A method of monitoring the quality of sampling for immunoassays

Abstract

The application provides a method for monitoring the quality of sampling of immunoassay. The application provides a method for judging the quality of a sample by detecting the presence of MUC5B. The method of the application judges the quality of a mucus or saliva sample by detecting the specific binding of an antibody to the accompanying marker MUC5B, and has the advantages of simple operation, rapid and intuitive detection.

Description

Technical Field

[0001] This invention relates to the field of biological detection. Specifically, this invention relates to a method for monitoring the quality of immunoassay samples. Background Technology

[0002] Immunoassay technology utilizes the specific reaction between antigens and antibodies for detection. It amplifies and displays the detection signal using isotopes, enzymes, chemiluminescent substances, etc., and is commonly used to detect trace substances such as proteins and hormones. Immunoassay plays a vital role in clinical diagnosis, animal disease control, and food safety. Common immunoassay techniques include radioimmunoassay, lateral flow immunochromatography, enzyme-linked immunosorbent assay (ELISA), chemiluminescence, electrochemiluminescence, and chemiluminescence using magnetic nanoparticles.

[0003] Lateral-flow immunochromatography (LC-IACI) can quickly and easily detect trace substances in samples without the need for complex instruments, providing qualitative assessments for clinically diagnosed diseases, animal diseases, and food, cosmetics, and pharmaceuticals awaiting release during production and customs clearance. Enzyme-linked immunochromatography (ELISA) allows for quantitative detection of samples using an ELISA reader, providing a more direct and clear determination of disease severity and product quality. With technological advancements, immunoassay has been applied to a wider range of areas, and more and more people are encountering it in their work and daily lives. However, immunoassay requires sampling and sample pretreatment before testing. Since immunoassay often detects trace substances, sampling quality and sample processing procedures have a significant impact on the final results, making it a crucial issue. Furthermore, LC-IACI often makes it difficult to visually assess sampling and sample processing quality at the time of testing. In some cases, non-professionals may need to perform sampling and sample processing themselves. If sampling quality is not guaranteed, false negatives are likely, reducing the reliability of the test.

[0004] Therefore, there is a need in this field to develop a simple and rapid method for judging the quality of immune detection samples. Summary of the Invention

[0005] The purpose of this invention is to provide a method for monitoring the quality of immunoassay samples.

[0006] In a first aspect of the present invention, a method for monitoring the quality of an immunoassay sample is provided, the method comprising the steps of:

[0007] 1) Contact the sample with an antibody that specifically binds to the accompanying marker;

[0008] 2) Detect the binding of the sample to the antibody to determine the quality of the sample.

[0009] In another preferred embodiment, the sample is selected from the group consisting of mucus, saliva, blood, serum, plasma, urine, feces, sweat, or combinations thereof.

[0010] In another preferred embodiment, the sample is mucus or saliva.

[0011] In another preferred embodiment, the accompanying marker has the following characteristics:

[0012] a) Commonly present in the aforementioned sample type;

[0013] b) The properties are stable during the testing process.

[0014] In another preferred embodiment, the accompanying biomarker is selected from the group consisting of proteins, nucleic acids, polysaccharides, or combinations thereof.

[0015] In another preferred embodiment, the accompanying marker is mucin (MUC).

[0016] In another preferred embodiment, the accompanying marker is the MUC5B protein.

[0017] In another preferred embodiment, the amino acid sequence of the MUC5B protein is shown in SEQ ID NO.1.

[0018] In another preferred embodiment, the antibody is obtained by immunizing animals using a concomitant marker as an antigen.

[0019] In another preferred embodiment, the animal is a mammal, bird, or fish; preferably a mammal.

[0020] In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.

[0021] In another preferred embodiment, the monoclonal antibody is obtained by a method selected from the group consisting of hybridoma cells, phage display libraries, yeast display libraries, and single B cell screening.

[0022] In another preferred embodiment, the antibody is an animal-derived antibody, a chimeric antibody, or a humanized antibody.

[0023] In another preferred embodiment, the antibody is a single-domain antibody, nanobody, single-chain antibody, double-chain antibody, or antigen-binding fragment.

[0024] In another preferred embodiment, the antibody is a humanized monoclonal antibody.

[0025] In another preferred embodiment, the antibody specifically binds to a mucin, preferably specifically binding to the MUC5B protein.

[0026] In another preferred embodiment, the antibody includes a heavy chain variable region and a light chain variable region, wherein the light chain variable region includes the following complementarity-determining regions (CDRs):

[0027] LCDR1 shown in SEQ ID NO:2

[0028] LCDR2 shown in SEQ ID NO:3, and

[0029] LCDR3 as shown in SEQ ID NO:4;

[0030] The heavy chain variable region includes the following complementary determinant region (CDR):

[0031] HCDR1, as shown in SEQ ID NO:9,

[0032] HCDR2 shown in SEQ ID NO:10, and

[0033] HCDR3 as shown in SEQ ID NO:11;

[0034] or,

[0035] The light chain variable region includes the following complementary determinant regions (CDRs):

[0036] LCDR1 shown in SEQ ID NO:16

[0037] LCDR2 shown in SEQ ID NO:17, and

[0038] LCDR3 as shown in SEQ ID NO:18;

[0039] The heavy chain variable region includes the following complementary determinant region (CDR):

[0040] HCDR1, as shown in SEQ ID NO:23,

[0041] HCDR2 shown in SEQ ID NO:24, and

[0042] HCDR3 as shown in SEQ ID NO:25.

