TWI294967B - Protein microarray for detecting pathogenic antigen inducing fever - Google Patents

Description

1294967 V. INSTRUCTION DESCRIPTION OF THE INVENTION (1) Technical Field of the Invention: The present invention relates to a protein microarray (Protein Microarray) for screening a protein in a rapid, large-scale and parallel manner in an in vitro environment (invitr). Refers to a clinical diagnostic (Clinical Diagnostics), drug screening (Drug Screening) and Vaccine Production (Vaccine Production), in order to detect the protein microparticles that induce human fever (Thermogen) in the early days of infection. Array. [Prior Art]: Biochips originated in the late 1980s. In a broad sense, biochips refer to microelectronics and micromachines on glass, nylon (Nylon Membrane), nitrocellulose (Ni trocel luiose) or plastic materials. Such precision technology is used to produce a product for detecting a gene sequence, biochemical analysis or processing a natural or/and artificially modified biological molecule such as a nucleic acid or a protein, and the object of action may be a gene, a protein or a cell tissue. Compared with traditional molecular biochemical analysis technology, biochips are mainly based on their reliability and accuracy. The analysis speed is fast, the sample and reagent dosage are small, and a large number of ensemble (parallelization) experimental results can be obtained. At present, the application of biochips is still in the development stage in the world. According to different objects and purposes, it is mainly divided into two types: Microfluidics-based Screening System (also known as processed biochip). Microarray Technology (Miccroarray Technology) 〇Microfluidic screening system combines biotechnology, semiconductor, micro-optoelectronics and

17425阎中原.Ptd Page 7 1294967 V. INSTRUCTIONS (2) Micro-mechanical and other technologies 'micro-machinery functions in the laboratory are miniaturized on the wafer for processing by 1 〇 to hundreds of micrometers wide Network (C ha η ne 1 S Ne twork) to connect crop i cropumps, Electroosmotic Flow, Integrated Valves and Mixing Devices Control Liquid Movement Perform in vitro testing together. When protein is used as the detection target of the microfluidic screening system, the channel network can be used to prevent the evaporation of the reagent to prevent the dehydration of the protein, but in the absence of reagent buffer, the interface between the protein and the reagent It is easy to have a high surface-to-volume ratio problem (generally the reagent volume is increased from 1 microliter to 〇. 〇1 μl, the surface to volume ratio will increase by 460%), resulting in protein in the liquid phase/solid phase. And deactivated at the liquid/gas phase interface to lose activity. In order to overcome the above problems, the scientific community has additionally developed a microarray type biochip (M i c r 〇 a r r a y Biochip) which neatly connects a sample to a glass (f) or nylon membrane. The so-called "microarray MA type wafer n" is connected to the south side of the wafer, the gene fragment, the Oligonucleotides or the modified nucleic acid fragment, using Deoxyr ibo-nucleic acid (DNA). Hybridization of A, T, G, and C (H ybridizati 〇n) to detect the gene to be detected. However, the application of d NA microarray technology may not be fully applicable to proteins, because proteins and DNA are in physical properties. The difference in chemical properties is very large. The DNA can still be dried and rehydrated (R ehydrati ο η) above 100 ° C, and can be directly used with microarray materials (eg glass or nylon membrane (Nylon Membrane)

17425阎中原.ptd Page 8 1294967 V. Description of the invention (3) -1^"^ ^ --- )) The organic coating coated on the surface does not damage the DNA activity, but θ 1 The protein is extremely sensitive to the environment, and the physical properties and chemical properties of the material in the humidity and temperature limit are very strict. Therefore, the target protein is inactivated in the microarray operation and affects the detection result. The required operating conditions at the liquid/solid phase interface are a further one. Because Biochip has the characteristics of fast, large, and parallel processing of samples, it can quickly detect the body of the patient. Internal physiological and biochemical values, to find the right cause to give appropriate treatment and to observe the disease course of the disease as a basis for drug use, therefore, both DNA Microarray Technology and Protein Microarray Technology can be applied. Clinical medical diagnosis including diagnosis of infectious diseases (such as bacteria, viruses, fungi, and parasites), cancer classification, hereditary diseases, or neonatal screening. Taking infectious diseases as an example, since most pathogens (path〇gen) invade the human body, they will activate the immune mechanism in the body and produce fever (Fever) symptoms at the beginning of the infection; and during the course of the disease, the symptoms of fever often become patients. Whether it has the key indicators of infectious power, therefore, if the doctor can detect the pathogen causing fever in the first time, it will be able to greatly reduce the dose of the broad-spectrum antibiotics, and the right medicine to avoid the disease and other diseases. The results of the original culture and serum student analysis (at least three to five days) and the timing of mistreatment. In general, the source of the antigen (Antigen) produced by the induction of fever, including the specific egg on the outer surface of the pathogen such as a virus, bacteria, fungus or parasite.

