CN114814243A - Quantitative detection kit and method applied to protein antigen - Google Patents
Quantitative detection kit and method applied to protein antigen Download PDFInfo
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- CN114814243A CN114814243A CN202210707753.0A CN202210707753A CN114814243A CN 114814243 A CN114814243 A CN 114814243A CN 202210707753 A CN202210707753 A CN 202210707753A CN 114814243 A CN114814243 A CN 114814243A
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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Abstract
The invention belongs to the technical field of in-vitro diagnosis and immunoassay, and provides a quantitative detection kit applied to protein antigens.
Description
Technical Field
The invention belongs to the technical field of in-vitro diagnosis and immunodetection, and particularly relates to a quantitative detection kit and a method applied to a protein antigen.
Background
An immunoassay method is commonly used for quantitative detection of protein antigens clinically, and the detection mode is a double-antibody sandwich method. The affinity of the paired antibodies in the double antibody sandwich method is a key factor determining the sensitivity of the assay and the detection range. The difference in affinity between the signal antibody and the coating antibody can determine the sensitivity and accuracy of the low region of the antigen to be detected. If a capture antibody with high affinity is used, the detection sensitivity can be improved, but a hook effect is easy to generate, and the upper limit of detection is reduced; the use of low affinity capture antibodies can broaden the upper detection limit of the detection method, but the detection sensitivity is low. And some special protein antigens, namely IGE, CEA, HCG, AFP, HBs and the like, have high requirements on functional sensitivity and detection range.
IgE is a secreted immunoglobulin that is present in very low levels in normal human serum (less than 0.001% of total serum immunoglobulin content). The IgE concentration in vivo is related to age, the content of newborn baby is lowest, and gradually increases with age, and reaches a stable level at 5-7 years, but reaches the highest content of IgE in the population of 10-15 years. IgE is mainly produced by plasma cells of the lamina propria at nasopharynx, tonsil, bronchus and the like, is a cytotropic antibody, and can be combined with IgE receptors on the surfaces of mast cells and basophils, so that the body is in a sensitized state. IgE is the primary antibody mediating type i hypersensitivity reactions, and the concentration of IgE in serum is often associated with the intensity of exposure to allergens and the severity of allergic symptoms. Serum IgE content measurement is often used for differential diagnosis and therapeutic efficacy monitoring of allergic diseases (such as asthma, hay fever, atopic dermatitis and urticaria) to distinguish between atopic and non-atopic allergic patients. Furthermore, accurate measurement of IgE concentration in serum during childhood is of great importance for predictive assessment of allergic symptoms. The upper limit of the detection range of the existing mainstream detection method is 2500IU/ml at most when the serum is not diluted again, and for the determination of high-level tIgE exceeding the upper limit of the detection, a specimen needs to be diluted and then determined, so that the workload of inspectors and the cost of reagents are increased, the determination time is long, and the rapid detection is not suitable.
Disclosure of Invention
The invention provides a quantitative detection kit aiming at the defects of the existing protein antigen immunodetection method, and when the kit is used for detecting the protein antigen, the kit has excellent sensitivity and wider detection range, and can meet two requirements of differential diagnosis and treatment curative effect monitoring of clinical patients.
In order to achieve the above objects, the present invention provides a quantitative assay kit for protein antigens, comprising a carrier, wherein a low concentration detection zone and a high concentration detection zone are sequentially disposed along a sample flow direction, the low concentration detection zone and the high concentration detection zone are capable of generating signals with different intensities based on the presence or absence of a target analyte, wherein the low concentration detection zone is coated with a capture antibody a, the high concentration detection zone is coated with a capture antibody B, and the affinity of the capture antibody a for the target analyte is higher than that of the capture antibody B for the target analyte.
In some embodiments of the invention, the signal antibody is an antibody labeled with a fluorescent microsphere, and the signal antibody and the capture antibody a or the capture antibody B simultaneously bind to the target analyte to form a sandwich complex with a fluorescent label.
