CN114660292A - Kit for detecting C5 complement and detection method and application thereof - Google Patents
Kit for detecting C5 complement and detection method and application thereof Download PDFInfo
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- CN114660292A CN114660292A CN202011527584.XA CN202011527584A CN114660292A CN 114660292 A CN114660292 A CN 114660292A CN 202011527584 A CN202011527584 A CN 202011527584A CN 114660292 A CN114660292 A CN 114660292A
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Abstract
The invention belongs to the field of immunodetection, and relates to a kit for detecting C5 complement, a detection method and application thereof. In particular, the invention relates to a kit for detecting C5 complement, and application thereof in preparing a diagnostic agent for detecting pathogen infection, a diagnostic agent for early diagnosis and screening of tumor markers, or a reagent or kit for detecting C5 complement. The kit provided by the invention has the advantages of high detection result accuracy, high precision, good stability and good linearity, can be used for primary screening and postoperative tracking of cancers, and provides reliable information for the risk degree of tumor progression; can also be used for detecting one or more pathogens in viruses, bacteria, fungi, mycoplasma, chlamydia and rickettsia, and has clinical guidance significance.
Description
Technical Field
The invention belongs to the field of immunodetection, and particularly relates to a kit for detecting C5 complement, and a detection method and application thereof.
Background
Complement component 5(The fifth component of complement, C5) is an important component in The complement system, and The C5 gene is reported in several organisms including human, mouse, rainbow trout and cattle. The human C5 gene is located in the region 32-34 of the long arm of chromosome 9. The C5 precursor protein consists of two polymorphic chains of alpha and beta through disulfide bonds, and has the molecular weight of 190 kDa. The 74 th-75 th arginine-leucine bond near the N-terminal is the site of action of the C5 convertase. Under the action of C5 convertase, the inactive C5 precursor protein is cleaved from the N-terminal of C5 enzyme chain to obtain a small fragment C5a, which enters the liquid phase, and the rest is C5b, which is still bound on the surface of cell membrane.
Infection is caused by invasion of bacteria and viruses into organisms, pathogens cannot be identified efficiently, the abuse phenomenon of antibiotics is more and more serious, drug resistance is caused by bacterial infection, and the drug resistance of antibiotics is generated continuously. The clinical bacterial culture needs 3-5 days, the waiting time is too long, the treatment is delayed, a doctor cannot give the medication scheme of a patient immediately, and the disease pathogenesis is delayed after the medicine is reused after the bacterial culture.
The C5 determination method is characterized in that the immunodiffusion method and the ELISA double-antibody sandwich technology are adopted in all countries in the world, including China, the method is time-consuming and labor-consuming, the accuracy is low, and 2 days are required for patients to take reports. The detection kit of human complement C5 on the market adopts a double-antibody two-step sandwich enzyme-linked immunosorbent assay (ELISA). Adding the standard substance and the sample to be detected into a transparent enzyme-labeled coated plate which is coated with complement component 5(C5) in advance, incubating for enough time, washing to remove the unbound components, adding an enzyme-labeled working solution, incubating for enough time, and washing to remove the unbound components. Adding a substrate A, B in sequence, converting the substrate (TMB) into a blue product under the catalysis of Horse Radish Peroxidase (HRP), changing the color into yellow under the action of acid, positively correlating the shade of the color with the concentration of the complement component 5(C5) in the sample, measuring the OD value at the wavelength of 450nm, and calculating the content of the complement component 5(C5) in the sample according to the OD values of the standard substance and the sample.
Due to the defects in the prior art, a kit for rapidly and accurately detecting the concentration of C5 complement is urgently needed to be provided.
Disclosure of Invention
Problems to be solved by the invention
Due to the problems in the prior art, the invention aims to provide a kit for detecting bacterial and/or viral infection, which is rapid, high in result accuracy, high in precision, good in stability and good in linearity.
Means for solving the problems
(1) A kit for detecting C5 complement, wherein the kit comprises:
reagent I: a first buffer solution and a first PEG buffer solution;
and (2) reagent II: the anti-C5 complement antibody comprises a monoclonal antibody and/or a polyclonal antibody, a second PEG buffer solution and IgG; the pH value of the first buffer solution is 3-10.