17425阎中原.ptd Page 9 1294967 V. INSTRUCTIONS (4) White matter structure 'We call these pathogens an external thermogen (Externai Thermogen). Taking the coronavirus idae, which mainly infects the upper respiratory tract, as an example, after the nuclear protein (Nucleocapsid) on the surface of the virus enters the human mucosa, the human immune system actively produces antibodies corresponding to the antigen in about two weeks. Traditional methods for diagnosing individuals with specific pathogenic proteins or corresponding specific pathogenic proteins include Immunofluorescence (Immun〇flu〇rescence Techniques) > ELISA (Enzyme-1inked Iramuno- sorbent Assay) ) and test paper coloring method. However, most of the general fluorescent immunoassays must use live virus to infect cells, which is dangerous and must be produced in a P3 laboratory, and is difficult to use widely. The EL ISA test can use the high-safety virus recombinant protein or antibody for the debt test, but the 96-well plate (9 6 - we 1 1 p 1 ate) is used as an example. The antigen/sample volume of each well must be at least 100 μl, and must be placed overnight or at a constant temperature of 3 for one hour to complete the antigen/sample reaction, so 'if large-scale clinical testing is performed' The consumption of the body and the reagents is extremely high, the cost is high, and the timeliness is poor. On the other hand, although the test paper coloring method can also use the recombinant protein to replace the live virus, the spraying test paper must use special equipment, and the antigen requirement is large, and the detection result cannot be quantified, so the accuracy and timeliness are It is also difficult to meet the requirements. To this end, '02 2 2 M e z z a s 〇 m a et al. in Clinical C h e m i s t r y 48:1, pp. 121-130, AntigenMic roarrays For Serodiagnosis Of Infectious Diseases’’ also reveals an egg

17425阎中原.ptd 1294967 V. Inventive Note (5) White matter microarray technology 'where' the microarray extracts human I g G and I g Μ antibodies by antigenic protein, and then calibrates with fluorescent antibody Confocal Microscopy was used to observe the binding of the antigen to the antibody. Although the EL I SA wafer method has begun to take advantage of the rapid, large-scale, and parallelization of biochips, but when foreign pathogens invade the human body, the detection results may be misjudged because the antibodies are not produced. Because the symptoms of fever are indicators of whether many infectious diseases are infectious, 'the correct type of antigen in the early stage of fever and the antigenic content of the antigen, and then the right medicines' often affect the patient's disease progression and the situation is very large. However, the human immune system usually produces the earliest antibody I g 7 7 to 14 days after infection, so before the symptoms of fever but the immune system has not yet produced antibodies, traditional immunofluorescence, EL I SA technology or test paper The color method is tested and may not achieve satisfactory results in terms of timeliness and precision. In addition, because part of the fever is not caused by infection, but by internal thermogen (Internal Thermogen), such as autoimmune diseases or malignant tumors induced by cytokines (Cytokine) or prostaglandins, These endogenous pyrogens were not included in the screening program during general fever screening and were not detectable. SUMMARY OF THE INVENTION The main object of the present invention is to provide a pathogenic agent (Path〇gen) or pyrogen which is induced to cause fever through a throat, a respiratory mucosa tissue or a blood sample immediately after the onset of fever. (Thermogen) type, a protein microarray for use by clinicians.

17425阎中原.ptd

Page 11 1294967 V. INSTRUCTION DESCRIPTION (6) Another object of the present invention is to provide a protein microarray made of a pathogenic antigen during fever to test for the presence or absence of a specific pathogenic antigen in a subject (SpecificaHy Pathogenic Antigen The corresponding antibody, to know if the individual has been infected. A further object of the present invention is to provide a rapid, correct and parallel screening of the cause (pathogenic antigen or pyrogen) for inducing fever, using only a very small amount of sample and test reagent, A protein microarray based on clinical testing. In view of the above and other objects, the present invention provides a detection platform for inducing pathogenicity-causing pathogens, in particular, a pathogenic antigen capable of detecting human body fever in the early stage of infection and fever (Pathogen ic Ant i) Gen) or a corresponding antibody produced by a person who induces infection, or a protein microarray that induces other pyrogens that produce fever. The protein microarray comprises a carrier coated on the surface with at least one aldehyde-containing coating; a plurality of Protein Array Regions, wherein the protein is spotted by an arrayer. On the aldehyde-based coating, and at least one pathogenic antigen that induces an increase in body temperature, or a corresponding antibody thereof, by a microwave having an intensity of between 1. 〇〇 and 5.00 GHz, or against an inducible temperature rise The antibody of the high pyrogen is immobilized on the protein array region; and a blocking region 0 formed in the adjacent protein array interval is caused by most of the pathogens causing fever symptoms, for example, severe acute respiratory distress syndrome (S) Evere A cu ΐ e

