CN113721023A

CN113721023A - Biological immunosensing system based on Barnase/barstar and application

Info

Publication number: CN113721023A
Application number: CN202111013055.2A
Authority: CN
Inventors: 刘畅; 宋海鹏; 何利中
Original assignee: Shenzhen Guochuang Nano Antibody Technology Co ltd
Current assignee: Shenzhen Guochuang Nano Antibody Technology Co ltd
Priority date: 2021-08-31
Filing date: 2021-08-31
Publication date: 2021-11-30

Abstract

The invention discloses a biological immunity sensing system, which is composed of two antibodies with different binding sites with target antigens, a protein binding agent barnase/barstar and a matrix material. The invention also provides a preparation process of the sensing system and application of the sensing system in the aspect of targeted antigen detection. The biological immunosensing system shows excellent detection effect in the detection of low-concentration antigen quantification.

Description

Biological immunosensing system based on Barnase/barstar and application

Technical Field

The invention discloses a sensing system and immunological application thereof, belonging to the technical field of polypeptides, and more specifically belonging to the technical field of immunoglobulin.

Background

Tumors are important factors seriously harming human health, so the detection of tumor markers plays an important role in early screening and disease diagnosis. An immunobiosensor is a novel technology for detecting functional proteins immobilized on a solid substrate by using specific interactions between antigens and antibodies, and can capture low concentrations of antigens from complex biological environments and quantitatively determine the amount of binding molecules by colorimetry, fluorescence or chemiluminescence. The immunobiosensor has been widely used in various fields such as food, industry, environmental detection, clinical medicine, etc. because of its advantages such as high specificity, low detection limit and stability. However, in a conventional immunosensor such as enzyme-linked immunosorbent assay (ELISA) or lateral flow immunoassay, due to steric hindrance between a solid substrate and a neighboring antibody, the antibody is restricted in recognizing an epitope, thereby greatly affecting the performance of the sensor. Therefore, in the design and preparation of the immunosensor, how to minimize the steric hindrance effect and thus exert the high sensitivity performance of the sensor itself becomes a difficult problem to be researched.

Currently, most of the biological companies including roche use the traditional streptavidin and biotin systems to engineer full-length antibodies for assembly of commercial kits. However, this method has two disadvantages: firstly, the full-length antibody and streptavidin have large volume and complex structure, and correct structure can be formed only by modification after transcription, so that only mammalian cells can be selected for expression, the time is long, and the cost is high; second, the auxiliary binder avidin has a complex chemical structure and therefore cannot be inserted into a protein after simple recombination. The current conventional methods for in vivo biotin conjugation in vitro use only chemical or enzymatic crosslinking (using biotin ligase), and require additional modification whatever the method chosen. Therefore, the development of a binding agent having a simple structure can greatly contribute to the development and application of an immunosensor.

The invention aims to provide an immunobiosensor with a novel binding agent, wherein the binding agent in the sensing system has a simple structure, can be introduced into a protein of a target antigen through a recombination technology, has high affinity, does not influence the activity of the protein of the target antigen combined with the binding agent, and lays a foundation for the application of the binding agent in early disease screening and diagnosis.

Disclosure of Invention

Based on the above-mentioned objects, the present invention first designs a set of immunobiosensing systems comprising a protein responsible for targeting and a specific binding partner for the protein to a carrier material. Wherein the protein responsible for targeting comprises an antibody, an immunologically active fragment, a receptor or binding fragment thereof, a lectin, and an enzyme. In the present specification, an antibody is an immunoglobulin of natural origin or produced synthetically. Unless otherwise indicated, the terms "antibody" and "immunoglobulin" are used synonymously throughout the specification. An immunologically active antibody fragment is a portion of an intact antibody that retains the ability to exhibit antigen binding activity, and the antigen binding site may be formed by the combination of antibody light and heavy chain variable domains or may comprise separate antibody variable domains. Binding fragments include Fab fragments, Fv fragments (comprising the variable domains of the heavy and light chains of an antibody associated with each other), or single chain Fv fragments (in which both the light and heavy chains are present as part of a fusion protein) comprising the two binding sites of the antibody linked together.

