CN117192112A - Method for monitoring immunodetection sampling quality - Google Patents

Abstract

The application provides a method for monitoring the sampling quality of immunodetection. The present application provides methods for determining the quality of a sample by detecting the presence of MUC5B. The method of the application judges the quality of mucus or saliva samples by detecting the specific combination of the antibody and the accompanying marker MUC5B, and has the advantages of simple operation, rapid and visual detection.

Description

Method for monitoring immunodetection sampling quality

Technical Field

The present application relates to the field of biological detection. In particular, the application relates to a method of monitoring the quality of immunodetection samples.

Background

The immunodetection technology is to use the specific reaction of antigen and antibody to detect, and uses isotope, enzyme, chemiluminescence substance, etc. to amplify and display the detection signal, and is often used to detect protein, hormone, etc. trace substances. Immunodetection occupies a very important position in the fields of clinical diagnosis, animal epidemic disease, food safety and the like, and common immunodetection technologies include radioimmunoassay, lateral flow immunochromatography, enzyme-linked immunoassay, chemiluminescence, electrochemiluminescence and nano magnetic particle chemiluminescence.

The lateral flow immunochromatography can simply and rapidly detect trace substances in a sample without any complicated instrument and equipment, and can carry out qualitative judgment on food, cosmetics, medicines and the like to be released in customs clearance in clinical diseases to be diagnosed, animal epidemic diseases or production. The ELISA can quantitatively detect the sample to be detected by an ELISA reader, and can more intuitively and clearly determine the disease degree and the quality of products such as food and the like. With the development of technology, immunoassays have been applied in a wider range of fields, and more people are exposed to immunoassays in work and life. However, the immunodetection needs sampling and pretreatment of the sample before detection, and since the immunodetection is often performed on trace substances, the influence of the sampling quality and the sample treatment process on the final result is very great, which has become a difficult problem to be ignored. Also, lateral flow immunochromatography is often difficult to intuitively judge whether the quality of sampling and the quality of sample processing are good or bad at the time of detection. Also, in some cases, it may be desirable for a non-professional to perform the sampling by himself or herself, and to perform the sample processing. If the sampling quality cannot be ensured, the obtained detection result is easy to generate false negative, and the reliability of detection is reduced.

Therefore, there is a need in the art to develop a method that can simply and quickly determine the quality of an immunodetection sample.

Disclosure of Invention

The application aims to provide a method for monitoring the sampling quality of immunodetection.

In a first aspect of the application, there is provided a method of monitoring the quality of an immunoassay sample, the method comprising the steps of:

1) Contacting the sample with an antibody that specifically binds to a companion marker;

2) Detecting the binding of the sample and the antibody, thereby judging the quality of the sample.

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

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

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

a) Ubiquitous in the sample types;

b) The performance is stable during the detection process.

In another preferred embodiment, the accompanying marker is selected from the group consisting of: proteins, nucleic acids, polysaccharides, or combinations thereof.

In another preferred embodiment, the accompanying marker is Mucin (MUC).

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

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

In another preferred embodiment, the antibody is obtained by immunizing an animal with a concomitant marker as an antigen.

In another preferred embodiment, the animal is a mammal, an avian or a fish; preferably mammalian.

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

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, single B cell screens.

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

In another preferred embodiment, the antibody is a single domain antibody, nanobody, single chain antibody, diabody, or antigen binding fragment.

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

In another preferred embodiment, the antibody specifically binds to mucin, preferably MUC5B protein.

In another preferred embodiment, the antibody comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises the following complementarity determining region CDRs:

LCDR1 shown in SEQ ID NO.2,

LCDR2 as shown in SEQ ID NO 3, and

LCDR3 shown in SEQ ID NO. 4;

the heavy chain variable region comprises the following complementarity determining region CDRs:

HCDR1 shown in SEQ ID NO. 9,

HCDR2 shown in SEQ ID NO 10, and

HCDR3 shown in SEQ ID NO. 11;

or,

the light chain variable region comprises the following complementarity determining region CDRs:

LCDR1 shown in SEQ ID NO. 16,

LCDR2 as shown in SEQ ID NO 17, and

LCDR3 as shown in SEQ ID NO. 18;

the heavy chain variable region comprises the following complementarity determining region CDRs:

HCDR1 shown in SEQ ID NO. 23,

HCDR2 shown in SEQ ID NO. 24, and

HCDR3 shown in SEQ ID NO. 25.

