Patent Application
20170074873
1. Field of the Invention
The present invention relates to the technology field of immunochromatographic test, and more particularly to an immunoassay kit consisting of a releasing strip and a reaction strip.
2. Description of the Prior Art
Recently, immunochromatographic test gets more and more attention because of having the advantages of simple structure and low manufacturing cost. For the maximum sensitivity of the immunochromatographic test being up to 10−12 g/mL, the immunochromatographic test has been broadly applied in following fields:
Lateral flow immunoassay is one kind of the immunochromatographic test assay, and commonly used for completing the detections of various liquid samples. Please refer to
In the lateral flow immunoassay test strip 1′ shown by
Therefore, after a specific sample is dropped onto the sample pad 14′, the antigen in the sample would move toward the conjugation pad 13′ under the capillarity effect, so as to conjugated with the antibody 1 having the marker (CGC-Ab1). Next, the antigen and the first antibody (Ab1) connecting with one terminal of the antigen would continuously move toward the membrane 12′; meanwhile, the antibody 2 (Ab2) on the test line 12T′ would conjugate to the other terminal of the antigen, such that the test line 12T′ would show a color reaction. Furthermore, the antigen and the first antibody (Ab1) which does not conjugate with the antibody 2 (Ab2) on the test line 12T′ would continuously move toward the control line 12C′, so as to connect with the second antibody for making the control line 12C′ show a color reaction. Herein, it needs to further explain that, the qualitative analysis result of the sandwich type immunochromatographic test can be obtained by determining whether the test line 12T′ does show the color reaction or not. To put that simply, the qualitative analysis result of the sandwich type immunochromatographic test is positive when the test line 12T′ show the color reaction, and the opposite is negative.
On the contrary, when the competitive type immunochromatographic test assay is implemented on the lateral flow immunoassay test strip 1′, the test line 12T′ is formed by a competing antigen (Ag) conjugate with carrier protein (carrier-Ag) and the control line 12C′ is also formed by the second antibody (anti-Ab1); in addition, the first antibody (Ab1) conjugating with a maker (CGC-Ab1) is further disposed in the conjugation pad 13′. Therefore, after a specific sample is dropped onto the sample pad 14′, the antigen in the sample would move toward the conjugation pad 13′ under the capillarity effect, so as to conjugated with the CGC-Ab1. Next, the antigen and the CGC-Ab1 connecting with one terminal of the antigen (Ag-Ab1-CGC) would continuously move to the membrane 12′; meanwhile, the Carrier-Ag on the test line 12T′ would compete with the CGC-Ab1. Furthermore, the free Ag-Ab1-CGC continuously moves toward the control line 12C′, so as to conjugate with the second antibody (anti-Ab1) for making the control line 12C′ show a color reaction. Herein, it needs to further explain that, the qualitative analysis result of the competitive type immunochromatographic test can be obtained by determining whether the test line 12T′ does show the color reaction or not. To put that simply, the qualitative analysis result of the competitive type immunochromatographic test is negative when the test line 12T′ show the color reaction, and the opposite is positive.
The commonly-used markers include latex and colloidal gold. Moreover, in order to facilitate the antibody having the marker be able to easily move from the conjugation pad 13′ to the test line 12T′ and/or the control line 12C′ on the membrane 12′, manufacturers of the immunoassay test strips particularly fabricate the conjugation pad 13′ by microporous materials. Therefore, the sensitivity of the immunoassay test strips is effectively enhanced.
However, in spite of that, the conventional lateral flow immunoassay test strip 1′ has been found following drawbacks in practical use:
(1) the use of the microporous materials cause the manufacturing cost of the immunoassay test strips be increase; moreover, the immunoassay test strips having the microporous materials must be stored under a low-temperature environment of 4° C., and the maximum storage life of the microporous materials is merely 1 year.