(2) The kit according to (1), wherein the anti-C5 complement antibody is selected from one or a combination of two or more of rabbit anti-human C5 complement antibody, sheep anti-human C5 complement antibody and mouse anti-human C5 complement antibody; preferably, the rabbit anti-human C5 complement antibody, the sheep anti-human C5 complement antibody and the mouse anti-human C5 complement antibody are monoclonal antibodies or polyclonal antibodies; optionally, the anti-C5 complement antibody has an antibody titer of 1:64 to 1:128 and a concentration of 100ml/L to 800 ml/L.
(3) The kit according to (1) or (2), wherein the lipophilic group of the anti-C5 complement antibody is changed to hydrophilic after the polyclonal antibody is modified by treatment with trace amounts of manganese dioxide or potassium permanganate.
(4) The kit according to (1) or (2), wherein the lipophilic group of the anti-C5 complement antibody is changed into hydrophilic after the C5 complement monoclonal antibody is modified by treatment with trace manganese dioxide or potassium permanganate.
(5) The kit according to any one of (1) to (4), wherein the anti-C5 complement antibody is covalently bound to a latex particle; preferably, the anti-C5 complement monoclonal antibody is bound first and then bound to the C5 complement polyclonal antibody by covalent binding to latex particles via a carbodiimide reaction.
(6) Use of the kit according to any one of (1) to (5) for the preparation of a diagnostic agent for detecting infection by a pathogen; optionally, the pathogen comprises one or more of a virus, a bacterium, a fungus, a mycoplasma, a chlamydia, a rickettsia.
(7) Use of the kit according to any one of (1) to (6) in the preparation of a diagnostic agent for early diagnosis and screening of a tumor marker, wherein the tumor marker is selected from thyroid cancer, liver cancer, gastric cancer, uterine cancer, cervical cancer, lung cancer, breast cancer, esophageal cancer, nasopharyngeal cancer, prostate cancer, colorectal cancer, renal cancer, pancreatic cancer, bladder cancer, and glioma.
(8) The use according to (6) or (7), wherein the detection is carried out using whole blood, peripheral blood or serum.
(9) Use of the kit according to any one of (1) to (8) for the preparation of a diagnostic agent for detecting C5 complement by a method comprising: immunotransmission turbidimetry, immunoscattering turbidimetry, immunolatex, chemiluminescence, colloidal gold filtration or chromatography, and real-time detection; preferably, the immunoturbidimetry comprises immunotransmission turbidimetry, an immunoscattering turbidimetry wavelength of 340nm, and an immunolatex method wavelength of 600nm-700 nm. Especially in the colloidal gold percolation method or chromatography, Poct (point of care), chemiluminescence method, microfluidic chip and magnetic particle method, the mouse monoclonal antibody is added to improve the sensitivity of the reagent, and the C5 complement polyclonal antibody is added to improve the linearity, wherein the monoclonal antibody comprises mouse, rabbit, sheep and horse cell strains, and the polyclonal antibody comprises mouse, rabbit, sheep and horse antibodies.
(10) The use according to (9), wherein human peripheral serum, whole blood and peripheral blood are detected.
(11) The use according to (9) or (10), wherein the immunoturbidimetry comprises the steps of:
sample reagent I addition step: adding the sample into the reagent I, incubating, and reading the sample A1 at the 1 st point;
sample reagent II addition step: after the sample reagent I addition step, reagent II is added, incubated, and sample A2 at point 2 is read.
Adding a calibrator reagent I: adding the calibrator into the reagent I, incubating, and reading the calibrator A1 at the 1 st point;
adding a calibrator reagent II: after the step of adding the calibrator reagent I, adding a reagent II, incubating, and reading and measuring a2 nd point calibrator A2; wherein,
the C5 complement concentration (mg/L) was calculated by the following equation:
(12) use of the kit according to any one of (1) to (5) in the preparation of a reagent or kit for detecting C5 complement.
