KR20230078558A - Composition including avidin-expressing microorganisms for improving the diagnostic accuracy in immunoassay - Google Patents

Abstract

The present invention relates to a composition for avidin-biotin interaction-mediated immunoassay containing a microorganism expressing avidin as an active ingredient. The present invention is based on the strong interaction of avidin-biotin, and by expressing and immobilizing avidin on the surface of the microorganisms to reduce free biotin in the blood, can be usefully used as an efficient immunoassay method that significantly reduces the possibility of false negatives or false positives in immunoassay tests and greatly improves test accuracy.

Description

Composition including avidin-expressing microorganisms for improving the diagnostic accuracy in immunoassay}

The present invention relates to a composition for improving the diagnostic performance of an immunoassay comprising an avidin-expressing microorganism.

Since the development of biomarkers is for early diagnosis of diseases and detection of pathogenic microorganisms, they are closely related to public health. Therefore, for early diagnosis of disease and detection of microorganisms, biomarker materials using organic or inorganic small molecule compounds, aptamers, peptides, antibodies, and the like are being developed. In order to detect a specific substance, only the target substance to be detected must be specifically detected and must not be reactive with other substances. These characteristics of antibodies are already used in various fields of the bioindustry. For example, detection of a specific protein in serum is used for diagnosis of a disease, and for this purpose, an antibody binding to the specific protein is used. In addition, in order to detect a specific virus or microorganism, the presence or absence of a pathogenic microorganism can be determined using an antibody that specifically binds to a protein possessed by the virus or microorganism.

Currently used intermolecular binding (interaction) or enzyme activity analysis can be largely divided into heterogeneous methods and homogeneous methods. Representative examples of the heterogeneity method include enzyme linked immunosorbent assay (ELISA), microarray, surface plasmon resonance (SPR), etc., and a representative example of the homogeneity method is fluorescence resonance energy transfer (fluorescence). Technologies such as resonance energy transfer (FRT) and fluorescence polarization (FP) exist. ELISA is a method of confirming an antigen-antibody reaction by binding an enzyme to an antibody, and is one of the most widely used antigen-antibody assays because the method is simple, inexpensive, and a large amount of analysis is possible. Although this is a very sensitive reaction like a radioimmunoassay (RIA), its use is increasing because it has the advantage of not using radioactivity as in the radioimmunoassay.

Among the enzymes that bind to the antibody, a color reaction is used when a simple substrate solution is added. Representative enzymes such as alkaline phosphatase and horseradish peroxidase are widely used. These enzymes are covalently linked to the Fc region of the antibody molecule by a chemical reaction. In this case, a process of displaying a signal when a substance is detected using the antigen-antibody reaction is required. For example, there is a method of detecting a change in a substrate when the substrate is treated by conjugating an antibody with an enzyme, and another method is a method of conjugating a fluorescent substance to an antibody and detecting the change with a laser. Currently, biotin and avidin, which are mainly used, exhibit excellent stability due to their specific binding. However, even if avidin is used, there is a limit to detection when the target substance is small, and above all, when free biotin introduced into the human body is present in a large amount in the blood, a diagnostic kit using avidin-biotin interaction There is a problem in that there is a possibility of false positive or false negative due to influence. Therefore, when detection is to be performed with only a small amount of the target material, a method for more effectively amplifying the detection signal and controlling the effect of free biotin present in large amounts in the blood are required.

The present inventors have made intensive research efforts to develop a reliable immunoassay method for clear diagnosis of disease and detection of pathogenic microorganisms. As a result, in order to overcome the limitations of the immunoassay method, microorganisms expressing avidin on the surface are introduced into the sample to reduce free biotin in the sample, thereby significantly reducing the possibility of false negative or false positive in the immunoassay test and improving test accuracy. Significant improvements have brought the invention to completion.

Accordingly, an object of the present invention is to provide a composition for improving the diagnostic performance of an immunoassay comprising an avidin-expressing microorganism.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, numerous specific details are set forth, such as specific forms, compositions and processes, etc., in order to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well known processes and manufacturing techniques have not been described in specific detail in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, the appearances of "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, particular features, forms, compositions, or properties may be combined in one or more embodiments in any suitable way.

