KR20170028637A

KR20170028637A - Recombinant Secondary Antibody Mimic as a Target-specific Signal Amplifier and an Antibody Immobilizer in Immunoassays

Info

Publication number: KR20170028637A
Application number: KR1020150125494A
Authority: KR
Inventors: 강세병; 민준선; 박태주; 송은경
Original assignee: 울산과학기술원
Priority date: 2015-09-04
Filing date: 2015-09-04
Publication date: 2017-03-14

Abstract

The present invention relates to a general-purpose recombinant secondary antibody analogue in charge of amplifying a signal in an immunochemical analysis and fixing the surface of an antibody. Provided is a GST-ABD recombinant secondary antibody analogue fused with a glutathione-S-transferase (GST) protein and an antibody-binding domain (ABD) protein bound with an Fc region of a primary antibody; or an HRP-GST-ABD recombinant secondary antibody analogue fused with a horseradish peroxidase (HRP) protein, a glutathione-S-transferase (GST) protein, and an antibody-binding domain (ABD) protein bound with an Fc region of a primary antibody. The general-purpose recombinant secondary antibody analogue of the present invention can be mass-produced at cheap costs, and can replace a secondary antibody in an immunochemical analysis. Thus, it is expected that the utilization thereof is very high.

Description

{Recombinant Secondary Antibody Mimic as a Target-specific Signal Amplifier and an Antibody Immobilizer in Immunoassays}, which is responsible for signal amplification and surface immobilization of an antibody in an immunochemical analysis,

The present invention relates to a universal recombinant secondary antibody analog that is responsible for signal amplification and surface immobilization of antibodies in immunochemical assays.

Immunochemical assays generally consist of primary antibodies specific to the target and enzyme-conjugated secondary antibodies conjugated to the enzyme. Both the primary antibody and the secondary antibody are generated and separated in small animals, which is costly and time consuming. In particular, the primary antibody determines the target specificity, while the secondary antibody binds to the Fc region of the primary antibody bound to the target irrespective of the target, amplifying the signal so that a very small sample can be measured with very high sensitivity do. Since the secondary antibody has no target specificity, the cost and time of immunochemical analysis can be drastically reduced if it is possible to produce an alternative substance with the property of binding to the primary antibody at low cost.

Conventional biosensor materials have focused on probes specific to each target, but most of them are used only for one or two targets. In the present invention, instead of developing a limited target specific probe, a system capable of amplifying a universal signal by recognizing all kinds of primary antibodies binding to a target is constructed, Instead, mass production using a cheap Escherichia coli expression system can dramatically save costs and time by maintaining or improving the benefits of immunochemical assays.

Korean Patent Laid-Open No. 10-2012-0070692 (2012.07.02)

An object of the present invention is glutathione - S - transferase raised (glutathione- S -transferase; GST) protein, and primary antibody that binds to the Fc region of an antibody domain (antibody-binding domain, ABD) ABD-GST fusion protein is a recombinant secondary antibody analogue or mustard radish peroxidase (horseradish peroxidase; HRP) protein, glutathione - S - transferase raised (glutathione- S -transferase; GST) domain antibody binding (antibody-binding proteins that bind to the Fc region of the primary antibody and 1 domain, ABD) fusion protein of HRP-GST-ABD recombinant secondary antibody.

It is another object of the present invention to provide a method for producing the recombinant secondary antibody analogue.

It is still another object of the present invention to provide an immunochemical analysis method using the recombinant secondary antibody analogue.

In order to achieve the above object, the present invention provides glutathione - S - transferase raised (glutathione- S -transferase; GST) protein, and antibody binding domains (antibody-binding domain, ABD) that binds to the Fc region of the primary antibody fusion protein RTI ID = 0.0 > GST-ABD < / RTI > recombinant secondary antibody analog.

The invention also mustard radish peroxidase (horseradish peroxidase; HRP) protein, glutathione - S - transferase raised (glutathione- S -transferase; GST) domain antibody binding (antibody-binding proteins that bind to the Fc region of the primary antibody and 1 domain, ABD) protein is fused to an HRP-GST-ABD recombinant secondary antibody analogue.

The present invention also provides a recombinant expression vector comprising a gene encoding GST protein and a gene encoding ABD protein, and a recombinant microorganism transformed with said recombinant expression vector.

The present invention also provides a method for producing the GST-ABD recombinant secondary antibody analog or the HRP-GST-ABD recombinant secondary antibody analog.

The present invention also provides an immunochemical analysis method using the GST-ABD recombinant secondary antibody analog or the HRP-GST-ABD recombinant secondary antibody analog.

The present invention relates to a general-purpose recombinant secondary antibody analogue that is responsible for signal amplification and surface immobilization of an antibody in an immunochemical assay, and comprises an antibody-binding domain (USA) that selectively binds strongly to the Fc region of the primary antibody ) And GST were fused to develop universal recombinant secondary antibody analogues that are cheap and mass-producible to replace secondary antibodies in immunochemical assays.