[0043] In another preferred embodiment, the heavy chain variable region and the light chain variable region further include a skeleton region (FR).

[0044] In another preferred embodiment, the CDR1, CDR2 and CDR3 of the light and heavy chain variable regions are separated by the skeletal regions FR1, FR2, FR3 and FR4, respectively.

[0045] In another preferred embodiment, the light chain variable region has FR1 as shown in SEQ ID NO.5, FR2 as shown in SEQ ID NO.6, FR3 as shown in SEQ ID NO.7, and FR4 as shown in SEQ ID NO.8; and / or

[0046] The heavy chain variable region has FR1 as shown in SEQ ID NO.12, FR2 as shown in SEQ ID NO.13, FR3 as shown in SEQ ID NO.14 and FR4 as shown in SEQ ID NO.15.

[0047] In another preferred embodiment, the light chain variable region has FR1 as shown in SEQ ID NO. 19, FR2 as shown in SEQ ID NO. 20, FR3 as shown in SEQ ID NO. 21, and FR4 as shown in SEQ ID NO. 22; and / or

[0048] The heavy chain variable region has FR1 as shown in SEQ ID NO.26, FR2 as shown in SEQ ID NO.27, FR3 as shown in SEQ ID NO.28 and FR4 as shown in SEQ ID NO.29.

[0049] In another preferred embodiment, the light chain and / or heavy chain of the antibody further include a constant region.

[0050] In another preferred embodiment, any of the above-mentioned amino acid sequences further includes a derived sequence that has optionally been added, deleted, modified and / or substituted at least one amino acid and is capable of retaining the binding affinity of the MUC5B protein.

[0051] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids does not exceed 30% of the total number of amino acids in the initial amino acid sequence, preferably 20%, and more preferably 10%.

[0052] In another preferred embodiment, the immunoassay is an immunochromatographic assay, an enzyme-linked immunosorbent assay (ELISA), an immunomagnetic bead assay, or a chemiluminescence assay.

[0053] In another preferred embodiment, the immune detection is lateral flow immunochromatography.

[0054] In another preferred embodiment, in step (2), the sample quality is determined according to the following criteria:

[0055] If the binding of the concomitant marker to the antibody is detected, the concomitant marker is interpreted as positive, indicating that the sample collection and processing are of good quality and the immunoassay results are reliable.

[0056] If no binding of the accompanying marker to the antibody is detected, the accompanying marker is interpreted as negative, indicating poor quality in the sample collection and processing, and the immunoassay result is unreliable.

[0057] In another preferred embodiment, the method is performed simultaneously with the immune detection; or before the immune detection.

[0058] In another preferred embodiment, the method involves detecting Mycoplasma pneumoniae by lateral flow immunochromatography while simultaneously detecting mucin MUC5B in nasal mucus with a mucin antibody, thereby monitoring the sampling quality of the sample for Mycoplasma pneumoniae detection.

[0059] In a second aspect of the invention, an immunochromatographic reagent strip is provided, the reagent strip comprising a sample pad, a conjugation pad, a chromatography membrane, and a substrate, wherein the chromatography membrane includes a quality control line.

[0060] The sample control line is coated with an antibody S1 that specifically binds to the accompanying marker, and the conjugate pad contains an antibody T that specifically binds to the target antigen to be detected and an antibody S2 that specifically binds to the accompanying marker.

[0061] In another preferred embodiment, the immunochromatographic reagent strip is used for mucus or saliva detection, and the accompanying marker is mucin (MUC), preferably MUC5B protein.

[0062] In another preferred embodiment, the antibodies S1 and S2 are the same or different antibodies.

[0063] In another preferred embodiment, the light chain variable region of antibody S1 includes the following complementarity-determining region (CDR):

[0064] LCDR1 shown in SEQ ID NO:2

[0065] LCDR2 shown in SEQ ID NO:3, and

[0066] LCDR3 as shown in SEQ ID NO:4;

[0067] The heavy chain variable region of antibody S1 includes the following complementarity-determining regions (CDRs):

[0068] HCDR1, as shown in SEQ ID NO:9,

[0069] HCDR2 shown in SEQ ID NO:10, and

[0070] HCDR3 as shown in SEQ ID NO:11.

[0071] In another preferred embodiment, the light chain variable region of antibody S2 includes the following complementarity-determining region (CDR):

[0072] LCDR1 shown in SEQ ID NO:16

[0073] LCDR2 shown in SEQ ID NO:17, and

[0074] LCDR3 as shown in SEQ ID NO:18;

[0075] The heavy chain variable region of antibody S2 includes the following complementarity-determining regions (CDRs):

[0076] HCDR1, as shown in SEQ ID NO:23,

[0077] HCDR2 shown in SEQ ID NO:24, and

[0078] HCDR3 as shown in SEQ ID NO:25.

