CN-121992147-A - Kit for ortho-ligation detection antibody and method for detecting antibody

CN121992147ACN 121992147 ACN121992147 ACN 121992147ACN-121992147-A

Abstract

The invention relates to the technical field of ortho-ligation detection, in particular to a kit for detecting an antibody through ortho-ligation and a method for detecting the antibody. The kit comprises a solid phase carrier, a first probe-labeled antibody against a protein label, a second probe-labeled recombinant protein, a connecting probe, an upstream primer and a downstream primer. The kit realizes high-sensitivity measurement of natural antibodies by connecting a plurality of antigen peptides (especially antigenic determinants) in series as active peptides connected to a solid carrier so as to cover more antigen epitopes. The solid phase carrier and the multicolor fluorescent probe are arranged in the same reaction system, so that the simultaneous detection of a plurality of antibodies can be realized. The kit provided by the invention has the technical advantage of wide application range, can meet the multi-epitope high-sensitivity detection of natural antibodies of various viruses, in particular to the multi-epitope high-sensitivity detection of natural antibodies of HIV viruses, and has very practical significance on blood transfusion safety and success rate of blocking treatment.

Inventors

MA ZHAN
ZHANG HONG
LIU CHUNFANG
TANG RUI
WENG WENHAO
DING GUOHUI

Assignees

上海市儿童医院

Dates

Publication Date: 20260508
Application Date: 20241107

Claims (10)

1. A kit for detecting an antibody through orthotopic ligation is characterized by comprising a solid phase carrier, a first probe-labeled antibody against a protein tag, a second probe-labeled recombinant protein, a ligation probe, an upstream primer and a downstream primer; The solid phase carrier is connected with active peptide, the active peptide comprises a plurality of antigen peptides and a plurality of protein tags, a protein tag is arranged between every two adjacent antigen peptides, a connecting peptide is arranged between the protein tag and the protein tag, and the protein tag is connected with the solid phase carrier in an affinity way or through a chemical covalent bond; the recombinant protein is used for specifically recognizing the antibody to be detected; And the connecting probe is subjected to a connecting reaction with the free 3 'end of the first probe and the free 5' end of the second probe, and an antigen peptide-connecting peptide-protein label-first probe-labeled antibody-connecting probe-second probe-labeled recombinant protein-antibody-antigen peptide to be detected complex is formed on the solid-phase carrier.
2. The kit according to claim 1, wherein the recombinant protein is of the same kind as the antigenic peptide.
3. The kit of claim 2, wherein the recombinant protein and the antigenic peptide are each of at least 2 species; preferably, the recombinant protein and the antigen peptide are 3 kinds.
4. A kit according to claim 3, wherein the antigenic peptide is selected from the group consisting of viral antigens and envelopes; preferably, the group of antigens is selected from the group consisting of matrix, capsid, nucleocapsid and trabecular polypeptide; the envelope is selected from the group consisting of envelope proteins, surface proteins, and transmembrane proteins; preferably, the antigenic peptide is selected from the group consisting of p24, gp41 and gp36 of a virus; Preferably, the virus is an HIV virus; Preferably, the amino acid sequence of the p24 is shown as SEQ ID NO.9, the amino acid sequence of the gp41 is shown as SEQ ID NO.10, and the amino acid sequence of the gp36 is shown as SEQ ID NO. 11; preferably, the amino acid sequence of the active peptide is shown as SEQ ID NO. 2.
5. The kit of claim 1, wherein the affinity linkage is selected from one of an antigen-to-antibody linkage or a ligand-to-receptor linkage.
6. The kit according to claim 5, wherein the number of amino acids of the connecting peptide is 1 to 30; preferably, the amino acid sequence of the connecting peptide is selected from (GGGGS) n, (GGGS) n, (GGS) n, (GS) n, (G) n or GGGGSGGGGSEAAAKEAAAKEAAAK, wherein n is 1,2, 3,4, 5 or 6.
7. The kit of claim 1, wherein the protein tag is selected from the group consisting of glutathione transferase, polyhistidine, V5 tag, MBP tag, mCherry tag, and Flag polypeptide; Preferably, the protein tag is GST.
8. The kit of claim 7, wherein the anti-protein tag is labeled 5' to the first probe and the nucleotide sequence of the first probe has at least 90% identity to the sequence set forth in SEQ ID No. 8; the recombinant protein is marked at the 3' end of the second probe, and the nucleotide sequence of the second probe has at least 90% identity with the sequence shown in SEQ ID NO. 12; the nucleotide sequence of the ligation probe has at least 90% identity to the sequence shown in SEQ ID NO. 13; Preferably, the 5 'end of the connecting probe is provided with a fluorescence reporting group, and the 3' end of the connecting probe is provided with a fluorescence quenching group; Preferably, the nucleotide sequence of the upstream primer has at least 90% identity with the sequence shown in SEQ ID NO.14 and the nucleotide sequence of the downstream primer has at least 90% identity with the sequence shown in SEQ ID NO. 15.
9. The kit of claim 1, wherein the solid support is selected from the group consisting of nanoparticles, magnetic beads, agarose gel microspheres, silica gel microspheres, latex microspheres, test tubes, EP tubes, multiwell plates, NC films, and PDMS films; Preferably, the kit further comprises a positive control, a negative control and a blank control.
10. A method for detecting antibodies using the kit according to any one of claims 1 to 9, characterized in that it does not aim at the diagnosis of a disease, comprising the steps of: S1, incubating a sample to be detected with the solid phase carrier connected with the active peptide; s2, adding a first probe-labeled antibody against a protein tag, incubating for 30 minutes at 37 ℃, centrifuging, discarding the supernatant, and washing; s3, adding a second probe-labeled recombinant protein, incubating for 30 minutes at 37 ℃, and washing; s4, after washing, adding a connection probe, an upstream primer, a downstream primer and ligase to perform fluorescent quantitative PCR reaction; Preferably, the conditions for the fluorescent quantitative PCR reaction include a pre-denaturation at 94℃for 3min followed by 40 cycles of 94℃for 5s and 60℃for 30s.