Unless otherwise defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention belongs.

Throughout the specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.

According to one aspect of the present invention, the present invention provides a composition for an avidin-biotin interaction mediated immunoassay comprising a microorganism expressing avidin as an active ingredient.

The present inventors have made intensive research efforts to develop a reliable immunoassay method for clear diagnosis of disease and detection of pathogenic microorganisms. As a result, in order to overcome the limitations of the immunoassay method, microorganisms expressing avidin on the surface are introduced into the sample to reduce free biotin in the sample, thereby significantly reducing the possibility of false negative or false positive in the immunoassay test and improving test accuracy. Significant improvements have brought the invention to completion.

10-15 M으로 측정되어 가장 강력한 비공유 결합으로 알려져 있다.As used herein, the term "avidin" refers to a tetrameric biotin binding protein produced in the fallopian tubes of birds, reptiles and amphibians and deposited in egg whites, and dimeric members of the avidin family are also found in some bacteria. In egg white, avidin constitutes approximately 0.05% of the total protein (approximately 1800 μg per egg), and the tetrameric protein contains four identical subunits (homotetramers), each containing biotin (vitamin) with high affinity and specificity. B7, vitamin H). The dissociation constant of the avidin-biotin complex is KD

It is known to be the strongest non-covalent bond, measured at 10-15 M.

On the other hand, "Streptavidin" is a thalicosylated avidin derivative that binds to biotin with high affinity like avidin, and has almost the same secondary, tertiary, and quaternary structures as avidin. Accordingly, since avidin and streptavidin may be used interchangeably with each other, the term "avidin" in the present specification is meant to include "streptavidin".

In the present specification, the term "biotin" refers to biotin, which is also called vitamin B7, is one of the B vitamins, has an alias of vitamin H, and is also called coenzyme R. It is involved in a wide range of metabolic processes both in humans and other organisms, primarily involving the utilization of fats, carbohydrates and amino acids.

According to a specific embodiment of the present invention, the microorganism is Streptomyces aureorectus, Streptomyces griseus, Streptomyces aureus, Streptomyces avermitilis, It is selected from the group consisting of Streptomyces avicenniae, Streptomyces avidinii, and Escherichia coli.

As used herein, the term "microorganism or microbe" is a microscopic organism that can exist in the form of a single cell or a colony of cells. The scientific study of microbes began with microscopic observation by Anton van Leewenhoek in the 1670s, and in the 1880s, Robert Koch discovered that the microbes cause tuberculosis, cholera, diphtheria, and anthrax. Microorganisms can be very diverse as they include most unicellular organisms in all three domains of life. Two of the three domains contain only archaea and bacteria, and the third domain, eukaryotes, includes all multicellular organisms as well as many single-celled protozoa, which are microorganisms.

As used herein, the term "microorganism expressing avidin" is meant to include microorganisms that endogenously express avidin as well as microorganisms transduced by inserting an avidin-encoding nucleic acid molecule into a gene carrier to express avidin. . More specifically, the microorganism of the present invention is a microorganism transduced with an avidin-encoding gene.

As used herein, the term "gene delivery system" refers to a medium for introducing and expressing a desired target gene into a target cell. An ideal gene delivery vehicle should be able to deliver genes efficiently and easily in mass production without distorting the sample.

In this specification, the term "gene transfer" means the transfer of a gene into a cell or microorganism, and has the same meaning as transduction of a gene into a cell. At the tissue or individual level, the term gene transfer has the same meaning as the spread of a gene. Accordingly, the gene delivery system of the present invention can be described as a gene penetration system and a gene diffusion system.

