CN110361547B - Reagent for chemiluminescence quantitative detection of fecal occult blood, detection method thereof and application of reagent in detection of lower digestive tract health - Google Patents
Reagent for chemiluminescence quantitative detection of fecal occult blood, detection method thereof and application of reagent in detection of lower digestive tract health Download PDFInfo
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- CN110361547B CN110361547B CN201910779607.7A CN201910779607A CN110361547B CN 110361547 B CN110361547 B CN 110361547B CN 201910779607 A CN201910779607 A CN 201910779607A CN 110361547 B CN110361547 B CN 110361547B
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/795—Porphyrin- or corrin-ring-containing peptides
- G01N2333/805—Haemoglobins; Myoglobins
Abstract
The invention discloses a reagent for chemiluminescence quantitative detection of fecal occult blood, a detection method thereof and application thereof in detecting digestive tract health; the invention obtains two specific antibodies by immunology, combines the antibodies with a hemoglobin-haptoglobin compound to obtain an immune complex of a marked first specific antibody-hemoglobin-haptoglobin compound-a marked second specific antibody, and then completes detection by using a magnetic bead chemiluminescence detection system; the design can specifically detect hemoglobin-haptoglobin compound in a human fecal sample, has the advantages of high sensitivity and good stability, can well reflect the lower gastrointestinal bleeding condition, assists in early screening of lower gastrointestinal tumors such as rectal cancer or colon cancer and the like, and has high clinical application value.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a reagent for quantitatively detecting fecal occult blood by chemiluminescence, a detection method thereof and application thereof in detecting the health of the lower digestive tract.
Background
Hemoglobin (Hb) in feces, also known as Fecal Occult Blood or Fecal Occult Blood (FOB, febac occlusion Blood), is currently clinically recognized as one of tumor markers for screening colorectal cancer and colon cancer. The color of the stool can not be changed due to a small amount of bleeding in the digestive tract, and the color can be determined only by a stool occult blood test. All digestive tract diseases cause a small amount of bleeding, and the digestive tract diseases can have occult blood stool, so occult blood examination has important value for finding and diagnosing various digestive tract hemorrhagic diseases, is an effective means for screening the digestive tract diseases by general investigation, and the fecal occult blood test becomes a hotspot of continuous and deep research of people.
Haptoglobin (Hp, Haptoglobin, also known as Haptoglobin or globin) is widely present in blood and other body fluids of humans and many mammals in relatively stable amounts in individuals. Haptoglobin has the ability to bind free hemoglobin, acting like a transporter for hemoglobin, which readily binds to haptoglobin to form a stable, irreversible Complex called hemoglobin-haptoglobin Complex (Hb-Hp Complex). In addition to being used as a diagnostic indicator of hemolysis, in recent years, haptoglobin has been highly correlated with nausea tumor, nephropathy, tissue damage, and the like, as research progresses.
The fecal hemoglobin-haptoglobin compound has the detection sensitivity and specificity equivalent to hemoglobin detection, has strong antibacterial ability in intestinal tracts, more stable property, is not easily influenced by components such as gastrointestinal mucus and the like, has longer activity duration time, has higher stability than hemoglobin which does not form a compound, and can overcome the false negative result after the hemoglobin in a detected specimen is decomposed or destroyed because of overlong retention time in the gastrointestinal tracts.
The main detection methods of fecal occult blood in the clinical at present are chemical methods and immunological methods: the chemical methods include o-toluidine method, reduction phenolphthalein method, benzidine method, Pilamivun method, Shuchuangzhi method, etc. The design principle is basically the same, and the iron-containing hemoglobin part in the hemoglobin has the effect of catalyzing the decomposition of peroxide, can catalyze the hydrogen peroxide in the reagent, decompose and release nascent oxygen, oxidize the chromogen substance to generate color, and the color development depth reflects the amount of the hemoglobin. However, in practical application, due to the large difference of the components of the feces, the specific operation details of each laboratory are different, the detection result by a chemical method is greatly different, and the specificity and the accuracy are lacked. If the exogenous food contains hemoglobin and myoglobin, the effect of the heme can make the experiment positive, and a large amount of uncooked vegetables containing active plant peroxide can also catalyze the decomposition of the peroxide to generate false positive reaction. If the blood stays in the intestinal tract for too long and hemoglobin is denatured, a negative result inconsistent with the disease condition will be obtained. In addition, if the patient takes a large amount of vitamin C or other drug with reducing properties, the peroxide can be reduced in the experiment, and thus the chromogen substance cannot be oxidized, and the experiment can also be false negative. Therefore, the chemical method is easily limited by conditions of multiple interference factors, high false positive rate, easy crystal formation of a reagent prepared from glacial acetic acid and the like, and a occult blood detection method which has good specificity, high sensitivity, quick detection and simplicity is urgently needed.
The immunological method is a commonly used method for detecting occult blood at present, and mainly comprises an immune monoclonal antibody method, an enzyme-linked immunosorbent assay, an immune spot method, a latex immune transmission turbidimetry, a radioimmunodification method, a reverse indirect hemagglutination method, a colloidal gold labeled sandwich immunoassay method and the like. The indexes of the immunity detection method are human hemoglobin or human erythrocyte stroma, and the antibody is an anti-human hemoglobin antibody or an anti-human erythrocyte stroma antibody. The chemical method is used for detecting the fecal occult blood, so that the bleeding of any part of the digestive tract can be detected, namely the upper digestive tract bleeding or the lower digestive tract bleeding cannot be distinguished; the immunoassay method mainly detects the lower gastrointestinal hemorrhage by detecting the human red blood cell matrix, thus having the advantages of high specificity and the like for screening diseases (colon, rectal polyp or tumor) related to the lower gastrointestinal hemorrhage and being not influenced by the upper gastrointestinal diseases. However, hemoglobin is denatured by the action of bacteria in the intestinal tract and by the mucus components produced by the large intestine mucosa, and thus a false negative reaction is likely to occur.
The limitations of immunological methods for detecting hemoglobin: the immunochromatography one-step method is to use an anti-human hemoglobin antibody to specifically bind a human hemoglobin antigen at present, and the method is simple, convenient and quick, only specifically aims at the epitope of the human hemoglobin antigen, and basically eliminates the interference of factors such as diet, medicines and the like. The immunocolloidal gold occult blood test has certain sensitivity, and false negative can occur when the sensitivity is higher or lower than the range. The monoclonal antibody colloidal gold method sometimes leaks diagnosis in detecting the bleeding of the digestive tract, and the reasons are as follows: A. the hemoglobin is degraded by digestive enzyme and denatured, and has no original immunogenicity; B. excessive massive hemorrhage causes excessive antigen in a reaction system and a posterior banding phenomenon; C. the structural change of hemoglobin antigen in a patient also affects the affinity of hemoglobin or red blood cell matrix for monoclonal antibodies. The company Eiken Chemical Co.Ltd. of Japan has already proposed the immune turbidimetry OC-SENSOR (latex enhanced immune turbidimetry) to detect human hemoglobin in stool samples, and the method also applies a fully automatic immune diagnostic system, and is simple and convenient to operate, however, the method is not specific to detect human haptoglobin-hemoglobin complex and is influenced by the factor that hemoglobin is easily degraded in stool. In addition, the above-mentioned immunochromatography and immunoturbidimetry detection procedures do not include any washing step, and are easily affected by fine solids such as fibers and particles contained in the stool sample, and in the immunochromatography, there may occur deposition and accumulation of stool fine particles in the detection strip region, and in the immunoturbidimetry, stool particles may directly interfere with the accuracy of turbidimetry results, thereby increasing the risk of false detection results.
The hemoglobin-haptoglobin complex detects occult blood reaction, which is detected by the presence or absence of occult blood rather than a tumor marker, and the occult blood reaction is positive and must be confirmed by other methods, such as enteroscopy and the like, because occasional bleeding positive possibly caused by other factors is possible, and the occult blood reaction negative is also possible to be false negative because a small amount of bleeding of early tumors and haptoglobin in stool form a complex, and an anti-hemoglobin antibody cannot recognize the complex, so that the detection result is negative. The method for detecting the haptoglobin-hemoglobin compound in the fecal sample is a novel marker for detecting fecal occult blood except hemoglobin and heme, and has the greatest advantage that the haptoglobin-hemoglobin compound is not easily degraded in the fecal sample and is more stable than hemoglobin, so that the risk of false negative results is reduced. The radioimmunoassay method is used for detecting Olb-Hp) and hemoglobin (Hb) of fecal hemoglobin-haptoglobin complex of 48 patients with colon cancer or rectal cancer in Japan, the positive results are 44 and 35 respectively, at least 9 patients with fecal occult blood are false negative, namely the positive coincidence rate of fecal occult blood for diagnosing rectal cancer or colon cancer is only about 70%, and the positive coincidence rate of the hemoglobin-haptoglobin complex for testing fecal occult blood for colorectal cancer or colon cancer is more than 90%, so that 20% -30% of false negative reasons of fecal occult blood for testing rectal cancer or colon cancer are caused: since a small amount of bleeding occurs in the digestive tract, the released hemoglobin (Hb) is combined with Hp in the feces to form a complex when passing through a long intestinal tract, so that occult blood false negative is caused, and the hemoglobin in the feces is obviously detected to diagnose whether a tumor marker of the rectal cancer or the colon cancer has serious omission.