[0079] In another preferred embodiment, the conjugate pad contains labeled antibodies, such as colloidal gold conjugate antibodies.

[0080] In another preferred embodiment, the chromatographic membrane further includes a sample detection line coated with an antibody T that specifically binds to the target antigen to be detected.

[0081] In another preferred embodiment, the target antigen is Mycoplasma pneumoniae, Chlamydia pneumoniae, adenovirus, influenza A virus, influenza B virus, SARS-CoV-2, or respiratory syncytial virus.

[0082] In another preferred embodiment, the chromatography membrane further includes a reagent control line, wherein the reagent control line is coated with antibody C that specifically binds to antibody T or antibody S2.

[0083] In another preferred embodiment, the immunochromatographic reagent strip further includes an absorbent pad.

[0084] In a third aspect of the invention, a detection kit is provided, the kit comprising the immunochromatographic reagent strips as described in the second aspect of the invention.

[0085] In another preferred embodiment, the kit is used for the detection of Mycoplasma pneumoniae in mucus or saliva samples.

[0086] In another preferred embodiment, the kit further includes a buffer solution.

[0087] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0088] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0089] Figure 1 The expression levels of MUC5B in various tissues are shown.

[0090] Figure 2 The expression levels of MUC5AC in various tissues are shown.

[0091] Figure 3 The expression levels of serum albumin (HSA) in various tissues were shown.

[0092] Figure 4 The expression levels of lactotransferrin in various tissues were shown.

[0093] Figure 5 The results of Western blot analysis of the purified MUC5B product are shown.

[0094] Figure 6 The HPLC results of the purified MUC5B product are shown.

[0095] Figure 7 The results show the ability of ELISA to detect the binding of cell supernatant expressing MUC5B antibody to saliva.

[0096] Figure 8 A schematic diagram of the structure of the lateral flow immunochromatographic reagent strip of the present invention is shown. Detailed Implementation

[0097] Through extensive and in-depth research, the inventors have developed, for the first time, a method for monitoring the quality of immunoassay samples. Specifically, this invention, after screening, selected MUC5B as a co-marker for saliva or mucus samples, and provides a method for assessing sample quality by detecting the presence of MUC5B. This invention also provides antibodies and antibody pairs that specifically target MUC5B. Based on these findings, this invention was completed.

[0098] the term

[0099] To provide a clear explanation of the invention, several terms are defined below.

[0100] Mucin 5B

[0101] As used in this article, the term "mucin 5B," also known as MUC5B or Muc5b, is a protein encoded by the MUC5B gene in humans. It is one of five mucins that form gels. Mucin MUC5B is widely found in the saliva, respiratory mucus, and cervical mucus of most humans.

[0102] Through screening, this invention discovered that MUC5B protein is simultaneously obtained when performing immunoassay sampling on the aforementioned sites, and that MUC5B protein is stable and present in high concentrations in the samples. Therefore, this invention selects MUC5B as a concomitant biomarker for immunoassay.

[0103] As used herein, the terms "marker," "marker," and "concomitant marker" refer to substances that are ubiquitous and stable in the sample to be tested, can be used for immunization, and are present throughout the entire immunization process. The concomitant markers selected in this invention can be substances such as proteins, nucleic acids, and polysaccharides contained in the sample, which can be collected together with the sample and are not completely lost during sample processing.

[0104] Antibody

[0105] This invention provides an antibody that specifically targets MUC5B. In one embodiment, the antibody of this invention is obtained by immunizing animals using MUC5B as an antigen.

[0106] As used in this article, the terms "antigen" and "immunogen" refer to substances that can elicit an immune response in the body and produce corresponding antibodies that specifically bind to it.

[0107] As used herein, the terms “antibody,” “monoclonal antibody,” and “polyclonal antibody” refer to immunoglobulin molecules, which are heterotetrameric proteins produced by binding two identical heavy chains and two identical light chains, and which perform antigen-specific binding through antigen-binding sites composed of a light chain variable region (VL) and a heavy chain variable region (VH), thereby evoking an antigen-specific humoral immune response.

[0108] As used herein, the terms “humanized antibody,” “single-domain antibody,” “single-chain antibody,” “nanobody,” “antigen-binding peptide,” “fragment,” “derivative,” and “analyte” refer to antibodies or antibody fragments that have antigen-binding capabilities, including fragments generated by cleaving antibodies with protein-cleaving enzymes and fragments generated in a recombinant manner, and examples of such fragments include Fab, F(ab')2, scFv, double-chain antibodies, triple-chain antibodies, sdAb, and VHH.

[0109] As used in this article, the term "specificity" refers to a highly specific and efficient interaction, such as an antigen binding specifically to its corresponding antibody but not to other antibodies.

[0110] As used in this article, the term "variable region (V region)" refers to the region of the immunoglobulin light and heavy chains near the N-terminus where the amino acid sequence changes significantly.

[0111] As used in this article, the term "constant region (C region)" refers to the region of the immunoglobulin light and heavy chains near the C-terminal amino acid sequence where the sequence is relatively stable.

