CN105548577A - Protein chip modified by succinyl-beta-cyclodextrin for detecting lyme disease and preparation and application of protein chip - Google Patents

Abstract

The invention belongs to the field of biomedical detection, and in particular relates to a succinyl-β-cyclodextrin-modified protein chip for Lyme disease detection and its preparation and application. The first aspect of the present invention provides a protein chip for detection of Lyme disease, comprising a solid phase carrier and a capture molecule immobilized on the solid phase support, the capture molecule contains the variable main protein of Borrelia burgdorferi Like sequence expresses E protein (VlsE). During detection, each chip can detect multiple cases of serum samples at the same time, with high sensitivity and specificity, reducing the probability of false positive and false negative detection between groups. Compared with traditional methods, using it to carry out large-scale screening of people in high-risk areas of Lyme disease can provide a simpler and more reliable detection method, and improve screening efficiency, positive detection rate and accuracy.

Description

A succinyl-β-cyclodextrin-modified protein chip for Lyme disease detection and its preparation and application

technical field

The invention belongs to the field of biomedical detection, and in particular relates to a succinyl-β-cyclodextrin-modified protein chip for Lyme disease detection and its preparation and application.

Background technique

Lyme disease (Lyme Disease, LD) is a disease caused by spirochete infection transmitted by ticks, involving skin, nerves, joints, heart and other multiple tissue and organ damage, also known as Lyme borreliosis (Lymeborreliosis). The main pathogen of Lyme disease is Borrelia burgdorferi, which is mainly divided into three subtypes: Borrelia burgdorferi sensustricto, Borrelia afzelii and Borrelia garnii. Lyme disease strains in my country are also divided into three genotypes, among which the ratios of B.burgdorferisensuestricto, B.afzelii, and B.garinii genotypes are 5.81%, 23.26%, and 66.28%, respectively. The incidence of Lyme disease is high in the United States and Europe, especially in Scandinavia and Central Europe; in China, the incidence of Lyme disease is mainly distributed in mountainous and forested areas, and the incidence of Lyme disease is higher in children than in adults.

Erythema migrans (EM) is a typical early symptom of Lyme disease. Untreated Lyme disease patients will develop neuropathy during the months to years of disease development, and both peripheral nerves and central nervous system will be damaged. Involvement, leading to neurological Lyme disease (neuroborreliosis, NB). However, due to the non-specific clinical manifestations of Lyme disease infection in the early stage, that is, the manifestations of its annular migratory erythema are similar and overlapping with many skin diseases, and clinicians have insufficient awareness of the dangers of Lyme disease. Lyme disease is easily misdiagnosed or missed clinically. Experimental studies have shown that host animals have different clinical symptoms caused by three genotypes of Borrelia burgdorferi infection. Borrelia burgdorferi in the narrow sense mainly causes diseases related to joints, Borrelia burgdorferi mainly causes skin diseases, and Borrelia burgdorferi mainly causes skin diseases. Borrelia mainly cause diseases related to the nervous system. Current methods for laboratory diagnosis of Borrelia burgdorferi infection include cell culture techniques, immunohistochemical techniques, polymerase chain reaction, and serum antibodies using enzyme-linked immunoassays with whole-cell lysates, recombinant proteins and peptides, or Western blot detection technology. Nevertheless, many questions remain about the value of routine serum immunoassays in the diagnosis of Lyme disease. The National Center for Disease Control and Prevention recommends that the preferred method for Lyme disease detection is to use ELISA or direct immunofluorescence to detect antibodies produced by Borrelia in serum, but the results still need to be confirmed by Western blot.

Contents of the invention

In order to overcome the problems existing in the prior art, the object of the present invention is to provide a protein chip for Lyme disease detection modified by succinyl-β-cyclodextrin and its preparation and application.

In order to achieve the above object and other related objects, the present invention adopts the following technical solutions:

The first aspect of the present invention provides a protein chip for detection of Lyme disease, comprising a solid phase carrier and a capture molecule immobilized on the solid phase support, the capture molecule contains the variable main protein of Borrelia burgdorferi Like sequence expresses E protein (VlsE).

Preferably, the fixation is lattice fixation.

Preferably, the solid phase carrier is a gold foil chip.

The base of the gold foil chip that adopts in the preferred embodiment of the present invention is a glass sheet, covers the pure gold (purity 99.9%) of one deck 10nm thickness on it, is the array of the TEFLON film (96 %) of a regionalized 50 μm on the gold foil holes*2, 8 rows*12 columns), the array aperture is 1.25mm.

Further preferably, the solid phase carrier is a gold foil chip whose surface is sequentially modified with 16-amino-1-hexadecanethiol and succinyl-β-cyclodextrin.

Preferably, the succinimide group activated by the carboxyl group on the succinyl-β-cyclodextrin combines with the amino group in the modified 16-amino-1-hexadecanethiol on the gold foil chip.

Preferably, the Borrelia burgdorferi variable major protein-like sequence expressed E protein (VlsE) is combined with succinyl-β-cyclodextrin.

Preferably, the density range of Borrelia burgdorferi variable major protein-like sequence expression E protein (VlsE) on the solid phase carrier is: 3.9x10 ^-4 ng/mm ² -0.41 ng/mm ² .

Further preferably, the density range of the Borrelia burgdorferi surface protein VlsE on the solid phase carrier is: 1.3x10 ^-2 ng/mm ² -0.41 ng/mm ² .