For the combination of specific binding of proteins responsible for specific binding to the carrier material, the Barnase/Barstar specific combination is selected by the present invention. Barnase is an extracellular small-molecule RNase (RNase) originally obtained from Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and has an amino acid sequence shown in SEQ ID No. 4. Barnase has a strong bactericidal effect, so that expression of the gene can cause death of the host cell. Barstar is a Barnase specific inhibiting protein in a bacillus amyloliquefaciens genome, the amino acid sequence of the Barstar is shown as SEQ ID NO.3, and the product of the Barstar can be specifically combined with Barnase at a ratio of 1:1 to form a stable complex, so that the Barnase loses activity. Therefore, the Barnase activity can be controlled by controlling the expression of Barstar through a certain technical means, so the Barnase/Barstar system is often used as a molecular switch in genetic engineering. The invention utilizes the specific binding property of Barnase/Barstar to construct the Barnase/Barstar in the immune biosensing system to be used as the function of specific binding with a carrier material, and provides a method for detecting a target antigen in a sample by using the Barnase/Barstar system, which comprises the following steps:

(1) placing a sample to be detected into a detection system containing a first antibody resisting the target antigen and a second antibody resisting the target antigen and having different antigen binding epitopes from the first antibody, wherein the first antibody is connected with barstar or barnase, and the second antibody is connected with a chemiluminescent marker;

(2) incubating the detection system obtained in step (1) under conditions that promote specific binding reactions of the antigen and the antibody;

(3) adding a matrix material coated with barnase or barstar, wherein the matrix material is coated with barnase when the first antibody is linked to barstar, and the matrix material is coated with barstar when the first antibody is linked to barnase, so that the barnase and barstar have specific binding reaction;

(4) obtaining the substrate material reacted in the step (3), and detecting the signal of the chemiluminescent marker.

In a preferred embodiment, the chemiluminescent label in step (1) and step (4) is the fluorescent dye ATTO 488.

In another preferred embodiment, the matrix material in step (3) is agarose microspheres.

In another alternative embodiment, the matrix material of step (3) is a solid matrix.

In a preferred embodiment, the first antibody and the second antibody in step (1) are nanobodies. The nanobodies are immunoglobulins that naturally lack a light chain or have only heavy chain variable domain fragments.

More preferably, the nanobody is a nanobody against CEACAM-5.

Particularly preferably, the amino acid sequence of the variable region of the first nano antibody for resisting CEACAM-5 is shown as SEQ ID NO.1, and the amino acid sequence of the variable region of the second nano antibody for resisting CEACAM-5 is shown as SEQ ID NO. 2. One specific example of the nanobody having a variable region sequence of SEQ ID NO.1 in the present invention is nanobody 2D5, and one specific example of the nanobody having a variable region sequence of SEQ ID NO.2 is nanobody 13A 5.

Still preferably, the first nanobody is linked to barstar whose amino acid sequence is shown in SEQ ID NO.3, and the amino acid sequence of barnase coated with the matrix material is shown in SEQ ID NO.4, as a fusion protein.

Most preferably, the barstar is attached as a tail to the N-terminus or C-terminus of the nanobody, forming a nanobody-barstar fusion protein structure. The amino acid sequence of the fusion protein 2D5-barstar is shown in SEQ ID NO. 5.

Secondly, the invention provides a kit for detecting a target antigen in a sample by using a barnase/barstar system, wherein the kit contains the following reagents:

(1) a first antibody and a second antibody having different binding sites to an antigen of interest, said first antibody being linked to barstar or barnase and said second antibody being linked to a chemiluminescent label;

(2) a matrix material coated with barnase or barstar, the matrix material being coated with barnase when the primary antibody is attached to barstar and coated with barstar when the primary antibody is attached to barnase.

