CN116068181A - Immunoassay kit, immunoassay method and immunoassay system - Google Patents

Immunoassay kit, immunoassay method and immunoassay system Download PDF

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CN116068181A
CN116068181A CN202111282854.XA CN202111282854A CN116068181A CN 116068181 A CN116068181 A CN 116068181A CN 202111282854 A CN202111282854 A CN 202111282854A CN 116068181 A CN116068181 A CN 116068181A
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measured value
reagent
sample
detected
concentration
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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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    • G01N33/57476—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncofetal proteins
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • 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
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Abstract

The invention relates to an immunoassay kit, an immunoassay method and an immunoassay system. The kit comprises a reagent 1 and a reagent 2 which have the same components, wherein the total content of specific capture molecules in the reagent 1 is different from that in the reagent 2, and the specific capture molecules can be specifically combined with target molecules to be detected. The method for carrying out immunoassay by using the kit can solve the problem of HOOK effect samples, is not limited by the detection range, and can directly detect up to 10 6 High sample concentrations at the ng/ml level and good reproducibility.

Description

Immunoassay kit, immunoassay method and immunoassay system
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to an immunoassay kit, an immunoassay method and an immunoassay system.
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
Aiming at the defects in the prior art, the invention aims to provide an immunoassay kit, an immunoassay method and an immunoassay system. The method and the system for carrying out immunoassay by using the kit can simply, conveniently, quickly and accurately calculate the concentration of the object to be detected.
In order to achieve the above object and other related objects, the present invention adopts the following technical scheme:
to this end, the first aspect of the present invention provides an immunoassay kit comprising reagents 1 and 2 of the same composition, and the total content of specific capture molecules in the reagents 1 is different from the total content of specific capture molecules in the reagents 2, the specific capture molecules being capable of specifically binding to target molecules to be detected; preferably, the specific capture molecules comprise a first antibody (or antigen) and a second antibody (or antigen) capable of specifically binding to the target molecule to be detected.
In some embodiments of the invention, the reagent 1 comprises a first antibody (or antigen) -coated luminescent particle at a concentration of α1, the reagent 2 comprises a first antibody (or antigen) -coated luminescent particle at a concentration of β1, and the α1 is greater than β1.
In other embodiments of the invention, the reagent 1 further comprises a second antibody (or antigen) labeled with a marker at a concentration of α2, while the reagent 2 further comprises a second antibody (or antigen) labeled with a marker at a concentration of β2, and the α2 is not less than β2; preferably, the α2 is greater than β2.
In a second aspect the invention provides a method of performing an immunoassay using a kit according to the first aspect of the invention, comprising the steps of:
s1, respectively carrying out two parallel immunoreaction detection on a sample to be detected containing target molecules to be detected and a reagent 1 and a reagent 2 in the kit, and exciting and recording detection results of the two parallel immunoreactions; the reading meter for detection by using the reagent 1 is a first measurement value, the reading meter for detection by using the reagent 2 is a second measurement value, and the ratio of the content of the specific capture molecules corresponding to the first measurement value to the content of the target molecules to be detected in the two parallel immunoreaction detection is larger than the ratio of the content of the specific capture molecules corresponding to the second measurement value to the content of the target molecules to be detected;
s2, calculating the ratio of the first measured value to the second measured value.
In some embodiments of the invention, the ratio of the content of the specific capture molecule corresponding to the first measurement to the content of the target molecule to be detected in the two parallel immunoreactions is greater than the ratio of the content of the specific capture molecule corresponding to the second measurement to the content of the target molecule to be detected by either:
mode 1: the amount of the sample to be tested containing the target molecule to be tested used in the two parallel immunoreaction tests is the same, and the same amount of the reagent 1 and the reagent 2 is used;
mode 2: in two parallel immunoreaction detection, the amount of a sample to be detected containing a target molecule to be detected by using the reagent 1 is different from the amount of the sample to be detected containing the target molecule to be detected by using the reagent 2, and the reagent 1 and the reagent 2 are used in equal amounts;
mode 3: in two parallel immunoreaction detection, the amount of a sample to be detected containing a target molecule to be detected by using the reagent 1 is different from the amount of a sample to be detected containing a target molecule to be detected by using the reagent 2, and the amount of the reagent 1 and the amount of the reagent 2 are also different;
mode 4: the amount of the sample to be tested containing the target molecule to be tested used in the two parallel immunoreactions tests is the same and the amount of reagent 1 used is different from the amount of reagent 2.