The second aspect of the present invention provides a method for preparing the solid-phase carrier in the above-mentioned protein chip for Lyme disease detection, comprising the steps of:

(1) activating the carboxyl group on the succinyl-β-cyclodextrin into a succinimide group to obtain the activated succinyl-β-cyclodextrin;

(2) modifying 16-amino-1-hexadecanethiol on the gold foil chip to obtain a gold foil chip modified with 16-amino-1-hexadecanethiol;

(3) The activated succinyl-β-cyclodextrin obtained in step (1) is modified on the 16-amino-1-hexadecanethiol-modified gold foil chip obtained in step (2) to obtain a solid phase carrier.

Preferably, in step (1), EDC and NHS are used to activate the carboxyl group on succinyl-β-cyclodextrin.

The third aspect of the present invention provides the above-mentioned method for constructing a protein chip for Lyme disease detection, comprising the steps of:

The capture molecule is diluted to obtain a capture molecule solution; the capture molecule solution is dotted on different positions on the surface of the solid phase carrier by conventional methods, and fixed.

The fourth aspect of the present invention also provides the use of the aforementioned protein chip for Lyme disease detection in the preparation of a Borrelia burgdorferi detection kit.

The fifth aspect of the present invention provides a Borrelia burgdorferi detection kit, which includes the aforementioned protein chip for detection of Lyme disease.

Preferably, the Borrelia burgdorferi detection kit also contains a negative control.

The negative control is PBST-BSA solution or negative serum.

The PBST-BSA solution is composed of 0.01M phosphate buffer solution PBS (PH7.2-7.4); 0.1% (v/v) Tween20; 0.1% (w/v) fetal bovine serum BSA.

The negative serum is the serum of a healthy person clinically confirmed not to be infected with Borrelia burgdorferi.

Preferably, the Borrelia burgdorferi detection kit also contains a positive control.

The positive control is selected from human immunoglobulin IgG, IgM or positive serum.

The positive serum is the serum of a patient with clinically confirmed Borrelia burgdorferi infection.

The selection of special antigen and gold foil chip is the key innovation point of the protein chip of the present invention. The kit based on the present invention adopts a fluorescence method for detection, so other reagents may also be included in the kit. For example: one or more of standards, diluents, and cleaning solutions. Specifically which reagents need to be assembled into the kit can be configured according to actual needs.

The standard includes IgG or IgM antibodies to Borrelia burgdorferi variable major protein-like sequence expressing E protein (VlsE).

Preferably, the kit also includes a fluorescein-labeled IgG secondary antibody and a fluorescein-labeled IgM secondary antibody.

Preferably, the fluorescein label is Cy3 label or Cy5 label.

Preferably, the kit also contains diluent PBST-BSA solution. The diluent can be used to dilute antigens, antibodies, serum, etc. The PBST-BSA solution is composed of 0.01M phosphate buffer solution PBS (PH7.2-7.4); 0.1% (v/v) Tween20; 0.1% (w/v) fetal bovine serum BSA.

The sixth aspect of the present invention also provides a method for using the kit, comprising the following steps:

Spot the serum sample on the aforementioned protein chip, incubate, wash, and then add the mixed solution of fluorescein-labeled IgG secondary antibody and fluorescein-labeled IgM secondary antibody.

Washing is used to remove incompletely reacted serum from the chip, and conventional washing solutions for antigen-antibody reactions in the art can be used. Preferably, PBST (pH7.4) can be used as the washing solution.

Preferably, when washing, wash with PBST 3 times, each time for 2 minutes, and blow dry with nitrogen.

Preferably, the fluorescein-labeled IgG secondary antibody is a Cy3-labeled IgG antibody. More preferably, it is a Cy3-labeled donkey anti-human IgG antibody.

Preferably, the fluorescein-labeled IgM secondary antibody is a Cy5-labeled IgM antibody. More preferably, it is a Cy5-labeled goat anti-human IgM antibody.

Compared with the prior art, the present invention has the following beneficial effects:

(1) In one aspect, the present invention utilizes gold foil as a substrate. Its advantage over traditional glass sheets, silicon sheets, and polymer material substrates is that gold foil itself is an inert metal, but its combination with chemical substances is stronger than traditional glass sheets and silicon sheets. Moreover, the bio-affinity of gold foil is low, and it is not easy to produce non-specific adsorption with genes or proteins, which greatly reduces non-specificity and avoids false positive test results.

(2) When it is used to actually detect the anti-Borrelia burgdorferi variable main protein-like sequence expression E protein (VlsE) antigen in the serum of clinical Lyme disease patients, each chip can detect multiple serum samples at the same time, and the sensitivity and specificity are average. Higher, reducing the probability of false positive and false negative detection between groups. Compared with traditional methods, using it to carry out large-scale screening of people in high-risk areas of Lyme disease can provide a simpler and more reliable detection method, and improve screening efficiency, positive detection rate and accuracy.

Description of drawings

Figure 1: Schematic diagram of the spotting array of gold foil chips.

Figure 2: Using atomic force microscope (AFM) to observe the characterization of gold foil chips before modification.

Figure 3: Using atomic force microscope (AFM) to observe the characterization of gold foil chips after modification.

Figure 4: A schematic representation of the chemically modified gold foil chip surface scanned by a Fourier transform infrared spectrometer.

FIG. 5A : the results obtained by scanning the protein chip obtained from the quality control experiment of the succinyl-β-cyclodextrin-modified gold foil chip in Example 2 for different protein compatibility using a chip scanner.

Fig. 5B: The fluorescence curve of the protein chip obtained from the quality control experiment of the compatibility of the gold foil chip modified with succinyl-β-cyclodextrin to different proteins in Example 2.