Description

Kit for ortho-ligation detection antibody and method for detecting antibody Technical Field The invention relates to the technical field of ortho-ligation detection, in particular to a kit for detecting an antibody through ortho-ligation and a method for detecting the antibody. Background Since the discovery of antisera at the end of the nineteenth century, antigen/antibody detection techniques using immune reaction have been used for over one hundred years, and as immunokinetics, antigen purification techniques, monoclonal antibody techniques, immunolabeling techniques and bioinformatics develop, detection techniques are mature, and have become the most important methods for detecting micro-bioactive substances such as pathogen antigen antibodies, specific micro-protein detection, hormones, and the like due to their detection capability up to picomolar level. However, the fields of early detection of pathogens, single cell research, histology research, tumor research and the like put higher demands on the sensitivity of antigen-antibody detection, and development of high-sensitivity antigen-antibody detection technology is necessary. The basic principle of the existing antigen-antibody detection method is based on heterogeneous labeled immune technology. Functionally, the technology comprises a carrier-antigen/antibody system and a signal amplification system, wherein the carrier-antigen/antibody system comprises a solid-phase carrier (such as a micro-pore plate and magnetic beads) and known antigens/antibodies combined with the solid-phase carrier (coated), the specific combination of the antigen (or the antibodies) to be detected and the carrier-antigen/antibody system is a specific source of a detection system, and isotopes, enzymes, colloidal gold, fluorescein, quantum dots, luminous substrates and the like combined with other known antigens/antibodies form the signal amplification system, and pathogen-specific antigen-antibody reactions are detected by virtue of the amplification effect of the signal amplification system. The efficiency and signal-to-noise ratio of the amplification system determine the sensitivity achievable by the detection. To achieve a high signal-to-noise ratio, a good amplification system should be able to be triggered to generate a detectable signal only after specific binding of an antigen-antibody occurs, and the generated signal has a high correlation with the number of molecules that react with the antigen-antibody. Due to the presence of free labels and non-specific binding, the erroneous amplification of these signals by the amplification system is critical in that the sensitivity of the prior art cannot be further improved, and more efficient and specific detection systems need to be sought. The immune amplification technology uses gene amplification as an amplification system to greatly improve the efficiency of the amplification system, however, due to the existence of non-specific amplification, the technology is mostly used for in-situ detection of tissue samples, and the specificity in detecting free substances is poor. The immunological PCR technology-derived ortholigation analysis technology (Proximity Ligation Assay, PLA) improves the specificity problem to a certain extent, besides the general antigen-antibody reaction, the hybridization of a second epitope orthonucleic acid probe is added, double confirmation is carried out on the specificity, the generation of non-specific signals can be greatly reduced theoretically, the detection product based on the technology is started to be used for proteomics research, but the technology is only used for the determination of substances with known antigen epitopes, and the detection omission caused by different antigen epitopes can occur when the detection of natural antigens with large variability and complex epitopes, especially natural antibodies. It is therefore of great importance to provide a kit for orthotopic ligation detection antibodies. Disclosure of Invention In order to solve the above problems, an object of the present invention is to provide a kit for ortho-ligation detection antibody and a method for detecting an antibody. The aim of the invention can be achieved by the following technical scheme: the first object of the invention is to provide a kit for orthotopic ligation detection of antibodies, comprising a solid support, a first probe-labeled anti-protein-tagged antibody, a second probe-labeled recombinant protein, a ligation probe, an upstream primer and a downstream primer; The solid phase carrier is connected with active peptide, the active peptide comprises a plurality of antigen peptides and a plurality of protein tags, a protein tag is arranged between every two adjacent antigen peptides, a connecting peptide is arranged between the protein tag and the protein tag, and the protein tag is connected with the solid phase carrier in an affinity way or through a chemical covalent bond; the recombina