KR20100101303A - Specific protein domain for the detection of salmonella contamination and manufacture method of its antibody and immuno-fluorescent psq nanoparticle - Google Patents

Abstract

PURPOSE: An antibody for detecting salmonella and immuno-fluorescent nanoparticle PSQ are provided to use in manufacturing immunobiosensor and photoimmunological biosensor. CONSTITUTION: An antibody for detecting salmonella is Salmonella HilA rabbit polyclonal antibody and Salmonella FliC rabbit polyclonal antibody. An immune-fluorescent nanopaticle PSQ is obtained by binding reaction of salmonella HilA antibody to PSQ or binding reaction of salmonella FliC antibody to PSQ.

Description

Specific Protein Domain for the Detection of Salmonella Contamination and Manufacture Method of its Antibody and Immuno-fluorescent PSQ nanoparticles

The present invention is to rapidly detect pathogenic microorganisms through signal amplification by producing an antibody for detecting Salmonella causing pathogenic food poisoning, intestinal infection and Immuno-fluorescent nanoparticle PSQ.

Conventional microbial detection has been developed by many immunoassay technologies such as biological methods, selective serological methods, DNA hybridization assays, polymerase chain reaction (PCR), radio-immunoassay, fluorescence labeled antibody assay, enzyme-link immunosorbent assays (ELISA), etc. Although the detection cost is expensive and time consuming, the pretreatment process is complex (Oh Byung-Keun. 2004 Surface plasmon resonance immunosensor for the detection of Salmonella typhimurium. Biosensors and Bioelectronics. 19: 1497-1504.) (SPR) is a well-known technique for measuring the interaction between biomolecules, and has the advantage of being able to search for high specificity and sensitivity of complex biological media samples in a short time and simply. Listeria monocytogenes (Paul Leonard. 2004. A generic approach for the detection of whole Listeria monocytogenes cells in contaminated samples using surf ace plasmon resonance.Biosensors and Bioelectronics. 19: 1331-1335.), Vibrio cholerae O1 (Jyoung Jy-Young. 2006. ), Salmonella ((Oh Byung-Keun. 2004 Surface plasmon resonance immunosensor for the detection of Salmonella typhimurium. Biosensors and Bioelectronics. 19: 1497-1504.)) Are used for the detection of various pathogenic bacteria. However, the SPR method also requires expensive equipment and skilled personnel. Therefore, we tried to develop a simpler and simpler method of detecting Salmonella strains by improving the weak signal amount of the labeled immune biosensor using fluorescent materials.

Conventional methods for microbial detection of Salmonella have problems such as high detection cost, long lead-time, complexity of pretreatment, skilled manpower, and low signal volume. Therefore, the present invention will solve the lack of signal amplification technology of fluorescent immunosensor.

Immunization was performed by immunoreactive binding of a flurorescent dye-labeled antibody to the prepared PSQ nanoparticles for the purpose of applying a labeling immunoassay using Salmonella HilA rabbit polyclonal antibody and Salmonella FliC rabbit polyclonal antibody. -Fluorescent nanopaticle PSQ was prepared. By improving the small signal volume of fluorescent immunosensor among bioimmune sensors, we propose a technique for non-researchers to easily and quickly detect pathogenic microorganisms.

The immuno-fluorescent nanoparticle PSQ combined with Salmonella HilA rabbit polyclonal antibody and Salmonella FliC rabbit polyclonal antibody, which are antibodies of Salmonella performed in the present invention, can be usefully used for the production of various immune biosensors and photoimmune biosensors. There is no need for expensive equipment, and the presence of pathogenic microorganisms can be detected quickly in large catering schools, large department stores, schools, and military facilities.

In order to achieve the above object, the present invention provides for the rapid detection of the presence of pathogenic microorganisms by making an antibody showing a specific response to Salmonella harming humans.

< Example  1> Antibody Production

Peptides with high specific antigenicity were selected for domain search for peptide sequences of Salmonella's Invasion (HilA) and Flagellin (FliC) proteins. The hydrophobicity and antigenecity of selected peptide regions were examined. In addition, the present invention was immunized by binding the carrier protein to increase the antibody titer because Peptide antigen is difficult to rise antigen titer. KLH (Keyhole Limpet Haemocyanin) was used as a carrier and bound using BSA. Emulsions were prepared using peptides conjugated to carriers and immunized. Subcutaneous injection was performed with 500 µg / 0.5 ml of emulsion per rabbit, and the primary and secondary tertiary boosting for the measurement of antigenic immunity were the same antigens at 4, 6 and 8 weeks, respectively, using IFA (Incomplete Freund's adjuvant, Difco, Rockford) was mixed in the same manner and injected by subcutaneous 200μg / 0.5ml per rabbit. A small amount of blood was collected from each boosting step to obtain serum, and antibody production and titer were measured. One week after the third boosting, blood was collected. When a small amount of blood is collected, the tail tip is cut and the blood is collected.