According to a specific embodiment of the present invention, the immunoassay is performed by enzyme immunoassay (EIA), fluorescent immunoassay (FIA), chemiluminescent immunoassay (CLIA), and radioimmunoassay (RIA). selected from the group consisting of

As used herein, the term "enzyme immunoassay (EIA)" refers to a rapid enzyme immunochemical method for determining the presence of antigens, antibodies or haptens in the blood, wherein the antigens or antibodies are bound to enzymes. A target molecule can bind and enzymatically highlight a specific immunological target in a bodily fluid sample. Enzyme-linked immunosorbent assay (ELISA), formerly known as enzyme-linked immunosorbent assay (ELISA), is one of the primary diagnostic tests for many infectious diseases, including Treponema pallidum (spirochetes that cause syphilis) and human immunodeficiency virus (HIV). used

In the present specification, the term "fluorescent immunoassay (FIA)" includes a method of attaching a first antibody to an antigenic determinant of a specific protein in immunocytochemistry and then attaching a second antibody that attaches to the first antibody. Since fluorescence is attached to the end of the second antibody, it can be confirmed that a specific protein is stained with fluorescence within the cell when observed through a microscope. When several proteins are attached to each other, immunofluorescence staining using different antibodies and fluorescent colors for these proteins can be performed to determine where these proteins are attached to each other.

As used herein, the term "chemiluminescent immunoassay (CLIA)" refers to an immunoassay technique in which a label, ie, a true indicator of an assay response, is a luminescent molecule. In general, luminescence is the emission of visible or near-radiation (rays = 300-800 nm) radiation that occurs when electrons transition from an excited state to a ground state. The resulting potential energy of an atom is released in the form of light, and in spectrophotometry, luminescence has an advantage over absorbance in that the former is an absolute measurement, whereas the latter is a relative one.

In the present specification, the term "radioimmunoassay (RIA)" refers to an immunoassay technique that minutely measures the concentration of a substance using a radioactive isotope. Radioimmunoassay is a highly sensitive in vitro test technique used to measure the concentration of a substance, and uses antibodies to measure the concentration of an antigen (eg, hormone levels in the blood). Almost all kinds of hormones including insulin, active substances, enzymes, vitamins, drugs, etc. Radioimmunoassay (RIA) is a RIA (competition reaction) that labels an antigen reagent with a radioisotope and an immunoradiometric assay (Immunoradiometric assay; IRMA). Immunoradiometric assay (IRMA) is essentially an excess reagent assay in which an excess concentration of radiolabeled antibody is used as a reagent. Excess labeled antibody and antigen (standard or sample) are allowed to react, and at the end of the assay, antigen-bound and free antibodies are separated and the antigen-bound fraction is assayed for radioactivity.