In a preferred embodiment, the chemiluminescent label is the fluorescent dye ATTO 488.

In another preferred embodiment, the matrix material is agarose microspheres.

In another alternative embodiment, the matrix material is a solid matrix.

In a preferred embodiment, the first antibody and the second antibody are nanobodies.

In a more preferable technical scheme, the sequence of the first nano antibody is shown as SEQ ID NO.1, and the sequence of the second nano antibody is shown as SEQ ID NO. 2.

More preferably, the first nanobody is linked to barstar whose amino acid sequence is shown in SEQ ID NO.3 and the amino acid sequence of barnase coated with the matrix material is shown in SEQ ID NO.4 as a fusion protein.

Particularly preferably, the amino acid sequence of the fusion protein (2D5-barstar) is shown as SEQ ID NO. 5.

The nano antibody-barstar system in the immunosensor can form strong action force with barnase in an aqueous environment. Sequence features may facilitate the formation of alpha-helical or beta-turn conformations, which in turn facilitate detection using conventional techniques. Barnase and barstar are small molecular weight proteins (12 kDa and 10kDa, respectively) with simple structures (no disulfide bonds) that can be inserted into proteins for recombination using conventional molecular biology techniques, saving a lot of time and cost. More rarely, barnase is an RNase in nature, and is normally not expressed in bacteria as a host. In the invention, barnase and the inhibitor barstar thereof are simultaneously expressed, and by constructing double plasmids of barnase and barstar, the toxicity of barnase to an expression host is inhibited, and double expression of barnase and barstar is successfully realized, thereby laying a solid foundation for the application of barnase/barstar in disease diagnosis.

Drawings

FIG. 1 is a diagram of the working principle of the Barnase/barstar immunosensor for detecting CEA;

FIG. 2 is a schematic diagram of the molecular construction of Barnase, barstar, GFP-barstar and 2D 5-barstar;

FIG. 3.Barnase and barstar interaction scheme;

FIG. 4 is a graph showing the results of detection of fluorescent signals of a GFP-barstar captured barnase microsphere;

FIG. 5 is a graph showing the result of detecting the fluorescence signal of CEA by the Barnase/barstar immunosensor;

FIG. 6 is a photograph of fluorescent microscope images of the Barnase/barstar immunosensor for CEA detection;

FIG. 7 is a graph showing the linear results of CEA detection by the Barnase/barstar immunosensor;

FIG. 8 is a graph showing the results of purification of recombinant proteins;

FIG. 9 is a comparison of the test results of different test conditions.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention.

FIG. 1 is a schematic diagram of the operation of one embodiment of the immunobiosensor system provided in the present invention. Taking the detection of CEA antigen as an example, the present invention first constructs a working component of the immune biosensor system, which includes a first nanobody, a second nanobody, and an agarose microsphere as a matrix material, where the first nanobody is linked to barstar to form a first nanobody-barstar (alternatively, the first nanobody may also be linked to barnase), the second nanobody is linked to a fluorochrome label to form a second nanobody-fluorescent label, and the agarose microsphere is coated with barnase (alternatively, the agarose microsphere may also be coated with barstar, when the first antibody is linked to barstar, the matrix material is coated with barnase, and when the first antibody is linked to barstar, the matrix material is coated with barstar, so that barnase and barstar perform a specific binding reaction). Mixing the two, adding the microsphere coated by barnase, and forming a microsphere-barnase/barstar-first nano antibody compound under the condition of no CEA, wherein the fluorescence signal is negative; under the condition of CEA, the first and second nanometer antibody-CEA complexes (fluorescent labels) are firstly formed after mixing, namely, the first nanometer antibody-CEA-second nanometer antibody-fluorescent labels, and the microspheres are added to form the microsphere-barnase/barstar-first nanometer antibody-CEA-second nanometer antibody-fluorescent labels, and the fluorescent signals are positive.