[0112] As used in this article, the term "complementarity-determining region (CDR)" refers to three highly variable regions within the variable regions of both the light and heavy chains of immunoglobulins that can form precise complementarity with antigenic determinants in spatial structure.

[0113] As used in this article, the term "framework region (FR)" refers to the region in the variable region of the immunoglobulin light and heavy chains where the amino acid composition and sequence are relatively conserved, except for the CDR.

[0114] This invention also provides other proteins or fusion expression products containing the antibodies of this invention. Specifically, this invention includes molecules having a CDR region of the light or heavy chain of the antibody provided by this invention, provided that its CDR has more than 90% (preferably more than 95%, most preferably more than 98%) homology with the CDR identified by this invention.

[0115] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies. The polypeptide fragments, derivatives, or analogs of this invention can be (i) polypeptides with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0116] The term "antibody of the present invention" refers to a polypeptide containing the aforementioned CDR region and exhibiting MUC5B protein-binding activity. This term also includes variants of polypeptides containing the aforementioned CDR region that have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically 20 to 25, preferably 10 to 25, more preferably 5 to 25) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids at the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the antibodies of the present invention. The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0117] The antibodies of the present invention include conserved variants thereof. A conserved variant of the antibody of the present invention refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to the table below.

[0118]

[0119]

[0120] The DNA sequences of the antibodies or fragments thereof of this invention can be obtained using conventional techniques, including but not limited to hybridoma cells, phage display libraries, yeast display libraries, and single B cell screening. The antibodies of this invention can be produced by immunizing animals with concomitant markers, for example, by immunizing mice.

[0121] As used in this article, the term "hybridoma cell" is a cell formed by fusing myeloma cells and B lymphocytes during the preparation of monoclonal antibodies.

[0122] As used herein, the term "phage display library" refers to a library in which a random oligonucleotide of a specific length is inserted to one side of the pⅢ gene of a phage, thereby displaying a random polypeptide fused with pⅢ on the surface of the phage. Since each phage presents a foreign polypeptide sequence, an aggregate of phages displaying various foreign polypeptide sequences of a specific length constitutes a complete phage display library.

[0123] As used in this article, the term "yeast display library" refers to a method of expressing and displaying various exogenous polypeptide sequences of specific lengths on the surface of yeast. This method, combined with certain screening conditions, can be used to obtain monoclonal antibody sequences with excellent affinity.

[0124] As used in this article, the term "single B cell screening" refers to the use of a special device to detect antibodies expressed on a single B cell and to screen for monoclonal antibody sequences with superior affinity.

[0125] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0126] As used herein, the terms "vector," "expression system," or "expression vector" refer to a nucleic acid sequence containing a desired coding and control sequence operatively linked together, such that a host transformed with these sequences can produce the encoded protein. For transformation, the expression system may be contained on a vector; however, the associated nucleic acid molecules may subsequently be integrated into the host chromosome as well.

[0127] As used herein, the term "host cell" is a cell that supports the replication or expression of an expression vector. Host cells can be prokaryotic cells such as Escherichia coli, or eukaryotic cells such as yeast cells, insect cells, amphibian cells, or mammalian cells.

[0128] As used herein, the terms “transfection,” “stable transfection,” or “transient transfection” refer to the uptake of an expression vector by a host cell, regardless of whether any coding sequence is actually expressed. Various transfection methods are known to those skilled in the art. For example, transfection can be performed in the presence of an expression vector and a high concentration of calcium phosphate, by electroporation, by inserting a phage or viral expression vector into a host cell, by mechanically inserting nucleic acid, or even by culturing host cells in the presence of unpackaged nucleic acid fragments. Successful transfection is typically confirmed when any indication of manipulation of the vector of interest appears in the host cell.

[0129] Detection methods

[0130] This invention provides a method that can detect concomitant biomarkers in samples simultaneously with immunoassay, and determine whether the sampling and sample processing meet the requirements by using the positive or negative results of the concomitant biomarkers, thereby verifying the reliability of the sampling and sample processing steps in immunoassay.

[0131] The detection method of the present invention is achieved through the following technical means:

[0132] First, by analyzing the sample components, stable and widely present substances in the sample are identified as accompanying biomarkers. Then, the biomarkers are purified and used as immunogens for immunization. The corresponding antibodies are obtained through purification, monoclonal screening and other experimental methods.

[0133] Secondly, by developing immunoassay methods, we can diagnose the target component and the accompanying biomarkers in the sample at the same time. We can determine the positive or negative of the target component and the biomarkers in the sample at the same time. A negative result indicates that the sampling or sample processing was unreasonable and the experimental result is unreliable. A positive result indicates that the sampling and sample processing was reasonable and the result is reliable.

[0134] This invention detects sample biomarkers simultaneously with the target component in an immunoassay. This allows for simultaneous interpretation of the sample biomarker and the target component. A positive result for the biomarker indicates sufficient biomarker content in the sample, demonstrating that the sample collection and processing procedures were compliant. Based on this, the qualitative or quantitative interpretation of the target component is then reliable. Conversely, a negative result indicates the absence or insufficient presence of the biomarker in the sample, suggesting that the sampling or processing procedures were inadequate, rendering the experimental results unreliable and requiring resampling or reprocessing. This method allows for monitoring the quality of sampling and sample processing in immunoassay tests, avoiding false negatives, and resulting in more accurate and reliable experimental results.