In another preferred embodiment, the heavy chain variable region and the light chain variable region each further comprise a Framework Region (FR).

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

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

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

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

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

In another preferred embodiment, the light chain and/or heavy chain of the antibody further comprises a constant region.

In another preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence, optionally with at least one amino acid added, deleted, modified and/or substituted, that is capable of retaining the binding affinity of the MUC5B protein.

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

In another preferred embodiment, the immunoassay is an immunochromatographic assay, an enzyme-linked immunoassay, an immunomagnetic bead assay, or a chemiluminescent assay.

In another preferred embodiment, the immunoassay is lateral flow immunochromatography.

In another preferred embodiment, in step (2), the sample mass is determined according to the following criteria:

if the combination of the accompanying marker and the antibody is detected, the accompanying marker is judged to be positive, the quality of the sample sampling and processing links is good, and the immune detection result is credible;

if the combination of the accompanying marker and the antibody is not detected, the accompanying marker is judged to be negative, the quality of the sample sampling and processing links is poor, and the immune detection result is not credible.

In another preferred embodiment, the method is performed simultaneously with the immunoassay; or prior to immunoassay.

In another preferred embodiment, the method is as follows: the detection of mycoplasma pneumoniae by lateral flow immunochromatography is performed while mucin MUC5B in nasal mucus is detected by mucin antibodies, thereby monitoring the sampling quality of samples for detecting mycoplasma pneumoniae.

In a second aspect of the present application, there is provided an immunochromatographic reagent strip comprising a sample pad, a conjugate pad, a chromatographic membrane comprising a same quality control line, and a base plate,

wherein the sample quality control line is coated with an antibody S1 which specifically binds to a concomitant marker, and the binding pad contains an antibody T which specifically binds to an antigen of interest to be detected and an antibody S2 which specifically binds to a concomitant marker.

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.

In another preferred embodiment, the antibodies S1 and S2 are identical or different antibodies.

In another preferred embodiment, the light chain variable region of antibody S1 comprises the following complementarity determining regions CDRs:

LCDR1 shown in SEQ ID NO.2,

LCDR2 as shown in SEQ ID NO 3, and

LCDR3 shown in SEQ ID NO. 4;

the heavy chain variable region of antibody S1 includes the following complementarity determining regions CDRs:

HCDR1 shown in SEQ ID NO. 9,

HCDR2 shown in SEQ ID NO 10, and

HCDR3 shown in SEQ ID NO. 11.

In another preferred embodiment, the light chain variable region of antibody S2 comprises the following complementarity determining regions CDRs:

LCDR1 shown in SEQ ID NO. 16,

LCDR2 as 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 shown in SEQ ID NO. 23,

HCDR2 shown in SEQ ID NO. 24, and

HCDR3 shown in SEQ ID NO. 25.

In another preferred embodiment, the binding pad contains an antibody that is labeled, such as a colloidal gold conjugated antibody.

In another preferred embodiment, the chromatographic film further comprises a sample detection line, and the sample detection line is coated with an antibody T which specifically binds to the target antigen to be detected.

In another preferred embodiment, the antigen of interest is mycoplasma pneumoniae, chlamydia pneumoniae, adenovirus, influenza a, influenza B, neocoronavirus, syncytial virus.

In another preferred embodiment, the chromatographic film further comprises a reagent quality control line, and the reagent quality control line is coated with an antibody C which specifically binds to the antibody T or the antibody S2.

In another preferred embodiment, the immunochromatographic reagent strip further comprises a water-absorbing pad.

In a third aspect of the application there is provided a detection kit comprising an immunochromatographic kit strip according to the second aspect of the application.

In another preferred embodiment, the kit is for the detection of mycoplasma pneumoniae in a mucus or saliva sample.

In another preferred embodiment, the kit further comprises a buffer.

It is understood that within the scope of the present application, the above-described technical features of the present application and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

The following drawings are illustrative of particular embodiments of the application and are not intended to limit the scope of the application as defined by the claims.

FIG. 1 shows the expression level of MUC5B in each tissue.

FIG. 2 shows the expression level of MUC5AC in each tissue.

FIG. 3 shows the expression level of serum albumin (HSA) in each tissue.

FIG. 4 shows the expression level of lactoferrin in each tissue.

FIG. 5 shows the WB detection results of MUC5B purified products.

Fig. 6 shows the HPLC detection results of MUC5B purified products.