Figure 1 is a schematic representation of the preparation and application of GST-ABD, a recombinant secondary antibody analog.
Figure 2 shows that GST-ABD binds to immobilized IgGs as well as to three different species-derived capture-free IgGs. (Δ F ) profile of GST-ABD (black line) or GST (red line) on a gold (AC) gold QCM sensor. (A), rabbit (B) and rat (C) IgGs on a monolayer of GST-ABD (black line) or GST (red line). (DF) IgG-immobilized gold SPR sensor GST-ABD (black line) and application curve (colored line). Mouse (D), rabbit (E) and rat (F). (G) represent associative ( k _a ) and dissociating ( k _d ) ratio constant values and associating ( K _a ) and dissociating ( K _d ) constant values for the indicated antibody.
Figure 3 is for an HRP-GST-ABD secondary antibody analog (AG) that amplifies the signal in an indirect ELISA, and in a directionally-controlled manner on the surface of a GSH-coated plate in a sandwich-type indirect ELISA with an antigen- For the GST-ABD, which is applied as a fixed medium of (HK). (A) Primary antibody and HRP-conjugated secondary antibody or HRP-GST-ABD. (B) or mouse anti-BSA primary antibody (C), rabbit anti-EpCAM (D) or anti-BSA (B) Mouse anti-EpCAM primary antibody (E) was applied. OPD and H ₂ O ₂ were added to HRP-GST-ABDs (black square, BE) or anti-rabbit secondary antibody (red circle, BE) and then absorbance was measured and plotted at 450 nm. The linear response of each measured value was plotted on each graph. The LODs of each measurement for BSA (F) and EpCAM (G) were plotted. (H) antigen-trapping antibody. (IJ) GST-ABDs were treated on the surface of GSH-coated plates and saturated with the capture antibodies anti-BSA IgGs (I) or anti-EpCAM rabbit IgGs (J). Various amounts of BSA (I) or EpCAM (J) were added and the absorbance was measured and plotted at 450 nm after adding OPD and H ₂ O ₂ to the HRP-conjugated anti-rabbit secondary antibody (IJ). The linear response of each measured value was plotted on each graph. (K) LODs of BSA and EpCAM in the above formats were plotted.
Figure 4 shows that HRP-GST-ABD can effectively and efficiently amplify signals in TSA-based immunostaining. (A) Anti-acetylated tubulin primary antibody (mouse) was used to detect nerve fibers in a cut Xenopus sample. The forebrain region was transected and nerve fibers were visualized as HRP-GST-ABD and tyramide-Alexa 488 (green). The nucleus was stained with DAPI (blue). Cerebellar nerve fibers were stained vividly in green (A ') and ciliary axonemes were specifically stained only in multi-ciliated cells (A "). The scale bars in A 'and A "are 20 μm and 5 μm, respectively. (B) To detect proliferating cells, anti-phospo-histone H3 primary antibody (rabbit) was used (green). After the TSA reaction, tubulin was stained with anti-tubulin antibody (rabbit) (red color) and stained with Alexa-555 conjugated anti-rabbit secondary antibody. The scale bars at B 'and B "are 20 μm and 5 μm, respectively. (C) In order to detect the extracellular matrix, anti-fibronectin primary antibody (mouse) was used (green). The scale bars at C 'and C "are 20 μm and 5 μm, respectively. (DE) anti-phospho-histone H3 primary antibody (rabbit, D) or anti-acetylated tubulin primary antibody (mouse, E) was used to stain whole mount embryos. Anti-actin antibodies were used to visualize cell boundaries (red) (D). D ' and D ' denote the confocal image of the cut surface shown. (E) HRP-GST-ABD successfully penetrated into whole mount embryos and amplified the signal of the target molecule specifically in the whole mount sample. The scale bar at D and E is 20 μm.

Accordingly, the present invention relates to a method for fusing an antibody-binding domain (ABD), which selectively binds strongly with the Fc region of a primary antibody, with various kinds of proteins, The universal recombinant secondary antibody analogue which can be mass produced inexpensively was developed and its usefulness was verified by ELISA, western blot, immunostaining, etc., and the present invention was completed.

The invention glutathione - S - transferase raised (glutathione- S -transferase; GST) protein, and primary antibody that binds to the Fc region of an antibody domain (antibody-binding domain, ABD) ABD-GST fusion protein is a recombinant secondary Antibody analogs.

Preferably, the GST protein may be represented by the amino acid sequence of SEQ ID NO: 1, and the ABD protein may be represented by the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

The present invention also provides a recombinant expression vector comprising a gene encoding GST protein and a gene encoding ABD protein.

