CN116068184A - Application method of immunoassay kit and detection system thereof - Google Patents

Application method of immunoassay kit and detection system thereof Download PDF

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CN116068184A
CN116068184A CN202111283742.6A CN202111283742A CN116068184A CN 116068184 A CN116068184 A CN 116068184A CN 202111283742 A CN202111283742 A CN 202111283742A CN 116068184 A CN116068184 A CN 116068184A
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reagent
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韩双亭
张黎明
李临
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Kemei Boyang Diagnostic Technology Shanghai Co ltd
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Kemei Boyang Diagnostic Technology Shanghai Co ltd
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57476—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncofetal proteins
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01—MEASURING; TESTING
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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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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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract

The invention relates to a use method of an immunoassay kit and a detection system thereof, wherein the kit comprises a reagent 1 and a reagent 2, and the use method of the kit comprises the following steps: adding a sample to be detected containing target molecules to be detected into a container 1 and a container 2 respectively, then adding an alpha dose of a reagent 1 and a reagent 2 into the container 1, and adding a beta dose of the reagent 1 and the reagent 2 into the container 2 so as to perform two parallel immune response detection on the sample to be detected; wherein the ratio of the sum of the amounts of the first antibody (or antigen) and the second antibody (or antigen) in the reagent added in the container 1 to the sum of the amounts of the first antibody (or antigen) and the second antibody (or antigen) in the reagent added in the container 2The ratio of the contents of the target molecules to be measured is different. The use method of the kit can directly measure up to 10 6 High sample concentrations at ng/ml level.

Description

Application method of immunoassay kit and detection system thereof
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to a use method of an immunoassay kit and a detection system thereof.
Background
Immunological detection is based on the principle of antigen-antibody specific reaction, and is often used for detecting trace amounts of bioactive substances such as proteins and hormones, because it can display the analyte or amplify the signal by using isotopes, enzymes, chemiluminescent substances, and the like.
The photoexcitation chemiluminescence method is one of the common methods of chemiluminescence analysis technology, can be used for researching the interaction between biomolecules, and is mainly used for detecting diseases clinically. The technology integrates the research of the related fields of polymer particle technology, organic synthesis, protein chemistry, clinical detection and the like. The technical principle of the photoexcitation chemiluminescence analysis technology is that a sensitizer can excite oxygen molecules in the surrounding environment into singlet oxygen molecules under the irradiation of laser, and the singlet oxygen molecules can react with a luminous composition with the distance of about 200nm to generate a light signal with a certain wavelength; when the sample contains the antigen or antibody to be detected, the immune reaction of the antigen and the antibody can enable the donor particles containing the sensitizer to be combined with the acceptor particles containing the luminous composition, so that a light signal with a specific wavelength is generated, and the content of the antigen or antibody to be detected can be detected by detecting the light signal.
In the antigen-antibody dose response curve, when the amount of antibody is fixed, the response signal shows a phenomenon of rising and then falling with the increase of the amount of antigen. The region in which the response signal rises with increasing antigen dose is referred to as the "front band" region, the region in which the response signal falls with increasing antigen dose is referred to as the "rear band" region, and the region where the front band and rear band are joined is referred to as the "equivalent band".
In the immune reaction, the reactivity of the antigen and the antibody shows a phenomenon of rising and then falling as the ratio of the antigen to the antibody rises, and is called a "HOOK effect" or a "HOOK effect". Clinically, the hook effect can lead to high value samples producing false negative results, i.e. "false negatives".
The current immunoassay method generally uses the front band region of the dose-response curve to calculate the concentration of the analyte according to the linear relationship between the analyte content and the response signal. This detection method has a number of drawbacks, such as:
the detection range is narrow: traditional immunodetection reagents can only be detected by using the front band section of the dose-response curve, and the detection concentration range is narrow. The samples beyond the detection range need to be detected after dilution, and the method has the advantages of complex operation, long time consumption and high dilution precision requirement;
HOOK effect: because the traditional immunodetection reagent lacks a means for identifying the HOOK effect, a clinician is often required to identify whether the HOOK effect exists in the sample by adopting a method for diluting a serum sample in combination with the clinical manifestation of a patient, and the traditional immunodetection reagent is complex in operation, long in time consumption and easy to cause missed detection.
Disclosure of Invention
In view of the shortcomings in the prior art, the invention aims to provide a method for using an immunoassay kit and a detection system thereof. The use method of the immunoassay kit can simply, conveniently, quickly and accurately calculate the concentration of the object to be detected, and the detection system can implement the use method of the kit.
In order to achieve the above object and other related objects, the present invention adopts the following technical scheme:
the first aspect of the invention provides a method of using an immunoassay kit comprising a reagent 1 and a reagent 2, the reagent 1 comprising a first antibody (or antigen) coated luminescent particle and the reagent 2 comprising a second antibody (or antigen) labelled with a marker; the using method of the kit comprises the following steps:
adding a sample to be detected containing target molecules to be detected into a container 1 and a container 2 respectively, then adding an alpha dose of a reagent 1 and a reagent 2 into the container 1, and adding a beta dose of the reagent 1 and the reagent 2 into the container 2 so as to perform two parallel immune response detection on the sample to be detected; wherein the ratio of the sum of the amounts of the first antibody (or antigen) and the second antibody (or antigen) in the reagent added to the container 1 to the amount of the target molecule to be measured is different from the ratio of the sum of the amounts of the first antibody (or antigen) and the second antibody (or antigen) in the reagent added to the container 2 to the amount of the target molecule to be measured.
