Novel immunodetection method and application

Abstract

The invention discloses a novel immunodetection method and application. The immunodetection method comprises the following steps: coating a captured antibody and a single-site recognition antigen on a solid-phase carrier; adding a to-be-detected sample and a biotin-labeled mixed detection antibody for incubation, so that the target antigen forms a double-antibody sandwich compound, and the single-site recognition antigen and the detection antibody form a competitive compound; after streptavidin marked by chemiluminescence is added for incubation, signal values of the two compounds are detected; whether interference exists or not is judged and detected by comparing the double signals, and the content of the target antigen is determined. The immunodetection method can effectively identify cross interference and hook effect, and improves the accuracy and reliability of multi-index detection.

Description

Technical Field

[0001] This application relates to the field of biodetection technology, specifically to novel immunodetection methods and their applications. Background Technology

[0002] In the field of in vitro diagnostics (IVD), immunoassays have become a crucial cornerstone of precision medicine due to their high specificity and sensitivity. Among them, the double-antibody sandwich method and the competitive method are two core technical approaches. Due to fundamental differences in their principles, they have long focused on different detection targets, forming insurmountable technical barriers.

[0003] The double-antibody sandwich assay is widely recognized as the "gold standard" for macromolecular detection, with both significant advantages and inherent limitations. It utilizes two antibodies for dual recognition of the target analyte, a unique mechanism that endows it with extremely high sensitivity and specificity, making it the top choice for detecting macromolecular targets such as proteins and glycoproteins (e.g., the tumor marker CA15-3). However, the limitations of the double-antibody sandwich assay are also clear: First, in high-concentration samples, the typical "hook effect" is prone to occur, where excess target analyte binds separately to the capture and detection antibodies, failing to form a complete complex, leading to falsely low signal and a serious risk of missed detection. Second, its application relies on a core prerequisite—the target analyte must possess at least two antigenic epitopes that can bind simultaneously. This firmly limits it to the scope of macromolecular detection, rendering it unsuitable for key small molecules such as hormones and drug metabolites. Furthermore, this method is susceptible to HAMA interference and cross-interference, which can affect test results.

[0004] Competitive assays fill the gap in small molecule detection and avoid the "hook effect," but they also have several performance limitations. They achieve detection by competing between the analyte and the label for a limited number of antibodies, perfectly solving the problem that small molecule haptens cannot be simultaneously bound by two antibodies. However, their core mechanism dictates that the detection signal is inversely proportional to the analyte concentration, resulting in lower sensitivity and precision in the low concentration range compared to the bispecific antibody sandwich assay, and a relatively narrower dynamic range. Therefore, in scenarios requiring ultra-high sensitivity, such as early disease screening and monitoring of trace drug concentrations, competitive assays struggle to meet increasingly demanding clinical needs.

[0005] The current technological landscape is thus stuck in a stalemate of "each going its own way": macromolecule detection relies on the double-antibody sandwich method, but is hampered by the "hook effect" and cross-contamination; small molecule detection can only rely on competitive methods, which are also limited by sensitivity bottlenecks. This clear-cut situation seriously hinders the development of IVD technology towards universality and precision.

[0006] Therefore, there is an urgent need in this field for an innovative technical solution that breaks down the traditional boundaries between the double-antibody sandwich method and the competitive method, creating a new detection paradigm that combines the high sensitivity of the double-antibody sandwich method with the small molecule detection capability of the competitive method, while effectively avoiding the "hook effect" and cross-contamination. Summary of the Invention

[0007] Therefore, it is necessary to provide novel immune detection methods and applications.

[0008] The first aspect of this application provides a novel immunoassay method, comprising the following steps:

[0009] The invention provides a capture antibody, a biotin-labeled mixed detection antibody, a unit spot recognition antigen, and a streptavidin labeled with a chemiluminescent marker, wherein the mixed detection antibody includes a detection antibody for a target antigen and a detection antibody for at least one other non-target antigen, the sequence of the unit spot recognition antigen contains a partial sequence of the target antigen, and the unit spot recognition antigen specifically binds only to the detection antibody for the target antigen.