[0135] The method of the present invention can be applied to a variety of immunoassay methods, such as immunochromatographic assay, enzyme-linked immunosorbent assay, immunomagnetic bead assay, or chemiluminescence assay.

[0136] As used in this article, the term "lateral flow immunochromatography," also known as lateral flow immunoassay, is a unique immunoassay method that emerged in the early 1960s. It uses strip-shaped fiber chromatography material as the solid phase and uses capillary adsorption to move the sample on the chromatography material. The analyte in the sample binds to antibodies in a certain area on the chromatography material, and the test results are obtained in a short time through enzymatic colorimetric reactions or by directly using colored markers.

[0137] As used herein, the term "enzyme-linked immunosorbent assay" (ELISA) refers to the ability of antigens or antibodies to maintain their immunological activity after binding to the surface of a solid-phase carrier, and the ability of antigens or antibodies to maintain their immunological and enzymatic activity after binding to an enzyme. The enzyme conjugate forms a complex with the corresponding antigen or antibody, which undergoes a colorimetric reaction catalyzed by the substrate. The intensity of the colorimetric reaction following the addition of the enzyme substrate solution indicates the presence or absence of a corresponding immune response and the amount of antigen or antibody.

[0138] As used in this article, the term "immunomagnetic beads" refers to composite materials with a superparamagnetic polymer surface covered by a core-shell synthesis method. These materials are stable and can be labeled later. Functional groups on the surface of these materials, such as amino, carboxyl, and thiol groups, can be used for covalent or non-covalent coupling with antibodies. They can be used to bind corresponding antigens or antibodies. Under the attraction of an external magnetic field, they can move in a directional manner, thereby achieving the purpose of separating, detecting, and purifying genes, proteins, cells, microorganisms, etc.

[0139] As used in this article, the term "chemiluminescence" is a type of molecular luminescence spectroscopy. It is a trace analysis method that mainly relies on the principle that the concentration of the analyte in a chemical detection system has a linear quantitative relationship with the chemiluminescence intensity of the system under certain conditions. The content of the analyte is determined by detecting the chemiluminescence intensity of the system using an instrument.

[0140] As used in this article, the term "negative" (-) in medical examinations typically indicates that the indicator is normal or that the pathogen is absent, while "positive" (+) indicates that the patient is ill or that a pathogen (such as a virus) is present. In medicine, the terms negative and positive are used generally to refer to the presence or absence of a test result.

[0141] In one embodiment, the method of the present invention can be applied to the detection of Mycoplasma pneumoniae using lateral flow immunochromatography. Simultaneously with the detection of Mycoplasma pneumoniae, the sampling quality is monitored by detecting mucin MUC5B in nasal mucus using mucin antibodies.

[0142] The main advantages of this invention include:

[0143] 1) This invention selects MUC5B as a co-marker for pathogen detection. MUC5B is widely present in the oral cavity, respiratory tract, and cervical mucus, with large secretion amounts and little inter-individual variation. Sampling from these sites according to standard procedures ensures that the sample will definitely contain MUC5B. Furthermore, MUC5B protein has high stability and is not easily lost during sample processing, ensuring that a sufficient amount of MUC5B will react with MUC5B antibodies during detection.

[0144] 2) This invention provides antibodies and antibody pairs that specifically target MUC5B. When used in combination in lateral flow chromatography, the antibody pairs of this invention can maximize the colorimetric effect of MUC5B detection and do not produce cross-reaction with Mycoplasma pneumoniae antibodies, thus exhibiting high sensitivity and high specificity.

[0145] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0146] Example 1: Accompanying biomarker screening

[0147] Saliva contains various proteins, mainly glycoproteins and mucins, as well as immunoglobulins, lactoferrin, inorganic salts, and salivary enzymes. The proteins in airway secretions are primarily mucins and immunoglobulins. Mucins include secreted polymeric mucins MUC5AC and MUC5B, and cell membrane-bound mucins MUC1, MUC4, MUC16, and MUC20. Immunoglobulins include IgG, IgM, and IgA. Cervical mucus is mainly composed of mucins, glycoproteins, and plasma proteins.

[0148] Among the aforementioned candidate biomarkers, the inventors focused on screening secretory mucins MUC5AC and MUC5B, serum albumin, and lactoferrin. The results are as follows: Figure 1-4 As shown.

[0149] A comparative analysis of human protein maps revealed that MUC5AC is highly expressed in the nasopharynx but not in the salivary glands, which limits its applicability to certain samples. Figure 2 Furthermore, the expression levels of MUC5AC differ significantly between healthy humans and patients, which may interfere with the accuracy of accompanying detection.

[0150] Serum albumin is mainly found in the blood and kidneys, and is also ubiquitous in the extracellular matrix of other tissues; it does not exhibit location specificity. Figure 3 Lactoferrin is expressed in the bronchi and salivary glands, but not in the nasopharynx, and at low levels. Figure 4 Both methods may lead to misjudgment of sample quality when sampling from the nasal cavity / pharynx due to excessively low levels of accompanying biomarkers, and are therefore unsuitable for nasal cavity / pharynx sampling and testing.