In some embodiments of the invention, the ratio of the sum of the amounts of the first antibody (or antigen) and the second antibody (or antigen) in the reagent added to the container 1 to the amount of the target molecule to be measured is achieved by any one of the following means:
mode 1: the amounts of the samples to be tested containing the target molecules to be tested added in the container 1 and the container 2 are the same, and alpha is not equal to beta;
mode 2: the amount of the sample to be measured containing the target molecule to be measured added in the container 1 is different from the amount of the sample to be measured containing the target molecule to be measured added in the container 2, and alpha is equal to beta;
mode 3: the amount of the sample containing the target molecule to be measured added in the container 1 is different from the amount of the sample containing the target molecule to be measured added in the container 2, and α is not equal to β.
In some embodiments of the invention, the method further comprises the steps of:
exciting and recording the detection results of two parallel immune reactions, respectively calculating a first measured value and a second measured value, and calculating the ratio of the first measured value to the second measured value; wherein the first measurement value is derived from a container 1, the second measurement value is derived from a container 2, and the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) corresponding to the first measurement value to the content of the target molecule to be detected in two parallel immunoreaction assays is larger than the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) corresponding to the second measurement value to the content of the target molecule to be detected.
In some embodiments of the invention, the method further comprises the steps of:
a1, detecting a series of standard substances with known target molecule content to be detected and different concentrations, wherein two parallel immune reaction detection is carried out on each standard substance, and the detection results of the two parallel immune reactions are excited and recorded, and are respectively calculated as a measured value a and a measured value a ', wherein the detection mode of the measured value a is the same as that of a first measured value of a sample to be detected, and the detection mode of the measured value a' is the same as that of a second measured value of the sample to be detected;
a2, calculating a ratio of the measured value a to the measured value a';
a3, a correlation standard curve of the ratio of the measured value a/the measured value a' and the concentration of the standard substance is made and stored.
In some embodiments of the invention, the method further comprises the steps of:
and calling the stored correlation standard curve, substituting the ratio of the first measured value to the second measured value of the sample to be tested containing the target molecule to the standard curve for calculation, and determining the concentration of the sample.
In some embodiments of the invention, the method further comprises the steps of:
b1, detecting a series of standard substances with known target molecule content to be detected and different concentrations, wherein two parallel immune reaction detection is carried out on each standard substance, and the detection results of the two parallel immune reactions are excited and recorded, and are respectively calculated as a measured value B and a measured value B ', wherein the detection mode of the measured value B is the same as that of a first measured value of a sample to be detected, and the detection mode of the measured value B' is the same as that of a second measured value of the sample to be detected;
b2, a reaction curve A of the measured value B and the concentration of the standard substance is made and stored;
b3, a reaction curve B of the measured value B' and the concentration of the standard substance is made and stored;
and B4, taking a point from the overlapping part of the standard substance concentration corresponding to the front zone area of the reaction curve A and the rear zone area of the reaction curve B, and recording the ratio of the measured value B/the measured value B' corresponding to the point as a critical point c and storing the critical point c.
In some embodiments of the invention, the method further comprises the steps of:
the stored reaction curve A, reaction curve B and critical point are retrieved, and the ratio of the first measured value to the second measured value of the measured sample and the critical point c are judged; when the ratio of the first measured value to the second measured value of the sample to be measured is less than or equal to the critical point c, calculating the concentration of the target molecules to be measured in the sample to be measured by using the front zone area of the reaction curve A; when the ratio of the first measured value to the second measured value of the sample to be measured is larger than the critical point c, the back band region of the reaction curve B is used for calculating the concentration of the target molecules to be measured in the sample to be measured.
In some embodiments of the invention, the target molecule to be tested is an antigen or an antibody.
In other embodiments of the invention, the first antibody (or antigen) and the second antibody (or antigen) are capable of specifically binding to a target molecule to be tested.
In a second aspect the invention provides a detection system for performing an immunoassay using a method according to the first aspect of the invention, comprising:
the immune reaction device comprises more than two containers, so that two parallel immune reaction detection is carried out on the same sample to be detected in the two containers at the same time; wherein container 1 is filled with alpha doses of reagent 1 and reagent 2 and container 2 is filled with beta doses of reagent 1 and reagent 2;
a chemiluminescent immunoreaction excitation and counting device for exciting and recording chemiluminescent readings and recording two parallel immunoreaction detection readings of the same sample to be tested as a first measured value and a second measured value respectively, wherein the first measured value is derived from a container 1, and the second measured value is derived from a container 2;
and the processor is used for calculating the ratio of the first measured value to the second measured value and calculating the concentration of the sample to be detected according to the ratio.
In some embodiments of the present invention, the processor stores a correlation standard curve of the ratio of the measured value a/the measured value a 'and the standard substance concentration, a reaction curve a of the measured value B and the standard substance concentration, a reaction curve B of the measured value B' and the standard substance concentration, and the critical point c, for measuring the concentration of the sample to be measured.