[0010] The capture antibody and the unit spot recognition antigen were coated onto a solid-phase support;

[0011] The test sample and the biotin-labeled detection antibody are added for a first incubation, wherein the target antigen in the test sample forms a double antibody sandwich complex with the capture antibody and the mixed detection antibody, and the unit spot recognition antigen binds to the detection antibody of the target antigen to form a competitive complex;

[0012] A second incubation was performed after adding streptavidin labeled with the chemiluminescent marker, and the first signal value of the dual-antibody sandwich complex and the second signal value of the competing complex were detected; and,

[0013] Based on the first signal value and the second signal value, it is determined whether there is interference in the detection result, and the content of the target antigen in the sample to be tested is determined.

[0014] In some embodiments, the concentration of the capture antibody is 0.01 mg / mL to 0.2 mg / mL.

[0015] In some embodiments, the concentration of the unit site recognition antigen is 0.01 mg / mL to 0.2 mg / mL.

[0016] In some embodiments, the concentration of the detection antibody for the target antigen is 0.25 µg / mL to 50 µg / mL.

[0017] In some embodiments, the solid support includes a protein chip;

[0018] Optionally, the capture antibody and the unit spot recognition antigen are spotted onto the protein chip in the form of a spatially separated microarray.

[0019] In some embodiments, the chemiluminescent label includes one or more of horseradish peroxidase, alkaline phosphatase, and acridine ester.

[0020] In some implementations, the conditions for the first incubation include: a temperature of 25°C to 35°C and a time of 35 min to 45 min.

[0021] In some implementations, the conditions for the second incubation include a temperature of 25°C to 35°C and a time of 35 min to 45 min.

[0022] In some implementations, the criterion for determining whether there is interference in the detection result based on the first signal value and the second signal value is: when using mixed detection antibodies, interference exists if the concentration values ​​corresponding to the first signal value and the second signal value differ by 3 times or more.

[0023] In some embodiments, the target antigen includes MUC1, and the other non-target antigens include one or more of CA199, CA724, CA125, and CEA.

[0024] In some embodiments, the amino acid sequence of the capture antibody is shown in SEQ ID NO: 1, the amino acid sequence of the detection antibody of the target antigen is shown in SEQ ID NO: 2, and the amino acid sequence of the unit site recognition antigen is shown in SEQ ID NO: 3.

[0025] In some implementations, the sample to be tested includes one or more of plasma and serum.

[0026] The second aspect of this application provides a novel detection immunoassay kit, the immunoassay kit comprising a solid-phase carrier as defined in the first aspect of this application, a capture antibody, a biotin-labeled detection antibody, a unit spot recognition antigen, and streptavidin labeled with a chemiluminescent marker.

[0027] The aforementioned immunoassay method can effectively identify and avoid the cross-interference and hook effect commonly found in traditional multi-indicator joint detection, significantly improving the accuracy and reliability of multi-indicator joint detection. It is especially suitable for high-sensitivity and high-specificity detection of tumor markers in complex samples, reducing the risk of false positives or false negatives in clinical diagnosis. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0029] Figure 1 This is a schematic diagram of a dual-method detection design in one embodiment of this application;

[0030] Figure 2 This application provides a method for analyzing the sample correlation between two methodological systems in one embodiment.

[0031] Figure 3 The correlation of the normal female core reaction system samples in one embodiment of this application. Detailed Implementation

[0032] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0034] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0035] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0036] The terms “having,” “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0037] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0038] In this application, if the unit of a data range is only followed by the right endpoint, it means that the units of the left and right endpoints are the same.

[0039] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0040] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0041] In this application, the terms "first aspect," "second aspect," "first peptide," "second peptide," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0042] In this application, when numerical intervals (i.e., numerical ranges) are mentioned, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0043] In this application, the terms "room temperature" or "normal temperature" generally refer to 4°C to 35°C, for example, 20°C ± 5°C. In some embodiments of this application, "room temperature" or "normal temperature" refers to 10°C to 30°C. In some embodiments of this application, "room temperature" or "normal temperature" refers to 20°C to 30°C.

[0044] The double-antibody sandwich method has the following defects and shortcomings:

[0045] 1. When there is an excess of antigen, the HOOK phenomenon occurs, resulting in false low-value results. Most manufacturers conduct tests by diluting samples, but in multi-indicator joint testing, dilution can affect other indicators, making dilution testing impossible. This leads to inaccurate test results and poor correlation for high-end samples.