ADVANTAGEOUS EFFECTS OF INVENTION
The kit provided by the invention has the advantages of high detection result accuracy, high precision, good stability and good linearity.
In one embodiment of the invention, the kit of the invention can be used to remove useless antibody, so that the affinity between the antibody and the antigen is enhanced, the binding speed is accelerated, and the end point is accelerated.
The immunoturbidimetry method can give a report in 10 minutes, is convenient and rapid, has high accuracy, shortens the waiting time of patients, and can see the report in 10 minutes.
The kit for detecting, diagnosing and screening thyroid cancer, liver cancer, stomach cancer, uterine cancer, cervical cancer, lung cancer, breast cancer, esophageal cancer, nasopharyngeal cancer, prostatic cancer, colorectal cancer, kidney cancer, pancreatic cancer, bladder cancer and glioma can improve the accuracy of cancer diagnosis, has good specificity, can be used for primary screening and postoperative tracking of the cancer, and provides reliable information for the risk degree of tumor progression; can also be used for detecting one or more pathogens in viruses, bacteria, fungi, mycoplasma, chlamydia and rickettsia, and has clinical guidance significance.
Drawings
FIG. 1 shows that the kit of the present invention 3 detects six concentration-gradient standards, each concentration gradient is detected 3 times, an average value is obtained, the regression analysis is performed on the average value of the measured values and the theoretical concentration of the standard, the X-axis represents the theoretical concentration, and the Y-axis represents the average value of the measured values.
Fig. 2 shows a schematic structural diagram of the colloidal gold test strip.
FIG. 3 shows the interpretation of the results of the C5 complement assay using a colloidal gold test strip.
Detailed Description
Other objects, features and advantages of the present application will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
Furthermore, in the following detailed description, numerous specific details are set forth in order to provide a better understanding of the present invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In other instances, methods, means, devices and steps which are well known to those skilled in the art have not been described in detail so as not to obscure the invention.
It should be noted that:
in the present specification, the numerical range represented by "a numerical value a to B numerical value B" means a range including the end point numerical value A, B.
In the present specification, "PEG" means polyethylene glycol; here, "PEG 1000" means PEG having a polymerization degree of 1000.
Reference in the specification to "some specific/preferred embodiments," "other specific/preferred embodiments," "embodiments," and so forth, means that a particular element (e.g., feature, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the described elements may be combined in any suitable manner in the various embodiments.
< first aspect >
The invention provides a kit for detecting C5 complement, which comprises:
reagent I: a first buffer solution and a first PEG solution;
and (2) reagent II: anti-C5 complement antibody, second PEG solution, IgG; the pH value of the first buffer solution is 3-10.
The inventor of the present invention found that the C5 antigen can be attached to a bacterial extract or a virosome, and the amount of bacterial or viral infection can be determined by measuring the C5 concentration, and the presence or absence of bacterial or viral infection in a patient can be determined.
The immunoturbidimetry method can give a report only in 10 minutes, is convenient and rapid, has high accuracy, shortens the waiting time of patients, and can see the report in 10 minutes. The colloidal gold infiltration method or the chromatography and the (Poct) immediate examination are faster, the report can be accurately output in 2 minutes, and the waiting time of the patient is reduced. The method is expected to replace an ELISA double-antibody sandwich method for detecting C5 complement, and the method is used for detecting C5 complement by using an immune transmission turbidimetric method, an immune scattering turbidimetric method, an immune latex method, a chemiluminescence method, a microfluidic chip method, a colloidal gold percolation method or a chromatography method, a Poct instant detection method and the like.
Pathogens detectable by the kit of the invention include: one or more of viruses, bacteria, fungi, mycoplasma, chlamydia, rickettsia.