Preferably, the gene encoding the GST protein may be represented by the nucleotide sequence of SEQ ID NO: 3, and the gene encoding the ABD protein may be represented by the nucleotide sequence of SEQ ID NO: 4, but the present invention is not limited thereto.

In the present invention, " vector " means a DNA molecule that is replicated by itself, which is used to carry the clone gene (or another fragment of the clone DNA).

In the present invention, an "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. The expression vector may preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include, but are not limited to, antibiotic resistance genes such as ampicilin, kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, It can be selected appropriately.

The present invention also provides a recombinant microorganism transformed with the recombinant expression vector. Preferably, the microorganism may be E. coli, but is not limited thereto.

The method of introducing the expression vector into a microorganism and transforming the expression vector is preferably carried out by a method known in the art such as, The present invention also relates to a method of transfection comprising the steps of injection, transduction, cell fusion, calcium phosphate precipitation, liposemmediated transfection, DEAE dextran-mediated transfection, polybrene- polybrene-mediated transfection, electroporation, and the like.

Also, the present invention provides a method for producing a recombinant microorganism, comprising: culturing the recombinant microorganism to express a GST-ABD recombinant protein; And separating the GST-ABD recombinant protein from the GST-ABD recombinant protein.

The present invention also provides a sandwich-type indirect ELISA immunoassay using a GST-ABD recombinant secondary antibody analogue as an anchoring adapter for antigen-capturing antibodies &Lt; / RTI >

Also, the present invention provides a method for producing a recombinant microorganism, comprising: culturing the recombinant microorganism to express a GST-ABD recombinant protein; Isolating the GST-ABD recombinant protein; And chemically binding an HRP protein to the separated GST-ABD recombinant protein. The HRP-GST-ABD recombinant secondary antibody analogue production method comprises the steps of:

In addition, the present invention provides a method for producing an HRP-GST-ABD recombinant secondary antibody, comprising: specifically binding a primary antibody to the HRP-GST-ABD recombinant secondary antibody analog; And detecting an antigen-antibody-specific binding by treating an antigen specific to the primary antibody.

The immunochemical analysis method may be any method capable of confirming the specific binding of an antigen-antibody, and may be an immunochemical analysis method through a visually, optically, or electrochemically method.

Preferably, it may be, but is not limited to, simple indirect ELISA or tyramide signal amplification (TSA) immunostaining.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Experimental Example >

The following experimental examples are intended to provide experimental examples that are commonly applied to the respective embodiments according to the present invention.

1. Recombinant secondary antibody analog ( GST - USA ), Expression and purification

The optimal gene encoding the antibody binding domain (58 amino acids) with 27 additional amino acids at the N-terminus was synthesized, and a glutathione-S-transferase containing a hexa-histidine tag at the N- terminal of the glutathione- S- transferase (GST) and inserted into the IPTG-induced pETDuet expression vector (Invitrogen). The amplified DNAs were transformed with competent E. coli strain BL21 (DE) and the recombinant secondary antibody analog (GST-ABD) was transformed with 500 μM isopropyl pD-1-thiogalactopyranoside ; IPTG) was added and over-expressed at 30 ° C overnight. Cells were obtained by high speed centrifugation and sequentially suspended in lysis buffer (50 mM phosphate, 100 mM NaCl and pH 6.5). Treated with resuspension solution containing lysozyme (50 μg / ml) for 1 hour at room temperature, and sonicated for 10 minutes at intervals of 30 seconds. Thereafter, centrifugation was performed at 12000 g for 1 hour to obtain a supernatant containing GST-ABD. The final GST-ABD was further purified by immobilized metal affinity chromatography (IMAC). Purity and molecular weight of GST-ABD were confirmed by SDS-PAGE, UV / visible spectrophotometer and mass spectrometer.

2. Quartz crystal microbalance ; QCM ) Measure

Standard gold QCM sensors and Q-Sense E4 (Biolin Scientific) were used. The overall system was run in a flow mode with a pump maintaining the temperature at 25.0 ± 0.1 ° C. Each sample solution was injected into a measuring chamber with a pump and phosphate buffer (50 mM phosphate, 100 mM NaCl, pH 6.5) was used for washing before each sample application. 0.1 mg / ml GST-ABD and GST, or rabbit, mouse and rat IgGs were applied to the same phosphate buffer. Resonance frequencies were simultaneously measured at 7 harmonics (5, 15, 25, 35, 45, 55 and 65 MHz) and only standardized frequencies of the third overtone were shown for clarity.

