer coupled with the N third antibodies.
In some embodiments of the invention, the chromogenic agent is a fluorescein, an enzyme, a metal ion, a quantum dot, or an isotope. In some embodiments of the invention, the enzyme is horseradish peroxidase, alkaline phosphatase, or a beta-glucosidase.
In some embodiments of the invention, the N is 2 to 5.
In some embodiments of the invention, the antigen binding portion is selected from the group consisting of a Fab fragment, a Fab 'fragment, a F (ab')2 fragment, a Fv fragment, a scFv fragment, a Fd fragment, and a single domain antibody. In some embodiments of the invention, the antigen binding portion is a Fab' fragment.
In some embodiments of the invention, the first and third antibodies are monoclonal antibodies, preferably rabbit or mouse monoclonal antibodies. The second antibody is a polyclonal antibody, preferably a goat anti-rabbit polyclonal antibody or a goat anti-mouse polyclonal antibody.
The ninth aspect of the present invention provides a method for simultaneously detecting N antigens, comprising the steps of:
s1, binding N first antibodies capable of specifically binding to the N antigens;
s2, combining N secondary antibodies against the N primary antibodies with the N primary antibodies, wherein the N secondary antibodies are respectively conjugated with N artificial polypeptides;
s3, combining N third antibodies or antigen binding parts thereof resisting the N artificial polypeptides with the N third antibody combination or antigen binding part combination, wherein the third antibodies or the antigen binding parts thereof are respectively conjugated with the polymers, and N color developing agents are conjugated on the polymers conjugated with the N third antibodies,
the N color developing agents are different from each other, so that the simultaneous detection of the N antigens is completed.
In some embodiments of the invention, the N is 2 to 5.
In some embodiments of the invention, the chromogenic agent is a fluorescein, an enzyme, a metal ion, a quantum dot, or an isotope. In some embodiments of the invention, the enzyme is horseradish peroxidase, alkaline phosphatase, or a beta-glucosidase.
In some embodiments of the invention, the antigen binding portion is selected from the group consisting of a Fab fragment, a Fab 'fragment, a F (ab')2 fragment, a Fv fragment, a scFv fragment, a Fd fragment, and a single domain antibody. In some embodiments of the invention, the antigen binding portion is a Fab' fragment.
In some embodiments of the invention, the first and third antibodies are monoclonal antibodies, preferably rabbit or mouse monoclonal antibodies. The second antibody is a polyclonal antibody, preferably a goat anti-rabbit polyclonal antibody or a goat anti-mouse polyclonal antibody.
The invention has the advantages of
Compared with the prior art, the invention has the following beneficial effects:
the artificial polypeptide sequence of the invention is completely designed artificially, is a sequence which does not exist in the body of a mammal, is produced by a chemical synthesis method, and has good stability and low cost.
The antibody produced by using the artificial polypeptide immunity of the invention can not generate non-specific reaction signals with samples of mammal origin, such as tissues, but only can generate signals by reacting with the artificial polypeptide, so that the antibody can be used for a second antibody detection system to avoid generating non-specific signals, and the specificity is better.
The pathological enhancement type secondary antibody detection method is established based on the specific combination of the antigen, the primary antibody and the polymer secondary antibody, has good stability and repeatability, is particularly suitable for detecting the protein with low expression abundance in immunohistochemistry, and effectively improves the sensitivity and specificity of detection.
Different artificial polypeptides are used for labeling different monoclonal antibodies-enzyme labeling-polymers and then matched with different substrate color developing solutions, and different artificial polypeptides and specific antibodies thereof are combined for use, so that double dyeing or multiple dyeing can be realized, a plurality of index detection results can be provided in the same experiment, and the judgment is more accurate.
The invention can also be applied to detection in aspects of ELISA, Westernblot, immunocytochemistry and the like, and has good specificity, high sensitivity and wide application prospect.
The artificial polypeptide and the antibody thereof prepared by the invention can realize signal amplification in pathological diagnosis, more toxic and harmful chemical reagents are avoided in the preparation process, the coupling process is simple, and the mass production is easy.
Drawings
Figure 1 shows a schematic for detection using an artificial polypeptide-enhanced secondary antibody. A: single antigen detection, B: both antigens were detected simultaneously.
Fig. 2 shows the binding curves of the artificial polypeptide PL and its antibody ELISA.
FIG. 3 shows the binding curves of the artificial polypeptide HM and its antibody ELISA.
Fig. 4 shows the binding curve of the artificial polypeptide LH and its antibody ELISA.
FIG. 5 shows the results of goat anti-mouse, goat anti-rabbit multiple antiserum titer ELISA.
FIG. 6 shows a graph of the results of identifying purified goat anti-mouse, goat anti-rabbit by reduced SDS-PAGE. The protein loading was 10. mu.g, and lane 1 was goat anti-rabbit IgG. Lane 2 is goat anti-mouse Ig.
FIG. 7 shows the result of Western blot identification of mouse immunoglobulin subtype recognized by goat anti-mouse polyclonal antibody.
Figure 8 shows that goat anti-rabbit polyclonal antibodies recognize rabbit IgG subclasses.
FIG. 9 shows the binding curves of the artificial polypeptide PL-goat anti-mouse antibody conjugate and anti-PL mab ELISA.
FIG. 10 shows the binding curves of the artificial polypeptide PL-goat anti-rabbit antibody conjugate and anti-PL mab ELISA.
FIG. 11 shows the binding curves of the artificial polypeptide HM-goat anti-mouse antibody conjugate and anti-HM monoclonal antibody ELISA.
FIG. 12 shows the binding curves of the artificial polypeptide HM-goat anti-rabbit antibody conjugate and anti-HM monoclonal antibody ELISA.
FIG. 13 shows the binding curve of artificial polypeptide LH-goat anti-mouse antibody conjugate to anti-LH monoclonal antibody ELISA.
FIG. 14 shows the binding curve of artificial polypeptide LH-goat anti-rabbit antibody conjugate to anti-LH monoclonal antibody ELISA.
Fig. 15 shows the results of the identification of anti-human polypeptide PL antibody-enzyme label-polymer.
FIG. 16 shows the results of the identification of anti-human polypeptide HM antibody-enzyme-labeled-polymer.