Respiratory Syndrome, SARS) virus, its nuclear protein

17425阎中原.ptd Page 12 1294967 V. Description of the invention (7) (Nucleocapsid) antigens are surrounded by a large number of positively charged amino acids, such as lysine and arginine. (Arginine), etc., when these positively charged amino acids are contacted with a carrier having a reactive aldehyde group on the surface (for example, a slide), the amine group (NH: ) on the protein molecule is generated with the carbon atom of the aldehyde group on the carrier. Schi ff covalently binds to cause the protein to be immobilized on the carrier. Moreover, the short-time, high-energy heating mode of the microwave does not destroy the covalent bond structure of the antigenic epitope (Antigen Determinant or E pit 〇pe) and causes protein denaturation. Therefore, the pathogenic antigen which induces an increase in body temperature can be The antigen binding site (Antigen Binding Site) on the human antibody (IgA, IgM or IgG) in the sample is combined to correctly diagnose the pathogen causing fever in the human body. The above protein microarrays can be used in clinical tests, mass drug screening, genetic disease testing, vaccine development or food safety, environmental sanitation inspection, and animal and plant quarantine. By using the protein microarray of the present invention, it is possible to confirm the source of the anti-pathogenic antigen and the type of the antibody against the fever of the human body by using a very small amount of the sample and the reagent amount, and the complex protein microarray is fast, correct, large and parallel. The characteristics further test the pathogenic cause of fever in the clinic, so that the clinician can correctly judge the cause of the disease at the initial stage of fever, and the right medicine is needed without using a broad-acting antibiotic to suppress the condition to wait for the test result. The protein microarray can be used in the human body, in addition to being used as a screening tool for finding the cause of fever, if it is used to diagnose digestive tract and respiratory diseases (such as influenza or severe acute respiratory syndrome (SARS)), it can also be used in the human body. The immune system is not producing antibodies I g A, I g G or I g ( (the earliest antibody I g in humans is 7 to 14 days after onset)

17425阎中原.ptd Page 13 1294967 V. Invention description (8) ---- can be produced), first through the mucosal tissue (such as the throat specimen) to test whether there is a specific pathogenic antigen in the patient' To determine if the patient has been infected. On the other hand, if the present invention is applied to the field of epidemic prevention, each carrier can simultaneously detect a large number of sample samples and reduce the cost of the sample. The present invention is described by way of specific embodiments, and those skilled in the art can readily appreciate the other advantages and advantages of the present invention from the disclosure. The present invention may be embodied or applied in various other specific embodiments, and various modifications and changes may be made without departing from the spirit and scope of the invention. Noun definition: ◎Protein: refers to the polymer molecule of Amin〇Acids, which is bonded and folded by Peptide Bond. The protein can be at least included. There are more than one month of single nucleotide or macromolecular complex (M acr 〇m ο 1 ecu 1 ar Complex), which may also be P〇iypept ide, Protein Fragments or An amino acid chain in the form of a short chain peptide. The protein referred to below includes any of Natural Protein and Artif icial Protein which chemically modifies one or more amino acids in the polypeptide chain, and the protein microarray of the present invention and The antigen μ (Antigen) and the antibody (Ant i body) covered by the detection method are all a protein structure.

1294967 V. INSTRUCTIONS (9) ◎ Antibody (A ntib 〇dy): refers to an immunoglobulin (Immunoglobulin) produced by natural or partial synthesis, which can be a monoclonal antibody (Monoclonal Antibody) or multiple antibodies ( P〇iyci〇nai Antibody), the antibody has a specific junction that can specifically bind to at least a portion of a particular molecular structure (generally an antigenic epitope (A ti D ΐ 〇 pe)) Binding Site), wherein the antibodies disclosed in the examples of the present invention include human antibodies I g A, I g Μ, I g G, I g D and I g E, mouse monoclonal antibodies, rabbits, goats, chickens or horses Multiple antibodies from the source. ◎Antigen (Ant i gen): is a molecule that can specifically bind to an antibody, including, for example, intermediate metabolites, hormones, lipid-filled proteins, proteins or other types of biomolecules, thereby triggering an immune response and inducing an infected host. Make antibodies. The protein microarray of the present invention uses a substance commonly causing fever symptoms as an antigen, and contains various types of endogenous (produced in a patient) or exogenous (infection) antigenic proteins, and the antigen protein may be in the form of a single protein molecule. The macromolecular complex (Macrorao 1 ecu 1 ar Comp lex) may also be an amino acid chain in the form of a polypeptide, a protein fragment (Protein Fragments) or a short chain peptide. ◎Thermogen: Fever means that the body temperature is higher than the normal range, because the brain itself occurs due to abnormal conditions or toxic substances affecting the body temperature regulation center. Frequently seen fever factors include (1) infection, (2) central nervous system diseases such as cerebrovascular disease, head trauma, cerebrospinal disease, (3) autoimmune diseases such as rheumatic fever, whole body