(2) because the commercial (competitive type) lateral flow immunoassay test strip 1′ cannot effectively inhibit the test line 12T′ from showing the color reaction, it needs to further use colorimetry to verify the chrominance of the test line 12T′ and the control line 12C′. However, the maximum sensitivity of the aflatoxin M1 colorimetry verification can only up to 0.5 ppb.
Accordingly, in view of the conventional lateral flow immunoassay test strip include many drawbacks, the inventor of the present application has made great efforts to make inventive research thereon and eventually provided an immunoassay kit.
The primary objective of the present invention is to provide an immunoassay kit. Differing from conventional lateral flow immunoassay test strips, this novel immunoassay kit comprises a release strip and a reaction strip. When using this immunoassay kit to complete a competitive-type immunochromatographic test or a sandwich-type immunochromatographic test for a test sample, it needs to firstly put the release strip into the test sample for releasing a specific antibody (or antigen) conjugating with a marker into the test sample, so as to make the first antibody connect to a target object in the test sample; after that, the reaction strip is eventually put into the test sample after the release strip is removed out of the test sample. Therefore, the qualitative analysis result of the competitive/sandwich-type immunochromatographic test can be easily obtained by determining whether the test line shows the color reaction or not.
Accordingly, in order to achieve the primary objective of the present invention, the inventor of the present invention provides a first embodiment for the immunoassay kit, used for detecting a target object from a test sample by way of a competitive-type immunochromatographic test, mainly comprising: a release strip and a reaction strip. In which, the release strip is provided with a first antibody (Ab1) conjugating with a marker (CGC-Ab1) thereon, and the reaction strip is provided with a membrane having a control line and a test line thereon. Moreover, the control line is formed by a second antibody (anti-Ab1), and the test line is formed by a conjugate consisting of a specific antigen and a biomolecule (e.g., Carrier-Ag), wherein the first antibody is used for connecting to the target object, and the said specific antigen is the antigen of the target object. When completing the competitive-type immunochromatographic test by using the immunoassay kit, the release strip needs to be firstly put into the test sample for releasing the first antibody conjugating with the marker (CGC-Ab1) into the test sample, and the reaction strip is eventually put into the test sample after the release strip is removed out of the test sample. Therefore, only the control line on the reaction strip would show a color reaction when the target object is included by the test sample.
Moreover, for achieving the primary objective of the present invention, the inventor of the present invention provides a second embodiment for the immunoassay kit, used for detecting a target object from a test sample by way of a sandwich-type immunochromatographic test, mainly comprising a release strip and a reaction strip. In which, the release strip is provided with a first antibody (Ab1) conjugating with a marker (CGC-Ab1) thereon, and the first antibody (Ab1) is used for connecting to the target object (e.g., Antigen (Ag)). In addition, the reaction strip is provided with a membrane having a control line and a test line thereon, wherein the control line is formed by a second antibody (anti-Ab1), and the test line is formed by an antibody2 (Ab2); moreover, the first antibody (Ab1) and antibody2 (Ab2) are used for connecting to the target object. when completing the sandwich-type immunochromatographic test by using the immunoassay kit, the release strip needs to be firstly put into the test sample for releasing the first antibody conjugating with the marker (CGC-Ab1) into the test sample, and the reaction strip is eventually put into the test sample after the release strip is removed out of the test sample. Therefore, the control line and the test line on the reaction strip would simultaneously show a color reaction when the target object is included by the test sample.
The invention as well as a preferred mode of use and advantages thereof will be best understood by referring to the following detailed description of an illustrative embodiment in conjunction with the accompanying drawings, wherein:
To more clearly describe an immunoassay kit according to the present invention, embodiments of the present invention will be described in detail with reference to the attached drawings hereinafter.