Fig. 17 shows the results of the identification of anti-human polypeptide LH antibody-enzyme-labeled-polymer.
Fig. 18 shows the cervical cell smear double staining results. A: p16/Ki-67 double-staining positive result; b: p16/Ki-67 double staining negative results.
FIG. 19 is a graph showing the identification of p63/CK5 double staining of prostate tissue with HM, LH artificial polypeptide and its enzyme-labeled polymer antibody.
Fig. 20 shows triple staining of kidney tissue using PL, LH, HM artificial polypeptides and their fluorescently labeled polymers of antibodies.
Figure 21 shows the use of pathologically enhanced secondary antibodies composed of artificial polypeptides and their antibodies in immunocytochemistry. A: detecting Raji cells by using the artificial polypeptide HM and the antibody thereof; b: detecting Daudi cells by using the artificial polypeptide PL and an antibody thereof; c: detecting Hela cells by using the artificial polypeptide LH and the antibody thereof.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.
Examples
The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
The experimental procedures in the following examples are conventional unless otherwise specified. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
Example 1 preparation of enhanced Secondary antibody System Using Artificial Polypeptides
The embodiment provides a method for preparing an enhanced secondary antibody by using an artificial polypeptide and application thereof in pathological detection.
Firstly, preparing artificial polypeptide, wherein the artificial polypeptide sequence is completely designed and synthesized by human, and the design principle is as follows: sequences that do not naturally occur in mammals and which produce antibodies that do not specifically cross-react with proteins on mammals, particularly human tissues.
Secondly, preparing a polyclonal antibody aiming at the primary antibody, and coupling the polyclonal antibody with the artificial polypeptide to form an artificial polypeptide-antibody conjugate, namely a primary antibody post-reagent. A polyclonal antibody can be coupled with a plurality of artificial polypeptides, thereby playing a role in amplifying a detection signal.
Then, preparing the anti-human polypeptide monoclonal antibody or antigen binding part thereof, and coupling with the polymer and the color-developing agent to form the anti-human polypeptide antibody-color-developing agent labeled-polymer, wherein the polymer can be combined with a plurality of color-developing agent molecules, thereby playing the role of amplifying the detection signal again. Meanwhile, the polymer can be combined with a plurality of anti-human polypeptide antibodies or antigen binding parts thereof, so that the sensitivity can be provided.
In the using process, as shown in fig. 1A, first, primary antibody is used for combining with antigen, then the artificial polypeptide-antibody conjugate is combined with the primary antibody, then the artificial polypeptide is identified by the anti-artificial polypeptide antibody-color reagent label-polymer, and the detection is completed through color reaction. The detection sensitivity can be improved by multiple amplification of the artificial polypeptide and the polymer, and the method is particularly suitable for detection of low-abundance antigen to be detected.
Aiming at the detection of two or more antigens, different artificial polypeptide-antibody conjugates can be prepared by using different polypeptides, and corresponding anti-artificial polypeptide antibody-color reagent labeled-polymer can be prepared, and the simultaneous detection of two or even more antigens can be realized by changing the type of the color reagent.
For example, as shown in fig. 1B, two different antigens are targeted: the first antigen and the second antigen can adopt two different obvious agents to complete double staining, thereby achieving the purpose of simultaneous detection. Specifically, the method comprises the following steps:
primary antibody incubation: first, a first primary antibody capable of specifically binding to a first antigen and a second primary antibody capable of specifically binding to a second antigen are dripped onto a tissue sample, separately or simultaneously.
Primary antibody post-incubation: separately or simultaneously adding a first artificial polypeptide-first multi-antibody conjugate (first post-primary-antibody reagent) and a second artificial polypeptide-second multi-antibody conjugate (second post-primary-antibody reagent).
And (3) secondary antibody incubation: an anti-first artificial polypeptide antibody-first chromogenic tag-polymer (a first secondary antibody) and an anti-second artificial polypeptide antibody-second chromogenic tag-polymer (a second secondary antibody) are added separately or simultaneously.
Because the colors of the first color developing agent and the second color developing agent or other detection display results are different, the simultaneous detection of the two antigens can be completed.
The first primary antibody and the second primary antibody are monoclonal antibodies, and the first primary antibody can only be combined with the first antigen and can not be combined with the second antigen; likewise, the secondary antibody is only able to bind to the secondary antigen and not to the primary antigen.
The first multi-antibody can only be combined with the first primary antibody and can not be combined with the second primary antibody; similarly, the second polyclonal antibody can only bind to the second primary antibody and not to the first primary antibody. Of course, the first polyclonal antibody and the second polyclonal antibody can be the same, but cannot be added simultaneously, namely, excessive first polyclonal antibody is added after the excessive first polyclonal antibody is removed, the excessive first polyclonal antibody is added, the second polyclonal antibody is added after the excessive first polyclonal antibody is removed, and then the second polyclonal antibody is added, so that the same purpose can be achieved.
The anti-first artificial polypeptide antibody binds only to the first artificial polypeptide and does not bind to the second artificial polypeptide; similarly, an antibody directed against the second artificial polypeptide will only bind to the second artificial polypeptide and will not bind to the first artificial polypeptide.
By using the same principle and method, a primary anti-post reagent and a secondary anti-post reagent for simultaneously detecting three or more antigens can be prepared, so that the simultaneous detection of three or more antigens is completed.
Example 2 Artificial Polypeptides
The term "artificial polypeptide" as used in this embodiment refers to a polypeptide that does not naturally occur in a mammalian body, and particularly does not occur in human tissue.
An artificial polypeptide PL: the amino acid sequence is TTT (PTT) n, and n is 1-10, namely:
TTTPTT(SEQ ID NO:1)，
TTTPTTPTT(SEQ ID NO:2)，
TTTPTTPTTPTT(SEQ ID NO:3)，
TTTPTTPTTPTTPTT(SEQ ID NO:4)，
TTTPTTPTTPTTPTTPTT(SEQ ID NO:5)，
TTTPTTPTTPTTPTTPTTPTT(SEQ ID NO:6)，
TTTPTTPTTPTTPTTPTTPTTPTT(SEQ ID NO:7)，
TTTPTTPTTPTTPTTPTTPTTPTTPTT(SEQ ID NO:8)，
TTTPTTPTTPTTPTTPTTPTTPTTPTTPTT (SEQ ID NO:9), or
TTTPTTPTTPTTPTTPTTPTTPTTPTTPTTPTT(SEQ ID NO:10)，
To facilitate coupling of the protein, 1 cysteine (C) may be added at the N-terminus or C-terminus of the amino acid.