According to a specific embodiment of the present invention, the microorganism is a composition characterized in that it is included in 0.1 x 10 ⁶ to 1.0 x 10 ¹⁰ CFU / 100μl in the sample to be analyzed. Specifically, 0.1 x 10 ⁶ to 1.0 x 10 ⁹ CFU/100 μl, 0.1 x 10 ⁶ to 2.0 x 10 ⁹ CFU/100 μl, 0.1 x 10 ⁶ to 4.0 x 10 ⁹ CFU/100 μl, 0.1 x 10 ⁶ to 6.0 x 10 ⁹ CFU/100 μl, 0.1 x 10 ⁶ to 8.0 x 10 ⁹ CFU/100 μl, 0.1 x 10 ⁶ to 9.0 x 10 ⁹ CFU/100 μl, 0.1 x 10 ⁶ to 1.0 x 10 ⁸ CFU/100 μl, 0.1 x 10 ⁶ to 2.0 x 10 ⁸ CFU/100μl, 0.1 x 10 ⁶ to 4.0 x 10 ⁸ CFU/100μl, 0.1 x 10 ⁶ to 6.0 x 10 ⁸ CFU/100μl, 0.1 x 10 ⁶ to 8.0 x 10 ⁸ CFU/100μl, 0.1 x 10 ⁶ to 1.0 x 10 ⁷ CFU/100μl, 0.1 x 10 ⁶ to 2.0 x 10 ⁷ CFU/100μl, 0.1 x 10 ⁶ to 4.0 x 10 7 CFU/100μl, 0.1 x 10 ^{6 to} 6.0 x 10 ⁷ CFU/100μl, 0.1 x 10 ⁶ to 8.0 x 10 ⁷ CFU/100μl, 0.1 x 10 ⁶ to 1.0 x 10 ⁶ CFU/100μl, 0.1 x 10 ⁶ to 2.0 x 10 ⁶ CFU/100μl, 0.1 x 10 ⁶ to 4.0 x 10 ⁶ CFU/100μl, 0.1 x 10 ⁶ to 6.0 x 10 ⁶ CFU/100 μl, or 0.1 x 10 ⁶ to 8.0 x 10 ⁶ CFU/100 μl, more specifically 0.1 x 10 ⁶ to 1.0 x 10 ¹⁰ CFU/100 μl, 1 x 10 6 to 1 x 10 ⁶ CFU/100 μl 1.0 x 10 ¹⁰ CFU/100μl, 2 x 10 ⁶ to 1.0 x 10 ¹⁰ CFU/100μl, 4 x 10 ⁶ to 1.0 x 10 ¹⁰ CFU/100μl, 6 x 10 ⁶ to 1.0 x 10 ¹⁰ CFU/100μl, 8 x 10 ⁶ to 1.0 x 10 ¹⁰ CFU/100μl, 1 x 10 ⁷ to 1.0 x 10 ¹⁰ CFU/100μl, 2 x 10 ⁷ to 1.0 x 10 ¹⁰ CFU/100μl, 4 x 10 ⁷ to 1.0 x 10 ¹⁰ CFU/100μl, 6 x 10 ⁷ to 1.0 x 10 ¹⁰ CFU/100μl, 8 x 10 ⁷ to 1.0 x 10 ¹⁰ CFU/100μl, 0.1 x 10 ⁸ to 1.0 x 10 ¹⁰ CFU/100μl, 0.2 x 10 ⁸ to 1.0 x 10 ¹⁰ CFU/100μl , 0.4 x 10 ⁸ to 1.0 x 10 ¹⁰ CFU ^/ 100μl, 0.6 x 10 ⁸ to 1.0 x 10 ¹⁰ CFU/100μl, 0.8 x 10 ⁸ to 1.0 x 10 10 CFU/100μl, 1 x 10 ⁸ to 1.0 x 10 ¹⁰ CFU /100μl, 2 x 10 ⁸ to 1.0 x 10 ¹⁰ CFU/100μl, 4 x 10 ⁸ to 1.0 x 10 ¹⁰ CFU/100μl, 6 x 10 ⁸ to 1.0 x 10 10 CFU/100μl, 8 x ^{10 8} to 1.0 x 10 ¹⁰ CFU/100 μl, 1 x 10 ⁹ to 1.0 x 10 ¹⁰ CFU/100 μl, 2 x 10 ⁹ to 1.0 x 10 ¹⁰ CFU/100 μl, 4 x 10 ⁹ to 1.0 x 10 ¹⁰ CFU/100 μl, 6 x 10 ⁹ to 1.0 x 10 ¹⁰ CFU/100 μl, or 8 x 10 ⁹ to 1.0 x 10 ¹⁰ CFU/100 μl, more specifically 0.5 x 10 ⁶ to 1.0 x 10 ⁹ CFU/100 μl, 0.5 x 10 ⁶ to 2.0 x 10 ⁹ CFU /100μl, 0.5 x 10 ⁶ to 4.0 x 10 ⁹ CFU/100μl, 0.5 x 10 ⁶ to 6.0 x 10 ⁹ CFU/100μl, 0.5 x 10 ⁶ to 8.0 x 10 ⁹ CFU/100μl, 0.5 x 10 ⁶ to 1.0 x 10 ⁸ CFU/100 μl, 0.5 x 10 ⁶ to 2.0 x 10 ⁸ CFU/100 μl, 0.5 x 10 ⁶ to 4.0 x 10 ⁸ CFU/100 μl, 0.5 x 10 ⁶ to 6.0 x 10 ⁸ CFU/100 μl, 0.5 x 10 ⁶ to 8.0 x 10 ⁸ CFU/100μl, 0.5 x 10 ⁶ to 1.0 x 10 ⁷ CFU/100μl, 0.5 x 10 ⁶ to 2.0 x 10 ⁷ CFU/100μl, 0.5 x 10 ⁶ to 4.0 x 10 ⁷ CFU/100μl, 0.5 x 10 ⁶ to 6.0 x 10 ⁷ CFU/100μl, 0.5 x 10 ⁶ to 8.0 x 10 ⁷ CFU/100μl, 0.8 x 10 ⁶ to 1.0 x ^{10 9} CFU/100μl, 0.8 x 10 ⁶ to 2.0 x 10 ⁹ CFU/100μl, 0.8 x 10 ⁶ to 4.0 x 10 ⁹ CFU/100 μl, 0.8 x 10 ⁶ to 6.0 x 10 ⁹ CFU/100 μl, 0.8 x 10 ⁶ to 8.0 x 10 ⁹ CFU/100 μl, 0.8 x 10 ⁶ to 1.0 x 10 ⁸ CFU/100 μl, 0.8 x 10 ⁶ to 1.0 x 10 ⁷ CFU/100 μl, 0.8 x 10 ⁶ to 2.0 x 10 ⁷ CFU/100 μl, 0.8 x 10 ⁶ to 4.0 x 10 ⁷ CFU/100 μl, 0.8 x 10 ⁶ to 6.0 x 10 ⁷ CFU/ 100 μl, 0.8 x 10 ⁶ to 8.0 x 10 ⁷ CFU/100 μl, 0.8 x 10 ⁶ to 1.0 x 10 ⁶ CFU/100 μl, 0.8 x 10 ⁶ to 2.0 x 10 ⁶ CFU/100 μl, 0.8 x 10 ⁶ to 4.0 x 10 ⁶ CFU/100 μl, 0.8 x 10 ⁶ to 6.0 x 10 ⁶ CFU/100 μl, or 0.8 x 10 ⁶ to 8.0 x 10 ⁶ CFU/100 μl, but is not limited thereto.