The immune response induced by injection of antigen was determined by ELISA to determine the degree of antibody present in rabbit serum. Antibody purification was performed by attaching the antigen to affinity-gel to prepare a column, separating the antibody, and using a column method using protein A. Finally, dialysis was performed. As a result, 15 ml (1 mg / ml) of HilA and 15 ml (10 mg / ml) of FliC were harvested. The prepared antibody was dissolved in PBS buffer containing 50% glycerol and stored at -20 ° C, and used as Ab that specifically reacts with Salmonella. Peptide antibody production process is shown in the Invasion protein HilA (Fig. 1) and Flagellin protein FliC (Fig. 2).

< Example  2> immobilization of antibodies

SPR sensor chip CM5 is coated with gold leaf on glass plate with 50nm thickness and dextran is applied on the gold leaf part. One of the test materials is used fixed to the dextran surface. The dextran surface is in contact with the flow-cell portion of the microchannel device and reacts with the sample flowing through the channel device. Depending on the model of the sensor chip, the cell contacts the cells of two or four independent flow path devices. If necessary, various experiments can be performed using different immobilized ligands for each cell. In this experiment, the carboxyl group has a negative charge on the surface of the sensor chip which is commonly used in contact with the solution. Dextran is a linear polymer of glucose, which has almost no nonspecific binding to the biological material, and the concentration of the biological material in the solution is very low. The experiment was carried out using a CM5 chip that can immobilize ligand very effectively. Prior to the experiment, the sensor chip surface was activated by preconcentrtion with 10mM sodium aetate (pH4.5-5.0) and injection of EDC / NHS for optimal Ab fixation. The antibody was diluted to 100 ug / ml in 10 mM sodium acetate (pH 5.0) and injected for 1 minute (5 μl / min). Blocking unreacted activators with 1M ethanolamine (pH 8.5) and immobilizing the CM5 chip Ab, the immobilization value is shown in FIG.

< Example  3> Salmonella cell  and HilA , FliC Ag Refractive Index Variation by Concentration of

Cell and Ag concentrations were diluted in PBS beffer to interact with the sensor chip surface for 15 minutes (1 μl / min). The results are shown in Figure 4 (HilA), 5 (FilC). The biosensor chip acted very proportionally with the change of cell concentration and the change of antigen (HilA, FliC) concentration.

< Example  4> Fluorascent nanoparticle PSQ Manufacture

Fluorescence-nanoparticle PSQ preparation is as follows. After adding vinyltrimethoxysilane and MPTMS to the trithylamine (TEA) solution, the mixture was stirred for one day at room temperature using a magnetic stirring bar. After Tween 80 was dissolved in distilled water, TEA and MPTMS were added and dissolved in turn at room temperature. In a typical preparation of PSQ, 1 g TEA was dissolved in distilled water 15 and then 1.55 g VTMS was added. The mixture was stirred for one day and then centrifuged. The harvested spheres were resuspended in 50 sterile distilled water. The spheres were washed twice with water and methanol and dried in 50 vacuum ovens for 4 days (Young Baek kim, 2006). The rhodamine labeling of PSQ spheres was performed by Van Blaaderen et al. And Fluorescence-nanoparticle PSQ spheres were prepared (Swadeshmukul Santra, 2001). The results are shown in FIG.

< Example  5> Antibody - labelled fluorascent nanoparticle PSQ Manufacture

Antibody-labeled fluorascent nanoparticle PSQ was prepared by bathing the fluorescence-nanoparticle PSQ prepared above in 10 mM PBS (pH 7.4). 25% glutaraldehyde was added, mixed for 1 hour, centrifuged and resuspended in pH 7.4 PBS buffer. Antibody was immobilized on fluorescence-nanoparticle PSQ spheres by adding 500 μl of Salmonella-rabbit polyclonal Ab (100 μg / ml). The unreacted activator was blocked by adding a quenching solution (1M ethanolamine, pH 8.5 + 0.5% BSA). Antibody-labeled fluorascent nanoparticle PSQ is shown in FIG.

<Example 6> Antibody - labelled fluorascent nanoparticle PSQ and Salmonella specific reactions

The interaction between the microorganism and the nanoparticle PSQ was analyzed by SEM, and the sensor chip was dried under vacuum to maintain the same shape in the vacuum. The results are shown in FIG.

1 is a schematic diagram illustrating a procedure for preparing Salmonella Invasion protein (HilA) peptide antibody,

Figure 2 is a schematic diagram showing the process for the manufacture of Salmonella Flagellin protein (FliC) peptide antibody,

Figure 3 is a schematic diagram showing the process of immobilizing the antibody produced in SPR,

4 is a schematic view showing the reaction of the sample with the immobilized HilA antibody,

5 is a schematic diagram showing the reaction of the sample with the immobilized FilC antibody,

6 is a schematic representation of SEM results of the prepared Fluorescent-PSQ,

Figure 7 shows the SEM results of the prepared Immunofluorescent-PSQ,

Figure 8 shows the reaction of the produced Immunofluorescent-PSQ with the produced antibody, a schematic representation of the specific reaction with Salmonella.

Claims (2)

Preparation and use of immuno-fluorescent nanoparticle PSQ which reacted Salmonella HilA antibody to PSQ Preparation and use of immuno-fluorescent nanoparticle PSQ which reacted Salmonella FliC antibody to PSQ

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