KR20030012130A - Protein chip-based hiv diagnostic system - Google Patents

Abstract

PURPOSE: Provided is a protein chip to detect HIV antibodies in a human serum sample. Also, provided are its manufacturing method and a diagnostic system using the protein chip. CONSTITUTION: The protein chip is manufacturing the steps of: arraying a plurality of antigen spots on the surface of a base plate which is partitioned; fixing antigen proteins to the base plate at room temperature at 30 minutes of time intervals; dipping the base plate into cooled acetone to strengthen the bind between the antigen and the base plate, and drying the base plate.

Description

PROCIN CHIP-BASED HIV DIAGNOSTIC SYSTEM}

The present invention relates to a protein chip for detecting the presence of an antibody against HIV in a human serum sample and a method for producing the protein chip. The present invention also relates to a diagnostic system comprising said protein chip, automated microarray and fluorescence reader.

The most common method for diagnosing viral infection is an enzyme-linked immunosorbent assay (ELISA) using 96 well-plates, which can detect antibodies to the virus using antigen-antibody reactions. Although ELISA is a useful method for diagnosis, it has the following disadvantages: 1) The maximum number of samples that can be analyzed in one reaction is 96, which is relatively small. 2) The time required for the reaction for analysis It takes a lot of time, about 3 hours or more. 3) The amount of sample required for analysis is relatively large.

To address this problem, the researchers studied how to use 384 well or 1536 well plates instead of 96 well-plates. However, such an attempt cannot solve the problem of excessively required amount of sample or reaction reagent. For example, even with 1536 well-plates, the amount of sample still requires microliters (about 5 μl). In addition, there is still a problem that the reaction time is not shortened. Therefore, there has been a need for another method for overcoming the disadvantages of ELISA, and the inventors of the present invention use a protein microarray and a protein chip using the same as an alternative to ELISA. It was developed.

Biological chips or biochips first appeared in the mid-19th century and have been used in some fields such as biology, biotechnology and medicine. The biochip has several advantages over other analysis systems, the most important of which is that a large amount of analysis can be performed in a short time using a small amount of sample. The most widely used biochips are DNA chips, which are only a few years from the beginning of the study (Lueking, A. et al ., Protein microarrays for gene expression and Antibody screening.Anal Biochem . 1999; 270 : 103-111, de Wildt, RMT et al ., Antibody arrays for high-throughput screening of antibody-antigen interactions.Nat Biotech 2000; 18 : 989-994, MacBeath, G., Schreiber SL, Printing proteins as microarrays for high-throughput function determination Science 2000; 289:. 1760-1763).

We have developed a protein chip that can be used for HIV diagnosis by arranging a plurality of HIV antigens on a solid substrate. Specifically, a very small scale Fluorescence immunoassay (FIA) was performed on the slide glass as shown in FIG. 1; 1) The primary antibody reaction was performed by dispensing the serum sample at the corresponding antigen spot location using an automated microarray, and 2) the anti-human antibody coupled with the fluorescent dye to the spot location using the same microarray. Antigen-antibody responses were detected by aliquoting, and 3) reactions were analyzed by reading fluorescence using a fluorescence reader.

An object of the present invention is to provide a protein chip that can be used for the diagnosis of HIV. Another object of the present invention is to provide a method for producing the protein chip. The final object of the present invention is to provide an HIV diagnosis system using the protein chip.

1 is a schematic diagram showing a diagnostic process using a protein chip of the present invention,

2 is a schematic showing the procedure flow of an assay using a protein chip, microarray and reader,

3 is a photograph showing accurate spot recognition and re-dispensing using an automated microarray.

A plurality of Cy3 labeled protein spots were arranged on a microscope slide glass (a) and a Cy5 labeled protein solution was dispensed onto the arranged spots (b). The images of a and b are superimposed on c.

4 is a schematic diagram showing an arrangement pattern of a protein chip,

5 is a photograph showing the results obtained using the FITC-bound anti-human antibody as a secondary antibody,

FIG. 6 is a photograph showing results obtained using an anti-human antibody conjugated with Alexa fluorescent 488 as a secondary antibody. FIG.

Protein chip for the diagnosis of HIV is a plurality of HIV antigens arranged on a solid substrate. The protein chip is prepared by arranging antigenic spots on a substrate using an automated microarray and immobilizing these spots with cold acetone.