Artificial polypeptide HM: the amino acid sequence is AAP (AADAAD) nAAPAAA, and n is 1-10, namely:
AAPAADAADAAPAAA(SEQ ID NO:11)，
AAPAADAADAADAADAAPAAA(SEQ ID NO:12)，
AAPAADAADAADAADAADAADAAPAAA(SEQ ID NO:13)，
AAPAADAADAADAADAADAADAADAADAAPAAA(SEQ ID NO:14)，
AAPAADAADAADAADAADAADAADAADAADAADAAPAAA(SEQ ID NO:15)，
AAPAADAADAADAADAADAADAADAADAADAADAADAADAAPAAA(SEQ ID NO:16)，
AAPAADAADAADAADAADAADAADAADAADAADAADAADAADAADAAPAAA(SEQ ID NO:17)，
AAPAADAADAADAADAADAADAADAADAADAADAADAADAADAADAADAADAAPAAA(SEQ ID NO:18)，
AAPAADAADAADAADAADAADAADAADAADAADAADAADAADAADAADAADAADAADAAPAAA (SEQ ID NO:19), or
AAPAADAADAADAADAADAADAADAADAADAADAADAADAADAADAADAADAADAADAADAADAAPAAA(SEQ ID NO:20)。
For ease of coupling to the protein, 1 cysteine (C) may also be added at the N-terminus or C-terminus of the amino acid.
An artificial polypeptide LH: the amino acid sequence is GQA (T) nAQ, and n is 1-10, namely:
GQATAQQ(SEQ ID NO:21)，
GQATTAQQ(SEQ ID NO:22)，
GQATTTAQQ(SEQ ID NO:23)，
GQATTTTAQQ(SEQ ID NO:24)，
GQATTTTTAQQ(SEQ ID NO:25)，
GQATTTTTTAQQ(SEQ ID NO:26)，
GQATTTTTTTAQQ(SEQ ID NO:27)，
GQATTTTTTTTAQQ(SEQ ID NO:28)，
GQATTTTTTTTTAQQ (SEQ ID NO:29), or
GQATTTTTTTTTTAQQ(SEQ ID NO:30)。
For ease of coupling to the protein, 1 cysteine (C) may also be added at the N-terminus or C-terminus of the amino acid.
EXAMPLE 3 preparation of anti-human polypeptide antibodies
Rabbit-derived monoclonal antibodies were further prepared by immunizing rabbits with the artificial polypeptide PL (TTTPTTPTT) of example 2. The antibody can specifically bind to the artificial polypeptide PL, the ELISA binding curve is shown in figure 2, and the EC50 is 9.94 ng/mL.
The anti-human polypeptide PL antibody is analyzed by using Kabat online software, and the result shows that the variable region sequence of the antibody has the following information:
(1) the CDR-H1 amino acid sequence includes: TNAMS (SEQ ID NO: 31).
(2) The CDR-H2 amino acid sequence includes: IISSSGSTYYARWAKG (SEQ ID NO: 32).
(3) The CDR-H3 amino acid sequence includes; GNI (SEQ ID NO: 33).
(4) The CDR-L1 amino acid sequence includes: QSSQSVYNNNLA (SEQ ID NO: 34).
(5) The CDR-L2 amino acid sequence includes: RASKLAS (SEQ ID NO: 35).
(6) The CDR-L3 amino acid sequence includes: LGGYDCSSADCGA (SEQ ID NO: 36).
Using the same method, rabbit-derived monoclonal antibodies were further prepared by immunizing rabbits with the artificial polypeptide HM (AAPAADAAADAAPAAA) of example 2. The antibody can specifically bind to the artificial polypeptide HM, the ELISA binding curve is shown in figure 3, and the EC50 is 11.6 ng/mL.
The anti-human polypeptide HM antibody is analyzed by using Kabat online software, and the result shows that the variable region sequence of the antibody has the following information:
(1) the CDR-H1 amino acid sequence includes: SYAMG (SEQ ID NO: 37).
(2) The CDR-H2 amino acid sequence includes: IATTGSSTYHASWAKG (SEQ ID NO: 38).
(3) The CDR-H3 amino acid sequence includes; DGDWTGWYFSI (SEQ ID NO: 39).
(4) The CDR-L1 amino acid sequence includes: QASQSISNRLA (SEQ ID NO: 40).
(5) The CDR-L2 amino acid sequence includes: DASDLAS (SEQ ID NO: 41).
(6) The CDR-L3 amino acid sequence includes: QQGYGGDNIENL (SEQ ID NO: 42).
Using the same method, rabbit-derived monoclonal antibody was further prepared by immunizing rabbit with the artificial polypeptide LH (GQATAQ) of example 2. The antibody can specifically bind to the artificial polypeptide LH, the ELISA binding curve is shown in figure 4, and the EC50 is 1.78 ng/mL.
The anti-human polypeptide LH antibody is analyzed by using Kabat online software, and the result shows that the variable region sequence of the antibody has the following information:
(1) the CDR-H1 amino acid sequence includes: RYAMC (SEQ ID NO: 43).
(2) The CDR-H2 amino acid sequence includes: IIGVSGTTYYTSWAKG (SEQ ID NO: 44).
(3) The CDR-H3 amino acid sequence includes; VMPGYDDYGDDGFDP (SEQ ID NO: 45).
(4) The CDR-L1 amino acid sequence includes: QASEDIYSNLA (SEQ ID NO: 46).
(5) The CDR-L2 amino acid sequence includes: AASYLAS (SEQ ID NO: 47).
(6) The CDR-L3 amino acid sequence includes: QCTYYSGSYELFT (SEQ ID NO: 48).
It is worth noting that although the monoclonal antibody is generated for one of the artificial polypeptide sequences, the generated epitopes are concentrated on several identical amino acids, all amino acids having the same epitope can be recognized, and the affinities are not significantly different.
