CN116068185A - Immunoassay device for detecting concentration of target molecules to be detected - Google Patents

Immunoassay device for detecting concentration of target molecules to be detected Download PDF

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CN116068185A
CN116068185A CN202111285000.7A CN202111285000A CN116068185A CN 116068185 A CN116068185 A CN 116068185A CN 202111285000 A CN202111285000 A CN 202111285000A CN 116068185 A CN116068185 A CN 116068185A
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measured value
detected
reagent
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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Abstract

The present invention relates to an immunoassay device for detecting the concentration of a target molecule to be detected. The device comprises an immunoreaction assembly, wherein the immunoreaction assembly comprises at least two reaction vessels, and the two reaction vessels are used for carrying out two parallel immunoreaction detection on the same sample to be detected; wherein, the ratio of the content of the specific capture molecules in the reaction container 1 for detecting the same sample to be detected to the content of the target molecules to be detected in the sample to be detected is different from the ratio of the content of the specific capture molecules in the reaction container 2 to the content of the target molecules to be detected in the sample to be detected. The device can solve the problem of a HOOK effect sample, is not limited by a detection range, and has good repeatability.

Description

Immunoassay device for detecting concentration of target molecules to be detected
Technical Field
The invention belongs to the technical field of immunodetection, and particularly relates to an immunoassay device for detecting the concentration of a target molecule to be detected.
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 of the prior art, the present invention aims to provide an immunoassay device for detecting the concentration of a target molecule to be detected. The measuring device can effectively avoid the HOOK effect, and can simply, conveniently, quickly and accurately calculate the concentration of the object to be measured.
In order to achieve the above object and other related objects, the present invention adopts the following technical scheme:
the first aspect of the present invention provides an immunoassay device for detecting a concentration of a target molecule to be detected, comprising an immunoreaction assembly comprising at least two reaction vessels for performing two parallel immunoreaction detections on the same sample to be detected; wherein, the ratio of the content of the specific capture molecules in the reaction container 1 for detecting the same sample to be detected to the content of the target molecules to be detected in the sample to be detected is different from the ratio of the content of the specific capture molecules in the reaction container 2 to the content of the target molecules to be detected in the sample to be detected.
In some embodiments of the invention, the device comprises a reagent compartment in which reagent 1 and reagent 2 are stored, and the reagent 1 and reagent 2 contain the same components in different concentrations; the component comprises a specific capture molecule; 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 present invention, the ratio of the content of the specific capture molecule in the reaction vessel 1 for detecting the same sample to be detected to the content of the target molecule to be detected in the sample to be detected is different from the ratio of the content of the specific capture molecule in the reaction vessel 2 to the content of the target molecule to be detected in the sample to be detected by any one of the following means:
mode 1: adding an equal amount of a sample to be tested containing target molecules to be tested into a reaction container 1 and a reaction container 2, and adding an equal amount of a reagent 1 and a reagent 2;
mode 2: adding unequal amounts of a sample to be tested containing target molecules to be tested into a reaction container 1 and a reaction container 2, and adding equal amounts of a reagent 1 and a reagent 2;
mode 3: adding unequal amounts of a sample to be tested containing target molecules to be tested into a reaction container 1 and a reaction container 2, and adding unequal amounts of a reagent 1 and a reagent 2;
mode 4: equal amounts of a sample to be tested containing a target molecule to be tested are added to the reaction vessel 1 and the reaction vessel 2, and unequal amounts of the reagent 1 and the reagent 2 are added. 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 marker-labeled secondary antibody (or antigen) at an α2 concentration, while the reagent 2 further comprises a marker-labeled secondary antibody (or antigen) at a β2 concentration, and the α2 is not less than β2; preferably, the α2 is greater than β2; further preferably, the marker is biotin.
In some embodiments of the invention, the device comprises an immunoassay component for recording the detection results of two parallel immunoreactions of the same test sample, a first measurement value and a second measurement value, respectively; wherein the reading in the reaction vessel 1 is a first measurement and the reading in the reaction vessel 2 is a second measurement; preferably, the ratio of the content of the specific capture molecule corresponding to the first measurement value to the content of the target molecule to be detected in the two parallel immunoreaction assays is greater than the ratio of the content of the specific capture molecule corresponding to the second measurement value to the content of the target molecule to be detected.
