KR20040064054A - Strips and fluoresence scanner for the measurement of high sensitivity c-reactive protein in serum and whole blood - Google Patents

Abstract

PURPOSE: Strips and fluorescence scanner for the measurement of high sensitivity C-reactive protein in serum and whole blood are provided, thereby rapidly detecting C-reactive protein, and easily and cheaply installing the strips and fluorescence scanner in small hospitals due to its small size. CONSTITUTION: A monoclonal antibody against C-reactive protein is provided, wherein the monoclonal antibody against C-reactive protein is mass produced by preparing the monoclonal antibody against C-reactive protein, producing a hybridoma cell line producing the monoclonal antibody against C-reactive protein, and selecting one monoclonal antibody having improved reaction to C-reactive protein. The strips for the measurement of high sensitivity C-reactive protein in serum and whole blood comprises a nitrocellulose membrane, a sample pad, an absorption pad which absorbs excessive sample; and a polystyrene support, wherein the nitrocellulose membrane contains a standard line containing a first fluorescence-labeled antibody and detection line containing a second fluorescence-labeled antibody.

Description

STRIPS AND FLUORESENCE SCANNER FOR THE MEASUREMENT OF HIGH SENSITIVITY C-REACTIVE PROTEIN IN SERUM AND WHOLE BLOOD}

The present invention relates to a method for quantitative determination of C-reactive protein using immunoassay and a method for testing C-reactive protein present in the blood at low levels using the method. More specifically, using monoclonal antibodies directed against C-reactive protein, they were reacted with liquid samples of subjects using lateral flow competing immunofluorescence chromatography and the results were reduced to several hundred pg / quantitatively measuring up to ml concentration; Using the selected monoclonal antibody and fluorescently labeled subjects, a method of quantitating a subject present in blood or serum samples by a competitive discrimination method, and a fluorescent substance labeled with the selected monoclonal antibody by fluorescent labeling. It provides a method of quantitatively measuring a subject in a blood or serum sample by a competitive discrimination method.

C-reactive protein has been used for more than 30 years as a measure of acute inflammatory response. Recently, a method for more sensitive and quantitative measurement of the concentration of C-reactive protein in serum or cerebrospinal fluid has been developed and is being re-evaluated for various diagnostic applications. C-reactive protein is an abnormal serum glycoprotein that is synthesized in the liver when an acute inflammatory reaction occurs. C-reactive protein is used as a measure of the progress of inflammation, since it increases dramatically almost simultaneously with the appearance of an inflammatory response and quickly disappears when the inflammatory response decreases or resolves. In addition, the measurement of C-reactive protein concentrations over time can provide important information on the termination or duration of the patient's inflammatory process.

C-reactive protein was named C-reactive protein (CRP) in the fact that it reacts with C-polysaccharide of Pneumococcus when calcium ion is present in serum of acute inflammatory patients. In all infectious and non-infectious inflammatory reactions and some malignancies, an increase in serum C-reactive protein is a common phenomenon. Because C-reactive protein increases nonspecifically in both infectious and non-infectious cases, C-reactive protein concentration alone is not suitable for use as a diagnostic tool for certain diseases. However, measurement of C-reactive protein concentration can provide very useful information in interpreting the patient's entire medical information.

C-reactive protein levels in blood or cerebrospinal fluid are always associated with pathological changes and are therefore important for diagnosis, prescription and observation of inflammation. C-reactive protein is a more sophisticated and reliable measure of the progression of the inflammatory response than the number of erythrocyte sedimentation rates (ESRs) or white blood cell counts used in the past. The blood C-reactive protein concentration increases faster than the erythrocyte sedimentation rate value in the same situation, and reaches a normal value several days before the erythrocyte sedimentation rate value returns to normal even when the situation ends and decreases. Serum C-reactive protein measurement is a good way to answer questions such as whether the patient is improving, worsening, complications, or other problems. Unlike other proteins and enzymes in serum, the increase in C-reactive protein always appears in one pattern, even for a variety of causes. However, it is also the first measurable factor due to its very rapid growth.