EXAMPLE 1 construction of recombinant proteins

1.1 plasmid construction: plasmid pMT416 (brand: Addgene; cat #8607) encoding barnase (GenBank: AAA86441.1) and plasmid pMT316 (brand: Addgene; cat #8608) encoding barstar (GenBank: AAM10782.1) are commercially available. Plasmids encoding 2D5, 13A5, GFP-barnase, 2D5-barstar, 2D5-barnase were synthesized by GenScript (hong Kong, China). Wherein the amino acid sequence of 2D5 is shown as SEQ ID NO.1, the amino acid sequence of 13A5 is shown as SEQ ID NO.2, the amino acid sequence of barstar is shown as SEQ ID NO.3, and the amino acid sequence of barnase is shown as SEQ ID NO. 4. The amino acid sequence of 2D5-barstar is shown in SEQ ID NO.5, the amino acid sequence of GFP-barnase is shown in SEQ ID NO.6, and the amino acid sequence of 2D5-barnase is shown in SEQ ID NO. 7. The GFP-barstar amino acid sequence is shown as SEQ ID NO. 8. The nano antibody gene phoA-2D5-barnase was first inserted into pETDuet-1 vector through restriction sites Nco I and Hind III, and then the barstar inhibitor gene was inserted through restriction sites Nde I and Xho I. The amino acid sequence of the added phoA secretion signal peptide (An N-terminal signal peptide sequence of alkaline phohatase from E.coli) is shown in SEQ ID NO. 9. The toxic 2D5-barnase can be transported to the periplasmic space for expression and is itself automatically removed by a signal peptidase. The genes encoding 2D5, 13A5, GFP-barnase and 2D5-barstar were inserted into pET28a vector through restriction sites Nco I and Xho I, respectively, to construct corresponding non-toxic recombinant vectors. We selected 2D5 and GFP as model molecules to map the molecular constructs, and the results are shown in figure 2. Wherein FIG. 2A is a commercially available base plasmid encoding barnase (GenBank: AAA 86441.1); FIG. 2B is a commercially available plasmid encoding barstar (GenBank: AAM 10782.1); the GFP-barstar amino acid sequence of FIG. 2C is shown in SEQ ID NO.8 (this molecular construction can be used for fluorescence verification of barnase and barstar interaction (see FIG. 3 for schematic), while the molecular design of FIG. 2D can be used to bind target antigen via Nanobody 2D5 and further bind antigen to the barnase-immobilized solid surface via the coupled barstar.

1.2 expression and purification of protein: first, plasmids encoding nanobodies 2D5, 2D5-barstar and 13a5 were transformed into host bacterium e.coli SHuffle T7, and the culture was inoculated such that the initial OD value was 0.1. Culturing at 30 deg.C for 3 hr, adding 1mM IPTG, and culturing at 20 deg.C for 36 hr; secondly, transforming the plasmid for coding 2D5-barnase into host bacteria BL21(DE3) and expressing at 37 ℃; third, the plasmid encoding GFP-barstar was transformed into BL21(DE3) and cultured at 37 ℃ for 20 hours under 1mM IPTG conditions.

After expression, the cell culture was centrifuged at 10,000rpm at 4 ℃ for 10 minutes. And extracting protein from the separated cells by a lysis or osmotic shock method. Cell lysis was performed by sonication of cytoplasmic expressed 2D5, 2D5-barstar, 13A5, GFP-barstar and barstar with lysis buffer (20mM Tris HCl, pH8, 0.5M NaCl, 0.5% Triton-X). For periplasmic expression of barnase and 2D5-barnase, the protein was extracted by osmotic shock. The specific operation is as follows: harvested cells were resuspended in precooled sucrose buffer (30mM Tris HCl, pH8, 20% sucrose) for 10 minutes, and after centrifugation and removal of the supernatant, the cells were resuspended using MilliQ water. Centrifuging again, and obtaining periplasmic protein from the supernatant. The extracted protein was purified using AKTA FPLC system (GE Healthcare). Wherein barnase expressed using the pMT416 vector was purified using a HiTrap SP HP cation exchange column (GE Healthcare), while the other proteins were purified using IMAC. The specific operation is as follows: the proteins were loaded onto a HisTrap column (GE Healthcare) and eluted with a gradient of 0-500mM imidazole in eluent (20mM Tris-HCl, pH8, 0.5M NaCl). The purified protein was desalted in DPBS and stored. 2D5-barstar was desalted into buffer (20mM Tris HCl, pH10.1, 140mM NaCl) to improve stability. Mass spectrometry analysis was performed on the expressed barnase, GFP-barstar and 2D5-barstar, and the results are shown in Table 1.