It can be known that the artificial polypeptide antibody prepared in the embodiment is combined with the corresponding artificial polypeptide amino acid sequence with high sensitivity and high specificity, and can be applied to various pathological detections, such as pathological detection by using ELISA, Western blot, immunocytochemistry, and the like.
Example 4 purification and characterization of goat anti-mouse Ig and goat anti-rabbit IgG
The goat is immunized by using a mouse immunoglobulin (Ig) compound and rabbit IgG respectively to obtain goat anti-mouse or goat anti-rabbit polyclonal antiserum. Serum titers of goat anti-mouse IgG (EC50) were 1: 1950000, serum titers of goat anti-rabbit IgG (EC50) are 1: 3550000. the results of the ELISA curve are shown in FIG. 5.
The Protein A purification is carried out on 2 polyclonal antibodies respectively, and the purification steps are as follows:
(1) adding equal volume of 2 × Binding Buffer into goat anti-mouse or goat anti-rabbit polyclonal antiserum to be purified, and mixing uniformly for later use.
(2) A new Protein A column is vertically fixed, and a Binding Buffer with the same volume is added according to the volume of the required column. Resuspending Protein A filler, sucking the filler suspension with the required column volume into the column, covering a gasket, adding 30CV deionized water to wash the host, adding 30CV Binding Buffer to balance the column, and adjusting the flow rate of the free flow column to be 1 mL/min.
(3) The Protein A column was taken out from 4 ℃ and fixed on a free-flowing plastic rack, and the column was returned to room temperature and used.
(4) When the alcohol in the column descends to the surface of the upper gasket, 30CV of deionized water is added to wash the column, 30CV of Binding Buffer balance column is added, and the flow rate of the gravity flow column is adjusted to be 1 mL/min. Multiple antiserum samples were loaded at room temperature at a loading rate of 1mL/min and flow-through samples were collected.
(5) The flow-through solution is concentrated by ultrafiltration and replaced by PBS buffer, and the flow-through solution is placed at 4 ℃ for standby after the concentration is detected.
The purified goat anti-mouse and goat anti-rabbit polyclonal antibodies are respectively identified by reduced SDS-PAGE, the identification result is shown in figure 6, and from figure 6, the reduced SDS-PAGE heavy chain used by the goat anti-mouse Ig polyclonal antibody and the goat anti-rabbit IgG polyclonal antibody is 50kD, the light chain is about 25kD, and the target bands are correct.
Purified goat anti-mouse polyclonal antibodies were run on reduced SDS-PAGE using 5 subtypes of monoclonal antibodies IgG1, IgG2a, IgG2b, IgG3, and IgM, respectively, and then transferred to membranes for Western blot detection. The primary antibody is a goat anti-mouse purified polyclonal antibody, the secondary antibody is donkey anti-goat marked HRP, a gel imager is used for photographing after color development is carried out by using a color development liquid, the identification result is shown in figure 7, and from figure 7, the goat anti-mouse polyclonal antibody recognizable mouse immunoglobulin is IgG1, IgG2a, IgG2b, IgG3 and IgM respectively, and 5 subtypes in total.
The goat anti-rabbit purified polyclonal antibody is subjected to enzyme labeling plate coating by using immunogen (purified rabbit IgG), after 5% skimmed milk powder is sealed, the goat anti-rabbit purified polyclonal antibody is diluted by 4 times from 1 mu g/mL, 7 gradients are obtained in total, the goat anti-rabbit purified polyclonal antibody is used as a primary antibody, the HRP-labeled donkey anti-goat IgG is used as a secondary antibody, and after TMB is subjected to color development reaction, 1M H2The plates were read after the termination reaction with SO 4. The ELISA experimental results are shown in FIG. 8, and it can be seen from FIG. 8 that goat anti-rabbit IgG can recognize rabbit IgG subclass, and the ELISA four-parameter fitting curve EC50 is 9.63 ng/mL.
Example 5 Artificial polypeptide-labeled antibody or antibody fragment
The artificial polypeptide PL, HM or LH is used as a conjugate of a polyclonal antibody and is coupled with goat anti-mouse Ig and goat anti-rabbit IgG, wherein the goat anti-mouse Ig comprises a mixture of immunoglobulin subclasses such as IgG1, IgG2a, IgG2b, IgG3, IgM and the like. Polyclonal antibodies include the full length of an antibody or antigen-binding portion thereof, wherein the antigen-binding portion includes F (ab ')2, Fab', ScFv, and the like.
Coupling of the artificial polypeptide PL to goat anti-rabbit IgG is taken as an example:
(1) after goat anti-rabbit IgG is primarily purified by protein A, the goat anti-rabbit IgG is respectively subjected to Mouse IgG, Rat IgG and Human IgG affinity chromatography columns, eluent is collected, and the eluent is dissolved in 5mM EDTA with a certain volume after the concentration is detected by OD 280.
(2) Sulf-SPDP is dissolved in a certain amount of DMSO, and then 1 XPBS solution is added to mix evenly.
(3) Slowly adding the Sulf-SPDP solution into the goat anti-rabbit IgG solution, stirring while adding, uniformly mixing, and standing at room temperature for 1h for activation.
(4) After 1h, the activated goat anti-rabbit IgG solution was filled into a cooled dialysis bag, placed into 2L of pre-cooled 1 XPBS solution, and dialyzed on a magnetic stirrer at 4 ℃ for 1 h.
(5) After 1h, 2L of fresh 1 XPBS dialysate is replaced, and dialysis is carried out for 2h at 4 ℃; after 2h, 2L of fresh 1 XPBS dialysate was replaced and dialyzed at 4 ℃ for 2 h.
(6) Dissolving artificial polypeptide PL with the same mass as the goat anti-rabbit IgG in a proper amount of DMSO, adding 1 XPBS, quickly mixing, immediately adding 1mL of activated and dialyzed goat anti-rabbit IgG solution, mixing, and performing cross-linking reaction at 4 ℃ overnight.
(7) The next day, dialyzing at 4 deg.C for 6 hr, and changing the solution 2-3 times. After the end, the vials were aliquoted and stored at-20 ℃.