Applications of the measurement of C-reactive protein include the diagnosis of diseases requiring rapid treatment, such as bacterial infections of newborns, neonatal sepsis, meningitis, neonatal infections, acute appendicitis, pneumonia, maternal infections, bacterial infections and protozoal infections, urinary It can serve as a tracking and diagnostic aid for various infectious diseases such as mechanical infections, osteomyelitis and the like. In addition, clinical tracking of diseases related to the immune system, such as rheumatoid arthritis and inflammatory bowel disease, has been studied as a predictor of survival after trauma, surgery, and treatment of malignant tumors. Recently, C-reactive proteins have been studied as predictors of heart attack, coronary artery disease and stroke. Low levels of C-reactive protein, but not disease levels, are always present in the blood. This low-level group of high C-reactive protein showed a three-fold higher incidence of myocardial infarction, two-times of ischemic complications, and four-times of atherosclerosis. In addition, the measurement of C-reactive protein together with high-density lipoprotein cholesterol could significantly predict vascular disease.

Due to these needs, many methods have been developed to detect C-reactive protein. Latex coagulation is the oldest method for measuring C-reactive protein and is a qualitative method that is positive in amounts around 10 mg / L. This method has the disadvantage of using several diluting serum to prevent false negatives, because when the C-reactive protein is present in excess of detectable levels, no aggregation occurs, resulting in false negatives. Although it is possible to set the concentration range by quantification, it has a disadvantage in that it is impossible to obtain accurate quantitative values.

The development of monoclonal antibody technology has led to the development of highly specific antibodies against C-reactive proteins. The use of these high specific antibodies has led to rapid, specific and highly sensitive C-reactive protein assays. New immunoassays, including the most widely used nephelometer, radioimmunoassay, enzyme immunoassay, and radioimmunoassay, are in the spotlight in various clinical settings. Due to advances in device manufacturing techniques, large readers using the Nephelometer method have now evolved to show accurate quantitation in tens of minutes, but most immunoassay kits (Eliza, Lia, etc.) The disadvantage is that it takes at least two hours to get a, a laboratory-level reader, and a professionally trained person to get the result through a complex experiment.

High sensitivity immunosuspension is a new C-reactive protein assay that measures the increase in suspension of reagents by antigen-antibody reactions. The lowest limit that can be measured is around 0.1 mg / L, and a variety of companies sell reagents that are ready for immediate use in large chemical analyzers. Due to the development of analyzers, the results can be viewed in minutes to tens of minutes and there are devices that can handle many samples at once. However, expensive and space-consuming analyzers are difficult to use in field detection or small labs. In addition, all of the above methods are difficult to use such samples because they may produce inaccurate results when using hemolysis, high lipid serum, or suspension serum.

In contrast to the time-consuming immunoenzyme method, the simple diagnosis kit using immunochromatography method takes about 15 minutes to obtain a result, which is convenient for various infectious diseases or cancers using whole blood, serum, and urine. It is widely used for diagnosis and detection of environmental hormones. The lateral flow immunochromatography method is based on the sandwich-type ELISA method, and mainly uses an analyte-gold particle conjugate as a detector and absorbs it into a release pad. If the sample contains a large amount of the detector, the amount of detector bound to the trap is relatively high, and the test line shows a strong positive reaction due to the gold particles, so that the sample can be easily read. The lateral flow analysis method is widely used in various fields such as pregnancy diagnosis, cancer diagnosis, microbial detection, and can be diagnosed very easily, but since the judgment is made with the naked eye, it is difficult to confirm the exact amount. Therefore, the situation is mainly used as a means for qualitative rather than quantifying the analyte.

The present invention can complement or replace the Eliza method by combining the accuracy and quantitative advantages of the conventional detection method with the advantages of a simple diagnostic kit that can be detected quickly, and can be detected immediately at the place where the sample is taken. And quantitative, and instead of the traditional method of requiring a large, dedicated reader, the use of a small mobile device enhances field diagnostics and ease of operation in small laboratories. More specifically, the detection kit using lateral flow fluorescence immunochromatography with improved lateral flow immunochromatography and its dedicated laser fluorescence reader, and more specifically, the blood at several hundred pg / ml Means a method for quantifying C-reactive protein present in serum samples.