[0046] 2. It is prone to serious cross-contamination.

[0047] Currently, most joint testing uses the double-antibody sandwich method, but for some proteins with special structures, cross-interference and hooking phenomena are prone to occur, resulting in inaccurate test results.

[0048] Based on this, the embodiments of this application at least provide an immunoassay method and its application. In some embodiments, the embodiments of this application combine a double-antibody sandwich method and a competitive method. First, a pair of antibodies with good correlation is screened for testing using the double-antibody sandwich method. Then, the antigen sequence is segmented into different small fragments for expression, and unit-site recognition antigens that bind only to the secondary antibody in the double-antibody sandwich method are screened out. The primary antibody selected in the first step and the unit-site recognition antigen selected in the second step are spotted on a chip. The unit-site competition antigen can effectively solve the phenomenon of cross-interference and does not produce hooks. The results of the double-antibody sandwich method and the competitive method corroborate each other, making the detection results more accurate and reliable.

[0049] In this application, "cross-interference" refers to the phenomenon that when using mixed detection antibodies for multi-indicator joint detection, non-target antigens or their analogues present in the sample to be tested bind non-specifically to the detection antibodies in the mixed antibody that target other antigens, thereby producing false positive signals.

[0050] In a first aspect of this application, a novel immunoassay method is provided, comprising: coating a capture antibody and a unit spot recognition antigen onto a solid-phase carrier; adding a sample to be tested and a biotin-labeled mixed detection antibody for incubation, thereby forming a double-antibody sandwich complex of the target antigen and a competitive complex of the unit spot recognition antigen and the detection antibody; adding chemiluminescently labeled streptavidin for incubation, and then detecting the signal values ​​of the two complexes; and determining the presence of interference in the detection and the content of the target antigen by comparing the two signals.

[0051] In some implementations, the above-described immune detection method specifically includes the following steps:

[0052] S100: Provides a capture antibody, a biotin-labeled mixed detection antibody, a unit spot recognition antigen, and a chemiluminescent label-labeled streptavidin, wherein the mixed detection antibody includes a detection antibody for the target antigen and a detection antibody for at least one other non-target antigen, the sequence of the unit spot recognition antigen contains a portion of the sequence of the target antigen, and the unit spot recognition antigen binds specifically to the detection antibody only.

[0053] S200: Capture antibodies and unit site recognition antigens are respectively coated onto a solid-phase support;

[0054] S300: The test sample and biotin-labeled detection antibody are added for the first incubation, in which the target antigen in the test sample forms a double antibody sandwich complex with the capture antibody and the mixed detection antibody, and the unit site recognition antigen binds to the detection antibody of the target antigen to form a competitive complex.

[0055] S400: A second incubation was performed after adding streptavidin labeled with a chemiluminescent marker, and the first signal value of the double-antibody sandwich complex and the second signal value of the competing complex were detected; and...

[0056] S500: Based on the first signal value and the second signal value, determine whether there is interference in the detection result and determine the content of the target antigen in the sample to be tested.

[0057] It should be noted that in multi-indicator detection scenarios such as protein chips, to simplify the operation process, detection antibodies targeting different antigens are usually pre-mixed to form a mixed detection antibody before being added to the reaction system. However, when the sample to be tested contains multiple antigens, this traditional mixed secondary antibody sandwich method is prone to cross-interference due to non-specific binding between antibodies, leading to false positive signals. Furthermore, for high-concentration samples, this method still cannot avoid the interference of the hook effect. The immunoassay method provided in this application is designed for the aforementioned mixed secondary antibody detection scenario. By running the double antibody sandwich method and the single-point competition method in parallel in the same reaction system and comparing their signals, it achieves, for the first time, simultaneous monitoring and effective avoidance of cross-interference and the hook effect in a single detection system, significantly improving the accuracy and reliability of multi-indicator joint detection results.

[0058] In some embodiments, the method further includes expressing small fragments of the target antigen at different segments and using conventional detection methods in the art (such as ELISA) to screen for single-site recognition antigens that bind only to the detection antibody of the target antigen.

[0059] In some embodiments, in step S200, the concentration ratio of the capture antibody to the unit site recognition antigen is (1~20):(1~20). Non-limitingly, the concentration ratio of the capture antibody to the unit site recognition antigen can be, but is not limited to, 1:1, 1:10, 1:20, 10:1, 20:1, or any ratio or range between two of the above.