EXAMPLE 6 identification of conjugates
And (3) identifying the conjugate by adopting an ELISA method:
the enzyme-linked plate was coated with the artificial polypeptide PL/artificial polypeptide HM/artificial polypeptide LH-goat anti-mouse or goat anti-rabbit antibody conjugate prepared in example 5 at 1. mu.g/mL, 50. mu.L/well and 4 ℃ overnight. The next day, after washing the plates, they were blocked with 5% skimmed milk powder at 100. mu.L/well for 1h at 30 ℃. Rabbit monoclonal antibody resisting the artificial polypeptide PL with different concentration gradients as a primary antibody, 50 mu L/well, and incubation for 1h at 30 ℃. Goat anti-rabbit IgG-labeled HRP was used as a secondary antibody, 50. mu.L/well, and incubated at 30 ℃ for 40 min. After color development with TMB developing solution, the reaction was terminated with 1M sulfuric acid, and the OD450 value was read at the microplate reader.
The detection result of the artificial polypeptide PL-goat anti-mouse or goat anti-rabbit antibody conjugate indirect ELISA is shown in figure 9 and figure 10. The results in FIG. 9 show that: the artificial polypeptide PL-goat anti-mouse antibody conjugate and the anti-human polypeptide PL monoclonal antibody have an immune color reaction, the ELISA binding curve EC50 of the goat anti-mouse and the anti-human polypeptide PL monoclonal antibody conjugated with the artificial polypeptide PL is 12ng/mL, the ELISA binding curve EC50 of the goat anti-mouse and the anti-human polypeptide PL monoclonal antibody before conjugation is 9.94ng/mL, the EC50 after conjugation is 83% of the EC50 before conjugation, and the loss of binding activity is less. The results also indicate that the artificial polypeptide PL is successfully coupled to the goat anti-mouse antibody. The results in FIG. 10 show that: the artificial polypeptide PL-goat anti-rabbit antibody conjugate and the anti-human polypeptide PL monoclonal antibody have an immune color development reaction, the ELISA binding curve EC50 of the goat anti-mouse conjugated with the artificial polypeptide PL and the anti-human polypeptide PL monoclonal antibody is 11.5ng/mL, the ELISA binding curve EC50 of the goat anti-mouse conjugated with the artificial polypeptide PL and the anti-human polypeptide PL monoclonal antibody before conjugation is 9.94ng/mL, the EC50 after conjugation is 86% of the EC50 before conjugation, and the loss of binding activity is less. The result also shows that the artificial polypeptide PL is successfully coupled on the goat anti-rabbit antibody. The obtained artificial polypeptide PL-goat anti-mouse antibody conjugate and the artificial polypeptide PL-goat anti-rabbit antibody conjugate can be prepared into a primary anti-post reagent.
The detection result of the artificial polypeptide HM-goat anti-mouse or goat anti-rabbit antibody conjugate indirect ELISA is shown in the figures 11 and 12. The results in FIG. 11 show that: the artificial polypeptide HM-goat anti-mouse antibody conjugate has an immune color development reaction with the anti-human polypeptide HM monoclonal antibody, an ELISA binding curve EC50 of the goat anti-mouse coupled with the artificial polypeptide HM and the anti-human polypeptide HM monoclonal antibody is 13.8ng/mL, an ELISA binding curve EC50 of the goat anti-mouse coupled with the artificial polypeptide HM and the anti-human polypeptide HM monoclonal antibody before coupling is 11.6ng/mL, EC50 after coupling is 84% of EC50 before coupling, and the loss of binding activity is less. The result also shows that the artificial polypeptide HM is successfully coupled on the goat anti-mouse antibody. The results in FIG. 12 show that: the artificial polypeptide HM-goat anti-rabbit antibody conjugate has an immunocolouring reaction, the EC50 is 12.6ng/mL, the ELISA binding curve EC50 before conjugation is 11.6ng/mL, the EC50 after conjugation is 92% of the EC50 before conjugation, and the loss of binding activity is less. The result also shows that the artificial polypeptide HM is successfully coupled on the goat anti-rabbit antibody. The obtained artificial polypeptide HM-goat anti-mouse antibody conjugate and the artificial polypeptide HM-goat anti-rabbit antibody conjugate can be prepared into a primary antibody post-reagent.
The detection result of the artificial polypeptide LH-goat anti-mouse or goat anti-rabbit antibody conjugate indirect ELISA is shown in the figure 13 and figure 14. The results in FIG. 13 show that: the artificial polypeptide LH-goat anti-mouse antibody conjugate has an immune color development reaction with the anti-human polypeptide LH monoclonal antibody, the ELISA binding curve EC50 of the goat anti-mouse conjugated with the artificial polypeptide LH and the anti-human polypeptide LH monoclonal antibody is 4.81ng/mL, the ELISA binding curve EC50 of the goat anti-mouse conjugated with the artificial polypeptide LH monoclonal antibody before conjugation is 1.78ng/mL, and the conjugated antigen antibody is still sensitive to binding EC 50. The result also shows that the artificial polypeptide LH is successfully coupled on the goat anti-rabbit antibody. The results in FIG. 14 show that: the artificial polypeptide LH-goat anti-mouse or goat anti-rabbit antibody conjugate has an immune color reaction, the EC50 is 3.23ng/mL, the ELISA binding curve EC50 before conjugation is 1.78ng/mL, although the EC50 after conjugation is 55% of the EC50 before conjugation, the antigen-antibody binding EC50 after conjugation is also sensitive. The result also shows that the artificial polypeptide LH is successfully coupled on the goat anti-rabbit antibody. The obtained artificial polypeptide LH-goat anti-mouse antibody conjugate and the artificial polypeptide LH-goat anti-rabbit antibody conjugate can be prepared into a primary anti-post reagent.
Example 7 preparation of anti-human polypeptide antibody-developer-labeled-Polymer
The color developing agent of the present embodiment is exemplified by horseradish peroxidase (HRP).
(1) Activating 50mg of horseradish peroxidase (dissolved in 0.01M PBS (pH7.4) containing 5mM EDTA, at a concentration of 25mg/mL) with Sulf-SMCC at room temperature for 30min, and ultrafiltering and concentrating at 10kD, wherein the molar ratio of the two is 20-40: 1.