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 other 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 other 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 a third aspect the invention provides a system for performing an immunoassay according to the method of the second aspect of the invention, comprising:
the immune reaction device comprises more than two reaction containers, so that two parallel immune reaction detection is carried out on the same sample to be detected in the two reaction containers at the same time; wherein the reaction vessel 1 is filled with the reagent 1 of the kit, and the reaction vessel 2 is filled with the reagent 2 of the kit;
the chemiluminescent immune response excitation and counting device is used for exciting and recording chemiluminescent readings, and respectively recording two parallel immune response detection readings of the same sample to be detected as a first measured value and a second measured value, wherein the first measured value is derived from the reaction container 1, and the second measured value is derived from the reaction 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, 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 are used for calculating the concentration of the sample to be measured.
Compared with the prior art, the invention has the beneficial effects that:
(1) The method and the system for detecting by using the kit can solve the problem of the HOOK effect, avoid missing detection caused by the HOOK effect, and are not limited by the detection range;
(2) The method and the system for detecting by using the kit directly use classical dose-response curve calculation, have good repeatability, can obtain the accurate measured value of the HOOK effect sample by single detection without multiple dilutions, and have high measuring speed;
(3) The detection range of the method and the system for detecting by using the kit of the invention is greatly larger than 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 calculation method 1 for performing an immunoassay using the kit of the present invention.
FIG. 3 is a schematic diagram of a calculation method 2 for performing an immunoassay using the kit of the present invention.
FIG. 4 is a graph showing the reaction of standard substance concentration with reagent 1 signal and reagent 2 signal 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 immunoassay kit according to the first aspect of the present invention comprises a reagent 1 and a reagent 2 which have the same components, wherein the total content of specific capture molecules in the reagent 1 is different from the total content of specific capture molecules in the reagent 2, and the specific capture molecules can be specifically combined with target molecules to be detected.
In some embodiments of the invention, the specific capture molecule is only one; in a preferred embodiment of the invention, the specific capture molecules comprise more than one, including a first antibody (or antigen) and a second antibody (or antigen) capable of specifically binding to the target molecule to be detected.
In some embodiments of the invention, the reagent 1 comprises a first antibody (or antigen) -coated luminescent particle at a concentration of α1, the reagent 2 comprises a first antibody (or antigen) -coated luminescent particle at a concentration of β1, and the α1 is greater than β1.
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).
In other embodiments of the invention, the reagent 1 further comprises a second antibody (or antigen) labeled with a marker at an α2 concentration, while the reagent 2 further comprises a second antibody (or antigen) labeled with a marker at a β2 concentration, and the α2 is not less than β2; preferably, the α2 is greater than β2. In the present invention, the marker may be biotin.
That is, in some preferred embodiments of the present invention, the kit comprises reagent 1 and reagent 2 of the same composition, and the reagent 1 comprises a first antibody (or antigen) -coated luminescent particle at an α1 concentration and a second antibody (or antigen) labeled with biotin at an α2 concentration; the reagent 2 comprises a first antibody (or antigen) coated luminescent particle with beta 1 concentration and a second antibody (or antigen) marked by biotin with beta 2 concentration; and the α1 is greater than β1 and the α2 is greater than β2.
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).