Compared with the prior art, the invention has the beneficial effects that:
(1) The application method of the kit can solve the problem of the HOOK effect and avoid missed detection caused by the HOOK effect, and the method and the system are not limited by the detection range;
(2) The using method of the kit directly uses classical dose-response curve calculation, has good repeatability, can obtain an accurate measured value of a HOOK effect sample by single detection without multiple dilutions, and has high measuring speed;
(3) The detection range of the using method of the kit greatly exceeds that of the conventional detection method.
Drawings
Figure 1 is a dose response curve for antigen-antibody.
FIG. 2 is a schematic diagram of a method 1 for calculating the method of using the kit of the present invention.
FIG. 3 is a schematic diagram of a method 2 for calculating the method of using the kit of the present invention.
FIG. 4 is a graph showing the reaction of standard substance concentration with the signals of reaction well 1 and reaction well 2 in example 2.
Detailed Description
In order that the invention may be readily understood, the invention will be described in detail. Before the present invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
The kit used in the method of using the immunoassay kit according to the first aspect of the present invention comprises a reagent 1 and a reagent 2, wherein the reagent 1 comprises a first antibody (or antigen) coated luminescent particle, and the reagent 2 comprises a second antibody (or antigen) labeled with a marker. The use method of the kit comprises the steps of carrying out two parallel tests on each sample, wherein the ratio of the content of the target molecule to be tested to the content of a specific capture molecule (such as a first antibody (or antigen) coated luminescent micro-object and a second antibody (or antigen) marked by a marker) in the two tests is different, and finally two different signals are generated, wherein the two signals are respectively a first measured value and a second measured value (the ratio of the sum of the content of the first antibody (or antigen) and the content of the second antibody (or antigen) corresponding to the first measured value/the content of the target molecule to be tested is larger than the ratio of the sum of the content of the first antibody (or antigen) and the content of the second antibody (or antigen) corresponding to the second measured value/the content of the target molecule to be tested). As the content of the target molecule to be detected increases, the ratio of the first measured value to the second measured value continuously rises and shows a certain linear relationship. According to the principle, the application method of the kit provided by the invention provides the following two methods for calculating the concentration of the target molecule to be detected, wherein the methods are as follows:
calculating method 1, directly calculating the concentration of the target molecules to be detected by the ratio of the first measured value to the second measured value:
as shown in fig. 2, according to a correlation standard curve of the ratio of the first measured value to the second measured value and the concentration of the standard substance, the concentration of the target molecule to be detected can be calculated by substituting the ratio of the first measured value to the second measured value into the curve.
Calculating method 2, using reaction curve A or reaction curve B to calculate the concentration of target molecules to be detected:
in the antigen-antibody dose response curve (fig. 1), when the amount of antibody is fixed, the response signal shows a phenomenon of ascending followed by descending with the increase of the amount of antigen. The region in which the reaction signal increases with increasing antigen amount is referred to as a front band region, the region in which the reaction signal decreases with increasing antigen amount is referred to as a rear band region, and the region in which the front band and the rear band are joined is referred to as an equivalent band.
As shown in fig. 3, the reaction curves a and B are reaction curves obtained from the first and second measured values and the standard substance concentration, respectively. In the portion where the front band region of the reaction curve a and the rear band region of the reaction curve B overlap in concentration (as in the portion in the dashed box in fig. 3), a point is taken in the above-mentioned portion where the concentrations overlap, and the ratio of the first measured value to the second measured value (as in a/b=15) corresponding to the point is taken as the critical point c.
When the ratio of the first measured value to the second measured value of the sample to be detected is less than or equal to the critical point c, the concentration of the sample is calculated by using the front zone area of the reaction curve A.
When the ratio of the first measured value to the second measured value of the sample to be measured is greater than the critical point c, the concentration of the sample is calculated by using the back band region of the reaction curve B.
Corresponding to the calculation method 1, the use method of the kit of the invention comprises the following steps:
first, a correlation standard curve of the ratio of the measured value a/the measured value a' to the standard substance concentration is obtained by a method comprising the steps of:
a1, detecting a series of standard substances with known target molecule content to be detected and different concentrations, wherein two parallel immune reaction detection is carried out on each standard substance, and the detection results of the two parallel immune reactions are excited and recorded, and are respectively calculated as a measured value a and a measured value a ', wherein the detection mode of the measured value a is the same as that of a first measured value of a sample to be detected, and the detection mode of the measured value a' is the same as that of a second measured value of the sample to be detected;
a2, calculating a ratio of the measured value a to the measured value a';
a3, a correlation standard curve of the ratio of the measured value a/the measured value a' and the concentration of the standard substance is made and stored.
Then, determining the concentration of the target molecule to be detected in the sample to be detected by a method comprising the following steps:
s1, respectively adding a sample to be detected containing target molecules to be detected into a container 1 and a container 2, then adding an alpha dose of a reagent 1 and a reagent 2 into the container 1, and adding a beta dose of the reagent 1 and the reagent 2 into the container 2 so as to perform two parallel immune response detection on the sample to be detected; wherein the ratio of the sum of the amounts of the first antibody (or antigen) and the second antibody (or antigen) in the reagent added in the container 1 to the amount of the target molecule to be measured is different from the ratio of the sum of the amounts of the first antibody (or antigen) and the second antibody (or antigen) in the reagent added in the container 2 to the amount of the target molecule to be measured;
s2, exciting and recording detection results of two parallel immune reactions, respectively calculating a first measured value and a second measured value, and calculating the ratio of the first measured value to the second measured value; wherein the first measurement value is derived from a container 1, the second measurement value is derived from a container 2, and the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) corresponding to the first measurement value to the content of the target molecule to be detected in two parallel immunoreaction assays is larger than the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) corresponding to the second measurement value to the content of the target molecule to be detected;
s3, the stored correlation standard curve is called, and the ratio of the first measured value to the second measured value of the sample to be detected is substituted into the correlation standard curve to be calculated, so that the concentration of the target molecules to be detected in the sample to be detected is determined.