1 is a side view of a lateral flow metering strip in one embodiment according to the present invention.

1 support plate 2 sample pad 3 nitrocellulose membrane 4 detection line 5 baseline 6 absorption pad

2 is a plan view of a lateral flow metering strip in one embodiment according to the present invention.

3 is a photograph of the strip assembled into the housing.

4 is a schematic diagram showing the principle of the laser fluorescence reader according to the present invention.

7 Laser 8 Incident Filter 9 Ellipsoidal or Spherical Reflector 10 Detection Strips 11 Spatial Filter

12 Parallel Optical System 13 Photodetector 14 Fluorescence Filter

5 is a schematic view as one embodiment according to the present invention.

6 is a graph illustrating the intensity of fluorescence read by a laser fluorescence reader by adding a sample to the strip according to the principle of FIG. 5.

7 is a graph for calculating an effective detection range of a strip according to the principle of FIG. 5.

The present invention first prepared a monoclonal antibody against the C-reactive protein, a hybridoma cell line producing the monoclonal antibody, and then C-reactive protein among the antibodies obtained from the hybridoma cell culture. The present invention relates to a method for selecting and mass-producing monoclonal antibodies with high reactivity to prepare lateral flow fluorescence immunochromatography strips and quantitative kits. Second, the kit can be used to quantify C-reactive protein. Provides content for dedicated readers.

The invention will be more specifically illustrated by the following examples. However, the following examples are one of various methods for implementing the present invention, and thus do not limit the present invention.

Experimental Example 1

Specifically, the preparation of monoclonal antibodies used in the present invention has been preceded. As for the preparation method of monoclonal antibodies, conventional methods in the monoclonal antibody art were used. Briefly, an aqueous C-reactive protein solution was mixed and emulsified with the same volume of the complete Freund's adjuvant and injected into the abdominal cavity of the Valb's line of mice. Immunize two or three times more antigens with incomplete Freund's adjuvant at intervals of two to three weeks. When a small amount of blood was collected from the mouse and confirmed that antibodies against C-reactive protein were produced by the direct Eliza method, lymphoma cell lines that can separate splenic cells from the mice to form hybridoma cell lines, e.g. Fusion with -14 (SP2 / o-Ag-14) produces a hybridoma cell line with infinite proliferation and producing the desired antibody.

Experimental Example 2

To select hybridoma cell lines producing antibodies that bind to C-reactive protein, 50-ul aliquots of C-reactive protein were diluted in ugly carbonate buffer (pH 9.6) at 2 ug / ml in 96-well microplates. Covered overnight at 4 ° C. and washed with PB-Twin20 buffer (PBST). In order to prevent nonspecific reaction, 50 μl / ml of a fish gelatin solution diluted to 5 mg / ml was added and blocked at room temperature for 1 hour. After washing three times with PB-Tween 20 buffer, 50ul of hybridoma cell line culture solution was added and reacted at room temperature for 2 hours. Mouse-derived anti-C-reactive protein antibodies in cell line cultures reacted with coated C-reactive protein were developed for TMB development using goat-derived anti-mouse immunoglobulin-echialpi-labeled antibodies. After 15 minutes, the color reaction was stopped by adding 10 mM sulfuric acid solution about 15 minutes, and the result was selected by hybridoma cell lines of the culture medium that reacted most strongly by using Eliza reader (Bio-Rad model 550). The cell lines reselected those that did not cross-react with amyloid blood in the serum and named two of them 1C1 and 18C2, respectively.

Experimental Example 3

Hybridoma cell lines producing the desired antibody were injected intraperitoneally. After 4-7 days, ascites were generated, the ascites was collected through a needle. After the ascites was precipitated by ammonium sulfate, the antibody was separated using the protein G column method. It is dialyzed in phosphate buffer and stored frozen until use.