[0060] It should be noted that controlling the concentration ratio of the capture antibody and the unit site recognition antigen within the above range can ensure that the signal intensity of the double antibody sandwich method and the unit site competition method in the same reaction system is within a suitable and comparable range. This avoids the inhibition of the reaction of the other method due to the excess of one component, thereby ensuring that the results of the two methods can effectively corroborate each other and improve the ability to identify cross-interference and hook effect.

[0061] In some embodiments, the concentration of the capture antibody is 0.01 mg / mL to 0.2 mg / mL. Non-limitingly, the concentration of the capture antibody can be, but is not limited to, 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, or any value or range between two of the above.

[0062] In some embodiments, the concentration of the unit site recognition antigen is 0.01 mg / mL to 0.2 mg / mL. Non-limitingly, the concentration of the unit site recognition antigen can be, but is not limited to, 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, or any value or range between two of the above.

[0063] In some embodiments, the concentration of the detection antibody for the target antigen is 0.25 µg / mL to 50 µg / mL. Non-limitingly, the concentration of the biotin-labeled detection antibody may be, but is not limited to, 0.25 µg / mL, 0.5 µg / mL, 1 µg / mL, 10 µg / mL, 20 µg / mL, 30 µg / mL, 40 µg / mL, 50 µg / mL, or any value or range between two of the above.

[0064] It should be noted that the concentration of the detection antibody for the target antigen has been optimized to provide sufficient antibody quantity for the formation of the double antibody sandwich complex, ensuring high sensitivity. At the same time, in the unit-site competition method, the detection antibody and the unit-site recognition antigen on the solid-phase carrier are made to reach a suitable binding balance, which avoids weakening the competition effect due to excessive antibody and also prevents the signal from being too low due to insufficient antibody.

[0065] In some implementations, the solid support includes, but is not limited to, protein chips.

[0066] In some implementations, the capture antibody and the unit spot recognition antigen are spotted onto a protein chip in the form of a spatially separated microarray. It is understood that the capture antibody and the unit spot recognition antigen are respectively fixed on opposite sides of the same protein chip.

[0067] In some embodiments, the chemiluminescent label includes one or more of horseradish peroxidase, alkaline phosphatase, and acridine ester.

[0068] In some embodiments, in step S300, the conditions for the first incubation include: a temperature of 25°C to 35°C and a time of 35 min to 45 min. Non-limitingly, the temperature for the first incubation can be, but is not limited to, 25°C, 30°C, 35°C, or any value or range between two of the above; the time can be, but is not limited to, 35 min, 40 min, 45 min, or any value or range between two of the above.

[0069] In some embodiments, in step S400, the conditions for the second incubation include: a temperature of 25°C to 35°C and a time of 35 min to 45 min. Non-limitingly, the temperature for the second incubation can be, but is not limited to, 25°C, 30°C, 35°C, or any value or range between two of the above; the time can be, but is not limited to, 35 min, 40 min, 45 min, or any value or range between two of the above.

[0070] In some implementations, in step S500, the criterion for determining whether interference exists in the detection result based on the first signal value and the second signal value is: when using a mixed detection antibody, interference exists if the concentration values ​​corresponding to the first signal value and the second signal value differ by a factor of three or more. It is understood that when interference is determined to exist, since the second signal value originates from the unit-point competition method unaffected by cross-interference, the content of the target antigen in the sample to be tested should be determined based on the second signal value. If interference is determined not to exist, the content of the target antigen can be determined based on either the first signal value or the second signal value.

[0071] It should be noted that when interference is detected, further verification testing can be performed using monoclonal or diclonal antibodies against other non-target antigens. Specifically, if the first signal value obtained by using this monoclonal or diclonal antibody test is greater than the threshold of 300, it confirms that the signal is indeed caused by the non-specific binding of the non-target interfering substance to the detection antibody, and the presence of the interfering substance can be determined.

[0072] In some implementations, the target antigen in the sample to be tested includes MUC1.

[0073] In some implementations, the detection antibodies for other non-target antigens include one or more of the detection antibodies for CA199, CA724, and CA125.