Corresponding to the calculation method 2, the use method of the kit of the invention comprises the following steps:
first, a reaction curve A, a reaction curve B and a critical point c are obtained by a method comprising the following steps:
b1, detecting a series of standard substances with known target molecule content to be detected and different concentrations, wherein two parallel immune reaction detection is carried out on each standard substance, and the detection results of the two parallel immune reactions are excited and recorded, and are respectively calculated as a measured value B and a measured value B ', wherein the detection mode of the measured value B is the same as that of a first measured value of a sample to be detected, and the detection mode of the measured value B' is the same as that of a second measured value of the sample to be detected;
b2, a reaction curve A of the measured value B and the concentration of the standard substance is made and stored;
b3, a reaction curve B of the measured value B' and the concentration of the standard substance is made and stored;
and B4, taking a point in the concentration overlapping part at a part (such as a part in a dotted line box in fig. 3) where the concentration overlapping part exists in the front zone region of the reaction curve A and the rear zone region of the reaction curve B, and recording the ratio of the measured value B and the measured value B' corresponding to the point as a critical point c and storing the critical point c.
Then, determining the concentration of the target molecule to be detected in the sample to be detected by a method comprising the following steps:
s1, respectively adding a sample to be detected containing target molecules to be detected into a container 1 and a container 2, then adding an alpha dose of a reagent 1 and a reagent 2 into the container 1, and adding a beta dose of the reagent 1 and the reagent 2 into the container 2 so as to perform two parallel immune response detection on the sample to be detected; wherein the ratio of the sum of the amounts of the first antibody (or antigen) and the second antibody (or antigen) in the reagent added in the container 1 to the amount of the target molecule to be measured is different from the ratio of the sum of the amounts of the first antibody (or antigen) and the second antibody (or antigen) in the reagent added in the container 2 to the amount of the target molecule to be measured;
s2, exciting and recording detection results of two parallel immune reactions, respectively calculating a first measured value and a second measured value, and calculating the ratio of the first measured value to the second measured value; wherein the first measurement value is derived from a container 1, the second measurement value is derived from a container 2, and the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) corresponding to the first measurement value to the content of the target molecule to be detected in two parallel immunoreaction assays is larger than the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) corresponding to the second measurement value to the content of the target molecule to be detected;
s3, retrieving the stored reaction curve A, the reaction curve B and the critical point c, and judging the ratio of the first measured value to the second measured value of the measured sample to the critical point c; when the processor judges that the ratio of the first measured value to the second measured value of the sample to be detected is less than or equal to c, calculating the concentration of the sample by using a front zone area of a reaction curve A; when the processor determines that the ratio of the first measured value to the second measured value of the sample to be measured is greater than c, the back band region of the reaction curve B is used to calculate the sample concentration.
In the invention, the amount of the sample to be measured and the amount of the reagent to be added in the container 1 and the container 2 containing the target molecule to be measured can be changed independently or simultaneously, and finally, the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) in the container 1 and the container 2 to the content of the target molecule to be measured is different.
In some embodiments of the invention, the ratio of the sum of the amounts of the first antibody (or antigen) and the second antibody (or antigen) in the reagent added to the container 1 to the amount of the target molecule to be measured is achieved by any one of the following means:
mode 1: the amounts of the samples to be tested containing the target molecules to be tested added in the container 1 and the container 2 are the same, and alpha is not equal to beta;
mode 2: the amount of the sample to be measured containing the target molecule to be measured added in the container 1 is different from the amount of the sample to be measured containing the target molecule to be measured added in the container 2, and alpha is equal to beta;
mode 3: the amount of the sample containing the target molecule to be measured added in the container 1 is different from the amount of the sample containing the target molecule to be measured added in the container 2, and α is not equal to β.
In some embodiments of the invention, the target molecule to be tested is selected from an antigen or an antibody. According to some embodiments of the invention, the antigen is any substance having immunogenicity. Including but not limited to those listed in the examples of target molecules described above that are immunogenic. According to some embodiments of the invention, the antibodies are used herein in the broadest sense and specifically cover monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments (e.g., fab regions, fc regions, single chain antibodies).
In other embodiments of the invention, the first antibody (or antigen) and the second antibody (or antigen) are capable of specifically binding to a target molecule to be tested.
In some embodiments of the invention, the marker may be biotin.
In the present invention, the luminescent particles contain a luminescent group capable of rapidly absorbing singlet oxygen and then emitting light of a certain wavelength (e.g., 500 to 615 nm).
The standard substance refers to a target molecule solution to be measured, the content of which is known or which can quantitatively determine and assign the target molecule to be measured.