The second aspect of the invention relates to a method of performing an immunoassay using a kit according to the first aspect of the invention. The method adopts the reagent 1 and the reagent 2 in the kit to respectively carry out two parallel tests on each sample, the ratio of the content of the target molecules to be detected in the two tests to the content of the specific capture molecules in the detection reagent is different, and finally two different signals are generated, namely a first measured value and a second measured value (the ratio of the content of the specific capture molecules corresponding to the first measured value to the content of the target molecules to be detected is larger than the ratio of the content of the specific capture molecules corresponding to the second measured value to the content of the target molecules to be detected). 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. The method for carrying out immunoassay by utilizing the kit provided by the invention provides the following two methods for calculating the concentration of target molecules to be detected, wherein the methods are respectively 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 method for performing immunoassay by using the kit of the invention specifically 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 carrying out two parallel immunoreaction detection on a sample to be detected containing target molecules to be detected and a reagent 1 and a reagent 2 in the kit, and exciting and recording detection results of the two parallel immunoreactions; the reading meter for detection by using the reagent 1 is a first measurement value, the reading meter for detection by using the reagent 2 is a second measurement value, and the ratio of the content of the specific capture molecules corresponding to the first measurement value to the content of the target molecules to be detected in the two parallel immunoreaction detection is larger than the ratio of the content of the specific capture molecules corresponding to the second measurement value to the content of the target molecules to be detected;
s2, calculating a ratio of the first measured value to the second measured value;
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 method for performing immunoassay by using the kit of the invention specifically 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 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.
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 carrying out two parallel immunoreaction detection on a sample to be detected containing target molecules to be detected and a reagent 1 and a reagent 2 in the kit, and exciting and recording detection results of the two parallel immunoreactions; the reading meter for detection by using the reagent 1 is a first measurement value, the reading meter for detection by using the reagent 2 is a second measurement value, and the ratio of the content of the specific capture molecules corresponding to the first measurement value to the content of the target molecules to be detected in the two parallel immunoreaction detection is larger than the ratio of the content of the specific capture molecules corresponding to the second measurement value to the content of the target molecules to be detected;
s2, calculating a ratio of the first measured value to the second measured value;
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 ratio of the content of the specific capture molecules in the reagent 1 to the content of the target molecules to be detected in the two parallel immunoreactions detection is realized in any one of the following modes to be larger than the ratio of the content of the specific capture molecules in the reagent 2 to the content of the target molecules to be detected:
mode 1: the amount of the sample to be tested containing the target molecule to be tested used in the two parallel immunoreaction tests is the same, and the same amount of the reagent 1 and the reagent 2 is used;
mode 2: in two parallel immunoreaction detection, the amount of a sample to be detected containing a target molecule to be detected by using the reagent 1 is different from the amount of the sample to be detected containing the target molecule to be detected by using the reagent 2, and the reagent 1 and the reagent 2 are used in equal amounts;
mode 3: in two parallel immunoreaction detection, the amount of a sample to be detected containing a target molecule to be detected by using the reagent 1 is different from the amount of a sample to be detected containing a target molecule to be detected by using the reagent 2, and the amount of the reagent 1 and the amount of the reagent 2 are also different;
mode 4: the amount of the sample to be tested containing the target molecule to be tested used in the two parallel immunoreactions tests is the same and the amount of reagent 1 used is different from the amount of reagent 2.
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 third aspect of the invention relates to a system for performing an immunoassay according to the method of the second aspect of the invention, comprising:
the immune reaction device comprises more than two reaction containers, so that two parallel immune reaction detection is carried out on the same sample to be detected in the two reaction containers at the same time; wherein the reaction vessel 1 is filled with the reagent 1 of the kit, and the reaction vessel 2 is filled with the reagent 2 of the kit;
the chemiluminescent immune response excitation and counting device is used for exciting and recording chemiluminescent readings, and respectively recording two parallel immune response detection readings of the same sample to be detected as a first measured value and a second measured value, wherein the first measured value is derived from the reaction container 1, and the second measured value is derived from the reaction 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 reaction vessel is not particularly limited. In some embodiments of the invention, the reaction vessel 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 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.
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: conventional method for detecting AFP sample by using conventional kit
The conventional 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 testing method comprises the following steps:
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 BDA0003331846650000111
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 by adopting the kit and the immunoassay method
The kit comprises: the invention relates to a double-reagent kit, which comprises the following main components:
reagent 1: luminescent microparticles coated with AFP antibody (concentration 100. Mu.g/mL), biotin-labeled AFP antibody (concentration 2. Mu.g/mL);
reagent 2: luminescent microparticles coated with AFP antibody (concentration 20. Mu.g/mL), biotin-labeled AFP antibody (concentration 0.4. Mu.g/mL).