Fig. 6: The fluorescence curve of the protein chip obtained from the antigen incubation temperature-time quality control experiment in Example 2.

FIG. 7A : The results obtained by scanning the protein chip obtained from the quality control experiment of rabbit anti-VlsE antigen IgG antibody concentration gradient incubation in Example 2 using a chip scanner.

Fig. 7B: The fluorescence curve of the protein chip obtained from the concentration gradient incubation quality control experiment of the rabbit anti-VlsE antigen IgG antibody in Example 2.

Fig. 8A: The fluorescence results of the three groups in the intra-group repeatability experiment of Example 3 are not limited by different sample wells and different time, and have good repeatability and high stability.

Fig. 8B: The fluorescence results of each group in the repeatability experiment between groups in Example 3 are not limited by different sample wells and different time, and have good repeatability and high stability.

FIG. 9A : a graph of scanning results of each protein chip obtained from the serum dilution experiment in Example 4 using a chip scanner.

FIG. 9B : In Example 4, the fluorescence intensity curve obtained is drawn with the serum dilution as the abscissa and the average value A of the fluorescence intensity as the ordinate.

FIG. 10A : the detection chart of IgM in serum of Lyme disease patients obtained by scanning the obtained protein chip in Example 5 with a chip scanner.

FIG. 10B : the protein chip obtained by scanning the obtained protein chip with a chip scanner in Example 5, and the detection chart of IgG in the serum of a Lyme disease patient.

detailed description

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are to describe specific specific embodiments, It is not intended to limit the protection scope of the present invention. The test methods for which specific conditions are not indicated in the following examples are usually in accordance with conventional conditions, or in accordance with the conditions suggested by each manufacturer.

When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to those skilled in the art's grasp of the prior art and the description of the present invention, the methods, equipment, and materials described in the embodiments of the present invention can also be used Any methods, apparatus and materials of the prior art similar or equivalent to the practice of the present invention.

Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field conventional technology.这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULARCLONING：ALABORATORYMANUAL，Secondedition，ColdSpringHarborLaboratoryPress，1989andThirdedition，2001；Ausubel等，CURRENTPROTOCOLSINMOLECULARBIOLOGY，JohnWiley&Sons，NewYork，1987andperiodicupdates；theseriesMETHODSINENZYMOLOGY，AcademicPress，SanDiego；Wolffe，CHROMATINSTRUCTUREANDFUNCTION， Thirdedition, Academic Press, San Diego, 1998; METHODSINENZYMOLOGY, Vol. 304, Chromatin (P.M. Wassarman and A.P. Wolffe, eds.), Academic Press, San Diego, 1999; and METHODSINMOLECULARBIOLOGY, Vol. Totowa, 1999 et al.

The source and preparation of each embodiment raw material of the present invention are as follows:

1. Gold foil chip source: the gold foil chip used in the present invention comes from Interactiva company (Germany, Ulm), and its substrate is a glass sheet covered with a layer of pure gold (purity 99.9%) with a thickness of 10nm on it, and above the gold foil is a region An array of 50 μm TEFLON membranes (96 holes*2, 8 rows*12 columns), the array aperture is 1.25mm, as shown in Figure 1.

2. Gold foil chip surface chemical modification reagent:

Modification solution 1: 16-amino-1-hexadecanethiol solution in ethanol with a concentration of 0.8 mM. The molecular formula of 16-amino-1-hexadecanethiol is C ₁₆ N ₃₆ ClNS;

The chemical structural formula is: Purchased from Japan DOJINDO company.

Modification solution 2: PBS aqueous solution of succinyl-β-cyclodextrin (Succinyl-β-CD, Mr=1135 g/mol) at a concentration of 1 mM. The specific preparation method may be as follows: Weigh 1.14mg of succinyl-β-cyclodextrin (Succinyl-β-CD, Mr=1135g/mol) and dissolve it in 10mMPBS (PH=7.4) to prepare a solution with a concentration of 1mM. Succinyl-β-cyclodextrin was purchased from SIGMA-ALDRICH, USA.

Modification solution 3: N-hydroxysuccinimide (NHS, Mr=115.09 g/mol) aqueous solution with a concentration of 430 mM. The specific preparation method may be: weigh 49.49 mg of N-hydroxysuccinimide (NHS, Mr=115.09 g/mol) and dissolve it in 1 ml of deionized water treated with high-pressure steam to obtain a solution with a concentration of 430 mM. N-hydroxysuccinimide was purchased from SIGMA-ALDRICH, USA.

Modification solution 4: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, Mr=191.70 g/mol) aqueous solution with a concentration of 50 mM. The specific configuration method can be: weigh 9.59mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, Mr=191.70g/mol) and dissolve it in 1ml of high-pressure steam-treated A solution with a concentration of 50 mM in deionized water was purchased from SIGMA-ALDRICH, USA.

Modification Solution 5: Mix equal volumes of EDC and NHS solutions, mix the mixed solution with Succinyl-β-CD solution at a volume ratio of 1/10, and incubate the resulting mixed solution at 4°C for 2 hours.