Experimental Example 4

To fluoresce the monoclonal antibody, 10 ul of 1 M chloride carbonate buffer (pH 8.3) is mixed with 100 ul of phosphate buffer in which 1 mg / ml of purified anti-C-reactive protein monoclonal antibody 18C2 is dissolved. To this was added 1 mg of fluorescent substance Alexa Fluor 647 (10 mg / ml) and reacted overnight at 4 ° C. This was centrifuged at 2500 rpm for 2 minutes using a Sephadex G25 column to remove excess unlabeled fluorescent material.

Fluorescent labels of anti-rabbit immunoglobulin G antibodies were also prepared according to the method described above.

Experimental Example 5

The fabrication of a kit containing an analysis strip for application to a laser fluorescent reader is as follows. The main material of the strip consists of a nitrocellulose film, a sample pad, an absorption pad, and a support plate (Figs. 1 and 2). On the adhesive side of the polystyrene support plate is placed a nitrocellulose membrane, a sample pad and an absorbent pad. The nitrocellulose membrane (Millipore HF 180) covered with a thin plastic film is the detection site. Standard lines and detection lines are divided into these detection areas. Standard lines were dispensed with rabbit immunoglobulin G at a concentration of 0.35 ug / ml, and detection lines were dosed with 0.45 ug / ml of anti-C-reactive protein 1C1. Two lines were dispensed at a volume of 1 ul per cm with a thickness of 1 mm using a divider at a position of 30.5 mm and a standard line of 33 mm from the sample pad.

Before placing the sample pad (S & S 903, 4 × 25 mm) onto the nitrocellulose membrane, pre-treat the sample pad thoroughly in phosphate buffer containing 1% bovine serum albumin and 0.05% Tween 20. This is dried for 1 hour in a 50 degree vacuum dryer. An absorbent pad (S & S 470 4 × 20 mm) is placed at the end of the nitrocellulose membrane. The assembled strip on the support plate is placed in a plastic housing designed to be inserted into a dedicated fluorescent reader to complete the assembly of the kit (FIG. 3). The reading part has a rectangular window, the sample inlet is composed of a 15mm diameter circle, and is 5mm deep and can hold about 100ul of sample. Initiate detection by mixing the sample and detection fluid in the sample inlet of the kit.

Experimental Example 6

The laser fluorescent reader of the present invention is composed of a laser, an exciter filter, an elliptical reflector or spherical mirror, a sample control means for emitting surface fluorescence, a spatial filter, a parallel optical system, a fluorescent filter, and a photodetector.

FIG. 4 shows a system for detecting surface fluorescence by arranging incident light and a sample focused on an ellipsoidal mirror or spherical mirror and processing a fluorescence emitted after being focused on a second focal point in parallel light according to the present invention. . The laser emits a wavelength that matches the excitation band maximum of a particular fluorophore. In one embodiment, the laser 7 is a 2 mW He-Ne laser that emits at a wavelength of 594 nm near the absorption maximum of the phosphor labeled Alexa. For example, a He-Ne laser model 05 LYR 173 (Melles Griot, Irvine, Calif.) May be used as the laser. The light of the laser 7 passes through the incident filter 8 and passes through a hole located at an intermediate point of the elliptical reflector 9 which is arranged between the incident filter and the detection strip 10. At this time, the laser, the incident filter and the ellipsoidal mirror are preferably arranged side by side so that the light of the laser can properly pass through the hole of the elliptical reflector 9 through the incident filter 8. The light passing through the ellipsoidal reflector is focused to an appropriate size on the sample surface fixed to the detection strip 10. The sample may be moved up and down or back and forth (direction perpendicular to the ground) to change the position by the control means so that the light can be optimally focused. This focusing point is located at the first focal point of the elliptical reflector 9. The fluorescence emitted from the first focus and the scattered light of the incident light are reflected by the ellipsoidal reflector 9 and focused on the spatial filter 11 as the second focus of the elliptical mirror. The spatial filter serves to remove noise caused by dust and the like located on the surface of the sample. However, the spatial filter may not be used. Light passing through the spatial filter is transformed into parallel light by a collimator 12. If the spatial filter is not used, the second focus of the elliptical mirror becomes a parallel optical system, focuses on it, and is immediately transformed into parallel light. The parallel light formed while passing through the parallel optical system passes through the fluorescence filter 14 and the scattered light is filtered and only the pure fluorescent component is incident to the photodetector 13.