17425阎中原ptd page 15 1294967 V. Invention description αο) System i c Lups Erythema to sns (SLE). In addition, cardiopulmonary infarction, malignant tumors, bleeding or endocrine diseases can cause fever of varying degrees. These substances that affect fever in the body temperature are commonly referred to as "Thermogen". ◎Exogenous Thermogen: In Thermogen, a fever-inducing substance that invades from outside the body is called an exogenous pyrogen, such as bacteria, viruses, rickettsia, mold, and parasites. , or bacterial capsule (C ap su 1 e), viral protein sheath (Proteocapsid), nuclear protein (Nucleocapsid), recombinant protein (Recombinant Protein), bacillus, biological tissue cell protein extract or nuclear extract protein Wait. ◎Endogenous Thermogen: Thermogenic Cytokine released by the body's own fever-inducing substances, such as macrophage and neutrophil (N eutr 〇ph i 1 e) activation. For example, prostaglandin E2), or autoantigen, etc., is called "Endogenous Thermogen". Body F1 uid: a liquid that is extracted, secreted, or excreted by a living tissue or organ. Substance, with or without cells. The body fluids defined by the present invention include, but are not limited to, blood, serum, urine, plasma, cerebrospinal fluid, tears, amniotic fluid, respiratory tract, digestive tract or secretions of the genitourinary tract. EXAMPLES The following examples are further details of the present invention, but

17425阎中原.ptd Page 16 1294967 V. INSTRUCTIONS (11) The scope of the present invention is not limited by any point of view. The present invention provides a detection platform for inducing an elevated temperature antigen (A ntige η Generated Fever), in particular, a pathogenic antigen (Pathogenic Ant i gen) which can detect a fever in a human body in an early stage of infection or fever Individual or large-scale screening is performed by inducing a corresponding antibody produced by the infected person, or a protein microarray that induces a pyrogen-producing pyrogen. First, the composition of the protein microarray (p r 〇 t e i η Microarray) of the present invention will be described with reference to Fig. 1. As shown in Figure i, the protein microarray comprises: a carrier coated with at least one layer containing an aldehyde-based coating; a plurality of Protein Array Regions arrays, which are arrayed by an arrayer The protein is spotted on the aldehyde-containing coating, and at least one pathogenic antigen that induces an increase in body temperature, or a corresponding antibody thereof, by a microwave having an intensity of from 1. 〇〇 to 5. 〇0 GHz, or An antibody against a pyrogen-inducible pyrogen is immobilized on the protein array region; and a blocking region (B 1 〇cking Region) formed in the adjacent protein array region. More specifically, the protein micro of the present invention Array (P r〇tein

Microarray) may be a surface-containing aldehyde-based slide produced by CEL Associates or Telechem International Inc., other acid-based coated slides with similar surface structure, nylon membrane (Nylon Membrane), poly-lysine coating Slides and nitrocellulose coated slides. However, according to the difference in structure and function of the carrier ligated protein, the following antigen-type microarray (also referred to as "antigen wafer") and carrier on which the antigen is conjugated to the carrier, respectively

17425阎中原.ptd Page 17 1294967 V. Description of the Invention (12) An antibody-type microarray (also referred to as "antibody wafer") for conjugated antibodies is taken as an example. A. Antigen Microarray 4 (Antigen Microarray) The protein microarray of the present embodiment is used to detect various antibodies against pyrogenic pyrogens and endogenous pyrogens that cause fever in the human body, and any of them can induce fever in the human body. The antigenic protein of the symptom, whether it is a pathogenic antigen or a non-pathogenic antigen, is included in the practice of the present invention. For example, a protein microarray for screening for Severe Acute Respiratory Syndrome (SARS) is shown. As shown in Fig. 2, the protein microarray is an arrayer (not shown) for SARS coronavirus. Nucleocapsid, cold virus antigen as a positive control, Hep-2 throat cancer cell extract as a negative control, and autoantigen (Aut〇antigen) and other common external causes A pyrogen, such as a chlamydia antigen protein, a mycoplasma antigen protein, or a dengue antigen, is prepared on the acid-containing coated slide by an Array method. A large number of positively charged amino acids, such as Lysine and Arginine, contact the active aldehyde group on the surface of the slide, and the amine group (NH2) on the antigen protein and the carbon on the base Atom produces Schiff covalent binding to not affect antigen and antibody activity junctions (Active Binding

Under the break-up structure of Site, it has a specific bond with the antibody in the sample. Figure 3 is a diagram showing the combination of antigen and antibody on an antigenic microarray. As shown in the figure, when the SARS coronavirus antigen is immobilized on a slide, the serum of the subject diluted (for example, a dilution ratio of 1:5〇〇〇) is used.