In some embodiments of the invention, the device comprises a processor for calculating a first measurement/second measurement ratio and determining the concentration of the target molecule to be measured in the sample to be measured.
In some embodiments of the invention, the apparatus performs the steps of:
a1: detecting a series of standard substances with known target molecule content to be detected, wherein two parallel immunoreaction detection is carried out on each standard substance, the detection results of the two parallel immunoreactions are recorded and respectively calculated into a measured value a and a measured value a ', the detection mode of the measured value a and the first measured value of the sample to be detected is the same, and the detection mode of the measured value a' and the second measured value of the sample to be detected is the same;
a2: calculating the ratio of the measured value a/the measured value a';
a3: and (5) a correlation standard curve of the ratio of the measured value a to the measured value a' and the concentration of the standard substance is made and stored.
In other embodiments of the invention, the apparatus performs the steps of:
the processor invokes the stored correlation standard curve, substitutes the ratio of the first measured value to the second measured value of the sample to be measured into the correlation standard curve to calculate so as to determine the concentration of the target molecules to be measured in the sample to be measured.
In some embodiments of the invention, the apparatus performs the steps of:
b1: detecting a series of standard substances with known target molecule content to be detected, wherein two parallel immunoreaction detection is carried out on each standard substance, the detection results of the two parallel immunoreactions are recorded and respectively calculated into a measured value b and a measured value b ', the measured value b and the first measured value of the sample to be detected are detected in the same mode, and the measured value b' and the second measured value of the sample to be detected are detected in the same mode;
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;
b4: taking a point in the part of the front zone of the reaction curve A, which is overlapped with the standard substance concentration corresponding to the rear zone 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 apparatus performs the steps of:
the processor retrieves the stored reaction curve A, the reaction curve B and the critical point c, and judges the ratio of the first measured value to the second measured value of the measured sample to the critical point c; 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.
A second aspect of the invention provides a computer-readable storage medium having stored thereon a computer program product for causing an apparatus according to the first aspect of the invention to perform the corresponding steps.
Compared with the prior art, the invention has the beneficial effects that:
(1) When the device is used for carrying out immunoassay, the problem of the HOOK effect can be solved, the detection omission caused by the HOOK effect is avoided, and the device is not limited by the detection range;
(2) When the device is used for carrying out immunoassay, classical dose-response curve calculation can be directly used, the repeatability is good, the accurate measurement value of a HOOK effect sample can be obtained through single detection without multiple dilutions, and the measurement speed is high;
(3) When the device provided by the invention is used for carrying out immune test, the detection range is greatly larger than that of a 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 apparatus of the present invention.
Fig. 3 is a schematic diagram of a calculation method 2 for performing an immunoassay using the device 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.
FIG. 5 is a schematic structural view of an immunoassay device according to the present invention.
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.
According to the immunoassay device for detecting the concentration of the target molecules to be detected, disclosed by the invention, two parallel tests can be carried out on each sample, and calculation is carried out according to the obtained two parallel test results, so that the concentration of the target molecules in the sample to be detected is finally obtained. The device has no limit of detection range, and can avoid missed detection caused by the HOOK effect.
In some embodiments of the invention, the immunoassay device for detecting the concentration of a target molecule to be detected comprises an immunoreaction assembly comprising at least two reaction vessels for performing two parallel immunoreaction assays on the same sample to be detected; wherein, the ratio of the content of the specific capture molecules in the reaction container 1 for detecting the same sample to be detected to the content of the target molecules to be detected in the sample to be detected is different from the ratio of the content of the specific capture molecules in the reaction container 2 to the content of the target molecules to be detected in the sample to be detected.