According to another aspect of the present invention, the present invention provides a target protein detection kit including a composition for immunoassay mediated by avidin-biotin interaction.

According to another aspect of the present invention, the present invention provides an avidin-biotin interaction mediated immunoassay method comprising the following steps:

(a) adding a microorganism expressing avidin in a biological sample obtained from a subject; and

(b) adding a biotinylated antibody that specifically recognizes the protein to be analyzed.

Since the immunoassay method and the avidin-expressing microorganism used in the present invention have already been described in detail, their descriptions are omitted to avoid excessive redundancy.

According to the present invention, it can be detected according to the immunoassay method using avidin-biotin of the present invention and used to analyze the concentration of a protein of a subject. Such an immunoassay can be performed according to various immunoassay or immunostaining protocols previously developed.

For example, when the method of the present invention is performed according to the radioimmunoassay method, antibodies labeled with radioactive isotopes (eg, C14, I125, P32 and S35) may be used. In the present invention, a biotinylated antibody is a polyclonal or monoclonal antibody, specifically a monoclonal antibody.

By analyzing the intensity of the final concentration by the above-described immunoassay process, the presence and concentration of the target protein in the human body can be predicted. The present invention significantly improved the reliability of conventional immunoassays using avidin-biotin interactions by reducing free biotin in the sample by introducing a microorganism expressing avidin on the surface into the sample.

According to a specific embodiment of the present invention, the sample is derived from a human body.

According to a specific embodiment of the present invention, the human-derived product is whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum ( Serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva saliva), peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions and cerebrospinal fluid ) is selected from the group consisting of

In the present invention, the term "sample" is any sample obtained from a mammal, including a human, containing a target protein or a cell expressing the target protein, and includes, but is not limited to, tissue, organ, blood, cell or cell culture medium.

According to the present invention, the immunoassay using the avidin-biotin interaction can be used to detect and purify a specific protein.

According to the present invention, the immunoassay using the avidin-biotin interaction is a thyroid function marker, hormone, tumor marker, cardiac marker, infection marker, nutritional marker, drug concentration, pregnancy-related markers, and other various proteins derived from the human body. can be used to analyze the concentration

According to the present invention, cytokeratin 19 (Cytokeratin 19) refers to an acidic cytoplasmic protein with a molecular weight of 40000 Da as a member of more than 20 diverse cytokeratin polypeptide families that form the intermediate filament structure of epithelial cells. Cytokeratin filaments are poorly soluble, but soluble fragments are formed after proteolysis and are released into bodily fluids.