3. Surface Plasmon Resonance plasmon resonance; SPR ) analysis

SPR experiments were carried out at 25 ° C using Carboxyl Dextran CM5 standard gold sensor chip on Biacore 3000 device. HBS-EP buffer solution and PBS buffer solution were used as immobilized solution. Rabbit, mouse, and rat IgGs were initially coupled to carboxyl functional groups exposed on the CM5 sensor chip surface via standard amine-coupling chemistry. Briefly, a mixture of EDC (0.5 mg / ml) and NHS (0.5 mg / ml) was gently mixed and injected onto the sensor chip at a flow rate of 10 μl / min to activate the carboxyl groups of the sensor chip. Then 0.1 mg / ml of rabbit, mouse and rat IgGs were individually injected into three different channels at the same flow rate for 7 minutes to fix on the surface of the sensor chip. The remaining unused reactor of the CM5 sensor chip was blocked with 1M ethanolamine (pH 8.0). Various concentrations of GST-ABD or GST (54, 108, 216 and 432 nM) were loaded onto the IgG-immobilized CM5 sensor chip at a flow rate of 10 μl / min for 7 minutes. Binding and release kinetics of GST-ABD to various IgGs were analyzed by Biaevaluation software using a 1: 1 Langmuir binding model.

4. GST - With USA Fluorescent dyes or Of HRP Chemical bonding

To bond GST-ABD with fluorescein-5-maleimide (F5M), a thiol-reactive fluorescent dye, GST-ABD was shaken vigorously with 10 mol equivalent of F5M at room temperature overnight . Unreacted free F5M overnight was removed by intensive dialysis against phosphate buffer (50 mM phosphate, 100 mM NaCl, pH 6.5). The degree of F5M binding to GST-ABD was measured by ESI-TOF mass spectrometry and UV / Vis spectrophotometer. On average, three fluororesins were attached to one GST-ABD. HRP-conjugated GST-ABD (HRP-GST-ABD) was prepared by conjugating EZ-Link Maleimide Activated Horseradish Peroxidase (Mal-HRP, Thermo Scientific) to GST-ABD. GST-ABD was reacted with 2.5, 5 or 10 mol equivalents of Mal-HRP by shaking vigorously overnight at room temperature. Unconjugated free Mal-HRPs and GST-ABDs were removed using centricon ultrafiltration (Millipore, MWCO: 100,000 Da). To produce HRP-GST-ABD, GST-ABD and 10 mol equivalent of Mal-HRP were treated to completely consume GST-ABD, leaving some residual free Mal-HRPs that could be easily removed by centric ultrafiltration.

5. Simple indirect ELISA (Simple indirect ELISA)

Model antigens BSA and EpCAM were purchased from Santa Cruz and Sino Biological Inc. To prepare the appropriate protein solution (final 2 nM), BSA and EpCAM were dissolved in PBS. Each protein solution (100 μL each) was loaded into the wells of an Immuno MicroWell 96-well plate (Maxisorp, flat-bottom, pinchibar, 400 μL) and incubated at 4 ° C Lt; / RTI > overnight. The next day, each well was washed 3 times with 250 μL PBS buffer (PBST) containing 0.1% Tween 20. Blocking buffer (PBS, 0.5% Tween 20, 5% goat serum) was loaded (200 μL each) and reacted at 37 ° C for 1 hour. After blocking, a primary antibody (rabbit antibody purchased from Santa Cruz Biotech and mouse antibody purchased from Abcam) or primary antibody against EpCAM (rabbit and mouse antibody purchased from Sino Biological Inc.) was dissolved in PBS buffer &Lt; / RTI > The preparation solution (100 [mu] L each) was loaded into the wells of BSA or EpCAM-immobilized plates and reacted at 37 [deg.] C for 1 hour. Each well was washed three times with 250 μL PBST. HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were then added (100 μL each) to make sample wells, and the mixture was reacted at 37 ° C for 1 hour. It was then washed 3 times with 250 μL PBST. HRP substrate solutions were prepared according to the manufacturer's instructions using SIGMAFAST OPD kit (Sigma-Aldrich) (0.4 mg / mL o-phenylendiamine (OPD), 0.4 mg / mL urea hydrogen peroxide, 0.05 M phosphate- ), 100 [mu] L of solution was loaded into each well. The reaction proceeded at room temperature for 1 minute or 5 minutes depending on the sample set, and 100 μL of 2N H ₂ SO ₄ solution was immediately added to each well to stop the reaction. The absorbance at 450 nm was observed in each well via a multimode microplate reader, infinite 200 PRO (TECAN). All experiments were performed independently 3 times and plotted with error.