17425阎中原.ptd Page 18 1294967 V. Description of invention (13) (subject to test) binds to each antigen and is resistant to human Ig A, Ig Μ or I labeled with a fluorescent agent (Cy 3 or Cy 5) The g G antibody (Anti-human Ig A / Ig G or Ig M Antibody) was ligated. Since the subject has been or has been infected with SARS virus, there are antibodies against the SARS virus in the serum I g A, I g I or I g G, therefore, if the slide with antigen protein is spotted and tested After the sample reaction, the fluorescence is detected under a Confocal Microscopy or a Laser Scanner, and the tester can know the antigen that induces the fever of the subject based on the position of the fluorescent light. Source and antibody type, and further determine whether the subject has or has been infected with the SARS virus. __Antibody Microarrav The above protein microarray can also develop an antibody type microarray (Ant i body M i c r o a r r a y) by utilizing the protein properties of the antibody itself. The antibody-type microarray detects a subject by, for example, treating a variety of different anti-pathogenic antigen antibodies as specific proteins and spotting them on a protein array region of the slide. The antigen (pathogen), thereby determining whether the individual has been infected. As shown in Fig. 4 and Fig. 5, such an antibody-type microarray is an antibody against a SARS coronavirus antibody or a positive control group by an Arrayer (not shown) (for example, when the sample is serum) An anti-albumin Ant i body), a negative control group (such as bovine serum albumin (BSA)) and other common anti-pathogenic antibodies, such as: Bacillus licheniformis, mycoplasma Antibody or dengue antibody, etc.

1294967 V. Description of the invention (14) on the base coated slide. The relationship between the antigen binding site (Antigen Determinant Site) and the antibody Fab fragment and the F(ab') fragment is specifically bonded to the antigen binding site (Antigen Binding Site). When anti-SARS virus antibodies and other pathogenic antibodies or non-pathogenic antibodies are immobilized on the slide, the body fluids (such as the laryngeal specimens) of the subjects diluted (eg, dilution ratio q:500) are combined. Antibody, multiplexed with biotin (β i 〇ti η) detection antibody (detecti ο n A ntib 〇dy) and biotin-specific protein labeled with fluorescent agent (Cy3 or Cy5) Streptavidin reacts, and if the antigen in the body fluid binds to the immobilized antibody on the slide, it can be judged from the fluorescence result whether the body fluid sample contains the SARS virus. In particular, because the antibody is produced as early as 14 days, it is difficult to detect the presence of antibodies in the early stage of the fever to detect whether the blood, throat, or nasal cavity has been infected with SARS virus. __ The protein microarray of the present invention (applied to the g-bed test, mass drug screen development or food safety, environmental sanitation inspection production method includes the following steps: the current I g Μ must wait until the onset 7 to detect by antigenic microarray Antibody, 'At this time, you need to use the type of antigen in the antibody-type micro-strand to judge 〇

Protein Microarray) can be selected, genetic disease detection, vaccine test and animal and plant quarantine. Its surface is coated with at least one layer containing a plurality of protein arrays containing Junyi, and (a) a carrier is provided on the surface. a base coating on the aldehyde-containing coating

Ϊ294967 ^^^___—5, (Protein Array Regions) and a blocking region formed in the adjacent protein array interval; (b) at least one specific protein is spotted onto the protein array region' Immobilizing the specific protein with a microwave having an intensity of 1 · 〇〇 to 5.0 GHz, wherein the at least a portion of the specific protein line comprises one or more pathogenic antigens that induce an increase in body temperature, or against inducible body temperature An antibody to an elevated endogenous pyrogen, or an antibody against a specific pathogenic antigen; (c) covering the aldehyde-containing coating with a blocking agent to sever the non-aldehyde-based coating in the subsequent reaction Specific binding; (d) removing excess blocker, contacting the treated subject with the particular protein; and (e) calibrating the subject's sample to which the particular protein is bound by at least one indicator, For reading by the reading device. The protein microarray utilizes microwave (M i cr owave) short-time, high-energy heating to avoid antigenic epitopes on the pathogenic antigen (Ant i gen De terminant or Epitope) or antigen junction of the antibody (Antigen Binding) Site) has a covalent bond structure that is disrupted; therefore, whether it is an antigenic microarray or an antibody-type microarray, the specific protein bound to the carrier can specifically bind to the subject's specimen for proper detection. The type of pyrogen that causes the body to have a fever. The following is an example of the preparation of an antigenic microarray using SAR-like viral nucleoprotein (Nucleocapsid) as an example: (a) Adding SARS coronavirus antigen, cold virus antigen and laryngeal cancer cell extract 20" 1 to 9 6 In the well plate (96 - well plate)