Kits for detecting hemoglobin-haptoglobin complexes are currently available on the market. For example, chinese patent application publication No. CN 104515859 a (2015, 4/15) provides a hemoglobin, hemoglobin-haptoglobin complex, transferrin assay kit, a preparation method thereof, and a detection method thereof, which apply an immunological principle, a lateral chromatography technique, and a chemical o-toluidine color development method to detect hemoglobin, hemoglobin-haptoglobin complex, and transferrin in human feces, thereby finding lower gastrointestinal bleeding symptoms and early diagnosing lower gastrointestinal tumors such as rectal cancer or colon cancer.
The invention aims to provide a hemoglobin, hemoglobin-haptoglobin compound and transferrin joint inspection kit, which is a test paper box consisting of a test paper strip for simultaneously detecting hemoglobin, hemoglobin-haptoglobin compound and transferrin, a chemical o-toluidine test paper, an upper card shell and a lower card shell, wherein the immune test paper strip comprises absorbent paper, a nitrocellulose membrane, a sample pad and a PVC bottom plate, the edge of the sample pad is adhered to the nitrocellulose membrane, the edge of the absorbent paper is adhered to the nitrocellulose membrane, and the nitrocellulose membrane is fixed on the PVC bottom plate. The nitrocellulose membrane is provided with 3 detection lines, a quality control line and a gold-labeled compound membrane scribing line close to the bottom end of the nitrocellulose membrane in parallel, the quality control line is coated with goat anti-mouse IgG, the 3 detection lines are simultaneously coated with a hemoglobin monoclonal antibody, a hemoglobin-conjugated globin compound monoclonal antibody and a transferrin monoclonal antibody, and the gold-labeled compound membrane scribing line contains a gold-labeled compound of the hemoglobin monoclonal antibody, the hemoglobin-conjugated globin compound monoclonal antibody and the transferrin monoclonal antibody. The gold-labeled compound is a marker of 60mm colloidal gold for resisting human hemoglobin monoclonal antibody, human conjugated protein monoclonal antibody and human transferrin monoclonal antibody. The colloidal gold and the marked protein are combined by means of electrostatic action, the binding force is unstable, and the transaction is interfered by external environments such as acid-base factors and the like, so the detection sensitivity is low.
The detection result of the invention is as follows: in the strip-shaped observation window of the test paper box, there are a quality control line and three detection lines corresponding to the detection substances, and at the same time, there is a result detected by a chemical o-toluidine color development method in the chemical test paper observation window. For the immunological method, in a strip-shaped observation window, purple red bands appear at the positions of a detection line and a quality control line, which indicate that hemoglobin, a hemoglobin-haptoglobin compound and transferrin exist in a sample; for the chemical method, the result is positive as the blue reaction appears in the observation window of the chemical test paper. The results of the test are interpreted as: the detection results show that hemoglobin, hemoglobin-haptoglobin compound and transferrin are all positive or hemoglobin, hemoglobin-haptoglobin compound are all positive or hemoglobin and transferrin are all positive, so that digestive tract bleeding is highly suspected, lower digestive tract tumors such as rectal cancer or colon cancer are likely to be suffered, and further diagnosis is recommended for confirmation; if the hemoglobin-haptoglobin complex, transferrin, is negative and hemoglobin positive may be due to paroxysmal inflammation; hemoglobin, hemoglobin-haptoglobin complex, transferrin were negative, indicating no bleeding in the digestive tract. The invention combines a chemical method and an immunological method, has more detection substances, complex detection results and a determination method thereof, possibly inconsistent determination of different methodologies and detection results of different substances, and has larger limitation on clinical application. Meanwhile, the detection result of the invention is a color development mode, and is judged by naked eyes only, and the accurate quantitative detection method is lacked, so that the digestive tract bleeding degree of a subject cannot be detected.
In order to solve the defects of the prior art, the market needs a detection reagent and a detection method capable of specifically detecting hemoglobin-haptoglobin compound in a human fecal sample, quantitatively reflecting the lower gastrointestinal hemorrhage condition and assisting in early screening of lower gastrointestinal tumors such as rectal cancer or colon cancer, and the invention solves the problems.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a reagent for quantitatively detecting fecal occult blood by chemiluminescence, a detection method thereof and application thereof in detecting the health of the lower digestive tract, wherein immunology is utilized to obtain two specific antibodies, the antibodies are combined with a hemoglobin-haptoglobin compound to obtain an immune complex of a marked first specific antibody, the hemoglobin-haptoglobin compound and a marked second specific antibody, and a magnetic bead chemiluminescence detection system is utilized to complete detection; the design can specifically detect hemoglobin-haptoglobin compound in a human fecal sample, has the advantages of high sensitivity and good stability, can quantitatively reflect the lower gastrointestinal hemorrhage condition, assists in early screening of lower gastrointestinal tumors such as rectal cancer or colon cancer and the like, and has high clinical application value.
In order to achieve the above object, the present invention adopts the following technical solutions:
a reagent for chemiluminescent quantitative detection of fecal occult blood comprising: an immune combination formed by an antibody detection system and a human hemoglobin-haptoglobin compound, and a magnetic bead chemiluminescence detection system of the immune combination; the fixed separation phase of the magnetic bead chemiluminescence detection system is magnetic particles, and the calibration sample and the quality control sample are human hemoglobin-haptoglobin compound; the immune combination body is 'magnetic bead-first specific antibody-human hemoglobin-haptoglobin compound-second specific antibody marked by luminescent marker'.
In the reagent for quantitatively detecting fecal occult blood by chemiluminescence, the first specific antibody and the second specific antibody are specific antibodies combined with a human hemoglobin monomer and specific antibodies combined with a human haptoglobin monomer; the specific antibody forms an immune combination with the human hemoglobin-haptoglobin compound, the specific antibody combined with the human hemoglobin monomer is not combined with any human haptoglobin monomer, and the specific antibody combined with the human haptoglobin monomer is not combined with any human hemoglobin monomer.
In the reagent for quantitatively detecting fecal occult blood by chemiluminescence, the first specific antibody and the second specific antibody are specific antibodies combined with a human hemoglobin-haptoglobin compound and specific antibodies combined with a human haptoglobin monomer; the specific antibody and the hemoglobin-haptoglobin compound form an immune combination body, and the specific antibody combined with the human hemoglobin-haptoglobin compound is not combined with any human haptoglobin monomer or human hemoglobin monomer; the specific antibody that binds to human haptoglobin monomer does not bind to any human hemoglobin monomer.
In the reagent for chemiluminescent quantitative detection of fecal occult blood, the first specific antibody and the second specific antibody are specific antibodies combined with the human hemoglobin-haptoglobin complex and specific antibodies combined with the human hemoglobin monomer, the specific antibodies can form an immune combination with the hemoglobin-haptoglobin complex, and the specific antibodies combined with the human hemoglobin-haptoglobin complex are not combined with any haptoglobin monomer or hemoglobin monomer; the specific antibody that binds to human hemoglobin does not bind to any haptoglobin monomer.
In the reagent for the chemiluminescent quantitative detection of fecal occult blood, the immobilized separation phase is streptavidin-coated magnetic beads, and the magnetic beads are combined with a biotin-labeled first specific antibody.
In the above-mentioned reagent for quantitatively detecting fecal occult blood by chemiluminescence, the immobilized separation phase is magnetic beads coated with a first specific antibody.
In the reagent for the chemiluminescent quantitative detection of fecal occult blood, the second specific antibody is an antibody marked by a luminescent marker, and the luminescent marker is acridinium ester.
In the reagent for quantitatively detecting fecal occult blood by chemiluminescence, the first specific antibody and the second specific antibody are polyclonal antibodies or monoclonal antibodies.
A detection method of a reagent for quantitatively detecting fecal occult blood by chemiluminescence comprises the following steps: calibrating the hemoglobin-haptoglobin compound quantitative detection by using the calibrator according to a calibration program; adding a quality control product and a sample to be detected into a specified reaction cup; adding a first specific antibody, a second specific antibody and magnetic beads to each reaction cup; forming an immune complex of magnetic beads, a first specific antibody, a human hemoglobin-haptoglobin compound and a second specific antibody marked by acridinium ester in the reaction cup through secondary incubation; washing each reaction cup by using a washing solution for multiple times to remove the unbound antibody; adding a luminous substrate solution A for a full-automatic immune inspection system into each reaction cup; adding a luminous substrate solution B for a full-automatic immune inspection system into each reaction cup; sequentially measuring on a full-automatic chemiluminescence apparatus, and carrying out data processing according to calibration curve information calibrated by the hemoglobin-haptoglobin compound reagent to obtain the concentration of the human hemoglobin-haptoglobin compound of the sample to be detected.