Testing standard substances: the concentration range is 0 ng/mL-4X 10 6 ng/mL of pureAFP 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)
Test method 1:
the two reaction vessels are the same sample test group, and the following liquid adding steps 1 and 2 are repeated for different sample test groups:
1. adding 10 μl of the sample to be tested and 25 μl of the reagent 1 into the reaction well 1;
2. adding 10 μl of the sample to be tested and 25 μl of the reagent 2 into the reaction well 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 BDA0003331846650000132
The analyzer takes readings.
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 reagent 1 and the reagent 2 are respectively prepared according to the numerical values of the table 4, as shown in figure 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 15.05-24.31, 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 BDA0003331846650000133
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 BDA0003331846650000131
/>
Figure BDA0003331846650000141
TABLE 5
Figure BDA0003331846650000142
As can be seen from the results shown in Table 5, the immunoassay method using the kit of the present invention can avoid the problem of lower sample measurement value due to 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 by adopting the kit and the immunoassay method
The experimental materials were the same as in example 2.
Test method 2:
the two reaction vessels are the same sample test group, and the following liquid adding steps 1 and 2 are repeated for different sample test groups:
1. adding 10 μl of the sample to be tested and 25 μl of the reagent 1 into the reaction well 1;
2. adding 20 μl of the sample to be tested and 25 μl of the reagent 2 into the reaction well 2;
3. incubating each reaction well at 37 ℃ for 15min;
4. 175 μl of photo-activated chemiluminescent fraction was added to each wellThe universal solution (donor reagent) for the analytical system was incubated at 37℃for 10min, using
Figure BDA0003331846650000143
The analyzer takes readings.
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 BDA0003331846650000144
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.
TABLE 6
Figure BDA0003331846650000151
The result shows that the kit and the immunoassay method can detect the ultra-high value AFP sample, have wide detection range and can simply, conveniently, quickly and accurately calculate the concentration of the object to be detected.
Example 4: AFP sample detection by adopting the kit and the immunoassay method
The experimental materials were the same as in example 2.
Test method 3:
the two reaction vessels are the same sample test group, and the following liquid adding steps 1 and 2 are repeated for different sample test groups:
1. adding 10 μl of the sample to be tested and 50 μl of the reagent 1 into the reaction well 1;
2. adding 20 μl of the sample to be tested and 25 μl of the reagent 2 into the reaction well 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 BDA0003331846650000163
The analyzer takes readings.
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 BDA0003331846650000164
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.
TABLE 8
Figure BDA0003331846650000161
TABLE 9
Figure BDA0003331846650000162
The result shows that the kit and the immunoassay method can detect the ultra-high value AFP sample, have wide detection range and can simply, conveniently, quickly and accurately calculate the concentration of the object to be detected.
Example 5: AFP sample detection by adopting the kit and the immunoassay method
The experimental materials were the same as in example 2.
Test method 4:
the two reaction vessels are the same sample test group, and the following liquid adding steps 1 and 2 are repeated for different sample test groups:
1. adding 10 μl of the sample to be tested and 50 μl of the reagent 1 into the reaction well 1;
2. adding 10 μl of the sample to be tested and 25 μl of the reagent 2 into the reaction well 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, use
Figure BDA0003331846650000172
The analyzer takes readings.
The test was performed on the gradient diluted AFP standard substances (Nos. 1 to 20) according to the test method described above, and the test results are shown in Table 10. 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 10. Storing the standard substance test result into
Figure BDA0003331846650000173
An analyzer.
Three sets of samples were tested according to the test methods described above, with the test results shown in table 11 below.
Table 10
Figure BDA0003331846650000171
Figure BDA0003331846650000181
TABLE 11
Figure BDA0003331846650000182
The result shows that the kit and the immunoassay method can detect the ultra-high value AFP sample, have wide detection range and can simply, conveniently, quickly and accurately calculate the concentration of the object to be detected.
Example 6: ultra-high end measurement precision verification
The experimental materials were the same as in example 2.