[0151] After screening, it was found that MUC5B is expressed in large quantities in the salivary glands and nasopharynx, and the expression level is stable. Figure 1 Therefore, MUC5B was chosen as the accompanying marker.

[0152] Example 2: Preparation of mucin MUC5B

[0153] This embodiment provides a method for separating and purifying MUC5B from saliva:

[0154] 1.200 mL of saliva was collected from 8 healthy adults (aged 25–34 years, with healthy oral cavity, no systemic diseases, and no antibiotic use within the past 3 months). Patients fasted and rinsed their mouths for 2 hours prior to saliva collection. The collected fresh saliva was placed in an ice bath, and 0.274 mol / L cetyltrimethylammonium bromide was immediately added. The mixture was gently stirred with a glass rod until a clot formed on the clot, at which point the MUC5B protein precipitate aggregated on the rod. The clot was transferred to another flask, and 250 mL of 3 mol / L NaCl was added to dissolve it. This solution is the crude MUC5B extract.

[0155] 2. Dialyze the crude MUC5B extract using a 14K dialysis bag for 48 hours. The dialysis buffer is 0.01M PBS. Change the dialysis buffer every 12 hours.

[0156] 3. MUC5B was purified by gel filtration chromatography. The Superdex 200 packing material was resuspended in pure water and packed into a 16×500mm chromatography column to a height of 400mm. The column was equilibrated with 0.01M PBS (pH 7.0) for 2 hours at a flow rate of 1mL / min. MUC5B was concentrated to 3mg / mL using ultrafiltration, and then loaded with 2mL samples at a flow rate of 1mL / min. The elution products were collected using an automated collector, and the products from multiple collections were combined and adjusted to a concentration of 1mg / mL.

[0157] 4. Perform Western blotting and HPLC to identify the purified MUC5B.

[0158] Figure 5 The image shows the purified MUC5B product as detected by Western blotting (WB), where the gray areas represent areas that can bind to the MUC5B antibody, indicating the presence of the MUC5B protein. Figure 6 The results show the HPLC analysis of the purified MUC5B product, with peaks at 8.3 min and 8.921 min both being MUC5B, corresponding to two bands in the Western blot.

[0159] Example 3: Method for screening anti-MUC5B monoclonal antibodies using single B cells

[0160] (1) The purified MUC5B was used as an immunogen to repeatedly immunize four mice.

[0161] (2) Collect a small amount of tail blood and determine the serum titer by indirect ELISA. The immunization is terminated when the serum titer reaches 100,000 or more.

[0162] (3) Select the mouse with the highest serum titer, sacrifice it, take its spleen, break it up, and obtain cell suspension.

[0163] (4) Flow cytometry was used to sort B cells expressing antibodies, the cells were lysed, and the antibody light and heavy chain genes were extracted.

[0164] (5) Construct the antibody gene onto a plasmid and express it transiently.

[0165] (6) Collect the expressed antibodies for subsequent screening.

[0166] Example 4: Detection of antibody binding to saliva samples

[0167] Eight positive monoclonal antibodies were screened by coating saliva onto ELISA plates and detecting the cell supernatant of transiently transfected antibody-expressing cells using ELISA. The specific implementation method is as follows:

[0168] (1) Collect saliva as antigen and dilute it tenfold with 0.05M sodium carbonate buffer at pH=9.0. Then add it to a 96-well microplate, 100uL per well, and incubate overnight at 4°C.

[0169] (2) Discard the incubated antigen, add 5% skim milk powder as blocking solution, 250uL per well, 37℃, and block for 3 hours;

[0170] (3) Discard the blocking solution, add 200uL PBST and wash three times, then pat dry;

[0171] (4) The obtained cell supernatant containing antibodies was serially diluted four times: 4, 16, 64, 256, 1024, 4096, 16384, and 65536 times. 100 μL of each supernatant was added to the above-mentioned ELISA plates and incubated at 37°C for 1 hour. After incubation, 200 μL of PBST was added, the plates were washed three times, and then patted dry.

[0172] (5) Add goat anti-human HRP secondary antibody diluted 3000 times with PBST, incubate at 37°C for 1 hour. After incubation, add 200uL PBST, wash three times, and pat dry.

[0173] (6) Add 50 μL of TMB colorimetric solution to each well, place in the dark, incubate for 5 minutes, and then add 50 μL of stop solution (0.2 M sulfuric acid) to terminate the reaction;

[0174] Read the values ​​at 450 nm using an ELISA reader within 30 minutes. The results are as follows: Figure 7 As shown in the figure. Based on the results, eight positive monoclonal antibodies were screened out: 1M2B3, 1M2C4, 1M2C5, 1M2F5, 1M2G6, 1M2B7, 1M2C10, and 1M2F12.

