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CN-121978327-A - Immunoassay kit, immunoassay method and immunoassay system

CN121978327ACN 121978327 ACN121978327 ACN 121978327ACN-121978327-A

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, can directly measure high-value sample concentration up to the level of 10 6 ng/ml, and has good repeatability.

Inventors

  • LI LIN

Assignees

  • 科美博阳诊断技术(上海)有限公司

Dates

Publication Date
20260505
Application Date
20211101

Claims (10)

  1. 1. An immunoassay kit comprising a reagent 1 and a reagent 2 of the same 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.
  2. 2. The kit of claim 1, wherein the specific capture molecule comprises a first antibody (or antigen) and a second antibody (or antigen) capable of specifically binding to a target molecule to be detected.
  3. 3. 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.
  4. 4. The kit according to claim 3, 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, and preferably the α2 is larger than β2.
  5. 5. A method of performing an immunoassay using the kit of any one of claims 1-4, comprising the steps of: s1, 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 respectively, and exciting and recording detection results of the two parallel immunoreactions, wherein a reading number detected by using the reagent 1 is a first measurement value, a reading number detected 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.
  6. 6. The method according to claim 5, 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 a sample to be detected containing a target molecule to be detected used in two parallel immunoreaction detection is the same, and the used reagent 1 and reagent 2 are equal; In the method 2, in two parallel immunoreaction detection, the amount of a sample to be detected containing a target molecule to be detected by adopting a reagent 1 is different from that of a sample to be detected containing a target molecule to be detected by adopting a reagent 2, and the used reagent 1 and the reagent 2 are equal in amount; In the method 3, in two parallel immunoreaction detection, the amount of a sample containing a target molecule to be detected by using the reagent 1 is different from the amount of a sample 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 the reagent 1 used is different from the amount of the reagent 2.
  7. 7. The method according to claim 5 or 6, 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.
  8. 8. The method according to claim 7, 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.
  9. 9. A system for performing an immunoassay according to the method of any one of claims 5-8, 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 container 1 is added with a reagent 1 of the kit, and the reaction container 2 is added with a 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.
  10. 10. The system of claim 9, wherein the processor stores a correlation standard curve of the ratio of the measured value a/the measured value a' and the concentration of the standard substance for calculating the concentration of the sample to be measured.

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, the singlet oxygen molecules can react with a luminous composition with a distance of about 200nm to generate a light signal with a certain wavelength, when a sample contains an antigen or an antibody to be detected, the immune reaction of the antigen and the antibody can enable donor particles containing the sensitizer to be combined with acceptor particles containing the luminous composition to generate the light signal with the specific wavelength, and the content of the antigen or the 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, and the traditional immunodetection reagent can only detect by using the front band section of the dose response curve, so that 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; Because the traditional immunity detection 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 clinical manifestations of a patient, and the detection reagent has complex operation, long time consumption and easy omission. 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 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 comprising a first antibody (or antigen) and a second antibody (or antigen) capable of specifically binding to target molecules 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