[0074] In some embodiments, the amino acid sequence of the capture antibody is as shown in SEQ ID NO: 1, and / or the amino acid sequence of the detection antibody is as shown in SEQ ID NO: 2, and / or the amino acid sequence of the unit site recognition antigen is as shown in SEQ ID NO: 3.

[0075] In some implementations, the sample to be tested includes one or more of plasma and serum.

[0076] In a second aspect of this application, an immunoassay kit for detecting MUCI is provided, comprising a solid-phase carrier as defined above, a capture antibody, a biotin-labeled detection antibody, a unit spot recognition antigen, and streptavidin labeled with a chemiluminescent marker.

[0077] The following are some examples.

[0078] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0079] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0080] Experimental Example 1:

[0081] (1) Screening double antibody sandwich method using paired antibodies

[0082] Existing CA15-3 antibodies were used as primary and secondary antibodies for spotting and enzyme labeling, and gradient samples were tested. By comparing the correlation between the test signal values ​​and the concentration of the reference system, the best pair of combinations was selected. The final pair of antibodies selected for the CA15-3 index were 5C0-023 (SEQ ID NO: 1) and 5C0-028 (SEQ ID NO: 2).

[0083] (2) Screening unit spot recognition antigen

[0084] Because MUC1 contains multiple repetitive sequences, the MUC1 antigen sequence needs to be segmented into different small fragments for expression testing when screening for single-site binding antigens. The expressed fragments are numbered, coated onto ELISA plates, and reacted with HRP-labeled secondary antibodies. Finally, the single-site recognition antigen that binds only to the 5C0-028 antibody is selected and named MUC1-A1 (SEQ ID NO: 3).

[0085] (3) Spot 5C0-023 and the selected MUC1-A1 onto a single chip for testing.

[0086] Preparation of spotting solution: Take 0.05~0.5% surfactant (Tween), 1~10% protectant (horse serum), 0.1~0.5% humectant (sodium heparin) and 0.05~0.5% biological buffer (PBS buffer), mix them evenly to obtain the spotting solution;

[0087] Spotting: 5C0-023 and the screened MUC1-A1 were diluted according to the prepared spotting solution and then spotted onto the chip matrix. After dilution, the concentration of 5C0-023 was 0.1 mg / mL and the concentration of MUC1-A1 was 0.1 mg / mL.

[0088] Blocking treatment: The chip was blocked with blocking buffer for 4 h after spotting. The solid support was removed, and the residual blocking buffer was removed by centrifugation to obtain the biochip. The blocking buffer is a buffer solution containing blocking protein, which is bovine serum albumin or ovalbumin. The buffer is selected from any one of PBS buffer, Tris buffer, HEPS buffer, and MOPS buffer.

[0089] Signal value test: The sample to be tested was aspirated into a sample cup and tested using a biochip analyzer (Jiangsu Sanlian Biotechnology Co., Ltd., SLXP-001B). (Step 1: Sample pretreatment, the sample was incubated with the treatment solution (containing 1% blocking agent and 2μg / mL CA153 labeled biotin antibody) at 30℃ for 30 minutes for pretreatment; Step 2: The pretreated sample was bound to the capture antibody or antigen on the same spot chip and incubated at 30℃ for 40 minutes; Step 3: The mixed detection antibody (CA153 0.5μg / mL; CA724 0.7μg / mL; CA125 0.1μg / mL; CEA 0.5μg / mL) was bound to the complex formed in Step 2 and incubated at 30℃ for 40 minutes. Finally, it was luminescent with the chemiluminescent substrate.)

[0090] The dual-method detection structure design in this case is as follows: Figure 1 As shown, the antibody spotted on the left is 5C0-023, and the antigen spotted on the right is MUC1-A1. In the first step of the reaction, the 5C0-023 antibody spotted in the double-antibody sandwich method on the left, along with the biotin-labeled 5C0-028 antibody in the treatment solution, binds to the antigen in the serum to form a sandwich, while the MUC1-A1 antibody on the right only binds to the biotin-labeled 5C0-028 antibody in the treatment solution. In the second step, they react together with streptavidin labeled with HRP enzyme, resulting in luminescence.