3. Antigens, antibodies, fluoresceins

Antigen: Borrelia burgdorferi variable major protein-like sequence expression E protein (recombinant) (variablemajorprotein-likesequenceEprotein, VlsE), Borrelia burgdorferi flagellin (recombinant) (Flagellin) were purchased from MYBIOSOURCE company; Borrelia burgdorferi outer membrane protein C (Recombination) (OuterspaceproteinC, OspC) was purchased from Aviva;

Antibody: rabbit anti-Borrelia burgdorferi variable main protein-like sequence expression E protein antigen IgG antibodies were purchased from ROCKLAND company, rabbit anti-Borrelia burgdorferi flagellin antigen IgG antibody, rabbit anti-Borrelia burgdorferi outer membrane protein C antigen IgG antibody ;

Fluorescein: Cy3-labeled goat anti-rabbit IgG antibody was purchased from Sangon Company; Cy3-labeled donkey anti-human IgG antibody was purchased from Sangon Company; Cy3-labeled goat anti-human IgM antibody was purchased from KPL Company.

4. PBST solution is composed of phosphate buffer PBS mixed with 0.01MTween20.

PBST preparation: Dissolve one packet of PBS in 1000ml deionized water, then add 1ml Tween-20, mix well, and place on a shaker overnight. PBS: 0.01M, pH7.2-7.4; Tween-20: 0.1% (v/v).

5. The PBST-BSA solution is composed of 0.01M, PH7.2-7.4 phosphate buffer PBS; 0.1% (v/v) Tween20 and 0.1% (w/v) fetal bovine serum BSA.

Preparation of PBST-BSA solution: Take 50 μl of BSA (200 mg/ml) in 10 ml of PBST solution, oscillate and let stand. To obtain a PBST-BSA solution (the mass concentration of BSA is required to be 0.1% (w/v)).

Phosphate buffer solution (PBS), Tween20 and fetal bovine serum (BSA) were purchased from SIGMA-ALDRICH Company.

The selection and surface chemical treatment of embodiment 1 carrier

The present invention selects a gold foil chip from Interactiva Company (Ulm, Germany). Its substrate is a glass sheet covered with a layer of pure gold (purity 99.9%) with a thickness of 10nm. On the gold foil is a regionalized 50μm TEFLON The membrane array (96 wells*2, 8 rows*12 columns), the array aperture is 1.25mm, as shown in Figure 1.

Step 1. Cleaning of gold foil chips

Prepare TL1 solution (H ₂ O:H ₂ O ₂ :NH ₃ ·H ₂ O=5:1:1) and pour it into a stainless steel box, put the chip into the box, bathe in 82°C water for 5 minutes, rinse with deionized water for 4~ 5 times, 2 times with ethanol, 3 minutes each time; air-dried with nitrogen, and stored in a dry place.

Step 2. Chemically modify the surface of the cleaned gold foil chip to obtain a solid phase carrier

After cleaning the gold foil chip, immerse it in the aforementioned modification solution 1, and incubate with shaking at room temperature for 12 hours in the dark. After taking it out, wash it with absolute ethanol solution and dry it with nitrogen to obtain the 16-amino-1-hexadecanethiol-modified Gold foil chip; after taking it out, soak it in the aforementioned modification solution 5 and incubate for 4 hours; then take out the chip and wash it with high-pressure steam-treated deionized water for 3 times, each time for 2 minutes, and dry it with nitrogen. The activated succinimide group is combined with the amino group in the 16-amino-1-hexadecanethiol modified by the gold foil chip to obtain the gold foil chip modified by succinyl-β-cyclodextrin, which is ready for use.

The atomic force microscope (AFM) was used to observe the characterization of gold foil chips before and after modification, as shown in Figure 2 and Figure 3, respectively. It can be seen that the plane surface after modification is rougher than the plane surface before modification, indicating that after modification, the The clusters are covalently attached to the surface of the gold foil chip.

Fourier transform infrared spectrometer was used to scan the surface of the modified gold foil chip, and the chemical characterization diagram is shown in Figure 4: the 1232cm ^-1 peak is attributed to the CO vibration in the hydroxyl group; the 1423 and 1560cm ^-1 peaks are respectively assigned to the CN and C= in the amide bond O vibration; the 1708cm ^-1 peak is attributed to the C=O vibration in the anhydride group; the 2935cm ^-1 peak is attributed to the CC vibration in hexadecane, and the 3278-3357cm ^-1 peak may be mainly attributed to the OH vibration in the hydroxyl group, the NH vibration in the amino group and The OH vibration in the carboxylic acid group.

Embodiment 2 quality control experiment

Preparation of incubation solution 1: Dissolve VlsE antigen in PBST-BSA solution, and prepare concentration gradients of 0.5μg/ml, 0.25μg/ml, 0.125μg/ml, 0.0625μg/ml, 0.0313μg/ml, 0.0156μg/ml , 0.0078μg/ml, 0.0039μg/ml, 0.0019μg/ml, 0.00095μg/ml, 0.000475μg/ml antigen solution.

Preparation of incubation solution 2: Dissolve human IgG in PBST-BSA solution, and configure the concentration gradient to be 0.5μg/ml, 0.25μg/ml, 0.125μg/ml, 0.0625μg/ml, 0.0313μg/ml, 0.0156μg/ml, 0.0078μg/ml, 0.0039μg/ml, 0.0019μg/ml, 0.00095μg/ml, 0.000475μg/ml solution;

Preparation of incubation solution 3: Dissolve Flagellin antigen in PBST-BSA solution, and prepare a concentration gradient of 0.5 μg/ml, 0.25 μg/ml, 0.125 μg/ml, 0.0625 μg/ml, 0.0313 μg/ml, 0.0156 μg/ml, 0.0078μg/ml, 0.0039μg/ml, 0.0019μg/ml, 0.00095μg/ml, 0.000475μg/ml solution;