6. Sandwich-type indirect ELISA (Sandwich-type indirect ELISA)

To obtain a final concentration of 4 nM, GST-ABD was prepared by dissolving in PBS. The prepared GST-ABD solution (100 μL each) was loaded onto a glutathione (GSH) -coated plate (Clear, 8-well strips, Thermo Scientific Pierce) and loaded with GST-ABD and GSH immobilized on the plate surface Lt; RTI ID = 0.0 > 37 C < / RTI > for 2 hours. To remove residual GST-ABD, each well was washed 3 times with 250 μL PBST. Capture-primary antibodies (rabbit IgG, Sino Biological Inc.) to BSA-derived primary antibodies (rabbit IgG, Santa Cruz Biotech) and EpCAM were prepared and added to GST-ABD-attached wells 100 [mu] L). After reacting for 2 hours at 4 ° C, each well was washed 3 times with 250 μL PBST. BSA and EpCAM solutions were prepared at various concentrations and loaded independently into the prepared wells (100 μL each). The mixture was reacted at 37 DEG C for 1 hour, and each well was washed again. Detection of primary antibodies (mouse IgG, Abcam) for BSA and detection of primary antibodies for EpCAM (mouse IgG, Sino Biological Inc.) were prepared and loaded into the prepared wells (100 μL each). The mixture was reacted at 37 ° C for 1 hour, and each well was washed 3 times with 250 μL PBST. The HRP-conjugated anti-mouse secondary antibody (Abcam) was added (100 μL each) and further reacted at 37 ° C for 1 hour, and the mixture was washed 3 times with 250 μL PBST. Then, 100 μL substrate solution was loaded into each well and the reaction was stopped after 2 minutes by adding 100 μL of 2N H ₂ SO ₄ solution to each well. The absorbance at 450 nm was observed in each well via a multimode microplate reader, infinite 200 PRO (TECAN). All experiments were performed independently 3 times and plotted with error.

7. TSA -Based cell immune staining

Tyramide signal amplification (TSA) kit was purchased from Life Technology and all reagents were prepared according to the manufacturer's instructions. Cell line SKBR3 and KB cells were purchased from Korean cell line bank (KCLB). SKBR3 and KB cells were treated with L-glutamine (300 mg / L), 10% fetal bovine serum (FBS, Invitrogen), antibiotics (1000 units / ml penicillin, 10000 μg / ml streptomycin and 25 μg / 0.0 > 37 C < / RTI > in the presence of 5% CO ₂ in RPMI 1640 medium containing 25 mM HEPES and 25 mM NaHCO ₃ .

For TSA analysis, SKBR3 and KB cells were cultured in 12-well microscopy chambers (1x10 ⁵ cells / well), washed with PBST, fixed with 4% paraformaldehyde for 20 min at 4 ° C, And washed again with PBST. Immobilized cells were permeabilized with 0.1% Triton ^® X-100 solution for 10 minutes at room temperature and rinsed with PBST. Fixed SKBR3 and KB cells were treated with anti-HER2 rabbit primary antibody and anti-integrin αβγ ₃ primary antibody at room temperature for 1 hour, washed with PBST to remove unbound or non-specific binding antibody, I got it. The HRP-conjugated rabbit or mouse secondary antibody was prepared by diluting the stock solution to 1/100 in 1% blocking solution, and HRP-GST-ABD was dissolved in the same solution and prepared in equal amounts. The primary antibody-treated cells were treated with HRP-conjugated secondary antibody or HRP-GST-ABD (100 μL each) solution for 1 hour at room temperature and rinsed again with PBST to remove the HRP-conjugate. A Tyramide working solution was prepared fresh by diluting the thiamide stock solution immediately before the reaction with 1/100 in an amplification buffer containing 0.0015% H ₂ O ₂ , and 100 μL was added to each sample . The reaction mixture was reacted at room temperature for 10 minutes and rinsed with PBST. In addition, the nuclei of the cells were independently stained with 6-diamidino-2-phenylindole (DAPI, Sigma). Fluorescent cell images were obtained using an Olympus Fluoview FV1000 confocal microscope (Olympus, UOBC).

8. Embryo sample preparation

To analyze the recombinant secondary antibody analog (HRP-GST-ABD), we used MEMFA (MEM salts and 4% formaldehyde) or Dent's fixative solution (methanol and 20% DMSO) The embryos were fixed. The immobilized embryos were then used for direct immunostaining or forebrain transverse sections fabricated to a thickness of 50 μm using a biomicroscope (vibratome).

9. Cut embryonic slices and whole embryos TSA -Based immunostaining

To block non-specific binding, embryonic or truncated embryo slices were reacted with blocking solution (5% BSA + 2% DMSO in TBS + 0.1% Triton X-100) for 30 minutes at room temperature. TSA-staining was performed for 3 hours at room temperature by diluting the following primary antibodies to 1: 300 or 1: 1000: anti-acetylated Tubulin (Sigma-Aldrich), anti- phospho-Histone H3 (Abcam) Fibronectin (DSHB), and anti-MHC (DSHB). Samples were rinsed with TBST (TBS + 0.1% Triton X-100). The peroxidase label was diluted 1: 1000 with HRP-conjugated secondary antibody (Sigma-Aldrich) or HRP-GST-ABD for 2 hours. For HRP-GST-ABD, the indicated primary antibodies were pre-reacted with HRP-GST-ABD in a 1: 1 ratio (0.8 ng / μl each) and tissue samples were mixed and reacted. Tyramide labeling was performed using tyramide Alex-488 dissolved in an amplification buffer containing 0.00015% hydrogen peroxide. The mixture was allowed to react at room temperature for 2 minutes, and the tissue samples were rinsed with TBST. The nuclei of the tissues were independently stained with DAPI (Abcam). Prior to clarification in BA: BB (benzyl alcohol / benzyl benzoate, 1: 2), all cut slices were rinsed with 100% methanol and reacted and mounted on slides for imaging. Images were captured using confocal microscopy analysis (LSM700).