According to the present invention, CYFRA 21.1 IRMA refers to an immunoassay method for determining the concentration of Cytokeratin 19 fragment in serum. The decision is based on the use of two monoclonal antibodies, BM 19.21 and KS 19.1, which recognize two different epitopes in the C-terminal helix region of the molecule. CYFRA 21.1 is helpful in monitoring disease progression, treatment efficiency, early detection of recurrence, and long-term follow-up of patients in patients with various solid cancers.

According to the present invention, CA19-9 is a glycoprotein antigen attached to O-glycans on the cell surface and is a tumor marker used for diagnosis, treatment response evaluation, and recurrence of various solid cancers such as digestive cancer and hepatobiliary cancer. CA19-9 IRMA recognizes a specific epitope of CA19-9 in serum.

According to the present invention, a standard sample refers to a material used as a standard for measuring the characteristics or calibration values of a certain material, and is also expressed as a standard material. Specifically, it may be CYFRA 21.1 or CA19.9, but is not limited thereto.

According to the present invention, the avidin-biotin interaction showing improved diagnostic performance uses an avidin-biotin assay that can affect biotin. Then, a standard sample whose concentration is already known is secured and the concentration is measured. Next, biotin is added to the standard sample to determine if the concentration of the sample is underestimated or overestimated. Biotin is consumed by pre-treating an avidin-expressing microorganism to a standard sample containing biotin and having an underestimated or overestimated concentration. Improved diagnostic performance is confirmed by evaluating whether the underestimated or overestimated standard sample concentration recovers to its original value after microbial treatment.

According to the present invention, when biotin was added to the standard samples of cyfra21-1 and CA19-9, it was confirmed that the concentration of the standard sample was lowered, and when biotin and avidin-expressing microorganisms were added, the concentration of the standard sample increased. By confirming a significant recovery, various immunoassay kits based on avidin-biotin interaction with improved diagnostic performance can be used to measure hormones, tumors, heart, infection markers, and drug concentrations with improved reliability for patients. can be used If a diagnostic kit containing an avidin-expressing microorganism with improved diagnostic performance is produced, it will be possible to reduce the possibility of false negatives and false positives and significantly improve test accuracy.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for avidin-biotin interaction-mediated immunoassay comprising a microorganism expressing avidin as an active ingredient.

(b) The present invention, based on the strong interaction of avidin-biotin, expresses and immobilizes avidin on the surface of microorganisms to reduce free biotin in the blood, significantly reducing the possibility of false negative or false positive results in immunoassay tests, It can be usefully used as an efficient immunoassay method with greatly improved test accuracy.

1 is a schematic diagram of a microorganism expressing avidin according to an embodiment of the present invention.
Figure 2 is a schematic diagram showing the lowering of the biotin concentration by administering an avidin-expressing microorganism before immunoassay according to an experimental example of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

[Example 1]

CYFRA21.1 assay

The CYFRA21.1 assay is capable of direct in vitro quantitative measurement of Cytokeratin19 fragments in human serum in the range of 0 to 60 ng/mL. The assay used the CYFRA 21.1 IRMA kit from Fujirebio, Inc. The test method is as follows. First, duplicate labels are applied to the coated tubes for each reference standard (S0-S5), serum and sample, and the two test tubes are labeled. Next, add 100 μL of standards, controls and samples. Then, add 100 μL of tracer to each tube and gently mix the contents of the tube. Thereafter, all tubes are sealed with plastic foil and incubated at 2 to 8° C. for 20±2 hours. Then, add 2.0 mL of wash buffer to each tube, drain the contents of the tube, and place the rack upside down on absorbent paper for 2 minutes. Repeat the previous step two more times. Next, count each tube for at least 60 seconds on a gamma counter, and calculate the CYFRA21.1 concentration in the sample as specified in the calculation of results, or use special software.