To diagnose an infection, the microarray must dispense serum samples at the exact location where the corresponding antigen spot is fixed. After washing by removing the antibodies that did not participate in the reaction, the anti-human antibody conjugated with the fluorescent dye is dispensed to the corresponding spot position using a microarray. The reaction is then detected by activating the fluorescent dye by exposure to light of a suitable frequency and reading the pattern of generated fluorescence with a fluorescence reader.

The HIV diagnostic system using the protein chip of the present invention is capable of: 1) first microarray arranging a plurality of HIV antigen spots on a micro substrate, and 2) dispensing subsequent samples (serum and secondary antibodies) accurately over the spot. Secondary microarrays, 3) readers for detecting reaction signals (e.g. fluorescence), and 4) means for converting the signals into computer readable data and analyzing these data.

The protein chip of the present invention consists of a small-sized solid substrate, on which a plurality of HIV antigen spots are precisely arranged. The amount of HIV antigen per spot is, for example, in picograms or nanoliters.

The substrate itself may be made from glass, modified silicone, or polymers or gels such as polycarbonate, polystyrene and polypropylene. The surface of the substrate may be encoded with a chemical that helps the protein to stably bind to the substrate. Preferably, the surface of the substrate may be coated with a chemical selected from the group consisting of polymers, plastics, resins, hydrocarbons, silicas, silica derivatives, carbons, metals, inorganic glasses and films.

The materials for coating the substrate preferably have a functional group to form a stable bond with the protein probe. For example, a material having an amino acid residue having an amino group such as lysine may be used.

HIV antigens are finely arranged on protein chip substrates. The antigen spot is preferably circular having a diameter of about 150-1000 μm. As a preferred example of the chip, in the present invention, a slide glass having a size of 2.5 cm x 7.5 cm may be used in which about 100 to 10,000 spots may be arranged per chip. Antigens are arranged in rows and columns on the substrate.

The present invention also provides two distinctive processes for making the protein chips.

The method of manufacturing a protein chip comprises the steps of: 1) arranging one or more antigens in a predetermined position on a substrate in an amount of several to several tens pg per spot; 2) immobilizing the protein by leaving the chip at room temperature for 30 minutes; 3) immobilizing the protein on a chip by dipping in 100% acetone; And 4) drying the chip.

Another method for producing a protein chip of the present invention comprises the steps of: 1) arranging one or more antigens in a predetermined position on a substrate in an amount of several to several tens pg per spot; 2) immobilizing the protein by leaving the chip at room temperature for 30 minutes; 3) immersing the chip in a PBS solution containing 0.1% casein to block regions of the chip that are not bound to the antigen; And 4) drying the chip.

The method for analyzing an antibody in a sample using the protein chip of the present invention comprises the steps of: 1) reacting the protein chip with a serum sample; 2) washing the protein chips; Reacting the protein chip with an anti-human antibody coupled with a fluorescent dye; 4) washing the protein chips; And 5) detecting fluorescence using a fluorescence reader. As the anti-human antibody conjugated with the fluorescent dye, an anti-human antibody conjugated with FITC or Alexa Fluoro 488 may be used.

Embodiments of the present invention describe diagnostic methods using protein chips. Positive and negative serum samples were reacted with protein chips immobilized with HIV antigen. Antigen-antibody responses were detected in all positive serum samples, but not in negative serum samples (FIG. 5). In addition, the specificity and sensitivity of the HIV antigen-antibody response was comparable to the ELISA method using 96 well plates (FIG. 6). These results show that the diagnostic system of the present invention and the diagnostic method using the same are useful for serologically diagnosing HIV infection.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the scope of the present invention is not limited thereto.

Example 1 Accurate Spot Recognition and Re-Spotting

The most important feature of the diagnostic system using the protein chip of the present invention is that the automated microarray can recognize the protein spot on the chip and dispense the sample at exactly the expected spot location.

To confirm that the microarray can perform this exact re-spotting, the protein solution labeled with Cy3 and Cy5 was immobilized on the slide glass, and then the images obtained with a fluorescence reader (Affymetrix 418 Array Scanner) were superimposed.

First, 0.1 M sodium carbonate buffer (pH 9.3) containing 250 μg of bovine serum albumin (BSA; Sigma, USA) and NHS-activated Cy3 or Cy5 solution (Amersham, UK) were mixed. The reaction solution was kept at room temperature for 45 minutes in the dark. Thereafter, the solution was filtered through a 10 kDa microcon (Microcon; Millipore, USA), and then centrifuged at 12,000 × g for 25 minutes. After centrifugation, the obtained precipitate was dissolved in 1 ml of 0.1 M sodium carbonate buffer (pH 9.3) and centrifuged again to obtain 50 µl of a protein solution in which fluorescent dyes were bound. After repeating the procedure two more times, the samples were recovered.