In some embodiments of the invention, the device comprises a reagent compartment in which reagent 1 and reagent 2 are stored, and the reagent 1 and reagent 2 contain the same components in different concentrations; the component comprises a specific capture molecule; 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 the present invention, the reagent 1 and the reagent 2 refer to not only a single reagent, but also a combination of a plurality of reagents. In some embodiments of the invention, reagents 1 and 2 each comprise 2 reagents, one of which is a luminescent particle coated with a specific capture molecule, and the other of which is a specific capture molecule labeled with a label, which are stored in separate compartments of the reagent chamber.
In some embodiments of the present invention, the ratio of the content of the specific capture molecule in the reaction vessel 1 for detecting the same sample to be detected to the content of the target molecule to be detected in the sample to be detected is different from the ratio of the content of the specific capture molecule in the reaction vessel 2 to the content of the target molecule to be detected in the sample to be detected by any one of the following means:
mode 1: adding an equal amount of a sample to be tested containing target molecules to be tested into a reaction container 1 and a reaction container 2, and adding an equal amount of a reagent 1 and a reagent 2;
mode 2: adding unequal amounts of a sample to be tested containing target molecules to be tested into a reaction container 1 and a reaction container 2, and adding equal amounts of a reagent 1 and a reagent 2;
mode 3: adding unequal amounts of a sample to be tested containing target molecules to be tested into a reaction container 1 and a reaction container 2, and adding unequal amounts of a reagent 1 and a reagent 2;
mode 4: equal amounts of a sample to be tested containing a target molecule to be tested are added to the reaction vessel 1 and the reaction vessel 2, and unequal amounts of the reagent 1 and the reagent 2 are added.
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 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; further preferably, the marker is biotin.
In some preferred embodiments of the invention, the reagent 1 and reagent 2 are identical in 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 luminous 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 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 shape of the reaction vessel is not particularly limited in the present invention. In some embodiments of the present invention, the reaction vessel may be a reaction well, a reaction cup, a reaction tank, or the like.
In some embodiments of the invention, the device comprises an immunoassay component for recording the detection results of two parallel immunoreactions of the same test sample, a first measurement value and a second measurement value, respectively; wherein the reading in the reaction vessel 1 is a first measurement and the reading in the reaction vessel 2 is a second measurement; preferably, the ratio of the content of the specific capture molecule corresponding to the first measurement value to the content of the target molecule to be detected in the two parallel immunoreaction assays is greater than the ratio of the content of the specific capture molecule corresponding to the second measurement value to the content of the target molecule to be detected.
In some embodiments of the invention, the immunoassay assembly may include a photon counting module and a light emitting diode for exciting and recording the detection results of two parallel immunoreactions of the same sample to be tested.
In some embodiments of the invention, the device comprises a processor for calculating a first measurement/second measurement ratio and determining the concentration of the target molecule to be measured in the sample to be measured.
In some embodiments of the present invention, the processor may be a computer, so as to process, map, store, etc. the detection result.
In some embodiments of the invention, the target molecule to be tested is 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 "antibody" is used herein in the broadest sense and specifically covers 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 the immunoassay device, in two parallel tests are carried out on each sample, the ratio of the content of target molecules to be detected in the sample to the content of specific capture molecules in the detection reagent (such as luminescent particles coated by a first antibody (or antigen) and a second antibody (or antigen) marked by a marker) 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 (concentration) of the target molecule to be measured increases, the ratio of the first measurement value to the second measurement value continuously rises and shows a certain linear relationship. According to the principle, the processor of the measuring device can calculate the concentration of the target molecules to be measured in the sample by executing the following two methods, namely, the following methods:
the calculating method 1 directly calculates the concentration of the target molecules to be detected in the sample 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 processor substitutes the ratio of the first measured value to the second measured value detected by the target molecule to be detected into the curve to calculate the concentration of the target molecule.
Calculating method 2, using reaction curve A or reaction curve B to calculate the concentration of target molecules to be detected in the sample:
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. A portion (e.g., a portion in a dashed box in fig. 3) where the front band region of the reaction curve a and the rear band region of the reaction curve B overlap each other is present, and a point is selected from the above-mentioned portion where the concentrations overlap, and a ratio of the first measured value to the second measured value (e.g., a/b=15) corresponding to the point is stored as a critical point c.