FIG. 7 shows the results of ELISA detection of the ability of cell supernatants transiently expressing MUC5B antibodies to bind saliva.

FIG. 8 shows a schematic structural diagram of a lateral flow immunochromatographic reagent strip of the present application.

Detailed Description

The inventor of the present application has developed a method for monitoring the quality of immunodetection samples for the first time through extensive and intensive studies. Specifically, the present application provides methods for determining sample quality by detecting the presence of MUC5B by screening, selecting MUC5B as a concomitant marker of saliva or mucus sample. The application also provides antibodies and antibody pairs that specifically target MUC5B. On this basis, the present application has been completed.

Terminology

In order to provide a clear description of the application, a number of terms are defined as follows.

Mucin 5B

As used herein, 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 gel-forming mucins. Mucin MUC5B is ubiquitous in saliva, respiratory mucus and cervical mucus in most humans.

Through screening, the application discovers that MUC5B protein can be simultaneously obtained when the parts are subjected to immunodetection sampling, and the MUC5B protein has stability and high content in a sample. Thus, the present application selects MUC5B as a concomitant marker for immunodetection.

As used herein, the terms "marker substance", "marker", "concomitant marker" all refer to substances that are prevalent and stably present in the sample to be tested, can be used to perform immunization, and are concomitant with the entire process of being detected by immunization. The accompanying markers selected by the application can be protein, nucleic acid, polysaccharide and other substances contained in the sample, can be collected together during sample collection, and cannot be completely lost during sample treatment.

Antibodies to

The present application provides antibodies that specifically target MUC5B. In one embodiment, the antibodies of the application are obtained by immunizing an animal with MUC5B as the antigen.

As used herein, the term "antigen", "immunogen" refers to a substance that is capable of eliciting an immune response in the body and producing corresponding antibodies that specifically bind thereto.

As used herein, the terms "antibody," "monoclonal antibody," "polyclonal antibody," and "polyclonal antibodies" refer to immunoglobulin molecules that are hetero-tetrameric proteins produced by binding two identical heavy chains and two identical light chains, and that perform antigen-specific binding through an antigen-binding site consisting of a light chain variable region (VL) and a heavy chain variable region (VH), thereby eliciting an antigen-specific humoral immune response.

As used herein, the terms "humanized antibody", "single domain antibody", "single chain antibody", "nanobody", "antigen binding peptide", "fragment", "derivative" and "analog" refer to an antibody or antibody fragment having antigen binding ability, and include fragments produced by cleavage of an antibody with a protein cleaving enzyme as well as fragments produced recombinantly, and examples include Fab, F (ab') 2, scFv, diabody, triabody, sdAb and VHH.

As used herein, the term "specific" is a highly specific and efficient manner of interaction, e.g., an antigen may specifically bind to a corresponding antibody but not to other antibodies.

As used herein, the term "variable region (V region)" refers to regions of immunoglobulin light and heavy chains that vary widely near the N-terminal amino acid sequence.

As used herein, the term "constant region (C-region)" refers to the region of an immunoglobulin light chain and heavy chain that is relatively stable near the C-terminal amino acid sequence.

As used herein, the term "Complementarity Determining Regions (CDRs)" refers to three highly variable regions within each of the variable regions of the immunoglobulin light and heavy chains that are spatially complementary to an epitope.

As used herein, the term "Framework Region (FR)" refers to a region of the immunoglobulin light and heavy chain that is relatively conserved in amino acid composition and arrangement order, except for CDRs.

The application also provides other proteins or fusion expression products having the antibodies of the application. In particular, the application includes those molecules having CDR regions of an antibody light or heavy chain provided by the application, so long as the CDRs have 90% or more (preferably 95% or more, most preferably 98% or more) homology with the CDRs identified by the application.

The application includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the application also includes fragments, derivatives and analogues of said antibodies. The polypeptide fragment, derivative or analogue of the application may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.

The antibody of the present application refers to a polypeptide having MUC5B protein binding activity and comprising the above CDR regions. The term also includes variants of polypeptides comprising the above-described CDR regions that have the same function as the antibodies of the application. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually within 20 to 25, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal end. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the application. The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes under high or low stringency conditions with the encoding DNA of an antibody of the application, and polypeptides or proteins obtained using antisera raised against an antibody of the application.

The antibodies of the application include conservative variants thereof. Conservative variants of an antibody of the application are those in which up to 10, preferably up to 8, more preferably up to 5, most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature, as compared to the amino acid sequence of the antibody of the application, to form a polypeptide. These conservatively mutated polypeptides are preferably produced by amino acid substitution according to the table below.