After obtaining the Cy3-labeled protein in the same manner as above, transfer the concentrations to the 96 well plates at 50 µg / ml, 5 µg / ml, 0.5 µg / ml and 0.05 µg / ml, and then transfer the plates to the microarray. Mounted inside. Cy3 labeled proteins were arranged on the slide glass surface using a microarray. Images of protein spots were read using a fluorescence reader (Affymetrix 418 Scanner) and then stored (FIG. 3A).

Cy3-labeled protein spot positions arranged on the same slide glass using a microarray after concentrations of the Cy5 labeled proteins were 0.05 µg / ml, 0.5 µg / ml, 5 µg / ml and 50 µg / ml, respectively. Was dispensed. Images of protein spots labeled with Cy5 were read with a fluorescence reader (FIG. 3B) and overlapped with images of spots labeled with Cy3 (FIG. 3C). Figure 3 shows that the fluorescence images obtained from the protein spots labeled with Cy3 and Cy5, respectively, overlap precisely, indicating that the automated microarray used in this experiment can perform accurate re-spotting operations. Can be.

Example 2 Preparation of Protein Chip for HIV Diagnosis I

384 HIV antigen spots were arranged on microscopic slide glass (Sigma, USA) coated with poly-L-lysine. The slide glass was divided into eight regions, each of which contained 48 (6 × 8) spots in a space of 1 mm ² (FIG. 4). Antigens were arrayed using an automated microarray (ArrayIt, USA) equipped with four iron pins with a diameter of 300 μm.

HIV-1 gp41, HIV-1 p24 and HIV-2 gp36 used as antigens were purchased from VenDiatech (Korea). These antigens were diluted with 10 mM sodium carbonate buffer (pH 9.6) to bring the antigen concentrations of gp41, p24 and gp36 to 150 μg / ml, 60 μg / ml and 75 μg / ml, respectively.

The antigen solution was transferred to a 96 well plate and then the plate was mounted in a microarray. The antigen solution was arranged on the slide glass for microscope by microarray.

The slide glass on which the HIV antigens were arranged was left at room temperature for 30 minutes in a humid chamber, and then taken out and dried. Thereafter, the slide glass was immersed in 100% cold acetone for 1 minute to firmly bind the antigen to the slide glass, and then taken out again and dried.

Example 3 Preparation of Protein Chip for HIV Diagnosis II

The antigen solution was arranged on a slide glass coated with poly-L-lysine using an automated microarray in the same manner as in Example 2. The antigen-arranged slide glass was removed from the wet room, dried, and then fixed in a 100% acetone solution, and then immersed in a PBS solution containing 0.1% casein for 30 minutes to block the non-antigen-binding portion of the slide glass. Thereafter, the slide glass was dried.

Example 4 HIV Diagnosis Using FITC-Bound Antibody as Secondary Antibody

The protein chips prepared in Examples 2 and 3 were fixed in the slide position in the microarray. HIV-negative and positive serum samples were transferred to separate 96 plates, and then the plates were mounted in microarrays. The binding reaction of the primary antibody was performed by dispensing each serum sample from the plate over the corresponding antigen spot present on the protein chip using an automated microarray. The pins used for dispensing the sample were sterilized by ultrasonic to prevent contamination of the sample, washed with water and dried with hot air. The protein chip was placed in a humid room temperature for 15 minutes, and then washed three times with PBS and dried.

Subsequently, in order to detect the bound antibody, an FITC-coupled anti-human IgG antibody (# 31529, Pierce, USA) was dispensed to each spot position of the protein chip using a microarray, and then 15 minutes in a dark room at room temperature. Reacted. After the reaction, the slide glass was removed, washed with PBS, and dried three times. The fluorescence represented by the spots on the slide glass was read with a fluorescence reader (Affymetrix 418 Scanner).

Representative results are shown in FIG. 5. In four independent experiments, all positive serum samples and positive controls showed strong fluorescence, whereas all negative samples and negative controls showed no response signal.

Example 5 HIV Diagnosis Using Alexa Fluor 488-Bound Antibody as Secondary Antibody

Diagnosis of HIV was carried out in the same manner as in Example 4 except that Alexa Fluor 488 bound antibody was used as the secondary antibody instead of FITC. Representative results are shown in FIG. 6. Similar results were obtained with the use of FITC-binding antibody in the case of using an anti-human antibody conjugated with Alexa Fluor 488.