The testing method 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 μl of the sample to be tested and 25 μl of the reagent 1 into the reaction well 1;
2. adding 10 μl of the sample to be tested and 25 μl of the reagent 2 into the reaction well 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 BDA0003331846650000184
The analyzer takes readings. />
The test results are shown in table 12 below (calculated using calculation method 1 of the present invention).
Table 12
Figure BDA0003331846650000183
Figure BDA0003331846650000191
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. An immunoassay kit comprising a reagent 1 and a reagent 2 which are identical in composition, wherein the total content of specific capture molecules in the reagent 1 is different from the total content of specific capture molecules in the reagent 2, and the specific capture molecules can be specifically combined with target molecules to be detected; preferably, the specific capture molecules comprise a first antibody (or antigen) and a second antibody (or antigen) capable of specifically binding to the target molecule to be detected.
2. The kit of claim 1, wherein the reagent 1 comprises a first antibody (or antigen) coated luminescent particle at an α1 concentration, the reagent 2 comprises a first antibody (or antigen) coated luminescent particle at a β1 concentration, and the α1 is greater than β1.
3. The kit according to claim 2, wherein the reagent 1 further comprises a second antibody (or antigen) labeled with a marker at an α2 concentration, and the reagent 2 further comprises a second antibody (or antigen) labeled with a marker at a β2 concentration, and the α2 is not smaller than β2; preferably, the α2 is greater than β2.
4. A method of performing an immunoassay using the kit of any one of claims 1-3, comprising the steps of:
s1, respectively carrying out two parallel immunoreaction detection on a sample to be detected containing target molecules to be detected and a reagent 1 and a reagent 2 in the kit, and exciting and recording detection results of the two parallel immunoreactions; the reading meter for detection by using the reagent 1 is a first measurement value, the reading meter for detection by using the reagent 2 is a second measurement value, and the ratio of the content of the specific capture molecules corresponding to the first measurement value to the content of the target molecules to be detected in the two parallel immunoreaction detection is larger than the ratio of the content of the specific capture molecules corresponding to the second measurement value to the content of the target molecules to be detected;
s2, calculating the ratio of the first measured value to the second measured value.
5. The method according to claim 4, wherein the ratio of the content of the specific capture molecules corresponding to the first measurement to the content of the target molecules to be detected in the two parallel immunoreactions is greater than the ratio of the content of the specific capture molecules corresponding to the second measurement to the content of the target molecules to be detected by either:
mode 1: the amount of the sample to be tested containing the target molecule to be tested used in the two parallel immunoreaction tests is the same, and the same amount of the reagent 1 and the reagent 2 is used;
mode 2: in two parallel immunoreaction detection, the amount of a sample to be detected containing a target molecule to be detected by using the reagent 1 is different from the amount of the sample to be detected containing the target molecule to be detected by using the reagent 2, and the reagent 1 and the reagent 2 are used in equal amounts;
mode 3: in two parallel immunoreaction detection, the amount of a sample to be detected containing a target molecule to be detected by using the reagent 1 is different from the amount of a sample to be detected containing a target molecule to be detected by using the reagent 2, and the amount of the reagent 1 and the amount of the reagent 2 are also different;
mode 4: the amount of the sample to be tested containing the target molecule to be tested used in the two parallel immunoreactions tests is the same and the amount of reagent 1 used is different from the amount of reagent 2.
6. The method according to claim 4 or 5, characterized in that 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.
7. The method according to claim 6, further comprising the step 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.
8. The method according to claim 4 or 5, 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.
9. The method according to claim 8, further comprising the step 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.
10. A system for performing an immunoassay according to the method of any one of claims 4-9, comprising:
the immune reaction device comprises more than two reaction containers, so that two parallel immune reaction detection is carried out on the same sample to be detected in the two reaction containers at the same time; wherein the reaction vessel 1 is filled with the reagent 1 of the kit, and the reaction vessel 2 is filled with the reagent 2 of the kit;
the chemiluminescent immune response excitation and counting device is used for exciting and recording chemiluminescent readings, and respectively recording two parallel immune response detection readings of the same sample to be detected as a first measured value and a second measured value, wherein the first measured value is derived from the reaction container 1, and the second measured value is derived from the reaction 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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