When the processor determines 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 the critical point c, the concentration of the sample to be detected is calculated by using the front zone area of the 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 the critical point c, the concentration of the sample to be measured is calculated by using the back band region of the reaction curve B.
Corresponding to the above calculation method 1, the immunoassay device provided by the present invention performs the following steps:
first the device obtains a correlation standard curve of the ratio of the measured value a/a' to the concentration of the standard substance by performing the following steps:
a1: the reagent arm respectively adds the reagent 1 and the reagent 2 in the reagent cabin into a reaction container 1 and a reaction container 2 containing standard substances with the same target molecule concentration to be detected so as to carry out two parallel immune reaction detection on each standard substance in a series of standard substances with different concentrations and known target molecule content to be detected;
a2: the immunoassay component records the detection results of two parallel immune reactions, namely a measured value a and a measured value a ', wherein the detection mode of the measured value a and the first measured value of the sample to be detected is the same, and the detection mode of the measured value a' and the second measured value of the sample to be detected is the same;
a3: the processor calculates the ratio of the measured value a to the measured value a ', and according to the calculated ratio of the measured value a to the measured value a ', a correlation standard curve of the ratio of the measured value a to the measured value a ' and the concentration of the standard substance is made and stored.
The device then determines the concentration of the target molecule to be tested in the test sample by performing the steps of:
s1: the reagent arm respectively adds the reagent 1 and the reagent 2 in the reagent cabin into the reaction container 1 and the reaction container 2 containing the sample to be detected so as to carry out two parallel immunoreaction detection on the same sample to be detected;
s2: the immunoassay component records the detection results of two parallel immune responses, and the detection results are respectively counted as a first measurement value and a second measurement value; the reading in the reaction container 1 is a first measurement value, the reading in the reaction container 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 immune reaction detection is greater 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;
s3: the processor calculates the ratio of the first measured value to the second measured value of the sample to be measured, and retrieves the stored correlation standard curve; and substituting the ratio of the first measured value to the second measured value of the sample to be measured into the correlation standard curve for calculation to determine the concentration of the target molecules to be measured in the sample to be measured.
Corresponding to the above-described calculation method 2, the immunoassay device provided by the present invention performs the steps of:
first the device obtains a reaction curve a, a reaction curve B and a critical point c by performing the following steps:
b1: the reagent arm respectively adds the reagent 1 and the reagent 2 in the reagent cabin into a reaction container 1 and a reaction container 2 containing standard substances with the same target molecule concentration to be detected so as to carry out two parallel immune reaction detection on each standard substance in a series of standard substances with different concentrations and known target molecule content to be detected;
b2: the immunoassay component records the detection results of two parallel immunoreactions, namely a measured value b and a measured value b ', wherein the measured value b is the same as the detection mode of a first measured value of the sample to be detected, and the measured value b' is the same as the detection mode of a second measured value of the sample to be detected;
b3: the processor makes a reaction curve A of the measured value b and the concentration of the standard substance according to the known measured value b of a series of standard substances with different concentrations, and stores the measured value b and the concentration of the standard substance; meanwhile, according to a known series of measured values B 'of standard substances with different concentrations, a reaction curve B of the measured values B' and the concentration of the standard substances is prepared and stored;
b4: 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), the processor takes a point in the above-mentioned portion where the concentrations overlap, marks the ratio of the measured value B and the measured value B' corresponding to the point as a critical point c, and stores the critical point c.
The device then determines the concentration of the target molecule to be tested in the test sample by performing the steps of:
s1: the reagent arm respectively adds the reagent 1 and the reagent 2 in the reagent cabin into the reaction container 1 and the reaction container 2 containing the sample to be detected so as to carry out two parallel immunoreaction detection on the same sample to be detected;
s2: the immunoassay component records the detection results of two parallel immune responses, and the detection results are respectively counted as a first measurement value and a second measurement value; the reading in the reaction container 1 is a first measurement value, the reading in the reaction container 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 immune reaction detection is greater 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;
s3: the processor retrieves the stored reaction curve A, the reaction curve B and the critical point c, and judges 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.
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 computer-readable storage medium, on which a computer program product is stored, which computer program product causes an apparatus according to the first aspect of the invention to perform the corresponding steps.