A second aspect of the invention relates to a detection system for performing an immunoassay using a method according to the first aspect of the invention, comprising:
the immune reaction device comprises more than two containers, so that two parallel immune reaction detection is carried out on the same sample to be detected in the two containers at the same time; wherein container 1 is filled with alpha doses of reagent 1 and reagent 2 and container 2 is filled with beta doses of reagent 1 and reagent 2;
a chemiluminescent immunoreaction excitation and counting device for exciting and recording chemiluminescent readings and recording two parallel immunoreaction detection readings of the same sample to be tested as a first measured value and a second measured value respectively, wherein the first measured value is derived from a container 1, and the second measured value is derived from a container 2;
and the processor is used for calculating the ratio of the first measured value to the second measured value and calculating the concentration of the sample to be detected according to the ratio.
In the present invention, the shape of the container is not particularly limited. In some embodiments of the invention, the container may be a reaction well or the like; the chemiluminescent immune response excitation and counting device can comprise a photon counting module and a light emitting diode; the processor may be a computer to process, map, store, etc. the readings.
In some embodiments of the present invention, a correlation standard curve of the ratio of the measured value a/the measured value a 'and the standard substance concentration, a reaction curve a of the measured value B and the standard substance concentration, a reaction curve B of the measured value B' and the standard substance concentration, and the critical point c are stored in the processor, and the processor retrieves the stored data according to the need for calculating the concentration of the sample to be measured.
In some embodiments of the invention, the correlation standard curve of the ratio of the measured value a/measured value a' stored in the process to the standard substance concentration is obtained by performing a method comprising the steps of:
a1, detecting a series of standard substances with known target molecule content to be detected and different concentrations, wherein two parallel immune reaction detection is carried out on each standard substance, and the detection results of the two parallel immune reactions are excited and recorded, and are respectively calculated as a measured value a and a measured value a ', wherein the detection mode of the measured value a is the same as that of a first measured value of a sample to be detected, and the detection mode of the measured value a' is the same as that of a second measured value of the sample to be detected;
a2, calculating a ratio of the measured value a to the measured value a';
a3, a correlation standard curve of the ratio of the measured value a/the measured value a' and the concentration of the standard substance is made and stored.
In other embodiments of the invention, the stress curve a, the reaction curve B and the critical point c stored in the process are obtained by performing a method comprising the steps of:
b1, detecting a series of standard substances with known target molecule content to be detected and different concentrations, wherein two parallel immune reaction detection is carried out on each standard substance, and the detection results of the two parallel immune reactions are excited and recorded, and are respectively calculated as a measured value B and a measured value B ', wherein the detection mode of the measured value B is the same as that of a first measured value of a sample to be detected, and the detection mode of the measured value B' is the same as that of a second measured value of the sample to be detected;
b2, a reaction curve A of the measured value B and the concentration of the standard substance is made and stored;
b3, a reaction curve B of the measured value B' and the concentration of the standard substance is made and stored;
and B4, taking a point in the part with overlapped concentration in the front zone area of the reaction curve A and the rear zone area of the reaction curve B, and recording the ratio of the measured value B and the measured value B' corresponding to the point as a critical point c and storing the critical point c.
Examples
In order that the invention may be more readily understood, the invention will be further described in more detail by the following examples, which are given by way of illustration only and are not limiting in scope of application. The starting materials or components used in the present invention may be prepared by commercial or conventional methods unless specifically indicated.
Alpha Fetoprotein (AFP) is a glycoprotein, also known as fetal alpha globulin, belonging to the albumin family. The AFP (alpha fetoprotein) value in serum of a Primary Liver Cancer (PLC) patient is greatly different, and the normal value and the pathological value are extremely bad and can reach 7 orders of magnitude. It has been reported in the literature that the concentration of AFP in primary serum of a primary liver cancer patient is directly measured by IEMA to be 29ng/mL, however, the actual concentration of AFP is measured and calculated to be 5.9X10 after serial dilution of serum samples 6 ng/mL. As can be seen, there are still significant drawbacks to the detection of AFP in the conventional art.
Example 1: detection of AFP samples using conventional methods of use of kits
The kit adopted is an Alpha Fetoprotein (AFP) detection kit (chemiluminescence method) produced by Komebo positive diagnostic technique (Shanghai) limited company (batch number: L2001), and the main components are:
reagent 1: luminescent particles coated with AFP antibodies;
reagent 2: biotin-labeled AFP antibodies.
The using method of the kit is as follows:
1. adding 25 μl of the sample to be tested, 25 μl of the reagent 1, 25 μl of the reagent 2 into the reaction well, and incubating at 37deg.C for 15min;
2. to the reaction well, 175. Mu.l of a universal solution for a photo-activated chemiluminescent assay system (donor reagent) was added, and incubated at 37℃for 10min, using
Figure BDA0003332216050000111
The analyzer takes readings.
Test sample (collected from clinical serum samples):
sample 1: negative serum sample (true measurement about 5 ng/mL)
Sample 2: low positive serum samples (true assay about 100 ng/mL)
Sample 3: strong positive serum samples (true assay about 2X 10) 6 ng/mL)
The test results are shown in Table 1.
TABLE 1
Initial measurement value Measured value ng/mL
Sample 1 7.41
Sample 2 109.43
Sample 3 11.79
The results shown in Table 1 are the results of direct detection using conventional kits, and the sample 3 measurement is only 11.79ng/mL, which is very easy to misjudge as a weak positive sample if not combined with clinical manifestation. In the case where sample 3 is known to be a strong positive sample, it is re-detected after 50-fold dilution with a diluent, and the detection results are shown in table 2.