17425阎中原.Ptd Page 21 1294967 V. Description of the invention (16) The arrayer (Arrayer) is made on the aldehyde-based coated slide; (b) After the point is completed, the microwave oven is placed and the energy intensity is between 1 00 to 5· 00 GHz (better 2.45 GHz), microwave heating of about 800 W for 60 seconds; (c) PBS transpiration (Phosphate Buffer + 2% Skim M i 1 k 'filled acid Salt buffer solution plus 2% w/v ratio skim milk powder) Microwave heating for 3 minutes to block the protein binding of the acid-containing coating in subsequent processes; (d) Room temperature PBST buffer (Phosphate Buffer + 0) · 0 2 5% Tween 20, phosphate buffer solution plus 〇. 0 2 5 % weight / weight ratio of Tween 20 (trade name)) agitated for 3 minutes, then infiltrated with phosphate buffer and centrifuged 1 2 0 0 RPM is dried; (e) adding the treated subject serum (primary antibody), reacting at room temperature for 30 minutes, repeating the washing action of step (d); (0 separately adding fluorescence (eg Cy3) Or Cy5) labeled anti-human Ig A antibody, anti-human I g G antibody or anti-human I g Μ antibody (secondary antibody) at room temperature The reaction is repeated for 30 minutes, and the cleaning operation of step (d) is repeated; and (g) the results are interpreted by a wafer scanner (Axon Genepix 4 0 0B), wherein the primary antibody is a test for the body fluid of the subject. Body, such as whole blood, serum, saliva, nasal and laryngeal secretions or mucosal tissue from endoscopy, must be collected by protein and diluted in PBSMT buffer (Phosphate Buffer + Skim Milk + Tween 20).

17425阎中原.ptd Page 22 1294967 V. Invention description (17) Can be used. The protein microarray is applied to two people for clinical examination. __ In this example, the coronavirus strain (C〇r οna viri da e) which causes upper respiratory tract infection is taken as an example to explain in detail the protein microarray of the present invention in the early stage of the patient's fever. To detect the pathogen causing fever in the human body and to correctly diagnose whether the maid has been infected with SARS virus. Acquisition of antigen: The nuclear factor (Nucleocapsid) gene of Cloning SARS coronavirus strain is selected by the severe acute respiratory distress syndrome (SARS) 病毒ί* virus strain causing fever in human body, and passed through Escherichia coli (E·

Col i ·) After performance and purification, the treated nuclear protein was used as an antigen, and the cold virus antigen and Hep-2 laryngeal cancer cell extract (Ce 1 1 Lysate) were spotted into the protein array region. Among them, the cold virus antigen is used as a positive control group of an exogenous antigen (E X 〇 g e η 〇 u s A n t i g e η) (e.g., S ARS coronavirus antigen). Since almost everyone has been infected with a cold virus, Ig G against the cold virus can be detected when testing the Ig G antibody in the individual's blood, so if the subject has a fever due to re-infection with a cold virus, The concentration of Ig G against the cold virus will increase. The extract of Hep-2 throat laryngeal carcinoma cells is used as a negative control group for exogenous antigens, and can also be used as an endogenous antigen (E nd 〇ge η 〇us A ntige η ) to detect autologous in the specimen. Autoant i body, such as anti-nuclear antibody (ANA). Because there is no antibody in the normal human serum, but in

17425阎中原.ptd Page 23 1294967 V. INSTRUCTIONS (18) Subjects who develop autoimmunity (Auto i mmune) often develop A ΝΑ and combine with Hep-2 laryngeal cancer cell extract. The use of a negative control group of laryngeal cancer cell extracts can converge the quality of different batches of protein microarrays to keep experimental error within a reasonable range. %: Compared to traditional immunofluorescence, ELISA or test paper coloring, the protein microarray preparation of the present invention requires less protein (generally only one hundred thousandth of the ISA method), samples and reagents. Low demand, simple process, short reaction time, replacement of live pathogens with recombinant proteins or antibodies (reducing the risk of infection in the preparation process of the manufacturer, and no need for P3 laboratories), and the same vector The advantages of a variety of standard antigens or antibodies. As shown in Fig. 6, the antigenic microarray of the SARS coronavirus is used to detect I g A, I g μ and I g ◦ antibodies in the serum of the formula, wherein the array, 1 acts as bovine serum albumin (Bovine Serum Albumin, BSA) ^ control group); array second behavior SARS virus nucleoprotein; array number [= for ^ 冒 = toxic antigen protein (positive control group), each group is ordered 6

Tg A _ is not, the tester has anti-SARS virus antibody stain in the serum; and in the third line of the array: :! ί 2 ί has indeed been affected by SAM virus to the needle η main / mesh In the case of the disease PV, the patient's blood can be clearly detected against the IgG antibody against the virus, to show the results of the microarray of the present invention and to monitor the serum resistance of the subject from the antigenic microarray (as shown in Fig. 7). Show), can also hold SARS virus antibodies (such as Ig G or Ig M)