< Example 1 > primary antibody Fc Establish a second antibody analogue platform that selectively binds to the site

Primary selective antibody that binds to the Fc region of an antibody domain (antibody-binding domain; ABD) and glutathione - S - transferase raised; and the (glutathione- S -transferase GST) coupled genetically build secondary antibody analogs platform , And constructed a system capable of mass production in E. coli. Such mass-produced recombinant secondary antibody analogs can be used to replace secondary antibodies in a variety of immunochemical assay systems, including ELISA and immuno-staining, or in antigen-trapped immobilized form in a sandwich-type indirect ELISA And to be applied as an anchoring adapter for antibodies (Fig. 1).

GST-ABD was produced in large quantities by a bacterial over-expression system and was simply separated by minimizing and optimizing cost and time by using a first stage Ni-NTA agarose column chromatography. High-purity GST-ABD (> 99%) was obtained, which was confirmed by SDS-PAGE, and the successful fusion of GST and ABD was confirmed by ESI-TOF MS.

< Example 2> GST - With USA Antibody Cohesion analysis

In order to confirm the binding ability of GST-ABD to antibodies derived from three different species (mouse, rabbit and rat), we first investigated the interaction between immobilized GST-ABD and pre-IgGs in the quartz crystal microbalance (QCM) (Fig. 2A, B and C). Independently adding three different types of IgGs significantly reduced the resonance frequencies of the GST-ABD-monolayered QCM sensor (black line), while the GST-monolayer samples did not (Figs. 2A, B, and C). To accurately determine the binding and dissociation properties of GST-ABD to various types of IgGs, we performed surface plasmon resonance (SPR) analysis (FIGS. 2D, E, and F). In contrast to the QCM study, the inventors first applied different concentrations of GST-ABD after immobilizing each IgG molecule (from mouse, rabbit or rat) onto standard SPR CM5 chips. Application of various concentrations of GST-ABD solution and wash buffer, respectively, observed a rapid increase and a very slow decrease in the SPR response unit (RU) (FIGS. 2D, E and F) It was confirmed that there was almost no change. GST-ABD was found to bind strongly to all types of IgGs from three different species. On the other hand, it was most strongly bound to rabbit IgG ( Kd 1.31 nM) and weakly bound to mouse IgG ( Kd 16.8 nM). The relatively fast dissociation rate constant of mouse IgGs may cause a difference in dissociation constant (Figure 2G). QCM and SPR studies with various IgGs have shown that GST-ABD can be applied as a universal IgG recognition molecule that binds immobilized IgGs as well as capture-free IgGs on a solid surface via simple molecular recognition.

< Example 3> In an indirect ELISA, an enzyme-linked recombinant secondary antibody analogue chain HRP - GST - Application of ABD

The GST region of GST-ABD has four cysteine groups that can chemically conjugate enzymes capable of signal amplification with a maleimide function. The HRP-GST-ABD is generated by conjugating the activated horseradish peroxidase (HRP) to GST-ABD and used as a secondary antibody substitute for HRP conjugation in various immunochemical assays. By chemically conjugating an average of three HRPs per molecule of GST-ABD, signal amplification and measurement sensitivity can be improved 2-3 times.

In order to evaluate the ability of HRP-GST-ABD as a commonly used HRP-conjugated secondary antibody substitute, the present inventors first performed a simple indirect ELISA. We fixed a fixed amount of BSA as an antigenic model on the surface of ELISA plates through physical adsorption and applied various concentrations of rabbit anti-BSA primary antibody. The inventors then washed intensively and then loaded HRP-conjugated anti-rabbit secondary antibody or HRP-GST-ABD. Next, the present inventors added o-phenylendiamine (OPD) substrate solution and measured the absorbance at 450 nm immediately or after 5 minutes. Absorbance readings were linearly increased in the range of about 40 pM to 5 nM as the concentration of the primary antibody increased in the HRP-GST-ABD and anti-rabbit HRP-labeled secondary antibody treated samples (Figures 3A and 3B) insertion). Interestingly, HRP-GST-ABD treated samples at medium and high concentrations showed significantly improved signals than anti-rabbit HRP-labeled secondary antibodies (FIG. 3A). This is expected to be due to the higher number of HRPs conjugated to HRP-GST-ABD (an average of 3 HRPs per GST-ABD molecule). Although we observed significantly improved signals in the intermediate and high concentrations of the primary antibody, we could not confirm a significant increase in the limit of detection (HRP-GST-ABD; 22 pM and anti-rabbit HRP-labeled secondary antibody; 25 pM) (Figures 3A and 3E).