[0091] Sequence Attachments:

[0092] SEQ ID NO: 1

[0093] EVQLVESGGGLVKPGGSLKLSCAASGFPFSSYAMSWVRQSPEKRLEWVAEISSGGTYFYYLHTVTGRFTISRDNAKNTLYLEMSSLRSEDTAMYFCAGETYGTPAWFPYWGQGTLV TVSADVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSIRFSGVPDRFSGSGSGTDFTLKISRVEADDLGLYYCFQGSHVPYTFGGGTKLEIK

[0094] SEQ ID NO: 2

[0095] QVQLKESGPGIVAPSQSLSITCTVSGFSLTTYGVHWIRQPPGKGLEWLGVIWAGGSTNYNSALMSRLSITKDNSKSQVFLKMNSLQTDDTAMYYCARDGAYETWAYWGQGTLVT VSADIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYWMSKLASGVPDRVSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLEIK

[0096] SEQ ID NO: 3

[0097] PAPGSTAPPAHGVTSAPDTR

[0098] Comparative Example 1:

[0099] In the Jiangsu Sanlian Biotechnology 12 Tumor Marker Assay Kit (JLY20240617, Microarray Chemiluminescence Immunoassay), the primary antibody (the same 5C0-023 antibody as in Example 1) is spotted on the chip. In the first step of the reaction, it binds to the antigen in the serum to form a complex. Then, it undergoes the second step of the reaction with the secondary antibody (the same 5C0-028 antibody as in Example 1) labeled with HRP enzyme, followed by luminescence.

[0100] Test results

[0101] Example 1 Test Results:

[0102] (1) Gradient sample testing

[0103] A dual-methodology approach was used to test 64 CA15-3 samples and compare their correlations. The results are as follows: Figure 2 As shown.

[0104] (2) Interference test

[0105] High-value samples of CA153 (less than 30 U / mL) were interfered with using CA199, CA724, and CA125 (concentrations shown in Table 1 below), respectively. The cross-interference signal was then detected using the corresponding monoclonal and secondary antibodies for CA199, CA724, and CA125. The test results of the dual-method system are shown in Tables 2 to 4 below.

[0106] Table 1

[0107]

[0108] Table 2. CA199 Interference with CA153 Sample Tests

[0109]

[0110] Table 3. CA724 Interference with CA153 Sample Tests

[0111]

[0112] Table 4. CA125 Interference with CA153 Sample Tests

[0113]

[0114] In addition, three interference samples from each of the above were selected, and the interference signals were detected using a mixed secondary antibody. The results of the two-method test are shown in Table 5 below:

[0115] Table 5. Samples of CA153 for mixed secondary antibody testing and CA-type interference.

[0116]

[0117] The results above show that when using mixed secondary antibodies for detection, if the results of the double antibody sandwich method and the competitive method are contradictory (i.e., the concentration detected by the double antibody sandwich method differs from the concentration detected by the competitive method by 3 times or more), it indicates that there is cross-interference.

[0118] (3) Detection limit verification

[0119] The results of the detection limit verification using the dual-method testing are shown in Table 6.

[0120] Table 6 Detection Limit Validation Results

[0121]

[0122] (4) Validation of the limit of quantitation

[0123] The results of the limit of quantitation verification using the dual-method testing method are shown in Table 7.

[0124] Table 7 Validation results of the limit of quantitation

[0125]

[0126] Comparative Example 1 Test Results:

[0127] (1) Gradient sample testing

[0128] Sixty-four CA15-3 samples were tested using a normal female core reaction system, and their correlation was compared. The results are as follows: Figure 3 As shown.

[0129] (2) Interference test

[0130] Similar to Example 1, CA199, CA724, and CA725 were used to interfere with the high-value samples of CA153, and the cross-interference signals were detected using the corresponding single and secondary antibodies of CA199, CA724, and CA725. The test results of the normal female core reaction system are shown in Tables 8 to 10 below:

[0131] Table 8. CA199 Interference with CA153 Sample Tests

[0132]

[0133] Table 9. CA724 Interference with CA153 Sample Tests

[0134]

[0135] Table 10 CA125 Interference with CA153 Sample Tests

[0136]

[0137] (3) Detection limit verification

[0138] The detection limit verification results using the normal female core reaction system are shown in Table 11.

[0139] Table 11 Detection Limit Validation

[0140]

[0141] (4) Validation of the limit of quantitation

[0142] The results of the limit of quantitation verification using the normal female core reaction system are shown in Table 12.