TABLE 2
50 times dilution Measured value ng/mL
Diluting the sample >1000
As above, the 50-fold diluted sample 3 was >1000ng/mL, which was determined to be a HOOK sample, and still no specific measurement was obtained. The diluted sample was diluted again with the diluent by 50 times, and the measured values are shown in table 3 below.
TABLE 3 Table 3
2500-fold dilution Measured value ng/mL
Diluting the sample 849.51
As above, the measured value of the sample after 2500-fold dilution is 849.51ng/mL, and the true concentration of the sample 3 obtained by back calculation is about 2.12X10 6 ng/mL。
Example 2: AFP sample detection using the kit of the present invention
The kit adopted is an Alpha Fetoprotein (AFP) detection kit (chemiluminescence method) produced by Komebo positive diagnostic technique (Shanghai) limited company (batch number: L2001), and the main components are:
reagent 1: luminescent particles coated with AFP antibodies;
reagent 2: biotin-labeled AFP antibodies.
The using method of the kit comprises the following steps:
the two containers were the same sample test set, and the following filling steps 1 and 2 were repeated for different sample test sets:
1. adding 10 mu l of a sample to be tested, 50ul of reagent 1 and 50ul of reagent 2 into the reaction well 1 respectively;
2. adding 10ul of a sample to be detected, 5ul of a reagent 1 and 5ul of a reagent 2 into the reaction hole 2 respectively;
3. incubating each reaction well at 37 ℃ for 15min;
4. to each reaction well, 175. Mu.l of a universal solution (donor reagent) for a photo-activated chemiluminescent assay system was added, and incubated at 37℃for 10min, using
Figure BDA0003332216050000121
The analyzer takes readings.
Testing standard substances: the concentration range is 0 ng/mL-4X 10 6 ng/mL of purified AFP antigen solution
Test sample (collected from clinical serum samples):
sample 1: negative serum sample (true measurement about 5 ng/mL)
Sample 2: low positive serum samples (true assay about 100 ng/mL)
Sample 3: strong positive serum samples (true assay about 2X 10) 6 ng/mL)
The test results of the gradient diluted AFP standards (Nos. 1-20) were shown in Table 4.
A reaction curve A and a reaction curve B of the standard substance concentration and the signals of the reaction well 1 and the reaction well 2 are respectively prepared according to the numerical values of the table 4, as shown in fig. 4; it can be seen that the front zone region of the reaction curve a corresponds to the standard substance 1-11, and the rear zone region of the reaction curve B corresponds to the standard substance 9-20, so that the portion where the front zone region of the reaction curve a overlaps with the concentration existing in the rear zone region of the reaction curve B is the concentration range of the standard substance 9-11, the corresponding a/B signal ratio is 14.90-25.53, and the central point a/B signal ratio=19 is taken as the critical point.
A correlation standard curve of the A/B signal ratio and the concentration of the standard substance is made according to the values in Table 4.
Storing the standard substance test result into
Figure BDA0003332216050000131
An analyzer.
Three sets of samples were tested according to the test methods described above and the test results are shown in table 5. The calculation method 1 is a test result obtained by calling the correlation standard curve, and the calculation method 2 is a test result obtained by calling the critical point.
TABLE 4 Table 4
Figure BDA0003332216050000132
TABLE 5
Figure BDA0003332216050000133
As can be seen from the results shown in Table 5, the method of using the kit of the present invention can avoid the problem of lower sample measurement value caused by the HOOK effect, and can directly obtain up to 2X 10 6 ng/mL detection results. The method is not limited by the detection range, and both calculation modes are feasible.
Example 3: AFP sample detection using the kit of the present invention
The kit adopted is an Alpha Fetoprotein (AFP) detection kit (chemiluminescence method) produced by Komebo positive diagnostic technique (Shanghai) limited company (batch number: L2001), and the main components are:
reagent 1: luminescent particles coated with AFP antibodies;
reagent 2: biotin-labeled AFP antibodies.
The using method of the kit comprises the following steps:
the two containers were the same sample test set, and the following filling steps 1 and 2 were repeated for different sample test sets:
1. adding 10 mu l of a sample to be tested, 50ul of reagent 1 and 50ul of reagent 2 into the reaction well 1 respectively;
2. respectively adding 20ul of a sample to be detected, 50ul of a reagent 1 and 50ul of a reagent 2 into the reaction hole 2;
3. incubating each reaction well at 37 ℃ for 15min;
4. to each reaction well, 175. Mu.l of a universal solution (donor reagent) for a photo-activated chemiluminescent assay system was added, and incubated at 37℃for 10min, using
Figure BDA0003332216050000141
The analyzer takes readings.
Testing standard substances: the concentration range is 0 ng/mL-4X 10 6 ng/mL of purified AFP antigen solution
Test sample (collected from clinical serum samples):
sample 1: negative serum sample (true measurement about 5 ng/mL)
Sample 2: low positive serum samples (true assay about 100 ng/mL)
Sample 3: strong positive serum samples (true assay about 2X 10) 6 ng/mL)
The test results of the gradient diluted AFP standards (Nos. 1-20) were shown in Table 6. A correlation standard curve of the A/B signal ratio and the concentration of the standard substance is made according to the values in Table 6. Storing the standard substance test result into
Figure BDA0003332216050000142
An analyzer.