Since GST-ABD binds to mouse IgG as well as rabbit IgG without any modification (Fig. 2), the present inventors have found that HRP-GST-ABD can be used as a universal secondary antibody analogue to various types of IgGs from various species . The present inventors substituted the rabbit primary anti-BSA IgG with mouse primary anti-BSA IgG and performed the same experiment as above. The samples treated with anti-rabbit HRP-labeled secondary antibodies did not show any signal at all of the concentration conditions applied, whereas the samples treated with HRP-GST-ABD showed almost the same graph as the results of rabbit primary anti-BSA IgG ( 3B). However, HRP-GST-ABD binding affinity for the mouse primary antibody was weak, requiring 5 min to obtain LOD (29 pM) and signaling results (FIG. 3B). In addition, we performed similar experiments using epithelial cell adhesion molecules (EpCAM), also known as biomarkers of circulating tumor cells (CTC) as other antigenic proteins. When we applied rabbit primary anti-EpCAM IgG and HRP-GST-ABD, we obtained approximately the same LOD (25 pM) and linear range for BSA (40 pM - 2.5 nM) (Fig. 3C and 3F). In addition, mouse primary anti-EpCAM showed almost the same result as BSA (FIG. 3D). Taken together, the above results suggest that HRP-GST-ABD can be used as a secondary antibody analog for signal amplification for any type of antigen molecule, if primary antibodies related to rabbit, mouse and rat species are available Can be applied.

< Example 4> Recombinant secondary antibody analogues GST - USA Applied as a fixed medium for antigen-trapping antibodies

The sandwich-type indirect ELISA system is one of the most popular formats. To capture a target antigen molecule in a complex biological sample, the antigen-capture antibody must be immobilized firmly on the surface of the ELISA plate with proper orientation. Chemical immobilization or related methods of antigen-trapping antibodies often impair the antigen-binding ability of the capture antibody due to alteration, denaturation, or erroneous orientation of the antigen-binding site of the antibody. Since the GST-ABD has a species-independent antibody-binding ability at one end (ABD) and a glutathione (GSH) -binding ability at the other end (GST) Can be bound to the surface of the coated plate and the remaining ABD moiety can bind to the Fc region of the capture antibody with proper orientation to detect the target antigen (Figures 3G and 3H). We treated several concentrations of GST-ABDs on commercially available GSH-coated plates. Unlike the control (mono-streptavidin-fused ABD), we observed a general curve that increased with increasing GST-ABD concentration and eventually saturates. Next, we determined the optimal dose of reagent used in each step of the sandwich-type indirect ELISA before applying it to the system for detecting antigens. We immobilized a certain amount of GST-ABD on the surface of GSH-coated plates and saturated respectively with capture antibodies against anti-BSA or anti-EpCAM rabbit IgGs, adding various amounts of BSA or EpCAM, The amount of antigen used in the normal sandwich type indirect ELISA was determined (Figures 3G and 3H). Similar results were obtained for LODs (BSA; 33 pM and EpCAM; 36 pM) and linear response when compared to previously tested simple indirect ELISA (Figure 3). The above results show that the GST-ABD of the present invention utilizes the interaction between molecules simply without chemical and physical modification, and thus it is possible to prevent the side effects such as the inactivation of the antibodies generally occurring in the complex antigen-trapping antibody immobilization process of the ELISA To simplify the sandwich-type indirect ELISA procedure and to ensure reproducibility of the results.

< Example 5> In immunochemical staining HRP - Substitution of secondary antibody HRP - GST - US apply