Example 6 Repeatability of the Assay

Three HIV positive samples and one HIV negative sample were tested to calculate repeatability of the assay using the protein chip of the present invention. That is, each of the four samples for each of the four samples to confirm the repeat reproducibility of the HIV diagnostic protein chip was performed. As the protein chip, one prepared in Example 3 was used, and experiments were performed using an antibody bound to Alexa Fluor 488. The results are shown in Table 1 below.

sample Analysis count Average fluorescence / baseline In-sample (% CV) Sample-to-sample (% CV) One 10 4.83 5.31 8.73 2 10 5.56 5.16 6.46 3 10 4.20 3.66 5.20 4 10 0.54 10.53 16.08

* CV (%) = Coefficient of variation: = standard deviation / mean x100

Example 7 Sensitivity and Specificity of Assays

Experiments were conducted on a large number of serum samples to confirm that the protein chip of the present invention can be successfully applied for the diagnosis of HIV. An antibody in which the protein chip prepared in Example 3 and Alexa Fluor 488 was bound was used in the experiment.

HIV positive serum samples were obtained from Seoul National University College of Medicine and Korea National Institutes of Health. HIV negative serum samples were obtained from healthy volunteers working at the Seoul National University Genetic Engineering Research Institute. All HIV positive serum samples were inactivated by heat treatment at 56 ° C. for 30 minutes prior to use.

In the analysis, the reaction was based on whether the fluorescence intensity was the same as or greater than the reference value, and the reference value was set to the average value of the fluorescence intensity in the negative control group plus 300.

The ELISA kit Murex HIV 1/2 / O (Abbott Lab.) Performed a diagnostic experiment using the HIV diagnostic protein chip of the present invention on a total of 62 samples confirmed to have an antigen against HIV.

Judgment result Number of samples responsiveness(%) Specificity (%) HIV positive (n = 51) HIV negative (n = 165) positivity 48 4 94.1 97.6 voice 3 161

By using protein chips for diagnostic purposes, it was possible to reduce the reaction time and the amount of reagent used, and to increase the amount of samples that could be analyzed in one reaction.

Claims (11)

1) dividing the compartments on the surface of the micro substrate so that a plurality of antigen spots are regularly arranged; 2) immobilizing the antigenic protein by leaving the substrate at room temperature for about 30 minutes; 3) immersing the micro substrate on which the antigen is arranged in cold acetone to strengthen the binding of the antigen to the substrate surface; And 4) manufacturing a protein chip for disease diagnosis, comprising the step of drying the micro-substrate. The method of claim 1, The microsubstrate is made from glass, modified silicone, or polymers or gels such as polycarbonate, polystyrene and polypropylene. The method of claim 1, Wherein said microsubstrate is coated with a chemical selected from the group consisting of polymers, plastics, resins, hydrocarbons, silicas, silica derivatives, carbons, metals, inorganic glasses and membranes. The method of claim 3, wherein Wherein the chemical coated on the substrate has an amino group. 1) dividing the compartments on the surface of the micro substrate so that a plurality of antigen spots are regularly arranged; 2) immobilizing the antigenic protein by leaving the substrate at room temperature for about 30 minutes; 3) blocking the reaction of the substrate region not bound to the antigen by immersing the micro substrate on which the antigen is arranged in a solution containing casein; And 4) manufacturing a protein chip for disease diagnosis, comprising the step of drying the micro-substrate. The method of claim 5, The solution of step 3 is a phosphate buffered saline (PBS) solution containing about 0.1% of casein. A protein chip prepared by the method of claim 1 or 5. The method of claim 7, wherein An HIV diagnostic protein chip, characterized in that at least one antigen selected from HIV-1 gp41, HIV-1 p24, HIV-2 gp36, and mixtures thereof is immobilized on a substrate. 1) reacting the protein chip of claim 7 and the serum sample; 2) washing the protein chip; 3) reacting the protein chip with an anti-human antibody coupled with a fluorescent dye; 4) washing the protein chip; And 5) detecting fluorescence using a fluorescence reader, Disease diagnosis method using protein chip. The method of claim 9, The fluorescent dye of step 3 is characterized in that the FITC or Alexa Fluor (Alexa Fluor). The method of claim 9, The reaction of the protein chip with the serum sample and the anti-human antibody combined with the fluorescent dye is carried out using an automated microarray.