1294967 V. Description of the invention (19) -~1 - The change is observed as the basis for drug evaluation by observing the development of the subject's disease course. L2·Egg fish + quality microarray applied to 籂烚: The above-mentioned antigenic microarray or body type microarray can also be applied to the large-scale inspection, so that multiple ΐ ϊ ΐ can be accepted in the same protein microarray. 1 test # ° as shown in Figure 8 'If you use the antigenic microarray of SARS virus nucleoprotein to detect the scent of SARS antibody, and only detect it at a time -: whether the second attack contains anti-caries or Ig G Etc.), the same vector carrier antibody type (for example, 4 A, k body; similarly, * use antibody type; can accommodate 12 subjects with SARS coronavirus, each piece of Christine Detecting whether the body fluid sample of the subject is the same as the body fluid sample of the subject, and can also accept the number of 12 protein array regions at the same time, and is examined: 'only the position of the sample disposed in the protein microarray can be visualized. Real = di-original (or antibody) type and subject without being restricted by the embodiment of the present invention, changing, replacing or adjusting, in addition, the protein region of the present invention, and different parts in the same area The column is more able to distinguish multiple subjects, the subject and the subject are: gentleman For the target, as shown in Fig. 8 and A, the two subjects can be separately accommodated in a protein microarray, and the regions detect the jg 血清 in the serum, and the four protein arrays of 1, 2, 3, and 4 respectively. Such as nasal cavity, larynx, trachea or lung % M, I g G or other secretion samples, etc. As shown in Figure 9, it is more positive. 卩) The corresponding antibody B of pathogenic antigen in Ig G and IS Blood, the main 1 I thick, ^ in the patient A and SARS patients within the uniform detection results, showing patients A and patients B &1; Haoli against the SARS virus Ig G and Ig Μ, but patients monthly ^

17425阎令原· _ 1294967 V. INSTRUCTIONS (20) Fluorescence reaction also occurs in the negative control group of Ig G (ie, the laryngeal cancer Hep-2 protein extract shown in the first line of the array) (patient No) It is obvious that the patient's unidentified autoimmune reaction in the B body leads to the detection of anti-nuclear antibody (ANA), which may be unfavorable for the prognosis. The above embodiments are merely illustrative of the principles of the invention and its advantages, and are not intended to limit the invention. Modifications and variations of the above-described embodiments can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be as set forth in the scope of the patent application to be described later.

17425阎中原.ptd Page 26 1294967 Schematic description of the figure [Simplified description of the drawings]: Figure 1 is a schematic perspective view of a protein microarray of the present invention; Figure 2 is a diagram of the protein microarray of the present invention. Schematic diagram of the pathogenic antigen (Pathogenic Ant igen) for detecting the antigenic microarray of SARS virus nucleoprotein; Figure 3 is a diagram of the protein microarray of the present invention for detecting whether the subject has an antibody Schematic diagram of the action of an antigenic microarray (for example, I g A, I g Μ or I g G); Figure 4 is an antibody microarray for detecting SARS virus nucleoprotein in the protein microarray of the present invention. Schematic diagram of an anti-pathogenic antigen antibody; Figure 5 is a schematic diagram of the action of an antibody-type microarray for detecting whether a subject has a specific pathogenic antigen in the protein microarray of the present invention. Figure 6 is a graph showing the results of a fluorescent microarray for detecting an anti-SARS virus antibody in an individual in a protein microarray of the present invention (line 1 shows bovine serum white) White (B〇vine Serum Albumin, BSA) (negative control group), array second behavior ^ viral nucleoprotein, array 3 is cold virus antigen protein (positive control group) ·, ^ Figure 7 The protein microarray of the present invention detects a change in the concentration of the antibodies 丨g G and 丨g M against the f ARS virus in the serum of the subject; FIG. 8 shows a large number of antigen-type microarrays in the protein microarray of the present invention. a top view of the /antibody type microarray;

17425阎中原.ptd

Page 27 1294967 Brief Description of the Drawing Figure 9 is a fluorescing result of a large number of screened SARS virus antigen-type microarrays in the protein microarray of the present invention (array 1 is used to detect anti-nuclear antibodies) (A nti - nuc 1 ear A ntib 〇dy, AHA), and as an antigen of autoimmune indicators), wherein the negative control group uses He ρ - 2 laryngeal cancer cell protein extract as an antigen source (array Line 2 indicates the nucleoprotein antigen of the SARS virus). (There is no component symbol in this case and the meaning it represents)

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Claims (1)