Prepare incubation solution 4: dissolve the OspC antigen in PBST-BSA solution, and configure a concentration gradient of 0.5 μg/ml, 0.25 μg/ml, 0.125 μg/ml, 0.0625 μg/ml, 0.0313 μg/ml, 0.0156 μg/ml, 0.0078μg/ml, 0.0039μg/ml, 0.0019μg/ml, 0.00095μg/ml, 0.000475μg/ml solution;

Prepare incubation solution 5: Dissolve rabbit anti-VlsEIgG antibody in PBST-BSA solution, and prepare antibody concentration gradients of 50 μg/ml, 25 μg/ml, 12.5 μg/ml, 6.25 μg/ml, 3.13 μg/ml, 1.56 μg/ml ml, 0.78μg/ml, 0.39μg/ml, 0.19μg/ml, 0.095μg/ml, 0.0475μg/ml solution;

Preparation of incubation solution 6: Dissolve Cy3-labeled donkey anti-human IgG antibody in PBST-BSA solution to prepare a solution with a fluorescent secondary antibody concentration of 2.5 μg/ml;

Preparation of incubation solution 7: Dissolve rabbit anti-Flagellin IgG antibody in PBST-BSA solution, and prepare a solution with an antibody concentration of 50 μg/mL;

Preparation of incubation solution 8: Dissolve rabbit anti-OspCIgG antibody in PBST-BSA solution respectively to prepare a solution with an antibody concentration of 50 μg/mL;

Preparation of incubation solution 9: Dissolve Cy3-labeled goat anti-rabbit IgG antibody in PBST-BSA solution to prepare a solution with a concentration of fluorescent secondary antibody of 2.5 μg/mL;

Prepare incubation solution 10: dissolve Cy3-labeled donkey anti-human IgG antibody in PBST-BSA solution, and prepare a solution with a concentration of fluorescent secondary antibody of 2.5 μg/mL;

Prepare incubation solution 11: Dissolve equal amounts of Cy3-labeled donkey anti-human IgG antibody and Cy5-labeled goat anti-human IgM antibody in PBST-BSA solution, and prepare Cy3-labeled donkey anti-human IgG antibody and Cy3-labeled donkey anti-human IgG antibody with a fluorescent secondary antibody concentration of 2.5 μg/ml Cy5-labeled goat anti-human IgM antibody mixed solution.

1. Quality control experiments on the compatibility of succinyl-β-cyclodextrin-modified gold foil chip to different proteins

The gold foil chip whose surface was chemically modified in Example 1 was used as a solid phase carrier, and four different gold foil cores modified with succinyl-β-cyclodextrin were taken, numbered 1, 2, 3, and 4 respectively. Incubation solution 1; Add incubation solution 2 to No. 2 chip spot; Add incubation solution 3 to No. 3 chip spot; Add incubation solution 4 to No. 4 chip spot; set two replicates for each gradient concentration, and incubate for 2 hours at room temperature (25°C) , washed with PBST for 3 times, each time for 2 minutes, and dried with nitrogen gas to obtain a protein chip for immunoserological diagnosis of Lyme disease.

Then spot the solutions of rabbit anti-VlsE IgG antibody, rabbit anti-Flagellin IgG antibody, and rabbit anti-OspCIgG antibody at a concentration of 50 μg/mL on the above-mentioned incubation with VlsE (number 1), Flagellin (number 3), OspC (number 4) antigens On the protein chip of the probe, Cy3-labeled donkey anti-human IgG antibody at a concentration of 2.5 μg/ml was spotted on the protein chip incubated with the human IgG antibody probe (numbered 2). Incubate for 1 hour in a humid chamber at room temperature (25°C). Take it out and wash it with PBST for 3 times, each time for 2 minutes, blow dry with nitrogen gas, and set aside.

Spot the Cy3-labeled goat anti-rabbit IgG antibody solution at a concentration of 2.5 μg/mL on the above-mentioned protein chips incubated with antibodies numbered 1, 3, and 4, keep away from light, and incubate at room temperature (25°C) for 0.5 hours . Remove and wash 3 times with PBST, 2 minutes each time. Blow dry with nitrogen.

The above protein chips were scanned with a chip scanner (Beijing Boao Company, model: Jingxin Luxscan10K-A), and the results are shown in FIG. 5A . As shown in Figure 5A, for the VlsE protein chip numbered 1, from left to right, the VlsE concentrations represented by columns 1 to 11 are 0.5 μg/ml, 0.25 μg/ml, 0.125 μg/ml, 0.0625 μg/ml, 0.0313μg/ml, 0.0156μg/ml, 0.0078μg/ml, 0.0039μg/ml, 0.0019μg/ml, 0.00095μg/ml, 0.000475μg/ml, the rightmost column, that is, from left to right Column 12 is PBST-BSA, negative control, and the antigen concentration is zero; for the IgG protein chip numbered 2, from left to right, the IgG concentrations represented by columns 1 to 11 are 0.5 μg/ml, 0.25 μg/ml, 0.125μg/ml, 0.0625μg/ml, 0.0313μg/ml, 0.0156μg/ml, 0.0078μg/ml, 0.0039μg/ml, 0.0019μg/ml, 0.00095μg/ml, 0.000475μg/ml, most The right column, that is, the 12th column from left to right, is PBST-BSA, negative control, and the antibody concentration is zero; for the Flagellin protein chip numbered 3, from left to right, from left to right, column 1 to column 1 The concentrations of Flagellin represented by the 11 columns are 0.5 μg/ml, 0.25 μg/ml, 0.125 μg/ml, 0.0625 μg/ml, 0.0313 μg/ml, 0.0156 μg/ml, 0.0078 μg/ml, 0.0039 μg/ml, 0.0019μg/ml, 0.00095μg/ml, 0.000475μg/ml, the rightmost column, that is, the 12th column from left to right, is PBST-BSA, negative control, and the antigen concentration is zero; for the OspC protein chip numbered 4 , from left to right, the concentrations of OspC represented by columns 1 to 11 are 0.5μg/ml, 0.25μg/ml, 0.125μg/ml, 0.0625μg/ml, 0.0313μg/ml, 0.0156μg/ml , 0.0078μg/ml, 0.0039μg/ml, 0.0019μg/ml, 0.00095μg/ml, 0.000475μg/ml, the rightmost column, that is, the 12th column from left to right, is PBST-BSA, negative control, antigen concentration to zero.