False negative evaluation according to biotin 1

First, interference according to the concentration of biotin was confirmed using a tumor marker (standard material) having a known concentration. 27.3 ng/mL of the CYFRA 21.1 standard material was treated with 50, 100, 150, and 200 ng of biotin to evaluate the concentration of the standard material according to each biotin concentration. As a result, serious interference, that is, false negatives, was confirmed at concentrations of 100 ng or more of biotin (Table 1).

experimental group Test / (sample concentration, ng/ml) Standard sample (Cyfra21.1) 27.3 Standard sample (Cyfra21.1) + Biotin 50ng 28.3 Standard sample (Cyfra21.1) + Biotin 100ng 3.5 Standard sample (Cyfra21.1) + Biotin 150ng 1.5 Standard sample (Cyfra21.1) + Biotin 200ng 1.1

False negative evaluation according to the addition of avidin-expressing microorganisms 1

After selecting the biotin concentration, the experiment was conducted using microorganisms ( E.coli MG1655), and the experimental groups are as follows (Table 2). In both the control and experimental groups below, 200 uL was dispensed from 1 mL of CYFRA21.1, and the total was adjusted to 300 uL. As for the expression and administration method, the expression of the microorganisms was induced with arabinose after 1 hour and 30 minutes of incubation, and the number of microorganisms was counted and administered to the sample after 3 hours of expression induction. The subjects used in the experiment were a tumor marker (standard sample) of known concentration as a control group, a standard sample (standard material) administered with biotin as an experimental group 1, and a microorganism without avidin expression ( E.coli ) in a standard sample administered with biotin. MG1655 1x10 ⁸ CFU/100 ul) was added to experimental group 2, and the sample to which avidin-expressing microorganisms ( E.coli MG1655 1x10 ⁸ CFU/100 ul) was added to biotin-administered standard material was used as experimental group 3. proceeded.

Assay results according to the addition of avidin-expressing microorganisms 1

The control concentrations of Test 1, Test 2, Test 3, and Test 4 of the CYFRA 21.1 standard were 46.7, 40.1, 47.5, and 48.3 ng/mL, respectively, and the values were 22.5, 18, 19.5, and 21.8 when 100 ng of biotin was added. lowered to ng/mL. When biotin and non-avidin-expressing microorganisms were added, the values were maintained similar to those obtained only with biotin, but when biotin- and avidin-expressing microorganisms were added, the values recovered to 31.2, 29.7, 32.8, and 35.7 ng/mL ( Table 2). This result confirmed that the concentration value of the sample, which had been lowered in the sample containing high concentration of biotin, was recovered considerably when the avidin-expressing microorganism was added.

experimental group Test 1 / (sample concentration, ng/ml) Test 2 /
(sample concentration, ng/ml) Test 3 / (sample concentration, ng/ml) Test 4 /
(sample concentration, ng/ml) Standard sample (Cyfra21.1) 46.7 40.1 47.5 48.3 Standard sample (Cyfra21.1) +
100ng biotin 22.5 18 19.5 21.8 Standard sample (Cyfra21.1) +
Biotin 100ng + Microorganisms
(Avidin non-expressing bacteria) 24.5 15 22.5 23.4 Standard sample (Cyfra21.1)+
Biotin 100ng+Microorganism
(Avidin expressing bacteria) 31.2 29.7 32.8 35.7

[Example 2]

CA19.9 assay

The CA19.9 assay can quantitatively measure the CA19-9 glycoprotein antigen in human serum in the range of 0 to 240 U/mL. The assay used the CA19.9 IRMA kit from Fujirebio, Inc. The test method is as follows. First, duplicate labels are applied to the coated tubes for each reference standard (S0-S5), serum and sample, and label the two test tubes. Next, add 100 μL of standards, controls and samples. Then, add 100 μL of tracer to each tube and gently mix the contents of the tube. Thereafter, all tubes are sealed with plastic foil and incubated at 2 to 8° C. for 20±2 hours. Then, add 2.0 mL of wash buffer to each tube, drain the contents of the tube, and place the rack upside down on absorbent paper for 2 minutes. Repeat the previous step two more times. Next, count each tube for at least 60 seconds on a gamma counter, and calculate the CA19.9 concentration in the sample as specified in the calculation of results, or use special software.