Tyramide signal amplification (TSA) assays are frequently used to detect low concentrations of biomolecules such as proteins, DNAs and RNAs in cells and tissues. The present inventors have confirmed that HRP-GST-ABD can be used as an HRP-conjugated secondary antibody substitute in an immuno-staining system. First, the present inventors first IgGs involved in two different cancer cell master SKBR3 and KB cells overexpressing HER2 and each integrin receptors on the cell surface αβγ ₃ (wherein each -HER2 rabbit and anti-integrin αβγ ₃ mouse IgGs) in TSA- based Were immunostained. The present inventors observed almost the same signal increase in both the HRP-GST-ABD and HRP-conjugated secondary antibodies treated with the sample in all the measured ranges. TSA-based immunostaining for truncated tissue or whole mount samples is more difficult than immunostaining for cultured cells, which not only requires higher specificity and signal-to-noise ratios, but also heterogeneity and complexity of the sample, Is also high. Thus, the present inventors prepared a frog (Xenopus) embryo and performed TSA-based immunostaining with HRP-GST-ABD. The 43-step frog (Xenopus) embryo is developed primarily into organs and tissues and is suitable for staining certain tissues from cut or whole mount samples. The present inventors have tested various tissue-specific antibodies, such as anti-acetylated tubulin antibodies and anti-myosin heavy chain (MHC) antibodies, in order to visualize nerve fibers and muscles, respectively (Figs. 4A and 4E). In addition, anti-phospho histone H3 (FIG. 4C) and anti-fibronectin antibody (FIG. 4D) were used to detect the intracellular location of the target in cut and whole mount samples . In all cases HRP-GST-ABD showed high target-specific signal amplification results in cuts (Figures 4A-4C) and whole mount samples (Figures 4D and 4E) and the origin of the primary antibody used Rabbit or mouse), it was almost similar to the HRP-conjugated secondary antibody. Unlike the conventional method of sequentially treating the primary antibody and the secondary antibody to avoid antibody aggregation, the time required for preparing a substantial amount of sample by simultaneously reacting the primary antibody and HRP-GST-ABD before the sample treatment . On the other hand, HRP-GST-ABD is more advantageous because it can penetrate deep into cells or tissues because its molecular size is much smaller than HRP-conjugated secondary antibody, Can be measured with high sensitivity.

<110> UNIST Academy-Industry Research Corporation <120> Recombinant Secondary Antibody Mimic as a Target-specific Signal Amplifier and an Antibody Immobilizer in Immunoassays <130> ADP-2015-0257 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 220 <212> PRT <213> Artificial Sequence <220> <223> GST <400> 1 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Ser Ile Ala Tyr Ser 100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125 Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp 210 215 220 <210> 2 <211> 57 <212> PRT <213> Artificial Sequence <220> <223> US <400> 2 Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu 1 5 10 15 His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser 20 25 30 Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys 35 40 45 Lys Leu Asn Asp Ala Gln Ala Pro Lys 50 55 <210> 3 <211> 660 <212> DNA <213> Artificial Sequence <220> <223> GST <400> 3 atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60 ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120 tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180 ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240 atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300 gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360 gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420 acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480 gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540 aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600 tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660 660 <210> 4 <211> 171 <212> DNA <213> Artificial Sequence <220> <223> US <400> 4 gataacaaat ttaacaaaga acagcagaac gcgttttatg aaattctgca tctgccgaac 60 ctgaacgaag aacagcgcaa cgcgtttatt cagagcctga aagatgatcc gagccagagc 120 gcgaacctgc tggcggaagc gaaaaaactg aacgatgcgc aggcgccgaa a 171

Claims

GST-ABD recombinant secondary antibody analogue fused with glutathione- S- transferase (GST) protein and an antibody-binding domain (ABD) protein binding to the Fc region of the primary antibody.

The GST-ABD recombinant secondary antibody analogue according to claim 1, wherein the GST protein is represented by the amino acid sequence of SEQ ID NO: 1.

The GST-ABD recombinant secondary antibody analogue according to claim 1, wherein the ABD protein is represented by the amino acid sequence of SEQ ID NO: 2.

A recombinant expression vector comprising a gene encoding GST protein and a gene encoding ABD protein.

5. The recombinant expression vector according to claim 4, wherein the gene encoding the GST protein is represented by the nucleotide sequence of SEQ ID NO: 3, and the gene encoding the ABD protein is represented by the nucleotide sequence of SEQ ID NO:

A recombinant microorganism transformed with the recombinant expression vector according to claim 4 or 5.

The recombinant microorganism according to claim 6, wherein the microorganism is E. coli.

Culturing the recombinant microorganism according to claim 6 to express a GST-ABD recombinant protein; And
And separating the GST-ABD recombinant protein from the GST-ABD recombinant protein.

A sandwich-type indirect ELISA (Sandwich-type) using a GST-ABD recombinant secondary antibody analogue according to any of claims 1 to 3 as an anchoring adapter for antigen-capturing antibodies, Immunochemical assay method.

Mustard radish peroxidase (horseradish peroxidase; HRP) protein, glutathione transferase raised -S- (glutathione- S -transferase; GST) domain antibody binding (antibody-binding domain, ABD) that binds to protein 1 and Fc portion of the primary antibody protein This fused HRP-GST-ABD recombinant secondary antibody analog.

Culturing the recombinant microorganism according to claim 6 to express a GST-ABD recombinant protein;
Isolating the GST-ABD recombinant protein; And
And chemically binding an HRP protein to the separated GST-ABD recombinant protein. The HRP-GST-ABD recombinant secondary antibody analog production method according to claim 1,

12. A method for producing a recombinant human HRP-GST-ABD recombinant secondary antibody, comprising: And
And detecting an antigen-antibody-specific binding by treating an antigen specific to said primary antibody.

The immunochemical assay method according to claim 12, wherein the immunochemical analysis method is a simple indirect ELISA or a tyramide signal amplification (TSA) immunostaining method.