[0175] Example 5: MUC5B detection for antibody pair screening

[0176] Eight MUC5B antibodies were screened using lateral flow immunochromatography to identify MUC5B antibody pairs suitable for antibody sandwich immunoassay, and then applied to colloidal gold test strips for the immunodiagnosis of Mycoplasma pneumoniae. The specific implementation method is as follows:

[0177] 1. Gold labeling of antibodies

[0178] 1) Take nine 10ml portions of gold solution, add 210uL of 0.5M potassium carbonate to adjust the pH, and add 90ug of eight kinds of mucin antibody or mycoplasma pneumoniae antibody solution dropwise, and stir for 30 minutes.

[0179] 2) Take 10% BSA and add it dropwise to the mixed solution until the final concentration is 0.5%, then stir for 15 minutes.

[0180] 3) Take 10% PEG (20,000) and add it dropwise to the mixed solution until the final concentration is 0.1%, then stir for 15 minutes.

[0181] 4) Centrifuge at 8000-9000 RPM, 4℃, for 10 minutes.

[0182] 5) Discard the supernatant, then add diluent to resuspend the colloidal gold particles, and adjust the volume to 1 mL.

[0183] 2. Pretreatment of colloidal gold pads and sample pads

[0184] 1) Determine the quantity to be processed, and stack 10 sheets of A4 paper (or cut to other sizes).

[0185] 2) Prepare the buffer solution (approximately 30 ml / A4 paper size). Adjust the solution formulation according to the thickness and performance of the material. Basic elements: buffer solution + NaCl + others.

[0186] 3) Soak the material completely in the buffer solution for 10 minutes, then remove it and dehydrate for 3 minutes, and place it flat on the sieve.

[0187] 4) Place in a 37℃ drying oven (humidity less than 20%) and dry until the humidity is constant at around 20%.

[0188] 5) After drying, pack the material into aluminum foil bags or plastic bags and seal them for storage. Label the bags and record the batch number. Note that the treated fiberglass should not be exposed to high humidity environments for a long time.

[0189] 3. Spotting

[0190] 1. Colloidal gold spray dots onto colloidal gold pads

[0191] 1) Dilute the labeled colloidal gold to 36 D / mL, and mix the colloidal gold labeled with Mycoplasma pneumoniae and the colloidal gold labeled with eight mucin antibodies separately. Add (final concentration): 10-20% sucrose, 1-3% BSA, 0.5-1% NaCl, and 0.5-1% surfactant.

[0192] 2) Under the conditions of 20-40% humidity and room temperature, spray the solution onto the colloidal gold pad using the AIRJET nozzle of the gold spraying and coating instrument, with the pressure set to 10 PSI and the spray point parameter to 7 μL / cm.

[0193] 3) Place the sprayed colloidal gold strips at 37°C to dry, and after the humidity is kept constant at 20%, collect and seal them for storage.

[0194] 4. Antibody crossing

[0195] 1) Remove the NC membrane and allow it to equilibrate for 1 hour at room temperature and normal humidity.

[0196] 2) Adhere the non-spotted side of the NC film to the PVC base plate.

[0197] 3) Dilute the eight mucin antibodies, Mycoplasma pneumoniae antibody, and goat anti-human Fc antibody to 1 mg / ml using buffer solution; pH range: 7.0.

[0198] 4) Using a gold-spraying membrane scrubbing instrument, set the scrubbing volume to 0.5ul / cm and scrub the membrane. Draw three lines on each membrane, from bottom to top: Mycoplasma pneumoniae, one of the eight mucin antibodies, and goat anti-human Fc antibody.

[0199] 5) Dry in an oven at 37 degrees Celsius for 2 hours until the humidity is below 20%, then seal and package for later use.

[0200] 5. Assembly, cutting, and packaging of test strips (Environmental requirements: room temperature, humidity below 20%)

[0201] 1) Combine colloidal gold pads and NC membranes with different mucin antibodies in pairs.

[0202] 2) The colloidal gold pad is pasted under the NC film, covering the NC film by 1mm.

[0203] 3) The absorbent pad is attached above the NC membrane, covering it by 2mm.

[0204] 4) The sample pad is attached below the colloidal gold pad, covering the colloidal gold pad by 2mm.

[0205] 5) Cut the pasted test plate into 4mm wide strips in a strip cutter.

[0206] 6) Pack the shell and desiccant together into an aluminum foil bag, seal it, and affix a label.

[0207] 6. Testing

[0208] 1) Positive test samples are pharyngeal mucus samples collected by pharyngeal swabs and treated with sample lysis buffer. Negative test samples are unused pharyngeal swabs treated with sample lysis buffer.

[0209] 2) Prepare two colloidal gold test strips for each combination, and load one positive sample and one negative sample onto each strip, with 50ul of sample loaded onto each strip.

[0210] 3) After standing for 5 minutes, observe the experimental results and compare the color depth with the color chart. Score the colors from darkest to lightest, with scores ranging from G10 to G0.

[0211] 4) Comparison revealed that colloidal gold labeled with antibodies 5, 7, and 8 showed cross-reactivity with Mycoplasma pneumoniae antibodies, therefore, none of these antibody pairings could be selected. After excluding the above conditions, pairing numbers 1+2, 1+4, and 2+4 were preferred, with 1+4 being the best.