An application of a reagent for quantitatively detecting fecal occult blood by chemiluminescence,
the step of detecting the health of the lower digestive tract comprises:
step one, physical examination sample collection and pretreatment: collecting and extracting a fresh human excrement sample to be detected by using the sample extraction liquid to obtain a clarified sample to be detected;
step two, pretreatment of a detection reagent: before use, placing the hemoglobin-haptoglobin compound detection reagent at room temperature for rewarming, preparing a first marked fecal occult blood antibody, a second marked fecal occult blood antibody, a hemoglobin-haptoglobin compound calibration product, a hemoglobin-haptoglobin compound quality control product, a sample to be detected, a coated magnetic particle solution, a cleaning solution, a substrate solution A for a full-automatic immune inspection system and a substrate solution B for the full-automatic immune inspection system;
the process for preparing the antibody comprises the following steps: step one, immune injection of a hemoglobin-haptoglobin compound to an experimental animal; step two, stopping immunization and bleeding; step three, preparing an immunochromatography affinity column; step four, extracting and purifying the antibody to obtain a hemoglobin-haptoglobin compound antibody with high concentration;
step three, pre-treating a calibration material and a quality control material;
step four, calibrating;
step five, detecting on a full-automatic chemiluminescence determinator,
adding 50 mu L of quality control materials and samples into a designated reaction cup;
adding 50. mu.L of the first labeled fecal occult blood antibody to each reaction cup;
25 μ L of the coated magnetic particle solution was added to each reaction cup;
incubation;
cleaning each reaction cup for many times by using a cleaning solution;
adding 100. mu.L of a second labeled fecal occult blood antibody to each reaction cup;
incubation;
cleaning each reaction cup for many times by using a cleaning solution;
adding 200 mu L of substrate solution A for the full-automatic immune test system into each reaction cup;
adding 200 mu L of substrate solution B for the full-automatic immune test system into each reaction cup;
sequentially measuring the relative light intensity RLU of each reaction cup on a full-automatic chemiluminescence apparatus;
step six, calculating: the chemiluminescence analyzer calculates concentration values of the sample and the quality control material through a calibration curve burnt in the fecal occult blood measuring reagent card and the measured relative light intensity RLU;
step seven, quality control: comparing the measured concentration of the quality control product with the marked value range, and if the measured concentration exceeds the marked quality control range, indicating that the detection is unqualified and needing to be carried out again;
step eight, determining the health condition of the lower digestive tract according to the reference range and the detection result:
the definition value of normal healthy population is lower than 200 mug/g;
positive population definition values greater than 50 μ g/g;
the strong positive population has a defined value of greater than 1000. mu.g/g.
The invention has the advantages that:
according to the invention, immunology is utilized to obtain two specific antibodies, and then a double-antibody sandwich technology that the two specific antibodies are combined with different antigenic determinants of a hemoglobin-haptoglobin compound is adopted to obtain an immune complex of a marked first specific antibody-hemoglobin-haptoglobin compound-a marked second specific antibody, so that the hemoglobin-haptoglobin compound in a human fecal sample can be specifically detected, and the human fecal sample has high sensitivity and good stability;
the detection method developed by the full-automatic magnetic bead chemiluminescence platform is simple in detection steps and short in time;
the result obtained by the detection of the invention can well reflect the lower gastrointestinal hemorrhage symptom, assist in early diagnosis of lower gastrointestinal tumors such as rectal cancer or colon cancer and the like, and has clinical application value for the detection of the lower gastrointestinal health;
the reagent is used for researching the concentration of the hemoglobin-haptoglobin compound in human excrement, and a percentile method is adopted for obtaining a reference range of a sample result, wherein the reference range can well assist the evaluation of individual lower gastrointestinal hemorrhage and the diagnosis of symptoms related to the lower gastrointestinal hemorrhage.
Drawings
FIG. 1 is a schematic diagram of the structure of the hemoglobin-haptoglobin complex of the present invention;
FIG. 2 is a flow chart of the preparation of a polyclonal antibody to the hemoglobin-haptoglobin complex of the present invention;
FIG. 3 is a flow chart of the preparation of hemoglobin-haptoglobin complex monoclonal antibodies of the present invention.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
A chemiluminescent reagent for quantitatively detecting fecal occult blood, comprising: immune complex, antibody detection system;
the immune complex comprises: the hemoglobin-haptoglobin compound in the calibration product, the quality control product and the sample to be detected is compounded with the marked first specific antibody and the marked second specific antibody to form a double-antibody sandwich immune complex of the marked first specific antibody-hemoglobin-haptoglobin compound-the marked second specific antibody;
the first specific antibody and the second specific antibody are generated by injecting the hemoglobin-haptoglobin compound into animals, and then preparing a hemoglobin-haptoglobin compound monoclonal antibody aiming at a specific antigenic determinant through an immune reaction or a hybridoma technology.
Human hemoglobin amino acid sequence:
human haptoglobin amino acid sequence:
as an example, a reagent for chemiluminescence quantitative determination of fecal occult blood comprises: an immune combination formed by an antibody detection system and a human hemoglobin-haptoglobin compound, and a magnetic bead chemiluminescence detection system of the immune combination; the fixed separation phase of the magnetic bead chemiluminescence detection system is magnetic particles, and the main components of the calibration product and the quality control product are human hemoglobin-haptoglobin compound; the immunoconjugate is a "magnetic bead," a first specific antibody, a human hemoglobin-haptoglobin complex, a second specific antibody labeled with a luminescent label. The luminescent marker can be acridinium ester, also can be luminol, alkaline phosphatase, horseradish peroxidase, preferably acridinium ester, the acridinium ester is excited to emit light and is a non-enzymatic reaction, a catalyst is not needed, the luminescent speed is high, the luminescent intensity is high, and the sensitivity is higher. As an example, the solid phase separator is streptavidin-coated magnetic beads, which are bound with a first specific antibody labeled with biotin; as another example, the solid phase separator is a first specific antibody coated magnetic bead.
The first specific antibody and the second specific antibody may be polyclonal antibodies or monoclonal antibodies.
The hemoglobin-haptoglobin complex structure of figure 1 formed by the hemoglobin molecule bound to a haptoglobin molecule, wherein two hemoglobin molecules form a dimer, bound to one haptoglobin molecule, having a hemoglobin molecule bound to haptoglobin molecule and a hemoglobin molecule unbound to haptoglobin molecule; wherein, the segment c and the segment d are the combination parts of the hemoglobin and the haptoglobin, the segment a and the segment b are the non-combination parts of the hemoglobin and the haptoglobin, the segment a is the unique part of the hemoglobin, and the segment b is the unique part of the haptoglobin.
There are three examples of the binding of antibodies to hemoglobin-haptoglobin complexes:
example 1: the first specific antibody and the second specific antibody are specific antibodies combined with human hemoglobin monomers and specific antibodies combined with human haptoglobin monomers; the specific antibody forms an immune combination with the human hemoglobin-haptoglobin compound, the specific antibody combined with the human hemoglobin monomer is not combined with any human haptoglobin monomer, and the specific antibody combined with the human haptoglobin monomer is not combined with any human hemoglobin monomer. This embodiment is combined in sections a and b as shown in fig. 1.
Example 2, the first specific antibody, the second specific antibody are specific antibodies binding to a human hemoglobin-haptoglobin complex and specific antibodies binding to a human haptoglobin monomer; the specific antibody and the hemoglobin-haptoglobin compound form an immune combination body, and the specific antibody combined with the human hemoglobin-haptoglobin compound is not combined with any human haptoglobin monomer or human hemoglobin monomer; the specific antibody that binds to human haptoglobin monomer does not bind to any human hemoglobin monomer. Such embodiment joints are at the joint of the segments c and d and at the segment b as shown in fig. 1.
Example 3, the first specific antibody, the second specific antibody are specific antibodies binding to the human hemoglobin-haptoglobin complex and specific antibodies binding to the human hemoglobin monomer, the specific antibodies can form an immunoconjugate with the hemoglobin-haptoglobin complex, and the specific antibodies binding to the human hemoglobin-haptoglobin complex do not bind to any haptoglobin monomer or hemoglobin monomer; the specific antibody that binds to human hemoglobin does not bind to any haptoglobin monomer. Such embodiment joints are at the joint of the segments c and d and at the segment a as shown in fig. 1.
The preparation process of the hemoglobin-haptoglobin compound antibody comprises the following steps: step one, immune injection of hemoglobin-haptoglobin compound to an experimental animal; step two, stopping immunization and exsanguination; step three, preparing an immunochromatography affinity column; and step four, extracting and purifying the antibody to obtain the hemoglobin-haptoglobin compound antibody with high concentration.
Further, the preparation process of the hemoglobin-haptoglobin compound polyclonal antibody comprises the following steps: step one, immune injection of hemoglobin-haptoglobin compound to an experimental animal; step two, measuring the titer of antiserum, and if the antiserum is qualified, stopping immunization and bleeding; step three, preparing an immunochromatography affinity column; and step four, extracting and purifying the antibody to obtain the hemoglobin-haptoglobin compound antibody with high concentration.
Further, the specific preparation process of the hemoglobin-haptoglobin compound polyclonal antibody comprises the following steps:
step one, immune injection of hemoglobin-haptoglobin compound to an experimental animal;
collecting blood from experimental animals, inactivating, coagulating, centrifuging, and collecting upper layer serum as negative reference; uniformly mixing the Freund's complete adjuvant with the high-concentration hemoglobin-haptoglobin compound, and injecting into an animal for experiment; taking blood 7-10 days after the first immunization cycle, and immunizing for 4-5 times;
step two, collecting blood of the immunized animal, performing antiserum titer determination, and stopping immunization and bleeding if the detection is qualified;
step three, preparing an immunochromatography affinity column;
(1) uniformly mixing agarose gel and a hydrochloric acid solution according to a certain proportion, and standing overnight at 2-4 ℃ to obtain swelling gel;
(2) filling the swelling glue into an affinity column, and washing media by using a hydrochloric acid solution and a coupling buffer solution respectively;
(3) taking three affinity columns, mixing high-concentration hemoglobin with a coupling buffer solution, and adding the mixture into swelling gel of the first affinity column; mixing high-concentration haptoglobin with a coupling buffer solution, and adding the mixture into swelling gel of a second affinity column; mixing the high-concentration hemoglobin-haptoglobin compound with a coupling buffer solution, adding the mixture into swelling gel of a third affinity column, mixing and shaking the affinity columns at room temperature, and washing the affinity columns by using the coupling buffer solution;
(4) solid phase antigen: BSA solution was added to the affinity column, incubated at room temperature, and the medium was washed with phosphate buffer and finally stored in a sealed ethanol solution.