Further, viruses detectable by the kit of the present invention include RNA viruses, DNA viruses and protein viruses. In some specific embodiments, the virus detected by the kit of the present invention is selected from one or more of hepatitis virus, enterovirus, measles virus, rubella virus, herpes virus, mumps virus, influenza virus, avian influenza virus, prion, coronavirus, adenovirus, respiratory syncytial virus. Illustratively, the hepatitis virus may be hepatitis a, b, c, d, e, etc.; the enterovirus can be norovirus, rotavirus, coxsackievirus, echovirus, astrovirus and the like; the herpesvirus may be herpes zoster virus, EB virus, etc.; the influenza virus can be influenza A virus, influenza B virus, parainfluenza virus, etc.;
further, bacteria detectable by the kit of the present invention include cocci, bacilli and spirochetes. In some specific embodiments, the viruses detected by the kits of the invention are selected from the group consisting of gram-positive aerobic cocci, gram-negative aerobic bacilli, gram-negative facultative anaerobes, gram-negative anaerobic bacilli, and the like. In some more specific embodiments, the bacteria detected by the kits of the invention are selected from one or more of the bacteria of the genera staphylococcus, streptococcus, enterococcus, neisseria, moraxella, acinetobacter, pseudomonas, alcaligenes, brunella, bordetella, legionella, escherichia, citrobacter, salmonella, shigella, klebsiella, serratia, proteus, plaffield, morganella, yersinia, haemophilus, vibrio, aeromonas, peptococcus, micrococcus, bacteroides, clostridium, anthrax, bacillus, clostridium, listeria, erysipelothrix, corynebacterium, actinomyces, mycobacterium, shigella.
Illustratively, the genus Staphylococcus includes Staphylococcus aureus, Staphylococcus epidermidis, etc., the genus Streptococcus includes alpha-hemolytic streptococcus, beta-hemolytic streptococcus, non-hemolytic streptococcus, pneumococcus, etc., the genus enterococcus includes enterococcus, etc., the genus Neisseria includes meningococcus, gonococcus, Neisseria, etc., the genus Moraxella includes Moraxella catarrhalis, etc., the genus Acinetobacter includes Acinetobacter nitorum, Acinetobacter lofoerium, etc., the genus Pseudomonas includes Pseudomonas aeruginosa, etc., the genus Alcaligenes includes Alcaligenes faecalis, the genus Bordetella includes Bordetella, etc., the genus Escherichia includes Escherichia coli, etc., the genus Yersinia includes Yersinia pestis, etc., the genus Haemophilus includes Bacillus influenzae, etc., the genus Vibrio includes Vibrio cholerae, Vibrio Eltor, Vibrio parahemolytic vibrio, etc., the genus Aeromonas includes Aeromonas hydrophila, etc., the genus Pediococcus includes peptococcus, etc., the genus Peptostreptococcus includes Streptococcus digestions and the like, the genus Peptococcus includes Micrococcus freudenreichii and the like, the genus Clostridium includes Bacteroides fragilis, Clostridium and the like, the genus Bacillus includes Bacillus anthracis and the like, the genus Bacillus includes Bacillus cereus, Bacillus tetani and the like, the genus Clostridium includes Clostridium botulinum, Clostridium difficile and the like, the genus Corynebacterium includes Corynebacterium diphtheria and the like, the genus Mycobacterium includes Mycobacterium tuberculosis, Bacillus leprae and the like, the genus Shigella includes Bacillus dysenteriae and the like, the genus Actinomyces includes Nocardia stelleri and the like, the genus Klebsiella includes Bacillus pneumoniae and the like, and the genus Legionella includes Legionella pneumophila and the like.
Furthermore, the kit can also detect fungi such as candida, aspergillus, cryptococcus neoformans and mucor, mycoplasma such as mycoplasma pneumoniae, rickettsia such as Q-fever, or pathogens such as chlamydia.
The kit of the invention can also detect tumor markers: thyroid cancer, liver cancer, gastric cancer, uterine cancer, cervical cancer, lung cancer, breast cancer, esophageal cancer, nasopharyngeal cancer, prostate cancer, colorectal cancer, kidney cancer, pancreatic cancer, bladder cancer, and glioma.
Reagent I
The reagent I comprises a first buffer solution and a first PEG solution; the pH value of the first buffer solution is 3-10.