The sequences of the DNA molecules of the antibodies or fragments thereof of the present application can be obtained using conventional techniques including, but not limited to, hybridoma cells, phage display libraries, yeast display libraries, single B cell screens. The antibodies of the application may be produced by immunizing an animal with a companion marker, for example, by immunizing a mouse.

As used herein, the term "hybridoma" is a cell that is fused with myeloma cells and B lymphocytes in the preparation of monoclonal antibodies.

As used herein, the term "phage display library" is a random oligonucleotide of a specific length inserted on one side of the phage piii gene, thereby displaying random polypeptides fused to piii on the phage surface. Since each phage presents a sequence of exogenous polypeptides, phage aggregates displaying a specific length of exogenous polypeptides of various sequences form a complete phage display library.

As used herein, the term "yeast display library" is a method of expressing exogenous polypeptides displaying various sequences of a specific length on the surface of yeast, and monoclonal antibody sequences of excellent affinity can be obtained by such a method, in combination with certain screening conditions.

As used herein, the term "single B cell screening" refers to the detection of antibodies expressed by single B cells by a particular device and the screening of monoclonal antibody sequences for excellent affinity.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

As used herein, the term "vector," "expression system," or "expression vector" refers to a nucleic acid sequence containing the desired coding and control sequences in operable linkage so that a host transformed with these sequences can produce the encoded protein. To effect transformation, the expression system may be contained on a vector; however, the relevant nucleic acid molecules may subsequently also integrate into the host chromosome.

As used herein, the term "host cell" is a cell that can support replication or expression of an expression vector. The host cell may be a prokaryotic cell, such as E.coli, or a eukaryotic cell, such as a yeast cell, an insect cell, an amphibian cell, or a mammalian cell.

As used herein, the term "transfection," "stable transfection," or "transient transfection" refers to the uptake of an expression vector by a host cell, whether or not any coding sequence is actually expressed. A variety of transfection methods are known to those skilled in the art. For example, transfection is accomplished by electroporation, by using phage or viral expression vectors to insert the host cells, by mechanical insertion of nucleic acids, or even by culturing the host cells in the presence of unpackaged (unpackaged) nucleic acid fragments in the presence of the expression vectors and high concentrations of calcium phosphate. The success of transfection is generally confirmed when any indication of the vector of interest is manipulated to occur in the host cell.

Detection method

The application provides a method for detecting the accompanying markers in a sample during immunodetection, and judging whether sampling and sample processing meet the requirements or not through the yin-yang property of the accompanying markers, so that whether sampling and sample processing links of immunodetection are credible or not is detected.

The detection method is realized by the following technical means:

firstly, by analyzing the components of a sample, stable and widely existing substances in the sample are determined as accompanying markers, then the markers obtained by purification are used as immunogens for immunization, and corresponding antibodies are obtained by experimental methods such as purification, monoclonal screening and the like.

Secondly, by developing an immunodetection method, the target component is diagnosed while the accompanying marker of the sample is diagnosed, and the negative and positive properties of the marker of the sample are judged while the negative and positive properties of the target component are judged, wherein the negative represents that the sampling or sample processing process is unreasonable, the experimental result is unreliable, the positive represents that the sampling and sample processing process is reasonable, and the result is reliable.

According to the application, the sample markers are detected while the target components are detected by immunodetection, the sample markers can be interpreted while the target components are interpreted, and if the sample markers are detected to be positive, the detection of the sample has enough sample markers, so that the collection process and the treatment process of the sample are proved to be in accordance with the requirements, and the qualitative or quantitative interpretation of the target components is achieved on the basis of the detection. Otherwise, if the detection of the sample marker is negative, the detection of the sample marker is that the sample marker is not present or the sample marker is too few, the sampling or sample processing process is not satisfactory, the experimental result is not credible, and the sample needs to be sampled again or processed again for the test. The method can monitor the quality of the immune detection test sampling and sample processing process, avoid false negative, and lead the test result to be more accurate and more credible.

The method of the present application can be applied to various immunoassay methods, for example, immunoassay is immunochromatography assay, enzyme-linked immunoassay, immunomagnetic bead assay, or chemiluminescent assay.