[0212] Table 1 Information on mucin antibodies

[0213]

[0214]

[0215] Table 2 Results of mucin antibody pairing detection

[0216]

[0217]

[0218] The sequences involved in this invention are shown in the table below:

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for monitoring the quality of immunoassay samples, characterized in that, The method includes the following steps: 1) Contact the sample with an antibody combination that specifically binds to the accompanying biomarker; 2) Detect the binding of the sample to the antibody to determine the quality of the sample; The accompanying biomarker is the MUC5B protein; The antibody combination comprises antibody S1 and antibody S2, wherein the light chain variable region of antibody S1 includes the following complementarity-determining regions (CDRs): LCDR1 shown in SEQ ID NO:2 LCDR2 shown in SEQ ID NO:3, and LCDR3 as shown in SEQ ID NO:4; The heavy chain variable region of antibody S1 includes the following complementarity-determining regions (CDRs): HCDR1, as shown in SEQ ID NO:9, HCDR2 shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:11; The light chain variable region of antibody S2 includes the following complementarity-determining regions (CDRs): LCDR1 shown in SEQ ID NO:16 LCDR2 shown in SEQ ID NO:17, and LCDR3 as shown in SEQ ID NO:18; The heavy chain variable region of antibody S2 includes the following complementarity-determining regions (CDRs): HCDR1, as shown in SEQ ID NO:23, HCDR2 shown in SEQ ID NO:24, and HCDR3 as shown in SEQ ID NO:25; The method is applied for non-diagnostic purposes.

2. The method as described in claim 1, characterized in that, The immunoassay is an immunochromatographic assay, enzyme-linked immunosorbent assay (ELISA), immunomagnetic bead assay, or chemiluminescence assay.

3. The method as described in claim 1, characterized in that, In step 2), the sample quality is determined according to the following criteria: If the binding of the concomitant marker to the antibody is detected, the concomitant marker is interpreted as positive, indicating that the sample collection and processing are of good quality and the immunoassay results are reliable. If no binding of the accompanying marker to the antibody is detected, the accompanying marker is interpreted as negative, indicating poor quality in the sample collection and processing, and the immunoassay result is unreliable.

4. An immunochromatographic reagent strip, characterized in that, The immunochromatographic reagent strip includes a sample pad, a conjugate pad, a chromatography membrane, and a substrate. The chromatography membrane includes a quality control line. The sample control line is coated with an antibody S1 that specifically binds to the accompanying marker, and the conjugate pad contains an antibody that specifically binds to the target antigen to be detected and an antibody S2 that specifically binds to the accompanying marker. The immunochromatographic reagent strip is used for mucus or saliva detection, and the accompanying marker is MUC5B protein; The light chain variable region of antibody S1 includes the following complementarity-determining regions (CDRs): LCDR1 shown in SEQ ID NO:2 LCDR2 shown in SEQ ID NO:3, and LCDR3 as shown in SEQ ID NO:4; The heavy chain variable region of antibody S1 includes the following complementarity-determining regions (CDRs): HCDR1, as shown in SEQ ID NO:9, HCDR2 shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:11; The light chain variable region of antibody S2 includes the following complementarity-determining regions (CDRs): LCDR1 shown in SEQ ID NO:16 LCDR2 shown in SEQ ID NO:17, and LCDR3 as shown in SEQ ID NO:18; The heavy chain variable region of antibody S2 includes the following complementarity-determining regions (CDRs): HCDR1, as shown in SEQ ID NO:23, HCDR2 shown in SEQ ID NO:24, and HCDR3 as shown in SEQ ID NO:

25.

5. The immunochromatographic reagent strip as described in claim 4, characterized in that, The target antigen is selected from: Mycoplasma pneumoniae, Chlamydia pneumoniae, adenovirus, influenza A virus, influenza B virus, SARS-CoV-2, and respiratory syncytial virus.

6. A test kit, characterized in that, The kit includes the immunochromatographic reagent strip as described in claim 4.

7. An antibody ensemble for monitoring the quality of immunoassay samples, said antibody ensemble being an antibody ensemble that specifically binds to the accompanying biomarker MUC5B protein, said antibody ensemble comprising antibody S1 and antibody S2, wherein, The light chain variable region of antibody S1 includes the following complementarity-determining regions (CDRs): LCDR1 shown in SEQ ID NO:2 LCDR2 shown in SEQ ID NO:3, and LCDR3 as shown in SEQ ID NO:4; The heavy chain variable region of antibody S1 includes the following complementarity-determining regions (CDRs): HCDR1, as shown in SEQ ID NO:9, HCDR2 shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:11; The light chain variable region of antibody S2 includes the following complementarity-determining regions (CDRs): LCDR1 shown in SEQ ID NO:16 LCDR2 shown in SEQ ID NO:17, and LCDR3 as shown in SEQ ID NO:18; The heavy chain variable region of antibody S2 includes the following complementarity-determining regions (CDRs): HCDR1, as shown in SEQ ID NO:23, HCDR2 shown in SEQ ID NO:24, and HCDR3 as shown in SEQ ID NO:25.

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