Aflatoxin is the most common toxic pollutants existing in mildewed crops, such as Corn, peanut, rice, wheat, and nuts. Aflatoxin is one kind of the carcinogenic substances belong to Category 1 classified by International agency for research on cancer (IARC). According to the classification of IARC, aflatoxin can be further divided into 4 types, including: aflatoxin B1, aflatoxin B2, aflatoxin G1, and aflatoxinG2, wherein the most common type is aflatoxin B1. After a human (or animal) takes the food (or feed) polluted by aflatoxin B1, the aflatoxin B1 would be metabolized to aflatoxin M1 in human body so as to damage the liver. So that, if a mammal takes the food (or feed) polluted by aflatoxin B1, the milk lactated by the mammal will include the aflatoxin M1. For above reason, U.S. Food and Drug Administration (FDA) especially limits that the concentration of the aflatoxin M1 containing by milk or dairy products cannot exceed 0.5 ppb.
The present invention provides an immunoassay kit for detecting aflatoxin M1 or wheat from a test sample by way of immunochromatographic test. Herein, the test sample can be urine, tissue fluid, gravy, soup, liquid dairy product, liquid sample obtained by dissolving solid sample in a solvent, and an extract of the aforesaid liquid sample, wherein the solvent can be water or sample buffer, and the sample buffer can be a phosphate solution or a high-concentration phosphate solution.
please refer to
As shown in
Therefore, the foundational structure of the immunoassay kit 1 shown in
The first embodiment of the immunoassay kit 1 is design for carrying out a competitive-type immunochromatographic test. As shown in
Herein, it needs to further explain that, the purpose of the related design for the first embodiment is to make the immunoassay kit 1 detect the aflatoxin M1 existing in the test sample effectively and rapidly. However, the above described related design for the first embodiment cannot be used for limiting any practicable embodiment of the immunoassay kit 1 proposed by the present invention. The primary technology feature of the present invention is that the immunoassay kit 1 consists of one release strip 11 and one reaction strip 12. So that, based on this technology feature, it reasonably believes that, the person ordinarily skilled in immunoassay kit art can based on their engineering experiences to properly change the kinds of the antibody and/or antigen, so as to facilitate the immunoassay kit 1 be able to detect any one kind of target objective, such as antigen, virus, protein, DNA, RNA, small molecule and so on. In which, the said molecule can be toxin, antibiotic, drug, or related derivatives. Moreover, the said toxin is like aflatoxin M1, aflatoxin B1, or aflatoxins. In addition, the said antibiotic may be β-lactam, for example, penicillin, penicillin derivatives, cephalosporin, carbapenem, and carbapenem inhibitors.
The immunoassay kit 1 provided by the present invention is suitable for detecting the target object from a liquid sample under immunochromatographic test, wherein the liquid sample can be a raw milk, a sterilized whole milk, a low-fat milk, a drink including dairy compositions, or a derivative product of milk. Herein, it needs to further explain that, the aforesaid solid sample can be milk powder, reconcile milk powder, or feed.
In order to verify the practicability of the first embodiment of the immunoassay kit 1, the inventors of the immunoassay kit 1 has complete related experiments for prove that.
Liquid dairy product is taken as the test sample in experiment 1, wherein the said liquid dairy product is whole milk, milk make from commercial powdered milk based on the manufacturer's suggestion, and commercial yogurt. Before executing the competitive-type immunochromatographic test, the milk make from commercial powdered milk needs to be cooled down to room temperature in advance. Moreover, because yogurt is a thick liquid sample, which needs to be treated with an extracting process, consisting of following steps:
After the liquid sample is cooled down to room temperature, 3 drops of the liquid sample are added into a small tube by using a first dropper; and then, 3 drops of dilution buffer (i.e., phosphate solution) are added into the small tube by using a second dropper. Next, after letting the front end (i.e., the conjugation pad 111) of the release strip 11 be put into the small tube, the small tube is slightly rotated for 30 seconds, so as to make the first antibody (Ab1) conjugating with the marker be released into the sample solution in the small tube from conjugation pad 111. Meanwhile, the sample solution would show a specific color, e.g., light pink. After statically standing the small tube for 3 minutes, the aflatoxin M1 included by the sample solution would connect to the first antibody (Ab1) (i.e., rabbit anti-aflatoxin antibody or mouse anti-aflatoxin antibody).