TABLE 1 results of Mass Spectrometry

Sequence coverage in mass spectrometry results shows that all recombinant proteins are successfully constructed. The optimization of the conditions for expressing the protein and the types of the vectors are shown in Table 2. The protein purification results are shown in FIG. 8.

TABLE 2 expression vectors for recombinant proteins

1.3 binding kinetics assay: binding kinetics detection is achieved by biofilm layer reflected light interference techniques using Aminopropylsilane (APS) sensors. Proteins were immobilized directly onto APS sensors by nonspecific hydrophobic adsorption, and kinetic assays were performed using DPBS unless otherwise indicatedIs a buffer solution (0.2g/L KCl, 0.2g/L KH)₂PO₄，8g/L NaCl，2.16g/L NaH₂PO₄)。

1.4 capture of GFP-barstar on Barnase coated microspheres: the microspheres coated with barnase were introduced into GFP-barstar, and the barnase and barstar interaction process is shown in FIG. 3. A specific procedure was to wash NHS activated commercial agarose microspheres (Sigma-Aldrich, Sydney, Australia) twice with DPBS. 50 μ L (about 50% m/m) of the microspheres were added to 0.5mL of barnase (0.3mg/mL) and coated overnight at 4 ℃. Microspheres used as negative controls were supplemented with 0.5mL BSA protein (3mg/mL) to simulate a complex biological environment. Blocking was performed using blocking solution (0.5mL of 1M Tris-HCl pH 8) for 10 min. DPBS wash was performed twice. To 200. mu.L of GFP (1mg/mL) was added 10. mu.L of barnase-coated microspheres (50% m/m), and after incubation for 5 minutes in the absence of light, the microspheres were centrifuged and washed twice. The centrifugally washed microspheres are resuspended by 100 μ L of DPBS, a Tecan microplate reader is used for reading OD value (relative value) of 509nm, the working principle is schematically shown in figure 3, the detection result is shown in figure 4, and as can be seen from figure 4, the signal value of the GFP-barstar group is obviously higher than that of the control group (about 5 times), which indicates that strong force exists between barnase and barstar in a complex aqueous phase environment. The invention also constructs GFP-barnase at the same time, and verifies that the GFP-barnase has strong action force as the microspheres coated by barstar.

Example 2 one-step detection of CEA

CEA can be detected by one-step method by using a nano antibody-barnase/barstar immunosensor, and the specific working principle is shown in figure 1.

2.1 fluorescent labeling of Nanobody 13A 5: NHS-ATTO488 fluorescent dye (Sigma-Aldrich, Sydney, Australia) was diluted to 2mg/mL and dissolved in DMSO. 50 μ L of Nanobody 13A5(1mg/mL), 3 μ L of NHS-ATTO488 and 7 μ L of NaHCO were taken₃The solution was incubated at room temperature for 1 hour. Purification was then performed using a MiniTrap desalting column (GE Healthcare).