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 immunoassay devices
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 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 BDA0003332681570000111
The analyzer takes readings.
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 devices, 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: detection of AFP samples Using the immunoassay device of the present invention
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 main components of the adopted reagent are as follows:
reagent 1: luminescent microparticles coated with AFP antibody (concentration 100 ug/mL), biotin-labeled AFP antibody (concentration 2 ug/mL);
reagent 2: luminescent microparticles coated with AFP antibody (concentration 20 ug/mL), biotin-labeled AFP antibody (concentration 0.4 ug/mL).
The detection steps performed by the measuring device are as follows:
setting two reaction vessels as the same sample test group, and repeating the following liquid adding steps 1 and 2 in different sample test groups:
1. the sample arm adds 10 mu l of the same sample to be tested into each of the two reaction containers;
2. reagent arm 25ul of reagent 1 and 25ul of reagent 2 are added to reaction vessel 1 and reaction vessel 2, respectively;
3. incubating each reaction vessel at 37 ℃ for 15min;
4. 175. Mu.l of a universal solution for a photo-activated chemiluminescent analysis system was added to each reaction vessel and incubated at 37℃for 10min;
5. the immunoassay component records detection results, and the detection results of two parallel immunoreactions of the same sample to be detected are respectively calculated as a first measurement value and a second measurement value of the sample; wherein the reading in the reaction vessel 1 is a first measurement and the reading in the reaction vessel 2 is a second measurement;
6. the processor calculates the ratio of the first measured value to the second measured value of the same sample to be detected, retrieves the stored data for judgment, and then calculates the concentration of the target molecules in the sample to be detected.
The gradient diluted AFP standard substances (Nos. 1 to 20) were tested according to the above test procedure, and the test results are 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.
And storing the standard substance test result into an immunoassay device.
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 BDA0003332681570000131
Figure BDA0003332681570000141
TABLE 5
Figure BDA0003332681570000142
As can be seen from the results shown in Table 5, the device of the present invention can avoid the problem of lower sample measurement value caused by 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: detection of AFP samples Using the immunoassay device of the present invention
The experimental materials were the same as in example 2.
The detection steps performed by the measuring device are as follows:
setting two reaction vessels as the same sample test group, and repeating the following liquid adding steps 1 and 2 in different sample test groups:
the detection steps performed by the measuring device are as follows:
setting two reaction vessels as the same sample test group, and repeating the following liquid adding steps 1 and 2 in different sample test groups:
1. sample arm 10 μl of sample to be tested is added into the reaction vessel 1, and 20 μl of sample to be tested is added into the reaction vessel 2;
2. reagent arm 25ul of reagent 1 and 25ul of reagent 2 are added to reaction vessel 1 and reaction vessel 2, respectively;
3. incubating each reaction vessel at 37 ℃ for 15min;
4. 175. Mu.l of a universal solution for a photo-activated chemiluminescent analysis system was added to each reaction vessel and incubated at 37℃for 10min;
5. the immunoassay component records detection results, and the detection results of two parallel immunoreactions of the same sample to be detected are respectively calculated as a first measurement value and a second measurement value of the sample; wherein the reading in the reaction vessel 1 is a first measurement and the reading in the reaction vessel 2 is a second measurement;
6. the processor calculates the ratio of the first measured value to the second measured value of the same sample to be detected, retrieves the stored data for judgment, and then calculates the concentration of the target molecules in the sample to be detected.
The gradient diluted AFP standard substances (Nos. 1 to 20) were tested according to the above test procedure, and the test results are 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. And storing the standard substance test result into an immunoassay device.
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 BDA0003332681570000151
TABLE 7
Figure BDA0003332681570000152
The result shows that the device 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: detection of AFP samples Using the immunoassay device of the present invention
The experimental materials were the same as in example 2.