Figure 5B is a graph of fluorescence intensity curves obtained when different concentrations of antigens are coated for detection: take the average value of the two fluorescence intensities obtained at the same concentration in each column in Figure 5A, take the antigen concentration as the abscissa, and the average value of the fluorescence intensity as the ordinate, Plot the fluorescence intensity curve, and the results are shown in Figure 5B. It can be seen from Figure 5B that: under the condition that the incubation conditions of rabbit anti-lyme disease antigen IgG antibodies and Cy3-labeled goat anti-rabbit IgG antibodies remain unchanged, when the incubation concentration of VlsE recombinant antigen is greater than or equal to 0.000475 μg/ml, the produced There is a significant difference in the intensity of the fluorescent signal from the fluorescent signal intensity generated by the negative control group. Therefore, it shows that the optimum concentration for incubation of VlsE recombinant antigen should be above 0.000475μg/ml. However, the binding rate of human IgG, Flagellin, OspC recombinant antigen to the protein chip modified by succinyl-β-cyclodextrin is very poor. It fully demonstrates that the binding of succinyl-β-cyclodextrin to protein has certain selectivity, and not all proteins can combine well with succinyl-β-cyclodextrin and realize detection.

2. Antigen incubation temperature-time quality control experiment

Spot the VlsE antigen solution with a concentration of 0.0156 μg/ml onto the gold foil chip solid-phase support whose surface has been chemically modified with succinyl-β-cyclodextrin in Example 1, respectively at 37°C, room temperature (25°C) and 4°C Incubate for 24 hours, 12 hours, 6 hours, 3 hours, 1 hour and 0.5 hours, take it out and wash it with PBST for 3 times, each time for 2 minutes, and dry it with nitrogen gas; The solution was spotted on the solid phase carrier incubated with the VlsE antigen probe, incubated at room temperature (25°C) for 1 hour, removed and washed with PBST for 3 times, each time for 2 minutes, and dried with nitrogen gas. Then dissolve the Cy3-labeled goat anti-rabbit IgG antibody with a dilution concentration of 2.5 μg/ml in PBST-BSA solution and spot on the solid phase carrier incubated with the antibody, incubate at room temperature in the dark for 0.5 hours, take it out and wash it 3 times with PBST-BSA , 2 minutes each time, blow dry with nitrogen.

The chip scanner (Beijing Boao Company, model: Jingxin Luxscan10K-A) was used to scan the above protein chips respectively, which is the quality control experiment of antigen incubation temperature-time, and the results are shown in Figure 6. Firstly, obtain the fluorescent scanning images obtained by coating the same concentration of VlsE antigen on the chip for different times and different temperatures; then calculate the fluorescence intensity obtained by coating the VlsE antigen on the chip for different times and different temperatures as shown in Figure 6 according to the fluorescent scanning images Graph.

According to the quality control experiment of antigen incubation temperature-time, it can be known that:

(1) The fluorescence intensity increases with the extension of the incubation time. When the incubation time is longer than 1 hour, the change of the fluorescence intensity is not obvious with the extension of the incubation time. Therefore, in the actual detection, in order to shorten the incubation time, it is recommended that the incubation time for probe production be longer than 1 hour to meet the demand.

(2) Incubation temperature, the fluorescence intensity at room temperature (25°C) is stronger than that at 37°C and 4°C. Therefore, in actual testing, it is recommended to perform incubation experiments at room temperature (25°C).

3. Rabbit anti-VlsE antigen IgG antibody concentration gradient incubation quality control experiment

The gold foil chip whose surface was chemically modified in Example 1 was used as a solid phase carrier and immersed in the VlsE recombinant antigen solution with a concentration of 0.0156 μg/ml, incubated at room temperature (25°C) for 2 hours, taken out and washed with PBST for 3 times, Blow dry with nitrogen for 2 minutes each time; spot the rabbit anti-VlsE antigen IgG antibody solution in incubation solution 5 on the chip incubated with VlsE and recombinant antigen probes, incubate at room temperature (25°C) for 1 hour, and take out Wash 3 times with PBST, 2 minutes each time, blow dry with nitrogen; add 2.5 μg/mL Cy3-labeled goat anti-rabbit IgG antibody, incubate at room temperature (25°C) for 0.5 hours in the dark, remove and wash 3 times with PBST, Blow dry with nitrogen gas for 2 minutes each time.

Scanning the chip with a chip scanner is a quality control experiment for rabbit anti-VlsE antigen IgG antibody concentration gradient incubation, and the results are shown in Figure 7A. As shown in Figure 7A, for the VlsE protein chip: from left to right, column 1 to column 11 represent, respectively, the concentrations of rabbit anti-VlsE antigen IgG antibodies are 50 μg/ml, 25 μg/ml, 12.5 μg/ml, 6.25 μg/ml, 3.13μg/ml, 1.56μg/ml, 0.78μg/ml, 0.39μg/ml, 0.19μg/ml, 0.095μg/ml, 0.0475μg/ml, the rightmost column, that is, from left to right Column 12 is PBST-BSA, negative control, and the antibody concentration is 0.