In some embodiments of the present invention, the carrier is a microfluidic chip, the capture antibody a and the capture antibody B are sequentially coated in the microchannel along the sample flow direction to form a low concentration detection region and a high concentration detection region, and the signal antibody is disposed in the microchannel and located at the front end of the capture antibody a to form a label region or directly disposed in a sample addition hole of the microfluidic chip.
In some embodiments of the present invention, a labeling zone, a low concentration detection zone, a high concentration detection zone, and a quality control zone are sequentially disposed in the microchannel along the flow direction of the sample, and the quality control zone is disposed conventionally.
In another aspect, the present invention also provides a method for quantitatively detecting protein antigens by using the kit of any one of the above technical solutions, comprising at least the following steps:
1) acquiring a calibration function;
2) adding a sample possibly containing a target analyte into a sample hole, firstly combining with a labeled antibody, sequentially flowing through a low-concentration detection area and a high-concentration detection area of a carrier, and acquiring a signal value of the low-concentration detection area and/or the high-concentration detection area by using a detection instrument after the reaction is finished;
3) determining the concentration of the target analyte in comparison to the calibration function: the concentration of the target analyte is determined using the calibration function T1 as a standard for the signal value from the lower concentration detection zone when only the low concentration detection zone detects a signal value, and the concentration of the target analyte is determined using the calibration function T2 as a standard for the signal value from the higher concentration detection zone when both the low concentration detection zone and the high concentration detection zone detect a signal.
The kit and the method applied to the protein antigen obtained by the technical scheme have the beneficial effects that:
1. the high-affinity capture antibody A and the low-affinity capture antibody B are sequentially arranged along the direction in which a sample flows, so that the antibodies with different affinities play different roles, the functional sensitivity is ensured, the detection range is widened, the accurate quantification of the protein antigen level with a large concentration range span is realized, and unnecessary dilution is reduced;
2. the micro-fluidic chip has small volume, consumes less reagent and sample, can complete the whole reaction process within 4 minutes, has simple operation and simple preparation of the chip at the early stage;
3. when determining the result, only the signal value of one of the lower concentration detection area and the high concentration detection area is needed to be determined separately, so that the analysis speed is improved.
Drawings
FIG. 1 is a structure of a kit for detecting IgE according to one embodiment of the present invention.
FIG. 2 is a test procedure using the kit of FIG. 1.
FIG. 3 shows the results of the test using the kit of FIG. 1.
Detailed Description
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The present invention relates generally to the field of immunoassay technology, and more particularly, to a method for obtaining concentration information of a target analyte in a sample by sequentially passing the sample possibly containing the target analyte through a high-affinity capture antibody and a low-affinity capture antibody and generating a signal that can be captured, and using two capture antibodies with different affinities in combination to give play to their advantages.
The calibration function is a dose response curve measured by passing a concentration standard containing a series of target analyte through a high affinity capture antibody and a low affinity capture antibody in sequence, thereby inferring the concentration of the unknown sample.
The invention will be further explained with reference to examples and figures, it being understood that the invention is not limited to the specific embodiments described.
The invention provides a quantitative detection kit applied to a protein antigen, which comprises a carrier, wherein a low-concentration detection area (LT) and a high-concentration detection area (HT) are sequentially arranged on the carrier along a sample flowing direction, the low-concentration detection area and the high-concentration detection area can generate signals with different intensities based on the existence or nonexistence of a target analyte, the low-concentration detection area is coated with a capture antibody A, the high-concentration detection area is coated with a capture antibody B, and the affinity of the capture antibody A for the target analyte is higher than that of the capture antibody B for the target analyte.
The low and high concentration detection zones generate a signal based on the sandwich of the target analyte, signal antibody and capture antibody, specifically from the signal carried by the signal antibody itself, which is captured by capture antibody a and/or capture antibody B after binding to the target analyte, to form an immobilized effective signal at the low and/or high concentration detection zones, such as the formation of an enzyme-labeled sandwich complex or the formation of a fluorescent microsphere-labeled sandwich complex, noting that the enzyme is capable of amplifying the target analyte chemical signal by converting the fifth to a detectable product.