The detection steps performed by the measuring device are as follows:
setting two reaction vessels as the same sample test group, and repeating the following liquid adding steps 1 and 2 in different sample test groups:
1. sample arm 10 μl of sample to be tested is added into the reaction vessel 1, and 20 μl of sample to be tested is added into the reaction vessel 2;
2. reagent arm 50ul of reagent 1 and 25ul of reagent 2 were added to reaction vessel 1 and reaction vessel 2, respectively;
3. incubating each reaction vessel at 37 ℃ for 15min;
4. 175. Mu.l of a universal solution for a photo-activated chemiluminescent analysis system was added to each reaction vessel and incubated at 37℃for 10min;
5. the immunoassay component records detection results, and the detection results of two parallel immunoreactions of the same sample to be detected are respectively calculated as a first measurement value and a second measurement value of the sample; wherein the reading in the reaction vessel 1 is a first measurement and the reading in the reaction vessel 2 is a second measurement;
6. the processor calculates the ratio of the first measured value to the second measured value of the same sample to be detected, retrieves the stored data for judgment, and then calculates the concentration of the target molecules in the sample to be detected.
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. And storing the standard substance test result into an immunoassay device.
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 BDA0003332681570000161
Figure BDA0003332681570000171
TABLE 9
Figure BDA0003332681570000172
The result shows that the device 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: detection of AFP samples Using the immunoassay device of the present invention
The experimental materials were the same as in example 2.
The detection steps performed by the measuring device are as follows:
setting two reaction vessels as the same sample test group, and repeating the following liquid adding steps 1 and 2 in different sample test groups:
1. the sample arm adds 10 mu l of the same sample to be tested into each of the two reaction containers;
2. reagent arm 50ul of reagent 1 and 25ul of reagent 2 were added to reaction vessel 1 and reaction vessel 2, respectively;
3. incubating each reaction vessel at 37 ℃ for 15min;
4. 175. Mu.l of a universal solution for a photo-activated chemiluminescent analysis system was added to each reaction vessel and incubated at 37℃for 10min;
5. the immunoassay component records detection results, and the detection results of two parallel immunoreactions of the same sample to be detected are respectively calculated as a first measurement value and a second measurement value of the sample; wherein the reading in the reaction vessel 1 is a first measurement and the reading in the reaction vessel 2 is a second measurement;
6. the processor calculates the ratio of the first measured value to the second measured value of the same sample to be detected, retrieves the stored data for judgment, and then calculates the concentration of the target molecules in the sample to be detected.
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. And storing the standard substance test result into an immunoassay device.
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 BDA0003332681570000181
TABLE 11
Figure BDA0003332681570000191
The result shows that the device 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 6: ultra-high end measurement precision verification
The main components of the adopted reagent are as follows:
reagent 1: luminescent microparticles coated with AFP antibody (concentration 100 ug/mL), biotin-labeled AFP antibody (concentration 2 ug/mL);
reagent 2: luminescent microparticles coated with AFP antibody (concentration 20 ug/mL), biotin-labeled AFP antibody (concentration 0.4 ug/mL).
The detection procedure performed by the measuring apparatus was the same as in example 2.
The test results are shown in table 12 below (calculated using calculation method 1 of the present invention).
Table 12
Figure BDA0003332681570000192
The results show that: the CV measured values of three high-value samples repeatedly measured for 10 times by adopting the device disclosed by the invention are all within a range of 10%, so that the accuracy 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 (12)

1. An immunoassay device for detecting the concentration of a target molecule to be detected, comprising an immunoreaction assembly comprising at least two reaction vessels for performing two parallel immunoreaction detections on the same sample to be detected; wherein, the ratio of the content of the specific capture molecules in the reaction container 1 for detecting the same sample to be detected to the content of the target molecules to be detected in the sample to be detected is different from the ratio of the content of the specific capture molecules in the reaction container 2 to the content of the target molecules to be detected in the sample to be detected.
2. The device of claim 1, comprising a reagent compartment in which reagent 1 and reagent 2 are stored, and wherein the reagent 1 and reagent 2 contain different concentrations of the same component; the component comprises a specific capture molecule; 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.