As used herein, the term "lateral flow immunochromatography", also known as lateral flow immunoassay, is a unique immunoassay that occurs in the early 60 s of the 20 th century, in which a strip-shaped fibrous chromatographic material is used as a solid phase, and a sample is moved over the chromatographic material by adsorption by capillary tubes, wherein an analyte in the sample binds to an antibody in a certain region on the chromatographic material, and visual test results are obtained by enzymatic chromogenic reaction or direct use of a colored marker for a short period of time.

As used herein, the term "enzyme linked immunosorbent" refers to an antigen or antibody that retains its immunological activity after binding to the surface of a solid support, and retains its immunological and enzymatic activity after binding to an enzyme. The enzyme conjugate forms a complex with the corresponding antigen or antibody, and a color reaction occurs under the catalysis of a substrate, and whether the corresponding immune reaction and the amount of the antigen or antibody exist or not can be judged according to the color reaction of the enzyme substrate solution.

As used herein, the term "immunomagnetic beads" is used to synthesize a polymer surface-coated composite material containing a superparamagnetic substance by a core-shell synthesis method, wherein the composite material has good stability and can be used for post-labeling, and functional groups such as amino groups, carboxyl groups, sulfhydryl groups and the like on the surfaces of the composite material are used for covalent or non-covalent coupling of antibodies, so that the composite material can be used for binding corresponding antigens or antibodies, and can be directionally moved under the attraction of an external magnetic field, thereby achieving the purposes of separating, detecting and purifying genes, proteins, cells, microorganisms and the like.

As used herein, the term "chemiluminescence" is a type of molecular luminescence spectrometry that is a trace analysis method for determining the content of an analyte by detecting the chemiluminescent intensity of a system with an instrument, mainly based on the principle that the concentration of the analyte in the chemiluminescent detection system and the chemiluminescent intensity of the system are in a linear quantitative relationship under certain conditions.

As used herein, the term "negative" (-) in a medical examination generally indicates that the indicator is normal or pathogen is absent and "positive" (+) indicates that a disease or pathogen (e.g., virus) is present. In medicine, the terms negative and positive refer broadly to the presence or absence, or are used to denote the outcome of an examination.

In one embodiment, the method of the application may be applied to the detection of mycoplasma pneumoniae by lateral flow immunochromatography. While detecting mycoplasma pneumoniae, mucin MUC5B in nasal mucus was detected by mucin antibodies to monitor sampling quality.

The main advantages of the application include:

1) The application selects MUC5B as a concomitant marker for pathogen detection. MUC5B is commonly found in oral cavity, respiratory tract and cervical mucus, secretion amount is large, individual difference is small, and sampling is performed according to standard procedure at the above parts, so that the sample can be ensured to contain MUC5B. Meanwhile, the MUC5B protein has high stability and is not easy to lose in the sample treatment process, and enough MUC5B can be ensured to react with MUC5B antibody during detection.

2) The application provides an antibody and an antibody pair of a specific target MUC5B, wherein the antibody pair can maximize the color development effect of MUC5B detection when being used in combination in lateral flow chromatography detection, does not generate cross reaction with mycoplasma pneumoniae antibody, and has high sensitivity and high specificity.

The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Example 1 concomitant marker screening

Saliva contains multiple proteins, mainly glycoprotein and mucin, and also contains immunoglobulin, lactoferrin, inorganic salt and sialidase. Proteins in secretions in the airways are mainly mucins and immunoglobulins. Mucins include secreted polymeric mucins MUC5AC and MUC5B, and cell membrane-bound mucins MUC1, MUC4, MUC16 and MUC20, immunoglobulins including IgG, igM and IgA. The main components in cervical mucus comprise mucin, glycoprotein and plasma protein.

Among the above candidate concomitant markers, the inventors focused on screening for secreted mucins MUC5AC, MUC5B, serum albumin and lactoferrin. The results are shown in FIGS. 1-4.

By searching and comparing in the human protein map, MUC5AC was found to be more expressed in the nasopharynx and not in the salivary glands, imposing a limit on the applicable samples (FIG. 2). Furthermore, the large difference in the expression level of MUC5AC in healthy humans and in patients may interfere with the accuracy of the concomitant detection.

Serum albumin is mainly present in blood and kidney, is also ubiquitous in extracellular matrix of other tissues, and has no site specificity (fig. 3). Lactoferrin was expressed in bronchi and salivary glands, not in nasopharyngeal sites, and was present in a low amount (fig. 4). Both are likely to misjudge the quality of the sample due to the excessively low content of the accompanying marker when sampling the nasal cavity/the pharynx, and are not suitable for nasal cavity/pharynx sampling detection.