In one embodiment, the signal antibody is an antibody labeled with a fluorescent microsphere, and the signal antibody and the capture antibody A or the capture antibody B are simultaneously bound with the target analyte to form a sandwich complex with a fluorescent label.
The signal antibody may be added dropwise to the carrier after mixing with the sample that may contain the target analyte, or may be provided at the front end of the low-concentration detection zone along the direction of flow of the sample to form a labeling zone.
The carrier can be a micro-fluidic chip driven by capillary force to drive a sample to flow or a chip driven by other mobile controllers to flow, and can also be a common detection test strip (comprising a sample loading pad, a marking pad, a chromatography pad and a sample sucking pad).
Specifically, the microfluidic chip comprises a microfluidic substrate and a microfluidic cover plate pressed on the microfluidic substrate, the substrate and the cover plate are enclosed to form a microchannel, one end of the microchannel is communicated with a sample adding hole formed in the cover plate, the other end of the microchannel is communicated with a waste liquid collecting pool, a marking area, a low concentration detection area (LT), a high concentration detection area (HT) and a quality control site C are sequentially arranged in the microchannel of the microfluidic substrate along the flow direction of a sample, a signal antibody marked with fluorescent microspheres is coated in the marking area, a capture antibody A is coated in the low concentration detection area, and a capture antibody B with low affinity relative to the capture antibody A is coated in the high concentration detection area.
The quality control site C is conventionally provided, for example, coated with an immobilized antigen that specifically binds to a signal antibody, and is used to determine whether the detection process is effective.
While figures 1-3 show a kit and process for detecting IgE using one of the embodiments of the present invention, it is to be understood that while the kit described herein is described for IgE determination, the kit described herein may also be used for content determination of protein antigens IgE, CEA, HCG, AFP, HBs, etc.
Taking IgE detection as an example, the signal antibody arranged in the labeling area of the carrier is an IgE antibody labeled by fluorescent microspheres, the high-affinity IgE monoclonal antibody a and the low-affinity IgE monoclonal antibody B are respectively coated in an LT area and an HT area after being coupled with biotin, and the IgE antigen is coated in a C area.
The serum to be detected is added into the sample adding hole and is driven by capillary force to enter the labeling area to be specifically combined with the fluorescent microsphere labeled IgE monoclonal antibody. The antigen-antibody compound continuously surges along the microchannel, and the high-affinity IgE monoclonal antibody A is encountered in the low-concentration detection area, so that the low-concentration IgE can be identified; if the content of IgE is too high, the excessive antigen-antibody complex continuously surges forwards to the high-concentration detection area to meet the low-affinity IgE monoclonal antibody B, and the paired antigen-antibody reaction plateau appears later, which is helpful for identifying the high-concentration IgE.
In another aspect, the present invention also provides a method for quantitatively detecting protein antigens by using the kit of any one of the above technical solutions, comprising at least the following steps:
1) obtaining a calibration function by sequentially flowing a concentration standard containing a target analyte through a low concentration detection zone and a high concentration detection zone to obtain a dose response curve, wherein the calibration function of the low concentration detection zone is T1, and the calibration function of the high concentration detection zone is T2;
2) combining a sample possibly containing a target analyte with a labeled antibody, sequentially flowing through a low-concentration detection area and a high-concentration detection area of a carrier (if the carrier is not provided with the labeled area, the sample needs to be mixed with the labeled antibody before being dripped into the carrier), and respectively acquiring signal values of the low-concentration detection area and the high-concentration detection area by using a detection instrument after the reaction is finished (the detection instrument is a conventional technology, such as a fluorescence detector, and is consistent with a signal carried by a signal antibody);
3) determining the concentration of the target analyte in comparison to the calibration function: when the signal value is detected at only the low concentration detection zone (the target analyte concentration in the sample is low, the target analyte-signal antibody bound complex is completely captured by capture antibody a, and no signal is generated at the high concentration detection zone), the concentration of the target analyte is determined using the calibration function T1 as a standard for the signal value at the lower concentration detection zone, and when the signal is detected at both the low concentration detection zone and the high concentration detection zone (the target analyte concentration in the sample is high, the capture antibody a is insufficient to capture all of the target analyte-signal antibody bound complex, and the signal is generated at the high concentration detection zone), the concentration of the target analyte is determined using the calibration function T2 as a standard for the signal value at the higher concentration detection zone.