Step four, extracting and purifying the antibody to obtain a hemoglobin-haptoglobin compound antibody with high concentration;
(1) washing the immunochromatography affinity column with a PBS solution;
(2) mixing the extracted immune rabbit serum with a PBS solution in equal proportion, respectively adding the mixture into three immunochromatography affinity columns, and mixing and shaking the affinity columns at room temperature;
(3) washing the affinity column with PBS solution to remove impurities;
(4) washing the affinity column with an antibody eluent, eluting the hemoglobin-haptoglobin compound antibody specifically bound on the affinity column, and collecting the eluent to obtain a specific antibody;
(5) and (3) concentrating the eluate containing the specific antibody by using sucrose or polyethylene glycol, and dialyzing and purifying in a PBS (phosphate buffer solution) solution to obtain the hemoglobin-haptoglobin compound polyclonal antibody with high concentration. The antibody detection system is a magnetic bead chemiluminescence detection system, and the stationary phase is magnetic particles;
the detection reagent comprises: the kit comprises a biotin-labeled fecal occult blood antibody, an acridinium ester-labeled fecal occult blood antibody, a hemoglobin-haptoglobin compound calibration material, a hemoglobin-haptoglobin compound quality control material, a sample to be detected, a streptavidin-coated magnetic particle solution, a fecal occult blood determination calibration card, a fecal occult blood determination reagent card, a cleaning solution, a sample extraction liquid, and a substrate liquid A and a substrate liquid B for a full-automatic immunoassay system.
As an example, the detection reagent is a streptavidin-biotin system; the method comprises the following reagents:
a biotin-labeled hemoglobin-haptoglobin complex monoclonal or polyclonal antibody;
a marker-labeled hemoglobin-haptoglobin complex monoclonal or polyclonal antibody;
a hemoglobin-haptoglobin complex calibrator comprises the following components: human fecal hemoglobin-haptoglobin complex antigen;
the hemoglobin-haptoglobin compound quality control product comprises the following components: human fecal hemoglobin-haptoglobin complex antigen;
a sample to be tested;
streptavidin-coated magnetic microparticles;
fecal occult blood determination calibration card;
fecal occult blood determination reagent card;
the cleaning solution comprises the following components: phosphates, sodium chloride, tween 20;
a sample extraction liquid, the composition of the sample extraction liquid comprising: calcium chloride, tween 20 and sodium azide;
the substrate liquid A for the full-automatic immunoassay system comprises the following components: hydrogen peroxide;
the substrate liquid B for the full-automatic immunoassay system comprises the following components: sodium hydroxide.
As an example, the detection reagent is an antibody directly coated magnetic particle system; the method comprises the following reagents:
magnetic particles coated with monoclonal or polyclonal antibodies of a hemoglobin-haptoglobin complex,
a marker-labeled hemoglobin-haptoglobin complex monoclonal or polyclonal antibody;
a hemoglobin-haptoglobin complex calibrator comprises the following components: human fecal hemoglobin-haptoglobin complex antigen;
the hemoglobin-haptoglobin compound quality control product comprises the following components: human fecal hemoglobin-haptoglobin complex antigen;
a sample to be tested;
measuring and calibrating the fecal occult blood;
fecal occult blood determination reagent card;
the cleaning solution comprises the following components: phosphates, sodium chloride, tween 20;
a sample extraction fluid, the sample extraction fluid comprising: calcium chloride, tween 20 and sodium azide;
the substrate liquid A for the full-automatic immunoassay system comprises the following components: hydrogen peroxide;
the substrate liquid B for the full-automatic immunoassay system comprises the following components: sodium hydroxide.
A detection method of a reagent for quantitatively detecting fecal occult blood by chemiluminescence comprises the following steps: calibrating the hemoglobin-haptoglobin compound quantitative detection by using the calibrator according to a calibration program; adding a calibration product, a quality control product and a sample to be detected into a designated reaction cup; adding a first specific antibody, a second specific antibody and magnetic beads into each reaction cup; after the secondary incubation, an immune complex of magnetic beads, a first specific antibody, a human hemoglobin-haptoglobin compound and a second specific antibody marked by acridinium ester is formed in the reaction cup; washing each reaction cup by using a washing solution for multiple times to remove the unbound antibody; adding a luminous substrate solution A for a full-automatic immune inspection system into each reaction cup; adding a luminous substrate solution B for a full-automatic immune inspection system into each reaction cup; sequentially measuring on a full-automatic chemiluminescence apparatus, and carrying out data processing according to calibration curve information calibrated by the hemoglobin-haptoglobin compound reagent to obtain the concentration of the human hemoglobin-haptoglobin compound of the sample to be detected. The full-automatic chemiluminescence apparatus has the functions of automatic sample adding, reagent mixing, incubation, washing, luminescence value detection and the like controlled by a computer program.
The further detection method comprises the following steps:
collecting and extracting a fresh human excrement sample by using a sample extraction liquid to obtain a clarified sample to be detected;
calibrating the hemoglobin-haptoglobin compound quantitative detection by using the calibrator according to a calibration program;
adding a quality control product and a sample into a designated reaction cup;
the process for preparing the antibody is as follows: step one, immune injection of a hemoglobin-haptoglobin compound to an experimental animal; step two, stopping immunization and exsanguination; step three, preparing an immunochromatography affinity column; step four, extracting and purifying the antibody to obtain a hemoglobin-haptoglobin compound antibody with high concentration;
adding a first labeled hemoglobin-haptoglobin complex antibody to each reaction cup;
adding the coated magnetic particle solution into each reaction cup;
incubation;
cleaning each reaction cup for many times by using a cleaning solution;
adding a second labeled hemoglobin-haptoglobin complex antibody to each reaction cup;
incubation;
cleaning each reaction cup for many times;
adding a substrate solution A for a full-automatic immune inspection system into each reaction cup;
adding a substrate solution B for a full-automatic immune inspection system into each reaction cup;
and sequentially measuring on a chemiluminescence instrument, and processing data results according to calibration curve information burnt in the hemoglobin-haptoglobin compound reagent card.
Δ as shown in fig. 2, the specific process for preparing the polyclonal antibody of the hemoglobin-haptoglobin complex is as follows:
1. animal for immunity experiment
(1) Selecting 2-3 healthy laboratory rabbits (raising time 6-7 weeks, weight about 2.0 kg);
(2) collecting blood from rabbit ear vein, inactivating, coagulating, centrifuging, and collecting upper layer serum as negative reference substance;
(3) mixing 250 μ L Freund's complete adjuvant and 250 μ L high concentration hemoglobin-haptoglobin complex, and injecting into rabbit shoulder subcutaneous muscle or hind leg muscle;
(4) the immunization period is 20 days, blood is taken 7-10 days after the first immunization (including test blood taking and final blood discharging), and the immunization is carried out for 4-5 times;
2. termination of immunity and exsanguination
(1) Taking a test sample: taking test blood within 7-10 days after the first immunization, and then taking test blood within 2-3 days after each immunization;
(2) and (3) detecting the immune effect: centrifuging an immunized rabbit blood sample, taking upper serum as antiserum, diluting the antiserum by using a dilution buffer solution, respectively setting five dilution gradients of 1:50, 1:100, 1:200, 1:400 and 1:1000, adding 100 mu L of diluted antiserum and a negative reference substance into a small hole of a hemoglobin-haptoglobin compound coated micropore plate, incubating at room temperature for 1 hour, washing the micropore plate by using a cleaning solution, then adding 100 mu L of HRP-labeled mouse anti-rabbit antibody, incubating at room temperature for 40 minutes, washing the micropore plate by using a cleaning solution, adding 50 mu L of Tetramethylbenzidine (TMB) into each hole, incubating at room temperature for 10-20 minutes, adding 50 mu L of stop solution into each hole, and reading the absorbance at 450nm on a microplate reader;
(3) and (3) termination of immunization: when the difference value of the absorbance value measured by the immune sample and the absorbance value of the negative reference substance meets the requirement of antibody preparation, stopping the immunity, killing the immune rabbit, discharging blood, centrifugally collecting upper serum, and refrigerating the serum sample at 2-8 ℃.
3. Preparation of an immunochromatographic affinity column
(1) Uniformly mixing agarose gel and a hydrochloric acid solution according to a certain proportion, and standing overnight at 2-4 ℃ to obtain swelling gel;
(2) filling the swelling glue into an affinity column, and washing media by using a hydrochloric acid solution and a coupling buffer solution respectively;
(3) taking three affinity columns, mixing a certain proportion of high-concentration hemoglobin with a coupling buffer solution, and adding the mixture into swelling gel of the first affinity column; mixing a certain proportion of high-concentration haptoglobin with a coupling buffer solution, and adding the mixture into swelling gel of a second affinity column; mixing a certain proportion of high-concentration hemoglobin-haptoglobin compound with a coupling buffer solution, and adding the mixture into swelling gel of a third affinity column. Mixing and shaking the affinity column at room temperature, and washing the affinity column with a coupling buffer solution;
(4) solid phase antigen: BSA solution was added to the affinity column, incubated at room temperature for about 3 hours, and the medium was washed with phosphate buffer and finally stored in a 20% ethanol solution under a seal.