The intensity of the internal standard fluorescence of the standard line and the fluorescence of the detection line are converted into area ratios, and the area ratios are converted into concentration values in comparison with the standard curves.

Experimental Example 7

The composition of the detection liquid may be modified to improve the performance of the invention as an example, and this example does not limit the scope of the invention and may be broadly interpreted.

The detection solution was prepared by dissolving fluorescently labeled anti-C-reactive protein monoclonal antibody 18C2 (2.4ng / ml) and fluorescently labeled anti-rabbit immunoglobulin G (16mg / ml) as a primary detection antibody in phosphate buffer. do. 10 ul of whole blood, or 5 ul of serum and 70 ul of buffer were added to the sample inlet, and after 12 minutes, the intensity of fluorescence at the detection line and the standard line was converted to the area value by a laser fluorescence reader. The area value at the detection line was A _T and the area value at the standard line was A _C. The ratio of A _T / A _C was quantified against the standard curve using the Microsoft Excel program.

5 is a schematic diagram of a process of detecting a subject in a strip in the above-described manner. The intensity of fluorescence reading samples of different concentrations using this kit was graphed, and FIG. 7 is a chart showing the amount of fluorescence according to a standard curve. It can be seen that the intensity of fluorescence is consistently increased from 100 pg / ml to 10000 pg / ml. More detailed experiments show that the kit has a valid measurement range of 133 pg / ml to 10000 pg / ml and a lower limit of 133 pg / ml. It is expected that the low-level C-reactive protein assay reagents on the market are generally in the range of 100 pg / ml to 20000 pg / ml. The standard curve derived from FIG. 7 is Y = 0.020 + 0.572X, and the regression coefficient R is 0.997.

Lateral flow immunofluorescence kit for detecting low-level C-reactive protein according to the present invention can detect C-reactive protein, which is a marker of inflammation and predictor of next-generation cardiovascular disease in blood or serum in a short time. Unlike large hematology analyzers, kits and peripherals are highly mobile, so they can directly test C-reactive proteins in blood or serum without request from small bottles, clinics, or public health centers that do not have large devices or precise laboratory equipment. It is expected to be used in various sectors in practical terms.

Claims (11)

Within the detection area, including a pad made of a porous material capable of transporting a fluid containing a subject, a pad capable of removing blood cells, a pad capable of absorbing extra samples, and a strip mainly composed of a chromatographic membrane, which is a detection area. Lateral flow chromatography that captures a subject combined with a labeled primary detection antibody in a detection solution by sandwiching a detection line and a reference line with a secondary detection antibody fixed to the detection line, and capturing the internal standard directly to the standard line. Graphi kit and reader for reading it. The device of claim 1, wherein the primary detection antibody and the internal standard are labeled with fluorescence The device of claim 1, wherein the subject is a C-reactive protein present in blood or serum. The device of claim 1, wherein the minimum detection limit of the subject is several hundred pg / L. The method of quantifying C-reactive protein in blood or serum according to claim 1, wherein the detection antibody comprises an anti-C-reactive protein goat antibody, rabbit antibody, or mouse antibody. The apparatus of claim 1, wherein the intensity of the signal appearing in the detection line and the standard line is converted into an area ratio, and the amount is proportionally quantified. Apparatus according to claim 1 or 6, wherein the substance causing the signal is a fluorescent substance Apparatus according to claim 1 and 6, wherein the device for reading out signals is a photodetector. The device according to claim 1 or 6, wherein a monoclonal antibody specific to the subject is used as the first capture antibody in the detection liquid, and the first capture antibody specific antibody is immobilized on the detection line. The device according to claim 9, wherein a mouse-derived monoclonal antibody specific for the subject is used as the first capture antibody in the detection liquid, and the mouse immunoglobulin-specific antibody is immobilized on the detection line. Device to increase the retention by adding capture antibody to the detection liquid, not inside the detection strip of claim 1