2.2 preparation and identification of the nano antibody-barnase/barstar: barnase-coated agarose microspheres were prepared according to the method of 1.4 in example 1. CEA (GenBank: CAE75559.1) was diluted to different concentrations using DPBS for detection. 20. mu.L of 2D5-barstar (0.1mg/mL), 20mu.L of CEA at various concentrations (10-10,000ng/mL) and 20. mu.L of ATTO 488-labeled nanobody 13A5(0.1mg/mL) were mixed and incubated for 10 minutes in the absence of light. mu.L of barnase-coated agarose microspheres (50 w/w%) were added to the nanobody-CEA complex for capture, the microspheres were separated by centrifugation at 14800rpm for 5 minutes and washed twice with DPBS. The washed microspheres were finally resuspended using 100. mu.L DPBS and transferred to a 96-well plate for fluorescence detection. The results are shown in FIGS. 5 and 9, where the results of FIG. 5 indicate that the nanobody-barnase/barstar immunosensor achieves a significantly enhanced signal value when detecting CEA at a concentration of 1 μ g/mL, whereas the nanobody-barnase/barstar immunosensor achieves substantially no signal value when detecting CEA at a concentration of 0 μ g/mL (indicating that the non-specific adsorption of the system is low in the absence of CEA). The results in FIG. 9 show that 2D5-barstar + CEA +13A5-ATTO488(barnase coated microspheres) achieved the highest signal values (approximately 5 times the low signal set), whereas under BSA conditions mimicking a complex biological environment, the signal values were essentially unchanged and were significantly higher than 2D5+ CEA +13A5-ATTO488(barnase coated microspheres) and 2D5-barstar + CEA +13A5-ATTO488(Tris blocking solution blocked microspheres). 2 μ L of microspheres were subjected to fluorescence microscopy (brand: Olympus; model: BX51) using a glass slide

PLUS (thermo scientific), 0.17mm coverslip (ZEISS), 0dB gain, 500ms exposure time. Fluorescence imaging results are shown in FIG. 6, where it can be seen that when the barnase/barstar immunosensor detects CEA at a concentration of 0ng/ml, the isolated microspheres are non-fluorescent under a fluorescence microscope (left side is a control under normal light). While in the detection of CE at a concentration of 1ng/mL, the separated microspheres were visibly fluorescent under a fluorescence microscope (the left side is a control under normal light source, i.e., the fluorescence signal is visible only in the presence of CEA. the linearity results of the Barnase/barstar immunosensor for CEA are shown in FIG. 7, which shows that the Barnase/barstar immunosensor still has good linearity values (see FIG. 7) when detecting low concentrations of CEA (10ng/mL, 50ng/mL and 100ng/mL) antigen, R is shown in FIG. 7²The lowest detection limit was 1ng/mL at 0.997, thus demonstrating its potential for detecting low concentrations of antigen. Despite the acquisition of signal valuesIs carried out by a secondary antibody 13A5 labeled by a fluorescent signal, but we still obtained a good linearity value in the range of low antigen concentration. If the signal amplification method is further optimized, such as using alkaline phosphatase or horseradish peroxidase coupled, electrochemical methods or trying different solid substrates, such as ELISA plates, silica magnetic beads or glass surfaces, etc., better detection effect can be obtained.

As an alternative of the invention, the combination of 2D5-barnase, 13A5-ATTO488 and microspheres coated with barstar can also be used for detecting CEA antigen with low concentration.

Sequence listing

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Ile Asn Thr Phe Asp Gly Val Ala Asp Tyr Leu Gln Thr Tyr His Lys