Subsequently, after removing the release strip 11 from the small tube, the front end of the reaction strip 12 is put into the small tube, so as to make the sample pad 122 be soaked in the sample solution for 10-15 minutes. Meanwhile, the conjugate consisting of a specific antigen and a bovine serum albumin (BSA) fixed on the test line 12T of the reaction strip 12 and the second antibody (i.e., Immunoglobulin G (IgG)) fixed on the control line 12C would compete to each other for the aflatoxin M1 in the sample solution.
Please refer to
With reference to the experimental data recorded in Table 1, and please simultaneously refer to
Thus, the first embodiment of the immunoassay kit 1 provided by the present invention and the practicability thereof have been described completely and clearly. Subsequently, a second embodiment designed for the immunoassay kit 1 will be further introduced in follows.
the second embodiment of the immunoassay kit 1 is designed for facilitating the immunoassay kit 1 be able to detect the bran wheat included in a test sample. Bran wheat, also known as gluten, gliadin, and gluten protein, is one kind of gluten existing in cereals, such as barley, wheat, oats, rye and so on. According to research and statistics data, a small number of people (˜1%) would be subject to celiac disease after eating the food including gluten. For above reason, U.S. Food and Drug Administration (FDA) especially limits that the concentration of the gluten containing by processed food cannot exceed 20 parts per million (ppm).
The first embodiment of the immunoassay kit 1 is design for carrying out a sandwich-type immunochromatographic test. As shown in
Herein, it needs to further explain that, the purpose of the related design for the first embodiment is to make the immunoassay kit 1 detect the gliadin existing in the test sample effectively and rapidly. However, the above described related design for the first embodiment cannot be used for limiting any practicable embodiment of the immunoassay kit 1 proposed by the present invention. The primary technology feature of the present invention is that the immunoassay kit 1 consists of one release strip 11 and one reaction strip 12. So that, based on this technology feature, it reasonably believes that, the person ordinarily skilled in immunoassay kit art can based on their engineering experiences to properly change the kinds of the antibody and/or antigen, so as to facilitate the immunoassay kit 1 be able to detect any one kind of target objective, such as antigen, virus, protein, DNA, RNA, small molecule and so on.
In order to verify the practicability of the second embodiment of the immunoassay kit 1, the inventors of the immunoassay kit 1 has complete related experiments for prove that.
Liquid dairy product is also taken as the test sample in experiment 2. After the liquid sample is cooled down to room temperature, 15 drops of the liquid sample are added into a bottle containing extracting solution A by using a first dropper; and then, the bottle is shaken up and down for 30 seconds, so as to evenly mix the liquid sample and the extracting solution A. Subsequently, 3 drops of the extracting solution A are moved from the bottle to small tube containing dilution solution B by using a second dropper; and then the small tube is shaken up and down for 30 seconds, so as to evenly mix the extracting solution A and the dilution solution B to a sample solution. Next, after letting the front end (i.e., the conjugation pad 111) of the release strip 11 be put into the small tube, the small tube is slightly rotated for 30 seconds, so as to make the first antibody (Ab1) (i.e., anti-gliadin antibody) conjugating with the marker (CGC-Ab1) be released into the sample solution in the small tube from conjugation pad 111. Meanwhile, the sample solution would show a specific color, e.g., light pink. After statically standing the small tube for 3 minutes, the gliadin included by the sample solution would connect to the first antibody (Ab1)
Subsequently, after removing the release strip 11 from the small tube, the front end of the reaction strip 12 is put into the small tube, so as to make the sample pad 122 be soaked in the sample solution for 10-15 minutes. Meanwhile, the antibody2 (Ab2) (i.e., anti-gliadin antibody Ab2) fixed on the test line 12T would connect to the gliadin included by the sample solution, and the test line 12T therefore shows a color reaction. Moreover, because the second antibody (anti-Ab1) (i.e., Immunoglobulin G (IgG)) would also connect to the antibody released by the release pad 11, the control line 12C does also show the color reaction. Briefly, when qualitative analysis result of the sandwich type immunochromatographic test is positive, both the test line 12T and the control line 12C would show the color reaction simultaneously. On the contrary, when qualitative analysis result of the sandwich type immunochromatographic test is negative, there is only that the control line 12C would show the color reaction.