Figure 7B is a graph of fluorescence intensity curves obtained when different concentrations of antibodies are used for coating detection: the average value of the four fluorescence intensities obtained at the same concentration in each column in Figure 7A is taken, with the antibody concentration as the abscissa, and the average value of the fluorescence intensity as the ordinate, Plot the fluorescence intensity curve.

It can be seen from Figure 7B: under the condition that the incubation conditions of VlsE recombinant antigen and Cy3-labeled goat anti-rabbit IgG antibody remain unchanged, when the incubation concentration of rabbit anti-VlsE antigen IgG antibody is greater than 0.39 μg/mL, the intensity of the fluorescent signal generated There was a discernible difference in the intensity of the fluorescent signal generated from the negative control group. It shows that this modified chip can detect the concentration of the rabbit anti-VlsE antigen IgG antibody visible to the naked eye at the μg/mL level.

Embodiment 3 repeatability experiment

Table 1 shows the repeatability experiment within a group. Each 16 wells is regarded as a group, and there are three groups in total. On the same day, the VlsE antigen with a concentration of 0.0156 μg/ml and the rabbit anti-VlsE antigen IgG antibody with a concentration of 50 μg/ml are incubated according to the steps and Cy3-labeled goat anti-rabbit IgG antibody at a concentration of 2.5 μg/ml.

Table 1

Table 2 shows the repeatability experiment between groups. Each 24 wells is regarded as a group, and there are three groups in total. The VlsE antigen with a concentration of 0.0156 μg/ml and the rabbit anti-VlsE antigen IgG with a concentration of 50 μg/ml were incubated according to the steps within three days. Antibody and Cy3-labeled goat anti-rabbit IgG antibody at a concentration of 2.5 μg/ml.

Table 2

In Tables 1 and 2, SD is the standard deviation, and CV is the coefficient of variation. These two indicators can reflect the stability of the biochip in the detection of E protein antibodies against protein-like sequences and will not cause large deviations.

As shown in Figure 8A: In the intra-group repeatability experiment, the fluorescence results of the three groups are not limited by different sample wells and different time, and have good repeatability and high stability.

As shown in Figure 8B, the fluorescence results of each group in the inter-group repeatability experiment are not limited by different sample wells and different time, and have good repeatability and high stability.

To sum up, the detection results of the protein chip of the present invention for the IgG antibody against the VlsE antigen are not limited by different spotting wells and different times, and have good repeatability and high stability.

Embodiment 4 serum dilution experiment

Sera from Lyme disease patients with clinically confirmed Borrelia burgdorferi infection and 3 cases of healthy human serum without clinically confirmed Borrelia burgdorferi infection were randomly collected.

Use PBST-BSA in each case according to the gradient 1:2.5, 1:5, 1:10, 1:20, 1:40, 1:80, 1:160; 1:320, 1:640, 1:1280.1: 2560 dilution. Then, according to the order from left to free, samples were spotted on the chip containing the probe of the primary VlsE antigen with an incubation concentration of 0.0156 μg/ml, and the remaining 6 blank sample solutions were PBST-BSA solution. That is, for the VlsE protein chip that detects positive serum: from left to right, columns 1 to 11 represent respectively, and the serum dilution ratios are 1:2.5, 1:5, 1:10, 1:20, 1: 40, 1:80, 1:160; 1:320, 1:640, 1:1280.1:2560, the rightmost column, that is, the 12th column from left to right, is PBST-BSA, negative control, serum concentration is 0 . For the VlsE protein chip for detecting negative serum: from left to right, columns 1 to 11 represent respectively, and the serum dilution ratios are 1:2.5, 1:5, 1:10, 1:20, 1:40,1 :80, 1:160; 1:320, 1:640, 1:1280.1:2560, the rightmost column, that is, the 12th column from left to right, is PBST-BSA, negative control, and the serum concentration is 0.

Incubate at room temperature for 1 hour, take it out and wash it with PBST 3 times for 2 minutes each time, and dry it with nitrogen gas. Add 2.5 μg/ml Cy3-labeled donkey anti-human IgG antibody, incubate in the dark for 0.5 hour at room temperature (25° C.), take it out and wash it with PBST for 3 times, each time for 2 minutes, and dry it with nitrogen gas.

Use the chip scanner to scan the chip, and the results show that as shown in Figure 9A, the upper 3 rows are positive serum groups (3 cases of positive serum groups are randomly selected), and the lower 3 rows are negative serum groups (3 cases of negative serum groups are randomly selected) . Figure 9B: Calculation of the ratio A of the mean fluorescence value of the positive serum group to the mean fluorescence value of the corresponding dilution negative serum control group. Take the serum dilution as the abscissa and the average fluorescence intensity A as the ordinate to draw a fluorescence intensity curve. We set the ratio A of the average value of the three serum fluorescence values of the positive serum group to the three serum fluorescence values of the negative serum group. A dilution greater than 4 can be used for experimental detection, so the range of serum dilution used for detection is ( 1:10) ~ (1:160).