4. Extraction and purification of antibodies
(1) Washing the immunochromatography affinity column with a high-concentration PBS solution to balance the affinity column;
(2) mixing the extracted immune rabbit serum with a PBS solution in equal proportion, respectively adding the mixture into three immunochromatography affinity columns, and mixing and shaking the affinity columns at room temperature;
(3) washing the affinity column with high-concentration PBS solution to remove other impurities such as impurity protein;
(4) washing the affinity column with an antibody eluent, eluting the hemoglobin-haptoglobin compound antibody specifically bound on the affinity column, and collecting the eluent to obtain a specific antibody;
(5) and (3) concentrating the eluate containing the specific antibody by using sucrose or polyethylene glycol, and dialyzing and purifying in a PBS (phosphate buffer solution) solution to obtain the high-concentration hemoglobin-haptoglobin compound polyclonal antibody.
Δ as shown in fig. 3, the preparation process of hemoglobin-haptoglobin complex monoclonal antibody:
1, animals for immunization experiments;
(1) 5 healthy laboratory BALB/c female mice were selected (rearing time 6-8 weeks);
(2) collecting blood of 0.2-0.3mL from a mouse, inactivating the blood, coagulating and centrifuging to collect upper serum as a negative reference substance;
(3) diluting the high-concentration hemoglobin-haptoglobin compound by using an antigen diluent, adding an equivalent Freund's complete adjuvant into 200 mu L of the diluted hemoglobin-haptoglobin compound, uniformly mixing, and injecting a mouse into an abdominal cavity;
(4) the immunization cycle is 20 days, blood sampling (including test blood sampling and final blood discharging) is started 7-10 days after the first immunization, and the immunization is carried out for 4-5 times;
2, stopping immunization and preparing hybridoma cells;
(1) taking a test sample: taking test blood within 7-10 days after the first immunization, and then taking test blood within 2-3 days after each immunization;
(2) and (3) detecting the immune effect: centrifuging an immunized rabbit blood sample, taking upper serum as antiserum, diluting the antiserum by using a dilution buffer solution, respectively setting five dilution gradients of 1:50, 1:100, 1:200, 1:400 and 1:1000, adding 100 mu L of diluted antiserum and a negative reference substance into a small hole of a hemoglobin-haptoglobin compound coated micropore plate, incubating at room temperature for 1 hour, washing the micropore plate by using a cleaning solution, then adding 100 mu L of HRP-labeled mouse anti-rabbit antibody, incubating at room temperature for 40 minutes, washing the micropore plate by using a cleaning solution, adding 50 mu L of Tetramethylbenzidine (TMB) into each hole, incubating at room temperature for 10-20 minutes, adding 50 mu L of stop solution into each hole, and reading the absorbance at 450nm on a microplate reader;
(3) termination of immunity and production of hybridoma cells: when the difference value of the absorbance value measured by the immune sample and the absorbance value of the negative reference substance meets the requirement of antibody preparation, the mice are killed by carbon dioxide gas, the spleen is taken out by aseptic technique, and the spleen cell suspension is prepared by extrusion grinding in a flat dish. Mixing prepared homologous myeloma cells and mouse spleen cells according to a certain proportion, and adding fusion promoter polyethylene glycol. Under the action of polyethylene glycol, various lymphocytes can be fused with myeloma cells to form hybridoma cells.
3, screening and preparing;
(1) screening of hybridoma positive clones: the hybridoma cells were cultured in HAT medium. Cloning and screening the hybridoma cells by using a hemoglobin monomer, a haptoglobin monomer and a hemoglobin-haptoglobin compound, screening out the hybridoma cells secreting a monoclonal antibody specifically bound with the hemoglobin monomer, wherein the secreted monoclonal antibody can be specifically bound with the hemoglobin-haptoglobin compound; screening out hybridoma cells secreting monoclonal antibodies specifically bound to haptoglobin monomers, wherein the secreted monoclonal antibodies can be specifically bound to hemoglobin-haptoglobin complexes; screening out hybridoma cells of the monoclonal antibody which can be specifically combined with the hemoglobin-haptoglobin compound, wherein the secreted monoclonal antibody cannot be combined with a hemoglobin monomer or a haptoglobin monomer;
(2) large-scale preparation of monoclonal antibodies: the selected hybridoma cells are placed in a culture flask for culture. During the culture process, the hybridoma cells produce and secrete monoclonal antibodies, culture supernatant is collected, and the cells and fragments thereof are removed by centrifugation, so that a large amount of the required monoclonal antibodies are obtained.
4, purifying the monoclonal antibody;
and purifying the monoclonal antibody obtained by cell culture by using a recombinant protein A affinity chromatography column to obtain the hemoglobin-haptoglobin compound monoclonal antibody.
The delta antibody detection system is a magnetic bead chemiluminescence detection system, and the stationary phase is magnetic particles. There are two solid phase separation embodiments:
(1) a "streptavidin-biotin" system;
a reagent for chemiluminescence quantitative determination of fecal occult blood, which comprises:
a biotin-labeled hemoglobin-haptoglobin complex monoclonal or polyclonal antibody;
a marker-labeled hemoglobin-haptoglobin complex monoclonal or polyclonal antibody, as an example, a luminescent signal marker may be acridinium lipid, HRP, alkaline phosphatase or rumi sodium;
a hemoglobin-haptoglobin complex calibrator comprises the following components: human fecal hemoglobin-haptoglobin complex antigen;
the hemoglobin-haptoglobin compound quality control product comprises the following components: human fecal hemoglobin-haptoglobin complex antigen;
a sample to be tested;
streptavidin-coated magnetic microparticles;
fecal occult blood determination calibration card: burning and recording related information of a hemoglobin-haptoglobin compound calibrator in a card;
fecal occult blood determination reagent card: burning and recording relevant information of a fecal occult blood measuring and calibrating curve in the card;
the cleaning solution comprises the following components: 0.01mol/L-0.2mol/L phosphate, 0.5mol/L-2mol/L sodium chloride and 0.05% -0.5% Tween 20;
a sample extraction liquid, the composition of the sample extraction liquid comprising: calcium chloride, tween 20 and sodium azide;
the substrate liquid A for the full-automatic immunoassay system comprises the following components: hydrogen peroxide;
the substrate liquid B for the full-automatic immunoassay system comprises the following components: sodium hydroxide.
(2) Antibody-directly coated magnetic microparticle systems;
a reagent for chemiluminescent quantitative detection of fecal occult blood comprising:
magnetic particles coated with monoclonal or polyclonal antibodies of a hemoglobin-haptoglobin complex,
monoclonal or polyclonal antibodies to hemoglobin-haptoglobin complex labeled with a label, which may be, as one example, acridinium lipid, HRP, alkaline phosphatase or rumi sodium;
a hemoglobin-haptoglobin complex calibrator comprises the following components: human fecal hemoglobin-haptoglobin complex antigen;
the quality control product of the hemoglobin-haptoglobin compound comprises the following components: human fecal hemoglobin-haptoglobin complex antigen;
a sample to be tested;
fecal occult blood determination calibration card: burning and recording related information of a hemoglobin-haptoglobin compound calibrator in a card;
fecal occult blood assay reagent card: burning and recording relevant information of a fecal occult blood measuring and calibrating curve in the card;
the cleaning solution comprises the following components: 0.01mol/L-0.2mol/L phosphate, 0.5mol/L-2mol/L sodium chloride and 0.05% -0.5% Tween 20;
a sample extraction liquid, the composition of the sample extraction liquid comprising: calcium chloride, tween 20 and sodium azide;
the substrate liquid A for the full-automatic immunoassay system comprises the following components: hydrogen peroxide;
the substrate liquid B for the full-automatic immunoassay system comprises the following components: sodium hydroxide.
An example of a method of using the hemoglobin-haptoglobin complex detection reagent-magnetic particle chemiluminescence;
the hemoglobin-haptoglobin complex detection reagent comprises: the kit comprises a biotin-labeled fecal occult blood antibody, an acridinium ester-labeled fecal occult blood antibody, a hemoglobin-haptoglobin compound calibrator, a hemoglobin-haptoglobin compound quality control product, a sample to be detected, a streptavidin-coated magnetic particle solution, a fecal occult blood determination calibration card, a fecal occult blood determination reagent card, a cleaning solution, a sample extraction liquid, and a substrate liquid A and a substrate liquid B for a full-automatic immunoassay system. The relevant information of a hemoglobin-haptoglobin compound calibrator burnt in the fecal occult blood determination calibrator card and the relevant information of a fecal occult blood determination calibration curve burnt in the fecal occult blood determination reagent card, wherein the main components of a cleaning solution are phosphate, sodium chloride and tween 20, the main components of a sample extraction liquid are calcium chloride, tween 20 and sodium azide, and the main component of a substrate liquid A for a full-automatic immunoassay system is hydrogen peroxide. The substrate liquid B for the full-automatic immunoassay system mainly comprises sodium hydroxide. The main component of the fecal occult blood determination standard substance and the fecal occult blood determination quality control substance is human fecal hemoglobin-haptoglobin compound antigen.