False negative evaluation 2 according to the addition of avidin-expressing microorganisms

After selecting the biotin concentration, the experiment was conducted using microorganisms ( E.coli MG1655), and the experimental groups are as follows (Table 3). In both the control and experimental groups below, 200 uL was dispensed from 1 mL of CA19.9, and the total was adjusted to 300 uL. As for the expression and administration method, the expression of the microorganisms was induced with arabinose after 1 hour and 30 minutes of incubation, and the number of microorganisms was counted and administered to the sample after 3 hours of expression induction. The subjects used in the experiment were the CA19.9 tumor marker (standard sample) of known concentration as the control group, the standard sample (standard material) administered with biotin as experimental group 1, and the microorganisms in which avidin was not expressed in the standard sample administered with biotin ( E.coli MG1655 1x10 ⁸ CFU/100 ul) was added to experimental group 2, and a sample to which avidin-expressing microorganisms ( E.coli MG1655 1x10 ⁸ CFU/100 ul) was added to biotin-administered standard material was experimental group 3. experiment was conducted.

Analysis result 2 according to the addition of avidin-expressing microorganisms

The control concentrations of Test 1 to Test 3 of the CA19.9 standard were 32.2, 58.5, and 142 ng/mL, respectively, and the values decreased to 14.7, 25.4, and 49.4 ng/mL when 100 ng of biotin was added. When biotin and non-avidin-expressing microorganisms were added, the values were similar to those obtained only with biotin, but when biotin and avidin-expressing microorganisms were added, the values recovered to 27.5, 38.1, and 69 ng/mL (Table 3). From this result, it can be seen that the concentration value of the sample, which had been lowered in the sample containing high concentration of biotin, recovered to a considerable extent when the avidin-expressing microorganism was added.

experimental group Test 1/
(sample concentration, ng/ml) Test 2 /
(sample concentration, ng/ml) Test 3 /
(sample concentration, ng/ml) Standard sample (CA19-9) 32.2 58.5 142 Standard sample (CA19-9) + Biotin 100ng 14.7 25.4 49.4 Standard sample (CA19-9) + Biotin 100ng
+ Microorganisms (bacteria that do not express avidin) 12.7 26.4 46.2 Standard sample (CA19-9) + Biotin 100ng
+Microorganism (Avidin-expressing bacteria) 27.5 38.1 69

Having described specific parts of the present invention in detail above, it is clear that these specific techniques are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (8)

A composition for an avidin-biotin interaction mediated immunoassay comprising a microorganism expressing avidin as an active ingredient.
According to claim 1,
The microorganisms are Staphylococcus aureus ( Streptomyces aureorectus ), Streptomyces griseus ( ( Streptomyces griseus ), Streptomyces aureus ( Streptomyces aureus ), Streptomyces avermitilis ( Streptomyces avermitilis ), Streptomyces aureus ( Streptomyces avicenniae ), Streptomyces Avidini ( Streptomyces avidinii ) And Escherichia coli ( Escherichia coli ) A composition, characterized in that selected from the group consisting of.
According to claim 1,
Characterized in that the immunoassay is selected from the group consisting of enzyme immunoassay (EIA), fluorescent immunoassay (FIA), chemiluminescent immunoassay (CLIA) and radioimmunoassay (RIA) composition.
According to claim 1,
The microorganism is a composition, characterized in that contained in the analysis target sample 0.1 x 10 ⁶ to 1.0 x 10 ¹⁰ CFU / 100μl.
A target protein detection kit comprising the composition of any one of claims 1 to 4.
Avidin-biotin interaction mediated immunoassay method comprising the following steps:
(a) adding a microorganism expressing avidin in a biological sample obtained from a subject; and
(b) adding a biotinylated antibody that specifically recognizes the protein to be analyzed.
According to claim 1,
The composition, characterized in that the sample is human-derived.
According to claim 3,
The human body-derived material is whole blood, leukocytes, peripheral blood mononuclear cells, leukocyte buffy coat, plasma, serum, sputum, tears Tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings , ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, characterized in that it is selected from the group consisting of nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions and cerebrospinal fluid composition to be.

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