In some embodiments of the invention, tris buffer may be used. The tris buffer is prepared by mixing tris solution and hydrochloric acid solution in proportion. In a preferred embodiment, the pH value of the tris buffer in the present invention is 7.5 to 9.5; in a more preferred embodiment, the tris buffer has a pH of 8.1.
In the present invention, the first PEG and the second PEG may be the same or different.
The reagent I comprises a first PEG solution (polyethylene glycol), when an anti-C5 complement antibody (antibody) is combined with C5 complement (antigen) to form an immune complex with a certain structure, the immune complex becomes microparticles suspended in a reaction solution under the action of the PEG, so that the turbidity of a sample is changed.
In some specific embodiments of the invention, the first PEG used in agent I comprises: PEG 1000; the second PEG used in the reagent II is PEG 6000-10000, and the reaction is accelerated and strengthened by utilizing the PEG with a great molecular weight difference between the reagent I and the reagent II. The absorbance at the end of the reaction is stable, and the affinity is stronger than that of the general reagent. In the reagent I, the concentration of the PEG1000 is 400 g/L-600 g/L of the PEG 1000; in a preferred embodiment, the concentration of PEG1000 is 500g/L, and the concentration of PEG1000 reduces non-specific reaction in the sample.
When the anti-C5 complement antibody used in the invention is anti-C5 complement antiserum, the other antibodies except the C5 complement antibody in the C5 complement antiserum are combined by the calcium chloride, so that useless antibodies can be removed, the affinity of the antibodies and the antigen is enhanced, the combination speed is increased, and the end point is accelerated. In a preferred embodiment, the concentration of the calcium chloride is 0.25g/mL to 0.35 g/mL; in a more preferred embodiment, the calcium chloride is present at a concentration of 0.294 g/mL. In the invention, CaCl is added2The affinity of the antibody and the antigen is enhanced, and the reaction and combination speed is accelerated. More importantly, the reaction linearity of the immunoturbidimetry is greatly improved.
Reagent II
The reagent II comprises an anti-C5 complement antibody, a second PEG solution and IgG.
The reagent II comprises an anti-C5 complement antibody, and the anti-C5 complement antibody can be a polyclonal antibody or a monoclonal antibody. In some embodiments of the invention, the polyclonal antibody is an antiserum containing anti-C5 complement antibodies. In a preferred embodiment, the anti-C5 complement antibody of the invention is a monoclonal antibody. In some embodiments of the invention, the anti-C5 complement antibody is selected from one or a combination of two or more of a rabbit anti-human C5 complement antibody, a goat anti-human C5 complement antibody, and a mouse anti-human C5 complement antibody. In a preferred embodiment, the rabbit anti-human C5 complement antibody, the sheep anti-human C5 complement antibody, and the mouse anti-human C5 complement antibody are monoclonal antibodies. In some specific embodiments of the invention, the concentration of the anti-C5 complement antibody is 100ml/L to 800 ml/L. In a preferred embodiment, the concentration of the anti-C5 complement antibody is 600 ml/L.
The second PEG is included in the reagent II, and the second PEG which can be used in the reagent II comprises: PEG 6000-10000. For example, the second PEG solution may be one or more of PEG with a molecular weight of 6000, 7000, 8000, 9000 or 10000. The concentration of PEG 6000-10000 in the reagent II is 50 g/L-70 g/L; preferably, the concentration of the PEG 6000-10000 is 60 g/L. When the anti-C5 complement antibody (antibody) is combined with C5 complement (antigen) to form immune complex with a certain structure, the immune complex becomes particles suspended in the reaction solution under the action of PEG, so that the turbidity of the sample is changed. In the invention, the PEG1000 is 400-600 g/L in the reagent I, the PEG 6000-10000 is 50-70 g/L in the reagent II, and the reaction is accelerated and strengthened by utilizing the great difference of the ratio of the reagent I to the reagent II. The absorbance at the end of the reaction is stable, and the affinity is stronger than that of the general reagent.