The detection method of the hemoglobin-haptoglobin compound detection reagent comprises the following steps of:
collecting and extracting a fresh human excrement sample by using a sample extraction liquid to obtain a clarified sample to be detected;
calibrating the hemoglobin-haptoglobin compound quantitative detection by using a hemoglobin-haptoglobin compound calibration product according to a calibration program;
adding 50 mu L of hemoglobin-haptoglobin compound quality control product and the sample into a designated reaction cup respectively;
adding 50 mu L of biotin-labeled fecal occult blood antibody into each reaction cup;
25. mu.L of streptavidin-coated magnetic microparticle solution was added to each reaction cup;
incubating at 37 ℃ for 15 min;
cleaning each reaction cup with cleaning solution for 3 times;
100. mu.L of acridinium ester-labeled fecal occult blood antibody was added to each reaction cup;
incubating at 37 ℃ for 10 min;
cleaning each reaction cup with cleaning solution for 3 times;
adding 200 mu L of substrate solution A for the full-automatic immune test system into each reaction cup;
adding 200 mu L of substrate solution B for a full-automatic immunoassay system into each reaction cup;
sequentially measuring on a full-automatic chemiluminescence apparatus, and processing data results according to calibration curve information burnt by the fecal occult blood measuring reagent card. The calibration curve of q-FOB reagent card burn is shown in the following table:
use of a hemoglobin-haptoglobin complex detection reagent for detecting lower gut health, the application steps comprising:
step one, physical examination sample collection and pretreatment; the inspection mechanism uses the sample extraction liquid to collect and extract regular excrement samples for physical examination personnel, and the extraction step is strictly carried out according to the instruction of the sample extraction liquid. The extracted sample to be tested can be preserved for 3 days at 15-25 ℃, 5 days at 2-8 ℃ and 3 months at-20 ℃, and is only frozen and thawed for 3 times. The specimen should be transported without violent shaking.
Step two, pretreatment of a detection reagent: before use, placing the hemoglobin-haptoglobin compound detection reagent at room temperature for rewarming, preparing a first labeled hemoglobin-haptoglobin compound antibody, a second labeled hemoglobin-haptoglobin compound antibody, a hemoglobin-haptoglobin compound calibration product, a hemoglobin-haptoglobin compound quality control product, a sample to be detected, coated magnetic particles, a cleaning solution, a substrate solution A for a full-automatic immunoassay system and a substrate solution B for the full-automatic immunoassay system; in one embodiment, the first labeled hemoglobin-haptoglobin complex antibody is a biotin-labeled fecal occult blood antibody, the second labeled hemoglobin-haptoglobin complex antibody is an acridinium ester-labeled fecal occult blood antibody, and the coated magnetic particles are streptavidin-coated magnetic particle solutions.
Step three, pretreatment of a calibration product, a quality control product and a cleaning solution: adding 1.0mL of distilled water or deionized water into each tube of the calibration material and the quality control material for dissolving, standing for 5 minutes, and turning upside down for uniformly mixing to ensure that all solids are completely dissolved and no bubbles are generated. After the use, the mixture is immediately stored at the temperature of-20 ℃ and is only frozen and thawed for 3 times; diluting the cleaning solution according to the specification requirement of the cleaning solution, and placing the diluted cleaning solution at the specified position of the full-automatic chemiluminescence tester.
Step four, calibration: putting the antibody and the magnetic particle solution into a reagent bottle sleeve, sweeping the instrument into a fecal occult blood measurement reagent card, and putting the reagent bottle sleeve into a specified position of the instrument; then, sweeping the fecal occult blood measuring calibration card into the instrument, and placing a calibration product into a specified reaction frame to calibrate the fecal occult blood quantitative detection;
putting the quality control product and the extracted sample to be detected into a reagent reaction frame designated on a full-automatic chemiluminescence tester, carrying out quantitative detection on fecal occult blood on the full-automatic chemiluminescence tester, and carrying out sample addition and operation (unit: mu L) according to the following programs:
step six, calculating: the chemiluminescence analyzer calculates concentration values of the sample and the quality control substance according to a calibration curve burnt in the fecal occult blood measuring reagent card and the measured relative light intensity (RLU).
Step seven, quality control: and comparing the measured concentration of the quality control product with the marked value range, and if the measured concentration exceeds the marked quality control range, indicating that the detection is unqualified and needing to be carried out again.
And step eight, judging the health condition according to the reference range and the detection result.
The reference ranges are as follows:
the definition value of normal healthy population is lower than 200 mug/g;
positive population definition values greater than 50 μ g/g;
the strong positive population has a defined value of greater than 1000. mu.g/g.
Application of reference range and interpretation of test results:
the hemoglobin-haptoglobin complex detection reagent explains the test results as follows:
the detection limit value of the healthy population is lower than 200 mug/g because the limit value is different in different antibodies and reaction systems, and the detection limit value of the normal population has certain fluctuation when detecting the population in different regions, for example, the detection limit value of the healthy population can be positioned as 180 mug/g, or 150 mug/g or 100 mug/g by detecting a certain amount of local healthy population, but the upper limit of the limit value must be lower than 200 mug/g. Therefore, when the result of the detection sample is higher than 200 mug/g, the sample can be judged to be positive, clinical explanation is that the symptom of lower gastrointestinal hemorrhage appears, endoscopic examination is needed to be carried out for further diagnosis, and treatment is needed under the guidance of a professional doctor; the definition value of the positive population is greater than 50 mug/g, the positive value can be determined by detecting a certain number of lower gastrointestinal hemorrhage patient stool samples and obtaining the detection result through a lower limit value obtained by a statistical method, different populations in different regions can determine the positive value according to the local positive sample result, for example, the positive sample detection definition value can be positioned as 60 mug/g, or 70 mug/g or 80 mug/g by detecting a certain number of local lower gastrointestinal hemorrhage patient stool samples, but the lower limit value of the definition value is necessarily greater than 50 mug/g. Therefore, when the detection result of the sample is lower than 50 mug/g, the sample can be judged to be negative, and clinical interpretation indicates that the lower gastrointestinal bleeding symptom does not appear;
the defining value of the strong positive population is more than 1000 mug/g, which is determined according to the content of hemoglobin-haptoglobin compound in the feces of the patient with lower gastrointestinal major hemorrhage. When the sample detection result is more than 1000 mug/g, the sample is judged to be strong positive, clinical explanation is severe gastrointestinal hemorrhage symptom, and timely intervention and treatment are required under the guidance of a professional doctor.
The reagent is used for carrying out the concentration research of the hemoglobin-haptoglobin compound of human feces, and then a percentile method is adopted to obtain a reference range for a sample result, wherein the reference range can well assist the evaluation of individual lower gastrointestinal hemorrhage and the diagnosis of symptoms related to the lower gastrointestinal hemorrhage, and is verified in the fourth experiment.
It should be noted that: the invention is a detection product, is not a method for diagnosing and treating diseases, but is a tool for assisting clinical judgment.
The defined values of 50. mu.g/g, 200. mu.g/g and 1000. mu.g/g are described below.
The invention takes 200 mug/g as the upper limit of the normal defined value, namely cut-off value, and has important significance. To establish the normal range for healthy population, in the laboratory, 3 cassettes of hemoglobin-haptoglobin complex detection reagents described in the present patent were used to determine the normal range for this item and to generate cut-off values for normal subjects. A total of 246 fresh stool samples from normal subjects were tested for this trial, ranging in age from 17-81 years. Statistics and analysis of the sample data were performed by the sps software, which detected sample data indicating subjects with a normal range of 0-590.4ng/mL and a 95% confidence interval of 33.34-57.38 ng/mL. The cut-off value of normal subjects is determined to be 400.04ng/mL according to the 95% confidence interval of normal human samples, and is converted into the content of hemoglobin-haptoglobin complex in each gram of feces according to the dilution multiple of the feces, and the cut-off value of the normal subjects is 200 mug/g.
The present invention uses 50. mu.g/g as the lower limit of the positive limit value. Through detecting fresh excrement samples of 120 patients with lower gastrointestinal hemorrhage, the patients with lower gastrointestinal hemorrhage are classified into mild, moderate and severe patients in the subject sample screening according to the clinical diagnosis result of the patients, wherein each degree is 40 samples, and the age range is 18-70 years old. In the laboratory, using 2 cases of the hemoglobin-haptoglobin complex detection reagent of the present invention, the test samples were measured in duplicate, and the mean value of the measurement results of each sample was taken as the final test result. And (3) carrying out data statistics and analysis on the detection result of the tested sample by using a sps software, and setting a 5% confidence interval of the detection result of the positive sample as a lower limit of a positive defining value, namely 50 mu g/g.
The invention defines 1000 mug/g as the strong positive definition value of fecal occult blood. The upper limit of the reagent linearity can reach 2000 mug/g, so that the strong positive result can be detected when the strong positive judgment value of 1000 mug/g is in the linear range of the detection. The strong positive judgment value of the occult blood of the stool is determined by 1000 mug/g according to the content of hemoglobin-haptoglobin compound in the feces of the patient with the gastrointestinal major hemorrhage. The hemorrhage of the digestive tract is more than 1000mL and is clinically regarded as the massive hemorrhage of the digestive tract, and about 10% of the hemorrhage of the digestive tract is discharged in the form of excrement, namely the excrement of a patient with the massive hemorrhage of the digestive tract has about 100mL of hemorrhage amount. In normal human blood, the hemoglobin content of women is 150g/L at 110-165 g/L and the hemoglobin content of men is 165g/L at 120-165g/L, the haptoglobin content in the normal human blood can be measured to be 0.3-2.0g/L through a hemoglobin binding method, the upper limit of the haptoglobin content can be defined as the hemoglobin-haptoglobin compound content in the blood, namely 2.0g/L, and then 0.2g of hemoglobin-haptoglobin compound exists in the excrement of a patient with gastrointestinal hemorrhage. The daily defecation amount of normal people is about 100-300g, 200g is the average level of the defecation of the normal people, and the content of the hemoglobin-haptoglobin compound in the excrement of the patient with the gastrointestinal hemorrhage is calculated to be 1000 mu g/g, so that the 1000 mu g/g is taken as the positive judgment value of the fecal occult blood.