[0143] Table 12 Validation of Limit of Quantitation

[0144]

[0145] The comparison of the above charts shows that using a dual-method approach to detect CA153 can identify cross-interference and hooking phenomena in the test, thus significantly improving the accuracy of the results. Figure 2 and Figure 3 The correlation comparison revealed that the correlation of the comparative method was not as strong as that of the dual-method system, and the dual-method system could identify the hook phenomenon. Furthermore, the comparison of interference signals in Tables 2-5 and Tables 8-10 showed that the comparative method exhibited significant cross-interference, while the competitive method in the dual-method system was largely unaffected by cross-interference. In conclusion, the dual-method system proposed in this technical solution can effectively identify cross-interference and the hook phenomenon in the reaction, resulting in more accurate and reliable test results and reducing the risk of false positives or false negatives in clinical testing.

[0146] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0147] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A novel immunoassay method, characterized in that, Includes the following steps: The invention provides a capture antibody, a biotin-labeled mixed detection antibody, a unit spot recognition antigen, and a streptavidin labeled with a chemiluminescent marker, wherein the mixed detection antibody includes a detection antibody for a target antigen and a detection antibody for at least one other non-target antigen, the sequence of the unit spot recognition antigen contains a partial sequence of the target antigen, and the unit spot recognition antigen specifically binds only to the detection antibody for the target antigen. The capture antibody and the unit spot recognition antigen were coated onto a solid-phase support; The test sample and the biotin-labeled detection antibody are added for a first incubation, wherein the target antigen in the test sample forms a double antibody sandwich complex with the capture antibody and the mixed detection antibody, and the unit spot recognition antigen binds to the detection antibody of the target antigen to form a competitive complex; A second incubation was performed after adding streptavidin labeled with the chemiluminescent marker, and the first signal value of the dual-antibody sandwich complex and the second signal value of the competing complex were detected. as well as, Based on the first signal value and the second signal value, it is determined whether there is interference in the detection result, and the content of the target antigen in the sample to be tested is determined.

2. The novel immunoassay method as described in claim 1, characterized in that, One or more of the following conditions must be met: The concentration of the capture antibody is 0.01 mg / mL to 0.2 mg / mL; The concentration of the unit site recognition antigen is 0.01 mg / mL to 0.2 mg / mL; The concentration of the detection antibody for the target antigen is 0.25µg / mL to 50µg / mL.

3. The novel immunoassay method as described in claim 1, characterized in that, The solid-phase support includes a protein chip; Optionally, the capture antibody and the unit spot recognition antigen are spotted onto the protein chip in the form of a spatially separated microarray.

4. The novel immunoassay method as described in claim 3, characterized in that, The chemiluminescent label includes one or more of horseradish peroxidase, alkaline phosphatase, and acridine ester.

5. The novel immunoassay method as described in claim 1, characterized in that, It also meets one or more of the following conditions: The conditions for the first incubation include: a temperature of 25℃~35℃ and a time of 35min~45min; The conditions for the second incubation include: a temperature of 25℃~35℃ and a time of 35min~45min.

6. The novel immunoassay method as described in claim 1, characterized in that, The criterion for determining whether there is interference in the detection result based on the first signal value and the second signal value is as follows: when using mixed detection antibodies, interference exists when the concentration values ​​corresponding to the first signal value and the second signal value differ by 3 times or more.

7. The novel immunoassay method according to any one of claims 1 to 6, characterized in that, The target antigen includes MUC1, and the other non-target antigens include one or more of CA199, CA724, CA125, and CEA.

8. The novel immunoassay method as described in claim 7, characterized in that, The amino acid sequence of the capture antibody is shown in SEQ ID NO: 1, the amino acid sequence of the detection antibody of the target antigen is shown in SEQ ID NO: 2, and the amino acid sequence of the unit site recognition antigen is shown in SEQ ID NO:

3.

9. The novel immunoassay method according to any one of claims 1 to 6, characterized in that, The sample to be tested includes one or more of plasma and serum.

10. A novel detection immunoassay kit, characterized in that, The immunoassay kit comprises a solid-phase carrier as defined in claims 1 to 9, a capture antibody, a biotin-labeled detection antibody, a unit-point recognition antigen, and streptavidin labeled with a chemiluminescent marker.

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