3. The device according to claim 1 or 2, wherein the ratio of the content of the specific capture molecules in the reaction vessel 1 to the content of the target molecules in the sample to be tested in the same sample to be tested is different from the ratio of the content of the specific capture molecules in the reaction vessel 2 to the content of the target molecules in the sample to be tested by any one of the following means:
mode 1: adding an equal amount of a sample to be tested containing target molecules to be tested into a reaction container 1 and a reaction container 2, and adding an equal amount of a reagent 1 and a reagent 2;
mode 2: adding unequal amounts of a sample to be tested containing target molecules to be tested into a reaction container 1 and a reaction container 2, and adding equal amounts of a reagent 1 and a reagent 2;
mode 3: adding unequal amounts of a sample to be tested containing target molecules to be tested into a reaction container 1 and a reaction container 2, and adding unequal amounts of a reagent 1 and a reagent 2;
mode 4: equal amounts of a sample to be tested containing a target molecule to be tested are added to the reaction vessel 1 and the reaction vessel 2, and unequal amounts of the reagent 1 and the reagent 2 are added.
4. A device according to claim 2 or 3, wherein the reagent 1 comprises a first antibody (or antigen) coated luminescent particle at an α1 concentration, wherein the reagent 2 comprises a first antibody (or antigen) coated luminescent particle at a β1 concentration, and wherein α1 is greater than β1.
5. The device of any one of claims 2-4, wherein the reagent 1 further comprises a concentration of a2 of a label-labeled secondary antibody (or antigen), while the reagent 2 further comprises a concentration of a2 of a label-labeled secondary antibody (or antigen), and the alpha 2 is not less than beta 2; preferably, the α2 is greater than β2; further preferably, the marker is biotin.
6. The device of any one of claims 1-5, comprising an immunoassay component for recording the results of two parallel immunoreactions of the same test sample, a first test value and a second test value, respectively; wherein the reading in the reaction vessel 1 is a first measurement and the reading in the reaction vessel 2 is a second measurement; preferably, the ratio of the content of the specific capture molecule corresponding to the first measurement value to the content of the target molecule to be detected in the two parallel immunoreaction assays is greater than the ratio of the content of the specific capture molecule corresponding to the second measurement value to the content of the target molecule to be detected.
7. The device of claim 6, comprising a processor for calculating a first measurement/second measurement ratio and determining a concentration of a target molecule to be measured in the sample to be measured.
8. The apparatus according to any of claims 1-7, wherein the apparatus performs the steps of:
a1: detecting a series of standard substances with known target molecule content to be detected, wherein two parallel immunoreaction detection is carried out on each standard substance, the detection results of the two parallel immunoreactions are recorded and respectively calculated into a measured value a and a measured value a ', the detection mode of the measured value a and the first measured value of the sample to be detected is the same, and the detection mode of the measured value a' and the second measured value of the sample to be detected is the same;
a2: calculating the ratio of the measured value a/the measured value a';
a3: and (5) a correlation standard curve of the ratio of the measured value a to the measured value a' and the concentration of the standard substance is made and stored.
9. The apparatus of claim 8, wherein the apparatus performs the steps of:
the processor invokes the stored correlation standard curve, substitutes the ratio of the first measured value to the second measured value of the sample to be measured into the correlation standard curve to calculate so as to determine the concentration of the target molecules to be measured in the sample to be measured.
10. The apparatus according to any of claims 1-7, wherein the apparatus performs the steps of:
b1: detecting a series of standard substances with known target molecule content to be detected, wherein two parallel immunoreaction detection is carried out on each standard substance, the detection results of the two parallel immunoreactions are recorded and respectively calculated into a measured value b and a measured value b ', the measured value b and the first measured value of the sample to be detected are detected in the same mode, and the measured value b' and the second measured value of the sample to be detected are detected in the same mode;
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;
b4: taking a point in the part of the front zone of the reaction curve A, which is overlapped with the standard substance concentration corresponding to the rear zone 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.
11. The apparatus of claim 10, wherein the apparatus performs the steps of:
the processor retrieves the stored reaction curve A, the reaction curve B and the critical point c, and judges the ratio of the first measured value to the second measured value of the measured sample to the critical point c; 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.
12. A computer readable storage medium having stored thereon a computer program product, characterized in that the computer program product causes an apparatus according to any of claims 1-11 to perform the corresponding steps.
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