(2) S-acetylmercaptosuccinic anhydride is used to activate polymer carrier (molecular weight is 7W-12W) (the polymer carrier is dissolved in 0.01M PBS solution with pH6.5. the reaction mixture is ultrafiltered and concentrated by 50kD ultrafiltering tube, the buffer solution is replaced to 0.01M PBS solution with pH7.4. the collected solution is ultrafiltered and concentrated, and is stored at 4 ℃ for standby.
(3) Mixing the products obtained in the step (1) and the step (2), and reacting for 16-20h at 4 ℃. 1/10 volumes of 0.1M mercaptoethanol were added to the reaction mixture, and the reaction was carried out at 30 ℃ for 20 min. This step produces a polymeric carrier-enzyme complex.
(4) The reaction product in step (3) was purified using a Sephadex G-200 column, and the solution at a high molecular weight at 403nm was collected and concentrated to about 3mL by ultrafiltration. This step produces a polymeric carrier-enzyme complex.
(5) Using pepsin to cut the anti-artificial polypeptide PL antibody/anti-artificial polypeptide HM antibody/anti-artificial polypeptide LH antibody, wherein the cutting buffer solution is citric acid buffer solution with the pH value of 3.2. The enzyme is cut at 37 ℃ for 2-3h to prepare F (ab') 2. Adding a proper amount of 0.1M cysteamine hydrochloride into the prepared F (ab ')2, reacting for 1.5h at the temperature of 37 ℃, wherein the molar mass ratio of the cysteamine hydrochloride to the antibody F (ab ')2 is 100:1-150:1, and preparing the Fab ' with sulfhydryl.
(6) Taking a proper amount of polymer carrier-enzyme complex prepared by activation of Sulf-SMCC, and reacting for 30min at room temperature. Ultrafiltering and concentrating with 50kD ultrafiltering tube, and replacing buffer solution with 0.01M PBS (pH7.4PBS); fractions at 403nm were collected.
(7) And (3) mixing the compound obtained in the step (6) with the anti-human polypeptide PL monoclonal antibody/anti-human polypeptide HM antibody/anti-human polypeptide LH antibody Fab', and reacting for 16-20h at 4 ℃. After the reaction is finished, adding a proper volume of 0.1M mercaptoethanol, and reacting for 20min at 30 ℃; the synthesized reaction mixture was then purified on Sephadex G-200 molecular sieves.
The absorbance was measured at 280nm and 403nm, respectively, and the results are shown in FIG. 15, FIG. 16 and FIG. 17.
Fig. 15 to 17 show the results of identification of anti-human polypeptide PL antibody-enzyme-labeled polymer, anti-human polypeptide HM antibody-enzyme-labeled polymer, and anti-human polypeptide LH antibody-enzyme-labeled polymer, respectively, in which the high molecular weight filtrate component having two peaks is the corresponding polymer, and a secondary antibody can be prepared.
Example 8 identification of tissue or cell types by Dual staining amplification signals
The detection method comprises the following steps:
(1) taking cervical cell slices (for simultaneously detecting p16 and Ki-67), placing in 95% ethanol, soaking and fixing for 30min, and air drying for later use. Paraffin-embedded prostate hyperplasia tissue sections (for simultaneous detection of p63 and CK5) were deparaffinized and hydrated.
(2) Using Tris-EDTA, pH9.0 antigen repairing buffer solution, boiling the tissue antigen at high temperature and normal pressure for 15-20min to repair. After 15-20min, the fire was turned off and the tissue sections were cooled to room temperature all the time in antigen retrieval buffer.
(3) The slices cooled to room temperature were rinsed 3 times with tap water for 10-30 seconds each.
(4) Use of 3% H2O2Endogenous peroxidase on the tissue sections was blocked and incubated for 10min at room temperature.
(5) Taking out the dyeing rack, and pouring out H in the plastic dyeing vat2O2Adding tap water, placing into a staining rack, rinsing, slicing, and rinsing for 3 times, 10 times each time.
(6) And (3) circling: wiping the water around the tissue on the section with dust-free paper, drawing a circle around the tissue by an immunohistochemical pen, wherein the distance between the circle and the tissue is 2-3mm, and soaking the circled sheet in PBST.
(7) Primary antibody incubation: the sections were arranged in a wet box and the first and second primary antibody reagents, 3 drops/sheet, were added drop wise to the sections (depending on the section size, it is appropriate to cover the section tissue completely). After incubation at room temperature for 30min, sections were collected and rinsed 3 times in PBST, 10 washes each time.
(8) Primary antibody post-incubation: the sections were arranged in a wet box, wherein 2 drops/sheet of the first and second primary anti-post reagents (depending on the size of the section, it is preferable to completely cover the tissue of the section) were dropped onto the sections of the experimental group, and the control primary anti-post reagent (ImmunoHistoProbe, Cat #1981-07, Advanced-Biosystems) was dropped onto the other group of sections, incubated at room temperature for 15-20min, then the sections were collected and rinsed in PBST.
(9) And (3) secondary antibody incubation: the sections are arranged in a wet box, 2 drops/piece of first secondary antibody reagent and second secondary antibody reagent are dripped on the section of the experimental group (the section size is preferably completely covered on the section tissue), and the section of the control group is dripped with the enzyme-labeled polymer secondary antibody. After incubation for 15-20min at room temperature, sections were collected and rinsed in PBST.
(10) DAB color development: and preparing fresh DAB color developing solution according to the required dosage. The sections were arranged in a wet box, 100. mu.L/piece of DAB staining solution (preferably, the sections were completely covered with the tissue) was dropped on the sections, and the sections were stained for 1 to 5min (microscopic staining), and the staining was stopped by washing with tap water. The sections were collected and rinsed in tap water.
(11) AP color development: and preparing a fresh AP color developing solution according to the required dosage. The sections were arranged in a wet box, 100. mu.L/piece of AP color developing solution (preferably, the cells of the sections were completely covered depending on the size of the sections) was dropped onto the sections, and color development was carried out for 5 to 10 minutes (microscopic staining control), and the sections were rinsed with tap water to terminate the color development. The sections were collected and rinsed in tap water.