Three sets of samples were tested according to the test methods described above and the test results are shown in table 7 below (calculated using calculation method 1 of the present invention).
TABLE 6
Figure BDA0003332216050000151
TABLE 7
Figure BDA0003332216050000152
The result shows that the using method of the kit can detect the ultra-high value AFP sample, has wide detection range and can simply, conveniently, quickly and accurately calculate the concentration of the object to be detected.
Example 4: AFP sample detection using the kit of the present invention
The kit adopted is an Alpha Fetoprotein (AFP) detection kit (chemiluminescence method) produced by Komebo positive diagnostic technique (Shanghai) limited company (batch number: L2001), and the main components are:
reagent 1: luminescent particles coated with AFP antibodies;
reagent 2: biotin-labeled AFP antibodies.
The using method of the kit comprises the following steps:
the two containers were the same sample test set, and the following filling steps 1 and 2 were repeated for different sample test sets:
1. adding 10 mu l of a sample to be tested, 50ul of reagent 1 and 50ul of reagent 2 into the reaction well 1 respectively;
2. respectively adding 20ul of a sample to be detected, 5ul of a reagent 1 and 5ul of a reagent 2 into the reaction hole 2;
3. incubating each reaction well at 37 ℃ for 15min;
4. to each reaction well, 175. Mu.l of a universal solution (donor reagent) for a photo-activated chemiluminescent assay system was added, and incubated at 37℃for 10min, using
Figure BDA0003332216050000161
The analyzer takes readings.
Testing standard substances: the concentration range is 0 ng/mL-4X 10 6 ng/mL of purified AFP antigen solution
Test sample (collected from clinical serum samples):
sample 1: negative serum sample (true measurement about 5 ng/mL)
Sample 2: low positive serum samples (true assay about 100 ng/mL)
Sample 3: strong positive serum samples (true assay about 2X 10) 6 ng/mL)
The test results of the gradient diluted AFP standards (Nos. 1-20) were shown in Table 8. A correlation standard curve of the A/B signal ratio and the concentration of the standard substance is made according to the values in Table 8. Storing the standard substance test result into
Figure BDA0003332216050000162
An analyzer.
Three sets of samples were tested according to the test methods described above and the test results are shown in table 9 below (calculated using calculation method 1 of the present invention).
TABLE 8
Figure BDA0003332216050000163
Figure BDA0003332216050000171
TABLE 9
Figure BDA0003332216050000172
The result shows that the using method of the kit can detect the ultra-high value AFP sample, has wide detection range and can simply, conveniently, quickly and accurately calculate the concentration of the object to be detected.
Example 5: ultra-high end measurement precision verification
The kit adopted is an Alpha Fetoprotein (AFP) detection kit (chemiluminescence method) produced by Komebo positive diagnostic technique (Shanghai) limited company (batch number: L2001), and the main components are:
reagent 1: luminescent particles coated with AFP antibodies;
reagent 2: biotin-labeled AFP antibodies.
The using method of the kit comprises the following steps:
the two reaction wells are of the same sample test set, and the following liquid adding steps 1 and 2 are repeated for different sample test sets:
1. adding 10 mu l of a sample to be tested, 50ul of reagent 1 and 50ul of reagent 2 into the reaction well 1 respectively;
2. adding 10ul of a sample to be detected, 5ul of a reagent 1 and 5ul of a reagent 2 into the reaction hole 2 respectively;
3. incubating each reaction well at 37 ℃ for 15min;
4. to each reaction well, 175. Mu.l of a universal solution (donor reagent) for a photo-activated chemiluminescent assay system was added, and incubated at 37℃for 10min, using
Figure BDA0003332216050000173
The analyzer takes readings.
The test results are shown in table 10 below (calculated using calculation method 1 of the present invention).
Table 10
Figure BDA0003332216050000174
Figure BDA0003332216050000181
The results show that: by adopting the determination method of the kit, the CV of 10 times of repeated determination of the measured values of three high-value samples is within the range of 10%, which shows that the precision result is good.
It should be noted that the above-described embodiments are only for explaining the present invention and do not constitute any limitation of the present invention. The invention has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined in the appended claims, and the invention may be modified without departing from the scope and spirit of the invention. Although the invention is described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, as the invention extends to all other means and applications which perform the same function.

Claims (11)

1. A method of using an immunoassay kit comprising a reagent 1 and a reagent 2, the reagent 1 comprising a first antibody (or antigen) coated luminescent particle and the reagent 2 comprising a second antibody (or antigen) labeled with a label; the using method of the kit comprises the following steps:
adding a sample to be detected containing target molecules to be detected into a container 1 and a container 2 respectively, then adding an alpha dose of a reagent 1 and a reagent 2 into the container 1, and adding a beta dose of the reagent 1 and the reagent 2 into the container 2 so as to perform two parallel immune response detection on the sample to be detected; wherein the ratio of the sum of the amounts of the first antibody (or antigen) and the second antibody (or antigen) in the reagent added to the container 1 to the amount of the target molecule to be measured is different from the ratio of the sum of the amounts of the first antibody (or antigen) and the second antibody (or antigen) in the reagent added to the container 2 to the amount of the target molecule to be measured.