It will be appreciated that the absence of a signal from the high concentration detection zone is relative, rather than absolute, and that when the signal from the high concentration detection zone is sufficiently weak compared to the signal from the low concentration detection zone, such as when the signal is fluorescence emitted by fluorescent microspheres, the signal from the high concentration detection zone is not visible to the naked eye, and the signal from the low concentration detection zone can be used to detect the signal from the low concentration detection zone, i.e., the concentration of the target analyte is still determined as the ratio of the signal from the low concentration detection zone to the calibration function T1; if the fluorescent signal from the high concentration detection zone is visible to the naked eye, a fluorescence detector may be used to detect the signal from the high concentration detection zone and the signal from the high concentration detection zone compared to the calibration function T2 to determine the concentration of the target analyte.
The detection method obtains the concentration of the target analyte by comparing the signal values of the low-concentration detection area and the high-concentration detection area with the calibration function, is simple to operate, does not need to carry out complex calculation, and has high analysis speed.
The technical solutions described above only represent the preferred technical solutions of the present invention, and some possible modifications to some parts of the technical solutions by those skilled in the art all represent the principles of the present invention, and fall within the protection scope of the present invention.
Claims (5)
1. The utility model provides a be applied to quantitative determination kit of protein antigen, includes the carrier, its characterized in that, along sample flow direction set gradually low concentration detection zone and high concentration detection zone on the carrier, low concentration detection zone and high concentration detection zone can be based on the existence or not produce the signal of different intensity of target analyte, wherein low concentration detection zone peridium has capture antibody A, and high concentration detection zone peridium has capture antibody B, and capture antibody A is higher than capture antibody B's affinity to the target analyte's affinity.
2. The quantitative detection kit applied to protein antigens as claimed in claim 1, wherein the signal antibody is an antibody labeled by fluorescent microspheres.
3. The quantitative detection kit applied to protein antigens as claimed in claim 1, wherein the carrier is a microfluidic chip, the capture antibody A and the capture antibody B are sequentially coated in a microchannel along a sample flowing direction to form a low concentration detection region and a high concentration detection region, and the signal antibody is disposed in the microchannel and positioned at the front end of the capture antibody A to form a labeling region or is directly disposed in a sample addition hole of the microfluidic chip.
4. The quantitative determination kit applied to protein antigens as claimed in claim 3, wherein a labeling zone, a low concentration detection zone, a high concentration detection zone and a quality control site are sequentially arranged in the micro-channel along the flow direction of the sample.
5. A method for the quantitative detection of protein antigens, characterized in that the detection is carried out using a kit according to any one of claims 1 to 4, comprising at least the following steps:
1) acquiring a calibration function;
2) firstly, combining a sample possibly containing target analytes with a labeled antibody, sequentially flowing through a low-concentration detection area and a high-concentration detection area of a carrier, and acquiring signal values of the low-concentration detection area and/or the high-concentration detection area by using a detection instrument after reaction;
3) determining the concentration of the target analyte in comparison to the calibration function: the concentration of the target analyte is determined using the calibration function T1 as a standard for the signal value from the lower concentration detection zone when only the low concentration detection zone detects a signal value, and the concentration of the target analyte is determined using the calibration function T2 as a standard for the signal value from the higher concentration detection zone when both the low concentration detection zone and the high concentration detection zone detect a signal.
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