Through screening, MUC5B was found to be expressed in salivary glands, nasopharynx in a large amount and stable in expression (FIG. 1). Thus, MUC5B was selected as the accompanying marker.

Example 2 preparation of mucin MUC5B

The present example provides a method for separating and purifying MUC5B from saliva:

1.200mL of saliva was obtained from 8 healthy adults (aged 25-34 years, oral health, no systemic disease, no antibacterial agent taken within 3 months). The saliva was fasted and rinsed 2 hours before collection. The collected fresh saliva was placed in an ice bath and 0.274mol/L cetyltrimethylammonium bromide was immediately added, and the glass rod was gently stirred until a clot formed thereon, at which time the MUC5B protein precipitate accumulated on the glass rod. The clot was transferred to another flask and 3mol/L NaC 1ml was added to dissolve the clot. The solution is MUC5B crude extract.

2. The MUC5B crude extract was dialyzed against a 14K dialysis bag for 48h with 0.01M PBS as dialysis buffer, and the dialysis buffer was changed every 12 h.

3. Gel filtration chromatography was performed to purify MUC5B, and after resuspension of the superdex 200 packing with pure water, the column was packed (16X 500 mm) with a packing height of 400mm, the column was equilibrated with 0.01M PBS having pH=7.0 for 2h, and the flow rate was controlled at 1mL/min. MUC5B was concentrated to 3mg/mL using ultrafiltration tube, then loaded with 2mL volumes each at a flow rate of 1mL/min. The eluted product was collected using an automatic collector, the collected products were pooled multiple times, and the concentration was adjusted to 1mg/mL.

4. The purified MUC5B was identified by WB and HPLC.

FIG. 5 shows purified MUC5B products of WB assay, wherein the gray region represents the binding to MUC5B antibody, indicating the presence of MUC5B protein. FIG. 6 shows HPLC analysis of MUC5B purified product, wherein both peaks at 8.3min and 8.921min are MUC5B, corresponding to two bands in WB.

Example 3 method of screening anti-MUC 5B monoclonal antibodies by Single B cells

(1) Four mice were immunized multiple times using purified MUC5B as immunogen.

(2) Trace tail blood is collected, serum titer is determined by indirect ELISA, and immunization is finished when the serum titer reaches more than 10 ten thousand.

(3) One of the mice with the highest serum titer was selected, sacrificed, spleen was taken, and crushed to obtain a cell suspension.

(4) The flow cytometry sorts B cells expressing the antibody, lyses the cells, and extracts the light and heavy chain genes of the antibody.

(5) The antibody gene is constructed on plasmid and transiently expressed.

(6) The expressed antibodies were collected for subsequent screening.

Example 4 detection of antibody binding to saliva samples

Saliva was coated by an ELISA plate, and the supernatant of cells transiently expressing the antibody was detected by ELISA, and 8 positive monoclonal antibodies were selected. The specific implementation mode is as follows:

(1) Saliva was collected as antigen and diluted ten times with 0.05M sodium carbonate buffer ph=9.0, then added to 96-well elisa plates and incubated at 4 ℃ overnight per well 100 ul;

(2) Pouring out the incubated antigen, adding 5% skimmed milk powder as a blocking solution, and blocking at 37 ℃ for 3 hours at 250uL per well;

(3) Pouring off the sealing liquid, adding 200uL of PBST, washing for three times, and beating to dryness;

(4) Four-time gradient dilution is carried out on the cell supernatant containing the antibody for 4 times, 16 times, 64 times, 256 times, 1024 times, 4096 times, 16384 times and 65536 times, the cell supernatant containing the antibody is respectively added into the ELISA plate, 100uL of each hole is incubated for 1 hour at 37 ℃, after the incubation is finished, 200uLPBST is added, and the cell supernatant is washed for three times and is patted dry;

(5) Adding goat anti-human HRP secondary antibody diluted with 3000 times of PBST, incubating for 1 hour at 37 ℃, adding 200uLPBST after incubation is finished, washing for three times, and drying by beating;

(6) 50uL of TMB color development solution is added to each hole, the mixture is placed in a dark place, incubated for 5 minutes, and 50uL of stop solution (0.2M sulfuric acid) is added to stop the reaction;

the results are shown in FIG. 7, which are read with a microplate reader at 450nm in 30 minutes. Eight positive monoclonal antibodies 1M2B3, 1M2C4, 1M2C5, 1M2F5, 1M2G6, 1M2B7, 1M2C10, 1M2F12 were selected according to the results.