2. The use method according to claim 1, wherein the ratio of the sum of the amounts of the first antibody (or antigen) and the second antibody (or antigen) in the reagent added to the container 1 to the sum of the amounts of the first antibody (or antigen) and the second antibody (or antigen) in the reagent added to the container 2 to the sum of the amounts of the target molecules to be measured is different by any one of the following means:
mode 1: the amounts of the samples to be tested containing the target molecules to be tested added in the container 1 and the container 2 are the same, and alpha is not equal to beta;
mode 2: the amount of the sample to be measured containing the target molecule to be measured added in the container 1 is different from the amount of the sample to be measured containing the target molecule to be measured added in the container 2, and alpha is equal to beta;
mode 3: the amount of the sample containing the target molecule to be measured added in the container 1 is different from the amount of the sample containing the target molecule to be measured added in the container 2, and α is not equal to β.
3. The method of use according to claim 1 or 2, characterized in that the method further comprises the steps of:
exciting and recording the detection results of two parallel immune reactions, respectively calculating a first measured value and a second measured value, and calculating the ratio of the first measured value to the second measured value; wherein the first measurement value is derived from a container 1, the second measurement value is derived from a container 2, and the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) corresponding to the first measurement value to the content of the target molecule to be detected in two parallel immunoreaction assays is larger than the ratio of the sum of the contents of the first antibody (or antigen) and the second antibody (or antigen) corresponding to the second measurement value to the content of the target molecule to be detected.
4. A method of use according to claim 3, wherein the method further comprises the steps of:
a1, detecting a series of standard substances with known target molecule content to be detected and different concentrations, wherein two parallel immune reaction detection is carried out on each standard substance, and the detection results of the two parallel immune reactions are excited and recorded, and are respectively calculated as a measured value a and a measured value a ', wherein the detection mode of the measured value a is the same as that of a first measured value of a sample to be detected, and the detection mode of the measured value a' is the same as that of a second measured value of the sample to be detected;
a2, calculating a ratio of the measured value a to the measured value a';
a3, a correlation standard curve of the ratio of the measured value a/the measured value a' and the concentration of the standard substance is made and stored.
5. The method of use according to claim 4, further comprising the steps of:
and calling the stored correlation standard curve, substituting the ratio of the first measured value to the second measured value of the sample to be tested containing the target molecule to the standard curve for calculation, and determining the concentration of the sample.
6. The method of use according to claim 1 or 2, characterized in that the method further comprises the steps of:
b1, detecting a series of standard substances with known target molecule content to be detected and different concentrations, wherein two parallel immune reaction detection is carried out on each standard substance, and the detection results of the two parallel immune reactions are excited and recorded, and are respectively calculated as a measured value B and a measured value B ', wherein the detection mode of the measured value B is the same as that of a first measured value of a sample to be detected, and the detection mode of the measured value B' is the same as that of a second measured value of the sample to be detected;
b2, a reaction curve A of the measured value B and the concentration of the standard substance is made and stored;
b3, a reaction curve B of the measured value B' and the concentration of the standard substance is made and stored;
and B4, taking a point from the overlapping part of the standard substance concentration corresponding to the front zone area of the reaction curve A and the rear zone area of the reaction curve B, and recording the ratio of the measured value B/the measured value B' corresponding to the point as a critical point c and storing the critical point c.
7. The method of use of claim 6, further comprising the steps of:
the stored reaction curve A, reaction curve B and critical point are retrieved, and the ratio of the first measured value to the second measured value of the measured sample and the critical point c are judged; when the ratio of the first measured value to the second measured value of the sample to be measured is less than or equal to the critical point c, calculating the concentration of the target molecules to be measured in the sample to be measured by using the front zone area of the reaction curve A; when the ratio of the first measured value to the second measured value of the sample to be measured is larger than the critical point c, the back band region of the reaction curve B is used for calculating the concentration of the target molecules to be measured in the sample to be measured.
8. The method of any one of claims 1-7, wherein the target molecule to be tested is an antigen or an antibody.
9. The method of use according to any one of claims 1 to 8, wherein the first antibody (or antigen) and the second antibody (or antigen) are capable of specifically binding to the target molecule to be tested.
10. A detection system for implementing the method of use of any one of claims 1-9, comprising:
the immune reaction device comprises more than two containers, so that two parallel immune reaction detection is carried out on the same sample to be detected in the two containers at the same time; wherein container 1 is filled with alpha doses of reagent 1 and reagent 2 and container 2 is filled with beta doses of reagent 1 and reagent 2;
a chemiluminescent immunoreaction excitation and counting device for exciting and recording chemiluminescent readings and recording two parallel immunoreaction detection readings of the same sample to be tested as a first measured value and a second measured value respectively, wherein the first measured value is derived from a container 1, and the second measured value is derived from a container 2;
and the processor is used for calculating the ratio of the first measured value to the second measured value and calculating the concentration of the sample to be detected according to the ratio.
11. The system of claim 10, wherein the processor stores a correlation standard curve of the ratio of the measured value a/the measured value a 'and the standard substance concentration, a reaction curve a of the measured value B and the standard substance concentration, a reaction curve B of the measured value B' and the standard substance concentration, and the critical point c, and is used for calculating the concentration of the sample to be measured.
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