Example 5 Detection of Serum Samples from Lyme Disease Patients

Prepare a succinyl-β-cyclodextrin-modified gold foil chip according to the method in Example 1. As a solid-phase carrier, apply the VlsE antigen solution with an incubation concentration of 0.0156 μg/ml on each well of the solid-phase carrier, and store at room temperature. Incubate at 25° C. for 2 hours, take it out, wash it with PBST 3 times for 2 minutes each time, and dry it with nitrogen gas to obtain a protein chip for immunoserological diagnosis of Lyme disease.

As shown in Figure 10A and Figure 10B, the sera of 188 patients with Lyme disease who had been clinically diagnosed with Borrelia burgdorferi infection were spotted on 188 wells of the indicated protein chip, and the other 2 wells were stained with sample solution. It is the serum of a healthy person clinically confirmed not to be infected with Borrelia burgdorferi. The sampling solution of the remaining 2 wells is PBST-BSA solution, incubated at room temperature for 1 hour, washed with PBST for 3 times, each time for 2 minutes, and dried with nitrogen gas. . The incubation solution 11 was then spotted on the chip incubated with the patient's serum antibody, incubated in the dark at room temperature (25°C) for 0.5 hours, removed and washed with PBST for 3 times, each time for 2 minutes, and dried with nitrogen.

The chip was scanned using a chip scanner, and the results are shown in Figure 10A and Figure 10B, respectively. Among them, Fig. 10A is a detection diagram of IgM in the serum of a Lyme disease patient; Fig. 10B is a detection diagram of IgG in the serum of a Lyme disease patient. It can be seen from the figure that in the actual detection of the serum of clinically diagnosed patients with Lyme disease, there is a significant difference between the fluorescence intensity of the serum of positive patients and the serum of healthy people and the fluorescence intensity of PBST-BSA spots sex.

Statistics were carried out according to the fluorescence detection results, and the detection method was compared with the serum of 188 clinically diagnosed patients with Lyme disease by using the protein chip (Biochip) of the present invention and the traditional method ELISA method respectively, and the results are shown in Table 3:

table 3

"+": positive; "-": negative

*:χ ² ＝0.44, p>0.05

Chi-square test: Biochip vs. ELISA, p>0.05, indicating that there is no significant difference between the chip method and the traditional ELISA method in detecting Lyme disease.

Calculated according to the results in Table 3: the positive rate detected by the Biochip method was 45.2% (85 cases), and the positive rate detected by the ELISA method was 43.6% (82 cases). It can be concluded that the positive rate of Lyme disease detection by Biochip method is higher than that by ELISA method.

In summary, the protein chip of the present invention can detect anti-VlsE antigen IgG and IgM antibodies in patients respectively, and the protein chip method can be used as an alternative to the traditional ELISA method to detect Lyme disease, especially in a large number of screening samples. big advantage.

The foregoing is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any form and in essence. Several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention. Those who are familiar with this field, without departing from the spirit and scope of the present invention, when they can use the technical content disclosed above to make some changes, modifications and equivalent changes of evolution, are all included in the present invention. Equivalent embodiments; at the same time, all changes, modifications and evolutions of any equivalent changes made to the above-mentioned embodiments according to the substantive technology of the present invention still belong to the scope of the technical solution of the present invention.

Claims (10)

1. for the protein-chip that Lyme disease detects, comprise solid phase carrier and be fixed on the capture molecules on described solid phase carrier, described capture molecules contains Borrelia burgdoyferi variable major protein sample sequence expresses E protein (VlsE).

2. protein-chip according to claim 1, is characterized in that, described solid phase carrier is goldleaf chip.

3. protein-chip according to claim 1, is characterized in that, described solid phase carrier is the goldleaf chip that surface is modified with 16-amino-1-hexadecanethiol and succinyl-beta-cyclodextrin successively.

4. protein-chip according to claim 3, is characterized in that, the amino in the 16-amino-1-hexadecanethiol that succinyl-beta-cyclodextrin is modified on solid phase carrier by the succinimido of activated carboxylic is combined.

5. protein-chip according to claim 3, is characterized in that, Borrelia burgdoyferi variable major protein sample sequence is expressed E protein (VlsE) and is combined with succinyl-beta-cyclodextrin.

6. protein-chip according to claim 1, is characterized in that, on described solid phase carrier, the density range of Borrelia burgdoyferi variable major protein sample sequence expression E protein (VlsE) is: 3.9x10 ^-4ng/mm ²~ 0.41ng/mm ².

7. the preparation method of solid phase carrier in the protein-chip detected for Lyme disease according to the arbitrary claim of claim 1 ~ 6, comprises step:

(1) be succinimido by the activated carboxylic on succinyl-beta-cyclodextrin, obtain the succinyl-beta-cyclodextrin after activation;

(2) on goldleaf chip, modify 16-amino-1-hexadecanethiol, obtain the goldleaf chip that 16-amino-1-hexadecanethiol is modified;

(3) succinyl-beta-cyclodextrin on the goldleaf chip that gained 16-amino-1-hexadecanethiol is modified in step (2) after the activation of modification step (1) gained, obtains solid phase carrier.

8. according to the arbitrary claim of claim 1 ~ 6 for the construction method of protein-chip that Lyme disease detects, comprise step: capture molecules diluted, obtain capture molecules solution; Adopt conventional method by capture molecules solution difference dot matrix in the diverse location of surface of solid phase carriers, and fixing.

9. the protein-chip detected for Lyme disease according to the arbitrary claim of claim 1 ~ 6 is preparing the purposes in Borrelia burgdoyferi detection kit.

10. a Borrelia burgdoyferi detection kit, described kit comprises the protein-chip for Lyme disease detection as described in claim as arbitrary in claim 1 ~ 6.