1294967 VI. Patent Application Range 1. A Protein Microarray comprising: a carrier having a plurality of Protein Array Regions defined thereon; at least one specific protein having an intensity of 1 a microwave of 0 0 to 5.0 GHz immobilizes the specific protein onto the protein array region, wherein the specific protein contains at least one antigenic protein capable of inducing an increase in body temperature to be associated with the sample to be tested The antibody produces a specific binding; and at least one calibration substance is bound to the antibody in the test subject for interpretation by a reading device. 2. The protein microarray according to claim 1, wherein the protein microarray is an antigenic microarray (A ntige η Microarray). 3. The protein microarray according to claim 1, wherein The carrier is a wake up: base coated slide. 4. The protein microarray of claim 1, wherein the carrier is a nylon membrane (Nylon Membrane). 5. The protein microarray of claim 1, wherein the carrier is a polylysine coated slide. 6. The protein microarray of claim 1, wherein the carrier is a stone coated with a stone (N i t r 〇 - c e 1 1 u 1 〇 s e). 7. The protein microarray of claim 1, wherein the specific protein is a pathogenic antigen protein (P a t h 〇 g e n i c 17425阎中原.ptd Page 29 1294967 6. Patent application scope Antigen) ° 8. The protein microarray of claim 7 wherein the pathogenic antigen protein is selected from the group consisting of bacteria, viruses, and ricketts. Pathogens such as secondary bodies, molds and parasites, bacterial capsules, Proteocapsid, Nucle〇capsid, Recombinant Protein, phage, biological tissue cell protein extracts and nuclear extraction Exogenous Thermogen, one of the group of proteins and the like. 9. The protein microarray of claim 1, wherein the specific protein is a negative control group of bovine serum albumin (BSA) 〇1 〇 · as claimed in claim 1 a protein microarray, wherein the specific protein line is a negative control group to detect an anti-nuclear antibody (eight 111:1-]111 (:1631 'person] 11:113 beaten, 〇幻之1^1)-2 Throat cancer cell extract. Π . For example, the protein microarray of the patent of the patent, wherein the system to be tested is a diluted body fluid of the subject. 12. A protein microarray according to claim 11 Wherein the body fluid of the subject is selected from the group consisting of secretions such as blood, serum, saliva, sweat, tears, urine, and throat, nasal cavity, lung, trachea, etc. 1 3 · Such as Shen Qing The protein microarray of the third aspect of the invention, wherein the anti-system is a human antibody I g A. 17425阎中原ptd page 30 1294967 VI. The scope of patent application is human antibody Ig M. The protein microarray of claim 1, wherein the antibody is a human antibody I g G. The protein microarray of claim 1, wherein the calibration substance is an antibody against a fluorescent agent (A n t i - a n t i b 〇 d y). 1 7 . The white matter microarray of claim 1, wherein the reading device is a Confocal Microscopy. The protein microarray of claim 1, wherein the reading device is a Laser Scanner. 19. A Protein Microarray comprising: a vector having a plurality of Protein Array Regions defined thereon; at least one specific protein having an intensity ranging from 丨.〇〇 to 5 The microwave of 0 0 GHz immobilizes the specific protein onto the protein array region, wherein the specific protein contains at least one antibody against the antigen which induces an increase in body temperature to specifically bind to the antigen in the sample to be tested. And at least one calibration substance is combined with the antigen in the sample to be tested for interpretation by a reading device. A protein microarray according to claim 19, wherein the protein microarray is an antibody type microarray (Ant ib〇dy Microarray) 〇 1294967 VI. Scope of Patent Application 2 1. A protein microarray according to claim 19, wherein the carrier system is an acid-based coated slide. 2 2. A protein microarray according to claim 19, wherein the carrier system is a nylon membrane (Nylon Membrane). A protein microarray according to claim 19, wherein the carrier system is a polylysine coated slide. 2. A protein microarray according to claim 19, wherein the carrier system is a coated glass of N i t r 〇 - c e 1 1 u 1 〇 s e. A protein microarray according to claim 19, wherein the specific protein is an anti-pathogen ic antigen antibody o 2 6 as claimed in claim 25 A protein microarray, wherein the pathogenic antigen is a pathogen selected from the group consisting of bacteria, viruses, rickettsia, molds, and parasites, a capsule, a protein sheath (Proteocapsid), and a nuclear protein. Exogenous Thermogen (Nucleocapsid), Recombinant Protein, bacillus, biological tissue cell protein extract, and nuclear extraction protein. 2. The protein microarray of claim 19, wherein the specific protein is an antibody against an intrinsic pyrogen (E n d 〇 g e η 〇 u s Thermo gen). 2 8. The protein microarray of claim 27, wherein the endogenous pyrogen is a thermogenic cytokine ( Thermogen Cytokine) 7422阎中原.Ptd Page 32 1294967 VI. Patent Application No. 2 9. The protein microarray of claim 27, wherein the endogenous pyrogen is an autoimmune antigen (A ut 〇antige) η). 3 Ο . The protein microarray 4 of claim 27, wherein the endogenous pyrogen is a prostaglandin. 3. A protein microarray according to claim 19, wherein the specific protein is a negative control group of Bovine Serum Albumin (BSA) o 3 2 . The protein microarray of the item, wherein the system to be tested is a diluted body fluid of the subject. 3. The protein microarray of claim 32, wherein the subject's body fluid is selected from the group consisting of, for example, blood, serum, saliva, sweat, tears, urine, and throat, nasal cavity, lung, trachea, etc. One of the groups of objects. 3. The protein microarray of claim 19, wherein the calibration material is an antibody with an illuminant (An t i body). 3 5. A protein microarray according to claim 19, wherein the calibration substance is a primary antibody with biotin (B i 〇ti η), and the phytosporin with a fluorescent cursor (Streptavidin) binds to the reaction. A protein microarray according to claim 19, wherein the reading device is a total coke microscope (C ο n f 〇 c a 1 M i c r 〇 s c 〇 p y ). 3 7. The protein microarray of claim 19, wherein the reading device is a Laser Scanner. 17425阎中原.ptd第33页