The effect of the present invention is verified by experiments below;
Δ experiment one, sensitivity experiment:
measuring relative light intensity value (xi) of the sample with zero concentration calibrator, repeating the measurement for 20 times to obtain relative light intensity value (xi) of 20 measurement results, and calculating average value
Calculating the Standard Deviation (SD) according to the formula (1) to obtain
Corresponding relative light intensity values. According to the calibration curve equation burned by the reagent card in the reagent kit, the method is to
Substituting the corresponding relative light intensity value into the equation to obtain a corresponding concentration value, namely the sensitivity, wherein the sensitivity is not more than 0.5 mu g/g according to the industrial standard and the design requirement.
According to the above method, the test was conducted using the zero concentration calibrator L1 for sensitivity of the hemoglobin-haptoglobin complex detection reagent. The experimental results are as follows:
the results show that the sensitivity of the hemoglobin-haptoglobin composite detection reagent is 0.12ng/mL and is lower than the standard of 0.5ng/mL, which indicates that the detection sensitivity of the kit is higher.
Experiment two, the accuracy experiment:
and (3) taking a working calibration product with a concentration level, repeatedly detecting for 3 times, calculating the relative deviation (B%) according to a formula (4), and judging to be qualified if the relative deviation (B%) of the results of the 3 times is not more than 15.0% according to the industrial standard and the design requirement. If the results of 2 times or more fail, the product is judged to be unqualified. If the results are not met for 1 time, the test is repeated for 20 times, the relative deviation is calculated according to the formula (4), and if the relative deviation (B%) of the results of more than or equal to 19 times of tests is not more than 15.0%, the accuracy is met.
B％＝(M-T)/T×100％…………………………(3)
B%: relative deviation;
m: a working calibrator test value;
t: working calibrant target value.
The test sensitivity of the hemoglobin-haptoglobin complex test reagent was measured by the method described above using the working calibrator L3 for this test. The experimental results are as follows:
the results show that the relative deviation (B%) of the results of the hemoglobin-haptoglobin compound detection reagent is lower than the standard of 15.0%, which indicates that the accuracy of the kit is better.
Experiment three, precision experiment in batch:
taking 2 different concentrationsRepeating the detection for 10 times to obtain average values
Calculating Standard Deviation (SD) according to formula (1), calculating Coefficient of Variation (CV) according to formula (2), and calculating Coefficient of Variation (CV) not more than 10% according to industry standard and design requirement.
According to the above method, the quality control product 1 and the quality control product 2 for the test are subjected to an in-batch precision test of the hemoglobin-haptoglobin complex test reagent. The experimental results are as follows:
the results show that the average CV of the hemoglobin-haptoglobin compound detection reagent in-batch precision is 3.9 percent and is lower than the standard of 10 percent, which indicates that the precision of the kit is better.
Experiment four, clinical usage data for comparative experiments with hemoglobin-haptoglobin complex detection reagents were as follows:
a comparative test of the hemoglobin-haptoglobin complex detection reagent was performed at a clinical institution of Jiaxing. The examination reagent is a fecal occult blood determination kit (magnetic particle chemiluminescence method) produced by Jiaxing Rongjian Biotechnology Limited, namely the hemoglobin-haptoglobin compound detection reagent disclosed by the invention, and a matched detection instrument is a Nanjing Diganis ACL2800 full-automatic chemiluminescence determinator; the reference reagent selects an OC-Auto Micro 80 fecal occult blood detector and a reagent thereof of Polymedco company of New York, USA, and the cut-off value of the reference reagent is 100 ng/mL. The test site is the clinical laboratory of the clinical institution. The subjects received a clinical endoscopic examination in advance to determine whether they developed symptoms of gastrointestinal bleeding. 26 fresh fecal samples of subjects who did not develop a hemorrhagic digestive tract and 77 fresh fecal samples of subjects who developed a hemorrhagic digestive tract were randomly selected. The test specimens were 103 in total, 56 males and 47 females, with the age range of 18-83 years. The clinical contrast test is a synchronous double-blind parallel contrast test, and the examination reagent and the reference reagent are simultaneously compared with the 'gold standard' -clinical endoscopy examination result to obtain a result.
In the test process, the collection, treatment and test process of the fecal sample are respectively carried out according to the specification requirements of a fecal occult blood determination kit (magnetic particle chemiluminescence method), the use specification requirements of a Nanjing Digance ACL2800 full-automatic chemiluminescence tester, and the specifications of an OC-Auto Micro 80 fecal occult blood tester and a reagent thereof.
The following results of clinical comparative tests of the detection reagent for the calprotectin in the excrement are as follows:
and (4) processing a result:
and drawing a clinical comparison four-grid table according to the detection data result, wherein the four-grid table of the examination reagent and the endoscopy examination (gold standard) result is shown in table 1, and the four-grid table data of the reference reagent and the endoscopy examination (gold standard) result is shown in table 2.
TABLE 1 examination reagent-endoscopy (gold standard) four-grid table
TABLE 2 reference reagent-endoscopy (gold Standard) four-Table
From the four tables above we can see: the detection sensitivity of the assessment reagent is 92.3%, the detection specificity is 98.7%, the positive predictive value is 96.0%, and the negative predictive value is 97.4%; the detection sensitivity of the reference reagent is 84.6%, the detection specificity is 93.5%, the positive predictive value is 81.5%, and the negative predictive value is 94.7%. The clinical indexes of the assessment reagent are superior to those of a reference reagent, the detection sensitivity and specificity are better, and the assessment reagent is closer to the actual clinical test result.
The invention provides a reagent for quantitatively detecting fecal occult blood by chemiluminescence, a detection method thereof and application thereof in detecting lower digestive tract health.A reagent for quantitatively detecting fecal occult blood by chemiluminescence is prepared by obtaining two specific antibodies by immunology, combining the antibodies with a hemoglobin-haptoglobin compound to obtain an immune complex of a labeled first specific antibody-hemoglobin-haptoglobin compound-labeled second specific antibody, and completing detection by using a magnetic bead chemiluminescence detection system; the design can specifically detect hemoglobin-haptoglobin compound in a human fecal sample, has the advantages of high sensitivity and good stability, can well reflect the lower gastrointestinal bleeding condition, assists in early screening of lower gastrointestinal tumors such as rectal cancer or colon cancer and the like, and has high clinical application value.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.