(12) Hematoxylin counterstaining: the sections were placed in hematoxylin stain for 4 min. Excess dye was removed by rinsing with tap water and then returned to blue in PBST solution for about 1 min.
(13) Gradient alcohol dehydration, transparency and sealing.
(14) The staining results were observed under a microscope.
Using the above method, the primary antibody post-reagent prepared in example 6 and the secondary antibody reagent prepared in example 7 were used, respectively, in combination with a specific primary antibody, to perform double staining on paraffin-embedded human tissue sections of different types, respectively, the specific protocol being shown in Table 1:
TABLE 1 Dual staining amplification Signal identification of tissue or cell types
The result of double staining of cervical cell smear is shown in FIG. 18. In the figure, the cervical cytoplasm/nucleus stained by p16 is red stained, the cervical nucleus stained by Ki-67 is brown stained, and the cervical nucleus stained by the Ki-67 is positive, and the cervical cytoplasm/nucleus stained by the p16 or the Ki-67 or no staining is negative. The results of double staining of prostate tissue are shown in FIG. 19. In the figure p63 stains the prostate epithelial cell nucleus, brown staining, and CK5 stains the prostate epithelial cytoplasm, red staining.
The results show that the detection system can be applied to double staining of pathological tissues and provide reference basis for clinical diagnosis.
Example 9 identification of tissue or cell types by triple staining
In this example, human kidney tissue sections were triple stained with COL4A1, PAX-8, and WT1 according to the protocol shown in Table 2.
TABLE 2 identification of tissue or cell types by triple staining
Human paraffin embedded tissue section kidney tissue 1 is selected for dewaxing and hydration. Antigen retrieval and the like were performed by the method described in example 8. Firstly, carrying out primary antibody incubation, primary antibody post-incubation and secondary antibody incubation by using the reagent of the 1 st group and the reagent of the 2 nd group; and then using the reagent of group 3 to perform primary antibody incubation, primary antibody post-incubation and secondary antibody incubation, wherein the adding amount of the artificial polypeptide PL-goat anti-rabbit antibody conjugate needs to be excessive, so that the phenomenon that part of the artificial polypeptide PL-goat anti-rabbit antibody conjugate is combined with COL4A1 antigen-derived COL4A1 monoclonal antibody, and the detection non-specificity is caused because the artificial polypeptide PL-goat anti-rabbit antibody conjugate is not combined with the artificial polypeptide PL-goat anti-rabbit antibody conjugate and is combined with the artificial polypeptide HM-goat anti-rabbit antibody conjugate added later.
After a second secondary antibody incubation, 1 drop of anti-fluorescence quencher (containing DAPI) was added dropwise to the sections, observed under a fluorescence microscope, and photographed. The results of the triple fluorescent staining are shown in FIG. 20, in which COL4A1 stained the basement membrane of the kidney tissue as green fluorescence; PAX-8 stains renal tissue renal tubular epithelial cells, and the cell nucleus is yellow fluorescence; WT1 stained renal tissue glomerular epithelial cell nuclei in red.
Example 10 immunocytochemistry applications
Different cells were tested using immunocytochemistry assay, the protocol is shown in table 3:
TABLE 3 enhanced two-antibody immunocytochemistry assay
Cell wax blocks prepared from the corresponding cells were individually sectioned, deparaffinized, hydrated, and then stained as described in example 8.
As a result of the staining, Raji cells (A) were stained brown in cell membrane after immunocyte staining using rabbit monoclonal antibody CD20, as shown in FIG. 21. After immunocyte staining of Daudi cells (B) and Hela cells (C) with FoxP1 rabbit mAb and Ki-67 rabbit mAb, respectively, the nuclei were stained brown.
The results show that the pathology enhanced secondary antibody consisting of the artificial polypeptides PL, HM and LH and the antibody thereof can realize the immunological staining of the cell wax block.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Hangzhou Bailing Biotechnology Ltd
<120> pathological detection kit for detecting multiple antigens, preparation method and application thereof
<130> AJ2010229
<140> 202011455212.0
<141> 2020-12-10
<150> 202011402424.2
<151> 2020-12-02
<160> 48
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<213> Artificial Sequence (Artificial Sequence)
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Thr Thr Thr Pro Thr Thr Pro Thr Thr Pro Thr Thr Pro Thr Thr
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<213> Artificial Sequence (Artificial Sequence)
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Thr Thr Thr Pro Thr Thr Pro Thr Thr Pro Thr Thr Pro Thr Thr Pro
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<213> Artificial Sequence (Artificial Sequence)
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Thr Thr Thr Pro Thr Thr Pro Thr Thr Pro Thr Thr Pro Thr Thr Pro
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Thr Thr Pro Thr Thr
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Thr Thr Pro Thr Thr Pro Thr Thr
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Thr Thr Pro Thr Thr Pro Thr Thr Pro Thr Thr
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Thr Thr Pro Thr Thr Pro Thr Thr Pro Thr Thr Pro Thr Thr
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<213> Artificial Sequence (Artificial Sequence)
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Thr Thr Pro Thr Thr Pro Thr Thr Pro Thr Thr Pro Thr Thr Pro Thr
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Thr
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Ala Ala Pro Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala
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Ala Pro Ala Ala Ala
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<210> 13
<211> 27
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Ala Ala Pro Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala
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Ala Asp Ala Ala Asp Ala Ala Pro Ala Ala Ala
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<210> 14
<211> 33
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Ala Ala Pro Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala
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Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Pro Ala Ala
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Ala
<210> 15
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Ala Ala Pro Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala
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Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala
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Asp Ala Ala Pro Ala Ala Ala
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<213> Artificial Sequence (Artificial Sequence)
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Ala Ala Pro Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala
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Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp
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Ala Ala Asp Ala Ala Pro Ala Ala Ala
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<210> 19
<211> 63
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Ala Ala Pro Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala
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Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala
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Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Ala Ala Pro Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala
1 5 10 15
Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala
20 25 30
Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp
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Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala Ala Asp Ala
50 55 60
Ala Pro Ala Ala Ala
65
<210> 21
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<213> Artificial Sequence (Artificial Sequence)
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<210> 22
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Gly Gln Ala Thr Thr Ala Gln Gln
1 5
<210> 23
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<213> Artificial Sequence (Artificial Sequence)
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Gly Gln Ala Thr Thr Thr Ala Gln Gln
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<210> 24
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Gly Gln Ala Thr Thr Thr Thr Ala Gln Gln
1 5 10
<210> 25
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Gly Gln Ala Thr Thr Thr Thr Thr Ala Gln Gln
1 5 10
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<213> Artificial Sequence (Artificial Sequence)
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1 5 10
<210> 27
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Gly Gln Ala Thr Thr Thr Thr Thr Thr Thr Ala Gln Gln
1 5 10
<210> 28
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Gly Gln Ala Thr Thr Thr Thr Thr Thr Thr Thr Ala Gln Gln
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<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Gly Gln Ala Thr Thr Thr Thr Thr Thr Thr Thr Thr Ala Gln Gln
1 5 10 15
<210> 30
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Gly Gln Ala Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Ala Gln Gln
1 5 10 15
<210> 31
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Thr Asn Ala Met Ser
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<210> 32
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Ile Ile Ser Ser Ser Gly Ser Thr Tyr Tyr Ala Arg Trp Ala Lys Gly
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<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Gly Asn Ile
1
<210> 34
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Gln Ser Ser Gln Ser Val Tyr Asn Asn Asn Leu Ala
1 5 10
<210> 35
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 35
Arg Ala Ser Lys Leu Ala Ser
1 5
<210> 36
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 36
Leu Gly Gly Tyr Asp Cys Ser Ser Ala Asp Cys Gly Ala
1 5 10
<210> 37
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 37
Ser Tyr Ala Met Gly
1 5
<210> 38
<211> 16
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<213> Artificial Sequence (Artificial Sequence)
<400> 38
Ile Ala Thr Thr Gly Ser Ser Thr Tyr His Ala Ser Trp Ala Lys Gly
1 5 10 15
<210> 39
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Asp Gly Asp Trp Thr Gly Trp Tyr Phe Ser Ile
1 5 10
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<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 40
Gln Ala Ser Gln Ser Ile Ser Asn Arg Leu Ala
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<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
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Asp Ala Ser Asp Leu Ala Ser
1 5
<210> 42
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 42
Gln Gln Gly Tyr Gly Gly Asp Asn Ile Glu Asn Leu
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<210> 43
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 43
Arg Tyr Ala Met Cys
1 5
<210> 44
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 44
Ile Ile Gly Val Ser Gly Thr Thr Tyr Tyr Thr Ser Trp Ala Lys Gly
1 5 10 15
<210> 45
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 45
Val Met Pro Gly Tyr Asp Asp Tyr Gly Asp Asp Gly Phe Asp Pro
1 5 10 15
<210> 46
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 46
Gln Ala Ser Glu Asp Ile Tyr Ser Asn Leu Ala
1 5 10
<210> 47
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 47
Ala Ala Ser Tyr Leu Ala Ser
1 5
<210> 48
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 48
Gln Cys Thr Tyr Tyr Ser Gly Ser Tyr Glu Leu Phe Thr
1 5 10
Claims (10)
1. A kit for simultaneously detecting N antigens is characterized by comprising:
a first antibody composition consisting of N first antibodies capable of specifically binding to said N antigens; a second antibody composition consisting of N second antibodies against the N first antibodies, the N second antibodies being conjugated to N artificial polypeptides, respectively;
and the third antibody or the antigen binding part thereof is respectively coupled with the polymer, and N color developing agents are coupled on the polymer coupled with the N third antibodies.
2. The kit of claim 1, wherein the visualization reagent is fluorescein, an enzyme, a metal ion, a quantum dot, or an isotope.
3. The kit of claim 2, wherein the enzyme is horseradish peroxidase, alkaline phosphatase, or β -glucosidase.
4. The kit of any one of claims 1 to 3, wherein N is 2 to 5.
5. The kit of any one of claims 1 to 3, wherein the first and third antibodies are monoclonal antibodies and the second antibody is a polyclonal antibody.
6. The kit of any one of claims 1 to 3, wherein the antigen binding moiety is selected from the group consisting of a Fab fragment, a Fab 'fragment, a F (ab')2 fragment, a Fv fragment, a scFv fragment, a Fd fragment, and a single domain antibody.
7. The kit of claim 6, wherein the antigen binding moiety is a Fab' fragment.
8. A method for simultaneously detecting N antigens is characterized by comprising the following steps:
s1, binding N first antibodies capable of specifically binding to the N antigens;
s2, combining N secondary antibodies against the N primary antibodies with the N primary antibodies, wherein the N secondary antibodies are respectively conjugated with N artificial polypeptides;
s3, combining N third antibodies or antigen binding parts thereof resisting the N artificial polypeptides with the N third antibody combination or antigen binding part combination, wherein the third antibodies or the antigen binding parts thereof are respectively conjugated with the polymers, and N color developing agents are conjugated on the polymers conjugated with the N third antibodies,
the N color developing agents are different from each other, so that the simultaneous detection of the N antigens is completed.
9. The method of claim 8, wherein N is 2-5.
10. The method of claim 8, wherein the first and third antibodies are monoclonal antibodies and the second antibody is a polyclonal antibody.
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| US20150031563A1 (en) * | 2011-07-28 | 2015-01-29 | Khanh Duc Huynh | Multi Component Antibody Based Detection Technology |
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| CN111077302A (en) * | 2019-12-26 | 2020-04-28 | 江苏美克医学技术有限公司 | Glucan signal amplification-based ki67 and p16INK4aDetection kit |
| CN111551709A (en) * | 2020-05-13 | 2020-08-18 | 河南赛诺特生物技术有限公司 | Enzyme-labeled secondary antibody compound and preparation method thereof |
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| US20020173053A1 (en) * | 2001-04-27 | 2002-11-21 | Bassam Damaj | Multiple simultaneous antigen detection by immunohistochemistry |
| US20050208529A1 (en) * | 2003-12-01 | 2005-09-22 | Lars Winther | Methods and compositions for immuno-histochemical detection |
| US20150031563A1 (en) * | 2011-07-28 | 2015-01-29 | Khanh Duc Huynh | Multi Component Antibody Based Detection Technology |
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