Example 5MUC5B detection antibody pair screening

The side-stream immunochromatography is used for screening the MUC5B antibody pair which can be paired for the immunological detection of the antibody sandwich method from eight MUC5B antibodies, and is applied to a colloidal gold test strip for the immunological diagnosis of mycoplasma pneumoniae. The specific implementation mode is as follows:

1. gold labelling of antibodies

1) Nine portions of 10ml gold solution are respectively measured, 210uL of 0.5M potassium carbonate is added to adjust the PH, 90ug of eight mucin antibody or mycoplasma pneumoniae antibody solution is added dropwise, and stirring is carried out for 30 minutes.

2) The mixed solution was taken in 10% BSA, added dropwise to a final concentration of 0.5%, and stirred for 15 minutes.

3) 10% PEG (20,000) was taken, the mixed solution was added dropwise to a final concentration of 0.1% and stirred for 15 minutes.

4) Centrifuging at 8000-9000RPM and 4 ℃ for 10min

5) The supernatant was discarded, and then the diluted solution was added to resuspend the gold particles, and the volume was adjusted to 1mL.

2. Pretreatment of colloidal gold pad and sample pad

1) The number of treatments, A4 sheets (or cut to other specifications), was determined and placed one stack of 10 sheets each.

2) Buffer (about 30ml/A4 paper size) was provided. According to the thickness and the performance of the material, the formula of the treatment fluid is adjusted, and the basic elements are as follows: buffer + NaCl + others.

3) Soaking the materials in buffer solution for 10min, taking out, dehydrating for 3min, and horizontally placing on a screen.

4) Placing into a 37 ℃ drying oven (humidity is less than 20%), and drying until the humidity is constant at about 20%.

5) And (3) placing the dried material into an aluminum foil bag or a plastic bag for sealing and storing, labeling and recording batch numbers, wherein the treated glass fiber cannot be exposed to the environment with high humidity for a long time.

3. Sample application

1. Spraying the colloidal gold to the colloidal gold pad

1) Diluting the labeled colloidal gold to 36D/mL, and mixing the mycoplasma pneumoniae labeled colloidal gold and the eight mucin antibody labeled colloidal gold respectively, and adding (final concentration) 10-20% sucrose, 1-3% BSA,0.5-1% NaCl, and 0.5-1% surfactant.

2) And spraying the solution onto the colloidal gold pad by using an AIRJET spray head of a metal spraying and film drawing instrument under the conditions of humidity of 20-40% and room temperature, wherein the pressure is set to be 10PSI, and the spray point parameter is 7ul/cm.

3) And (3) drying the sprayed colloidal gold strips at 37 ℃, and after the humidity is constant at 20%, packing and sealing for storage.

4. Antibody scratch film

1) The NC film was removed and equilibrated at room temperature and normal humidity for 1 hour.

2) And (5) adhering the NC film non-sample application surface to the PVC bottom plate.

3) Diluting eight mucin antibodies, mycoplasma pneumoniae antibodies and goat anti-human Fc antibodies to 1mg/ml with a buffer solution; pH range: 7.0,

4) And (3) setting the film dividing amount to be 0.5ul/cm by using a metal spraying film dividing instrument, dividing films, dividing three lines on each film, wherein the three lines are respectively mycoplasma pneumoniae and one of eight mucin antibodies from bottom to top, and are goat anti-human Fc antibodies.

5) Oven drying at 37deg.C for 2 hr to humidity below 20%, and sealing and packaging.

5. Test paper strip assembly, cutting and packaging (environmental requirement: room temperature, 20% humidity below)

1) The colloidal gold pads spotted with different mucin antibodies and NC films were combined two by two.

2) The colloidal gold pad was stuck under the NC film and covered with the NC film by 1mm.

3) The water absorbing pad is stuck above the NC film and covers the NC film by 2mm.

4) The sample pad is adhered below the colloidal gold pad and covers the colloidal gold pad by 2mm.

5) The stuck test plate was cut into strips 4mm wide in a slitter.

6) Packaging, packaging with aluminum foil bag together with desiccant, sealing, and labeling.

6. Testing

1) The positive test sample is a pharyngeal mucus sample collected with a pharyngeal swab and the sample is treated with a sample lysate. The negative test sample was an unused pharyngeal swab and was treated with sample lysate.