165 170 175

Leu Pro Asp Asn Tyr Ile Thr Lys Ser Glu Ala Gln Ala Leu Gly Trp

180 185 190

Val Ala Ser Lys Gly Asn Leu Ala Asp Val Ala Pro Gly Lys Ser Ile

195 200 205

Gly Gly Asp Ile Phe Ser Asn Arg Glu Gly Lys Leu Pro Gly Lys Ser

210 215 220

Gly Arg Thr Trp Arg Glu Ala Asp Ile Asn Tyr Thr Ser Gly Phe Arg

225 230 235 240

Asn Ser Asp Arg Ile Leu Tyr Ser Ser Asp Trp Leu Ile Tyr Lys Thr

245 250 255

Thr Asp His Tyr Gln Thr Phe Thr Lys Ile Arg His His His His His

260 265 270

His

<210> 8

<211> 346

<212> PRT

<213> Artificial

<400> 8

His His His His His His Ser Ser Gly Val Ser Lys Gly Glu Glu Leu

1 5 10 15

Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val Asn

20 25 30

Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr Tyr

35 40 45

Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro Val

50 55 60

Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys Phe

65 70 75 80

Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser Ala

85 90 95

Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp Asp

100 105 110

Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr Leu

115 120 125

Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly Asn

130 135 140

Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val Tyr

145 150 155 160

Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys Ile

165 170 175

Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr Gln

180 185 190

Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn His

195 200 205

Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys Arg

210 215 220

Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr Leu

225 230 235 240

Gly Met Asp Glu Leu Tyr Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly

245 250 255

Ser Lys Lys Ala Val Ile Asn Gly Glu Gln Ile Arg Ser Ile Ser Asp

260 265 270

Leu His Gln Thr Leu Lys Lys Glu Leu Ala Leu Pro Glu Tyr Tyr Gly

275 280 285

Glu Asn Leu Asp Ala Leu Trp Asp Ala Leu Thr Gly Trp Val Glu Tyr

290 295 300

Pro Leu Val Leu Glu Trp Arg Gln Phe Glu Gln Ser Lys Gln Leu Thr

305 310 315 320

Glu Asn Gly Ala Glu Ser Val Leu Gln Val Phe Arg Glu Ala Lys Ala

325 330 335

Glu Gly Ala Asp Ile Thr Ile Ile Leu Ser

340 345

<210> 9

<211> 21

<212> PRT

<213> Escherichia coli

<400> 9

Met Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr

1 5 10 15

Pro Val Thr Lys Ala

20

Claims

1. A method for detecting an antigen of interest in a sample using the barnase/barstar system, said method comprising the steps of:

2. The method of claim 1, wherein the chemiluminescent label in steps (1) and (4) is the fluorescent dye ATTO 488.

3. The method of claim 1, wherein the matrix material of step (3) is agarose microspheres.

4. The method of claim 1, wherein the matrix material of step (3) is a solid matrix.

5. The method according to claim 1, wherein the first antibody and the second antibody of step (1) are nanobodies.

6. The method according to claim 5, wherein the sequence of the first nanobody is shown as SEQ ID No.1, and the sequence of the second nanobody is shown as SEQ ID No. 2.

7. The method according to claim 6, wherein the first nanobody is linked to a barstar whose amino acid sequence is shown in SEQ ID No.3 and the amino acid sequence of barnase coated with the matrix material is shown in SEQ ID No.4 as a fusion protein.

8. The method according to claim 7, wherein the amino acid sequence of the fusion protein is as shown in SEQ ID No. 5.

9. A kit for detecting an antigen of interest in a sample using the barnase/barstar system, said kit comprising the following reagents:

10. The kit of claim 9, wherein the chemiluminescent label is the fluorescent dye ATTO 488.

11. The kit of claim 9, wherein the matrix material is agarose microspheres.

12. The kit of claim 9, wherein the matrix material is a solid matrix.

13. The kit of claim 9, wherein the first antibody and the second antibody are nanobodies.

14. The kit according to claim 13, wherein the sequence of the first nanobody is shown in SEQ ID No.1, and the sequence of the second nanobody is shown in SEQ ID No. 2.

15. The kit according to claim 14, wherein the first nanobody is linked to a barstar whose amino acid sequence is shown in SEQ ID No.3 and the amino acid sequence of barnase coated with the matrix material is shown in SEQ ID No.4 as a fusion protein.

16. The kit according to claim 15, wherein the amino acid sequence of the fusion protein is shown in SEQ ID No. 5.