Furthermore, a comparison experiment between commercial mono-strip-type gliadin kits and the immunoassay kit of the present invention has been made by the inventors, and related experimental data are therefore recorded in following Table 2.
Through Table 2, it can find that, the maximum sensitivities of the mono-strip-type gliadin kits provided by manufacturer C is 1.0 ppm. However, the maximum sensitivity of the immunoassay kit 1 provided by the present invention for gliadin is 0.1 ppm. That is, the maximum sensitivity of the immunoassay kit is greater than the commercial mono-strip-type gliadin kits by at least 10 folds.
Therefore, through above descriptions, the immunoassay kit 1 provided by the present invention have been introduced completely and clearly; in summary, the present invention includes the advantages of:
(1) Differing from conventional lateral flow immunoassay test strips, the present invention provides an immunoassay kit comprising a release strip and a reaction strip. When using this immunoassay kit to complete a competitive-type immunochromatographic test or a sandwich-type immunochromatographic test for a test sample, it needs to firstly put the release strip into the test sample for releasing a specific antibody (or antigen) conjugating with a marker into the test sample, so as to make the first antibody connect to a target object in the test sample; after that, the reaction strip is eventually put into the test sample after the release strip is removed out of the test sample. Therefore, the qualitative analysis result of the competitive/sandwich-type immunochromatographic test can be easily obtained by determining whether the test line shows the color reaction or not.
(2) Inheriting to above point (1), the experimental data obtained from the EXPERIMENT 1 and EXPERIMENT 1 have proved that the maximum sensitivity of the immunoassay kit is greater than the commercial mono-strip-type aflatoxin/gliadin kits by at least 10 folds.
(3) In addition, the conjugation pad the conventional lateral flow immunoassay test strip 1′ shown as
Herein, it needs to further explain that, if the test sample is urine, the sample buffer will not be used when carrying out the immunochromatographic test. That is, when using this immunoassay kit to complete a competitive-type immunochromatographic test or a sandwich-type immunochromatographic test for a urine sample, the release strip 11 can be directly put into the urine sample for releasing a specific antibody (or antigen) conjugating with a marker into the sample, without using any buffer to dilute the urine in advance.
Continuously, please refer to
Furthermore, please refer to
When using this novel immunoassay kit 1 to detect whether a milk sample includes antibiotics enzyme of β-lactam or not, it needs use the pre-process strip 13 for treating a pre-process to the milk sample. To complete the said pre-process, the front end of the pre-process strip 13 is put into the milk sample, so as to release the pre-processing object (i.e., ampicillin) into the milk sample. Next, after the pre-process strip 13 is removed from the milk sample, the front end of the release trip 11 is put into the milk sample for releasing the penicillin enzyme antibody connecting with a maker disposed in the conjugation pad 111 into the milk sample. Meanwhile, when the milk contains the penicillin enzyme antibody, the ampicillin would be hydrolyzed by the penicillin enzyme, such that there has no penicillin enzyme for connecting to the penicillin enzyme antibody. Eventually, after the reaction strip 12 is put into the milk sample, the penicillin enzyme antibody connecting with the maker in the milk sample would connect to the antigen fixed on the test line 12T, so as to make the test line 12T show a color reaction.
The above description is made on embodiments of the present invention. However, the embodiments are not intended to limit scope of the present invention, and all equivalent implementations or alterations within the spirit of the present invention still fall within the scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201510574230.3 | Sep 2015 | CN | national |