2) 2 colloidal gold test strips of each combination were prepared, and 1 positive sample and 1 negative sample were respectively loaded, and each sample was loaded with 50ul.

3) After standing for 5 minutes, observing an experimental result, comparing the experimental result with a colorimetric card, scoring from deep to light according to the color, and sequentially: G10-G0.

4) By comparison, the colloidal gold marked by the antibodies 5, 7 and 8 has cross reaction with the mycoplasma pneumoniae antibody, so that the antibodies can not be selected as the marked pairing. After the above conditions are eliminated, the pairing numbers 1+2, 1+4 and 2+4 are better, wherein the pairing numbers are further optimized by 1+4.

TABLE 1 mucin antibody information

TABLE 2 mucin antibody pairing assay results

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The sequences involved in the application are shown in the following table:

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all documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (10)

1. A method of monitoring the quality of an immunoassay sample, the method comprising the steps of:

1) Contacting the sample with an antibody that specifically binds to a companion marker;

2) Detecting the binding of the sample and the antibody, thereby judging the quality of the sample.

2. The method of claim 1, wherein the accompanying marker is Mucin (MUC), preferably MUC5B protein.

3. The method of claim 1, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises the following complementarity determining region CDRs:

LCDR1 shown in SEQ ID NO.2,

LCDR2 as shown in SEQ ID NO 3, and

LCDR3 shown in SEQ ID NO. 4;

the heavy chain variable region comprises the following complementarity determining region CDRs:

HCDR1 shown in SEQ ID NO. 9,

HCDR2 shown in SEQ ID NO 10, and

HCDR3 shown in SEQ ID NO. 11;

or,

the light chain variable region comprises the following complementarity determining region CDRs:

LCDR1 shown in SEQ ID NO. 16,

LCDR2 as shown in SEQ ID NO 17, and

LCDR3 as shown in SEQ ID NO. 18;

the heavy chain variable region comprises the following complementarity determining region CDRs:

HCDR1 shown in SEQ ID NO. 23,

HCDR2 shown in SEQ ID NO. 24, and

HCDR3 shown in SEQ ID NO. 25.

4. The method of claim 1, wherein the immunoassay is an immunochromatographic assay, an enzyme-linked immunoassay, an immunomagnetic bead assay, or a chemiluminescent assay.

5. The method of claim 1, wherein in step (2), the sample mass is determined according to the following criteria:

if the combination of the accompanying marker and the antibody is detected, the accompanying marker is judged to be positive, the quality of the sample sampling and processing links is good, and the immune detection result is credible;

if the combination of the accompanying marker and the antibody is not detected, the accompanying marker is judged to be negative, the quality of the sample sampling and processing links is poor, and the immune detection result is not credible.

6. An immunochromatographic reagent strip, which is characterized by comprising a sample pad, a binding pad, a chromatographic film and a bottom plate, wherein the chromatographic film comprises a same quality control line,

wherein the sample quality control line is coated with an antibody S1 which specifically binds to a concomitant marker, and the binding pad contains an antibody T which specifically binds to an antigen of interest to be detected and an antibody S2 which specifically binds to a concomitant marker.

7. The immunochromatographic strip according to claim 6, which is used for mucus or saliva detection, and the accompanying marker is Mucin (MUC), preferably MUC5B protein.

8. The immunochromatographic reagent strip as defined in claim 6, wherein the light chain variable region of the antibody S1 comprises the following complementarity determining regions CDRs:

LCDR1 shown in SEQ ID NO.2,

LCDR2 as shown in SEQ ID NO 3, and

LCDR3 shown in SEQ ID NO. 4;

the heavy chain variable region of antibody S1 includes the following complementarity determining regions CDRs:

HCDR1 shown in SEQ ID NO. 9,

HCDR2 shown in SEQ ID NO 10, and

HCDR3 shown in SEQ ID NO. 11;

the light chain variable region of antibody S2 comprises the following complementarity determining regions CDRs:

LCDR1 shown in SEQ ID NO. 16,

LCDR2 as 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 shown in SEQ ID NO. 23,

HCDR2 shown in SEQ ID NO. 24, and

HCDR3 shown in SEQ ID NO. 25.

9. The immunochromatographic strip as defined in claim 6, wherein the antigen of interest is mycoplasma pneumoniae, chlamydia pneumoniae, adenovirus, influenza a virus, influenza B virus, neocoronavirus, syncytial virus.

10. A test kit comprising the immunochromatographic strip of claim 6.