Sequence listing
<110> Jiaxing xing Jian Biotech Co., Ltd
<120> reagent for chemiluminescence quantitative detection of fecal occult blood, detection method thereof and application thereof in detecting digestive tract health
<141> 2019-08-22
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<170> SIPOSequenceListing 1.0
<210> 1
<211> 321
<212> PRT
<213> human hemoglobin
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Gly Pro Gly Glu Leu Gln Gly Ser Ala Ala Arg Ala Phe His Gly Asp
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Thr Ser Arg Trp Asn Pro Glu Gln Leu Leu Leu Ala Ala Leu Ala Glu
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Cys His Ile Leu Ser Tyr Leu Phe Val Ala Gly Gln Glu Lys Leu Ala
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Val Ser His Ile Glu Val Gly Gly Glu Leu Leu Leu Ser His Asp Leu
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Lys Glu Gly Gly Gln Val Ala Ser Ala Val Leu Arg Pro Gln Val Trp
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Leu Asp Glu Ala Thr His Asp Gln Asp Pro Gln Ala Tyr Gln Arg Ala
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Gly Glu Leu His Glu Thr Ala His Gln Gln Cys Phe Val Ala Arg Ser
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Val Asn Phe Pro Val Ala Ile Lys Pro Val Thr Thr Gly Glu Pro Lys
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Arg Lys Ser Pro Gln Phe Thr Thr Arg Leu Gly Ala Pro Ile Ala Leu
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Arg Pro Pro Arg Pro Ala Lys Lys Pro Val Ser Val Thr Gly Ala Thr
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Gly Ser Pro Ala Val Glu Gln Arg Ser Phe Tyr Asp Gln Val Gly Gly
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His Glu Thr Phe Ala Lys Leu Ala Arg Val Phe Tyr Glu Gln Val Ala
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Thr Asp Pro Glu Phe Arg Ala Leu Tyr Pro Glu Glu Asp Leu Gly Pro
225 230 235 240
Ala Glu Ala Arg Leu Arg Met Phe Leu Glu Gln Tyr Trp Gly Gly Pro
245 250 255
Thr Thr Tyr Ser Gln Gln Arg Gly His Pro Arg Leu Arg Lys Arg His
260 265 270
Met Pro Phe Glu Ile Gly Pro Gln Glu Arg Asp Thr Trp Leu Lys Tyr
275 280 285
Met Lys Val Ala Val Lys Ser Leu Asp Leu Ala Pro Leu Gln Glu Glu
290 295 300
Thr Leu Trp Asp Tyr Leu Glu Arg Ala Ala His Ala Met Gln Asn Arg
305 310 315 320
Ser
<210> 2
<211> 406
<212> PRT
<213> human haptoglobin
<400> 2
Met Ser Ala Leu Gly Ala Val Ile Ala Leu Leu Leu Trp Gly Gln Leu
1 5 10 15
Phe Ala Val Asp Ser Gly Asn Asp Val Thr Asp Ile Ala Asp Asp Gly
20 25 30
Cys Pro Lys Pro Pro Glu Ile Ala His Gly Tyr Val Glu His Ser Val
35 40 45
Arg Tyr Gln Cys Lys Asn Tyr Tyr Lys Leu Arg Thr Glu Gly Asp Gly
50 55 60
Val Tyr Thr Leu Asn Asp Lys Lys Gln Trp Ile Asn Lys Ala Val Gly
65 70 75 80
Asp Lys Leu Pro Glu Cys Glu Ala Asp Asp Gly Cys Pro Lys Pro Pro
85 90 95
Glu Ile Ala His Gly Tyr Val Glu His Ser Val Arg Tyr Gln Cys Lys
100 105 110
Asn Tyr Tyr Lys Leu Arg Thr Glu Gly Asp Gly Val Tyr Thr Leu Asn
115 120 125
Asn Glu Lys Gln Trp Ile Asn Lys Ala Val Gly Asp Lys Leu Pro Glu
130 135 140
Cys Glu Ala Val Cys Gly Lys Pro Lys Asn Pro Ala Asn Pro Val Gln
145 150 155 160
Arg Ile Leu Gly Gly His Leu Asp Ala Lys Gly Ser Phe Pro Trp Gln
165 170 175
Ala Lys Met Val Ser His His Asn Leu Thr Thr Gly Ala Thr Leu Ile
180 185 190
Asn Glu Gln Trp Leu Leu Thr Thr Ala Lys Asn Leu Phe Leu Asn His
195 200 205
Ser Glu Asn Ala Thr Ala Lys Asp Ile Ala Pro Thr Leu Thr Leu Tyr
210 215 220
Val Gly Lys Lys Gln Leu Val Glu Ile Glu Lys Val Val Leu His Pro
225 230 235 240
Asn Tyr Ser Gln Val Asp Ile Gly Leu Ile Lys Leu Lys Gln Lys Val
245 250 255
Ser Val Asn Glu Arg Val Met Pro Ile Cys Leu Pro Ser Lys Asp Tyr
260 265 270
Ala Glu Val Gly Arg Val Gly Tyr Val Ser Gly Trp Gly Arg Asn Ala
275 280 285
Asn Phe Lys Phe Thr Asp His Leu Lys Tyr Val Met Leu Pro Val Ala
290 295 300
Asp Gln Asp Gln Cys Ile Arg His Tyr Glu Gly Ser Thr Val Pro Glu
305 310 315 320
Lys Lys Thr Pro Lys Ser Pro Val Gly Val Gln Pro Ile Leu Asn Glu
325 330 335
His Thr Phe Cys Ala Gly Met Ser Lys Tyr Gln Glu Asp Thr Cys Tyr
340 345 350
Gly Asp Ala Gly Ser Ala Phe Ala Val His Asp Leu Glu Glu Asp Thr
355 360 365
Trp Tyr Ala Thr Gly Ile Leu Ser Phe Asp Lys Ser Cys Ala Val Ala
370 375 380
Glu Tyr Gly Val Tyr Val Lys Val Thr Ser Ile Gln Asp Trp Val Gln
385 390 395 400
Lys Thr Ile Ala Glu Asn
405
Claims (5)
1. A reagent for chemiluminescence quantitative determination of fecal occult blood, which is characterized by comprising: an immune combination formed by an antibody detection system and a human hemoglobin-haptoglobin compound, and a magnetic bead chemiluminescence detection system of the immune combination; the fixed separation phase of the magnetic bead chemiluminescence detection system is magnetic particles, and the calibration product and the quality control product are human hemoglobin-haptoglobin compound; the immune combination body is 'magnetic bead-first specific antibody-human hemoglobin-haptoglobin compound-second specific antibody marked by luminescent marker';
the first specific antibody and the second specific antibody are generated by injecting a hemoglobin-haptoglobin compound into an animal, and then preparing a hemoglobin-haptoglobin compound polyclonal antibody obtained by immune reaction or preparing a hemoglobin-haptoglobin compound monoclonal antibody aiming at a specific antigenic determinant by a hybridoma technology;
the first specific antibody and the second specific antibody are any one of the following three antibodies:
the first specific antibody and the second specific antibody are specific antibodies combined with human hemoglobin monomers and specific antibodies combined with human haptoglobin monomers; the specific antibody and the human hemoglobin-haptoglobin compound form an immune combination body, the specific antibody combined with the human hemoglobin monomer is not combined with any human haptoglobin monomer, and the specific antibody combined with the human haptoglobin monomer is not combined with any human hemoglobin monomer;
the first specific antibody and the second specific antibody are specific antibodies combined with a human hemoglobin-haptoglobin compound and specific antibodies combined with a human haptoglobin monomer; the specific antibody and the hemoglobin-haptoglobin compound form an immune combination body, and the specific antibody combined with the human hemoglobin-haptoglobin compound is not combined with any human haptoglobin monomer or human hemoglobin monomer; the specific antibody that binds to human haptoglobin monomer does not bind to any human hemoglobin monomer;
the first specific antibody and the second specific antibody are specific antibodies combined with a human hemoglobin-haptoglobin compound and specific antibodies combined with a human hemoglobin monomer, the specific antibodies can form an immune combination body with the hemoglobin-haptoglobin compound, and the specific antibodies combined with the human hemoglobin-haptoglobin compound are not combined with any haptoglobin monomer or hemoglobin monomer; the specific antibody that binds to human hemoglobin does not bind to any haptoglobin monomer.
2. The reagent for chemiluminescent quantitative detection of fecal occult blood according to claim 1, wherein the immobilized separation phase is streptavidin coated magnetic beads, and the magnetic beads are conjugated with biotin labeled first specific antibody.
3. The reagent for chemiluminescent quantitative detection of fecal occult blood according to claim 1, wherein the immobilized separation phase is a first specific antibody coated magnetic bead.
4. The reagent for the quantitative chemiluminescent detection of fecal occult blood according to claim 1, wherein the second specific antibody is an antibody labeled with a luminescent marker which is acridinium ester.
5. The reagent for chemiluminescent quantitative detection of fecal occult blood according to claim 1, wherein the first specific antibody, the second specific antibody is polyclonal antibody or monoclonal antibody.
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| CN201910779607.7A CN110361547B (en) | 2019-08-22 | 2019-08-22 | Reagent for chemiluminescence quantitative detection of fecal occult blood, detection method thereof and application of reagent in detection of lower digestive tract health |
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| CN201910779607.7A CN110361547B (en) | 2019-08-22 | 2019-08-22 | Reagent for chemiluminescence quantitative detection of fecal occult blood, detection method thereof and application of reagent in detection of lower digestive tract health |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112979799B (en) * | 2019-12-18 | 2022-11-08 | 东莞市朋志生物科技有限公司 | Binding protein containing hemoglobin antigen structural domain |
| CN111044470A (en) * | 2019-12-31 | 2020-04-21 | 大连德泰克森生物医药有限公司 | Hemoglobin semi-quantitative detection reagent and method for collecting and detecting fecal occult blood specimen |
| CN111381046A (en) * | 2020-03-12 | 2020-07-07 | 迪瑞医疗科技股份有限公司 | Calprotectin chemiluminescence immunoassay kit and preparation method thereof |
| CN112557647A (en) * | 2020-12-10 | 2021-03-26 | 宁波华仪宁创智能科技有限公司 | Detection method based on immunochromatography technology and application |
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| JPH05172809A (en) * | 1991-01-03 | 1993-07-13 | Masashi Funayama | Feces occult blood uniquely detecting method and feces occult blood quantitative kit using it |
| CN101236210A (en) * | 2008-01-29 | 2008-08-06 | 王学生 | Rapid joint inspection reagent for hemoglobin -haptoglobin complex and occult blood in stool |
| CN202903803U (en) * | 2012-07-27 | 2013-04-24 | 王学生 | Kit for quick combined detection of hemoglobin-haptoglobin complex and fecal occult blood |
| CN104515859A (en) * | 2014-12-17 | 2015-04-15 | 杭州慧缘泰医疗器械有限公司 | Hemoglobin, hemoglobin-haptoglobin composite and transferrin joint examination kit and preparation method and detection method thereof |
| CN107543932A (en) * | 2017-10-31 | 2018-01-05 | 太原瑞盛生物科技有限公司 | The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of calcitonin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080227208A1 (en) * | 2007-03-16 | 2008-09-18 | Hsiao-Ching Yee | Devices and Methods for Detection of Occult Blood |
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| JPH05172809A (en) * | 1991-01-03 | 1993-07-13 | Masashi Funayama | Feces occult blood uniquely detecting method and feces occult blood quantitative kit using it |
| CN101236210A (en) * | 2008-01-29 | 2008-08-06 | 王学生 | Rapid joint inspection reagent for hemoglobin -haptoglobin complex and occult blood in stool |
| CN202903803U (en) * | 2012-07-27 | 2013-04-24 | 王学生 | Kit for quick combined detection of hemoglobin-haptoglobin complex and fecal occult blood |
| CN104515859A (en) * | 2014-12-17 | 2015-04-15 | 杭州慧缘泰医疗器械有限公司 | Hemoglobin, hemoglobin-haptoglobin composite and transferrin joint examination kit and preparation method and detection method thereof |
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