In some embodiments of the invention, the reagent II further comprises a second buffer. The pH value of the second buffer solution is 3-10; preferably 8.1. In a preferred embodiment, the second buffer is tris buffer. The pH of the buffer was 8.1, the reaction was accelerated, and the endpoint arrival rate was fast.
In some embodiments of the invention, a trace amount of manganese dioxide or potassium permanganate, or manganese dioxide and potassium permanganate is added to modify the antibody before the antibody is treated with PEG, and the effect is achieved before the saturation of manganese dioxide or potassium permanganate, or manganese dioxide and potassium permanganate, and in practical applications, the effect is best when the concentration of manganese dioxide and potassium permanganate is 5-15 mg/L. After the polyclonal antibody is treated and modified by trace manganese dioxide or potassium permanganate, the lipophilic group of the anti-C5 complement antibody is changed into hydrophilic, so that the antibody has higher efficiency and stronger efficiency, the reagent has better stability, can be stored for 24 months with longer storage period, has better linearity and stronger anti-interference performance.
In some embodiments of the invention, a trace amount of manganese dioxide or potassium permanganate or manganese dioxide and potassium permanganate is added to modify the antibody before the antibody is treated with PEG, and the effect is achieved before the saturation of manganese dioxide or potassium permanganate or manganese dioxide and potassium permanganate, but in practical applications, the effect is best when the concentration of manganese dioxide and potassium permanganate is 5-15 mg/L. After the C5 complement monoclonal antibody is modified by trace manganese dioxide or potassium permanganate treatment, the lipophilic group of the anti-C5 complement antibody is changed into hydrophilic. Can improve the sensitivity of the reagent, lead the reagent to be fast and stable, have small error and correspondingly drive the polyclonal C5 complement antibody to have better linear reaction.
In some embodiments of the invention, the anti-C5 complement antibody is covalently bound to the latex particle. As a preferred embodiment, the anti-C5 complement antibody is covalently bound to the latex particle by a carbodiimide reaction. The kind and size of the latex particles are not particularly limited in the present invention, and latex particles having a diameter of 15 to 120nm can be used.
< second aspect >
In a second aspect the invention provides a kit according to the first aspect for use in the preparation of a diagnostic for the detection of C5 complement by a method comprising: immunoturbidimetry and/or chemiluminescence. The immunoturbidimetry in the invention is selected from the group consisting of immunotransmission turbidimetry, immunoscattering turbidimetry, and immunolatex.
The immunoturbidimetry method of the invention comprises the following steps:
sample reagent I addition step: adding the sample into the reagent I, incubating, and reading the sample A1 at the 1 st point;
sample reagent II addition step: after the sample reagent I addition step, reagent II is added, incubated, and sample A2 at point 2 is read.
Adding a calibrator reagent I: adding the calibrator into the reagent I, incubating, and reading the calibrator A1 at the 1 st point;
adding a calibrator reagent II: after the calibrator reagent I addition step, reagent II is added, incubation is performed, and the calibrator A2 is read at point 2.
After immunoturbidimetric determination, the C5 complement concentration (mg/L) was calculated by the following equation:
specifically, the immunoturbidimetry in the present invention comprises: adding a reagent I160 ul into the sample, incubating at 37 ℃ for 5min, reading a sample A1 at the 1 st point, adding a reagent II 40ul, incubating at 37 ℃ for 5min, reading a sample A2 at the 2 nd point, wherein the sample A is a sample A2-a sample A1; the method also comprises a step of measuring the calibrator by a biochemical analyzer, wherein the calibrator is added into the reagent I160 ul, incubated at 37 ℃ for 5min, the calibration A1 at the 1 st point is read, the reagent II 40ul is added, incubated at 37 ℃ for 5min, the calibration A2 at the 2 nd point is read, and the calibration A is calibration A2-calibration A1;
in one embodiment, the ratio of the reagent I and the reagent II added in the above method may be appropriately adjusted in a ratio of 4: 1.
The temperature measured by the immunoturbidimetry is 36.8-37.2; in a preferred embodiment, the temperature of the immunoturbidimetric assay is 37 ℃, the dominant wavelength of the immunoturbidimetric assay is 340nm, the minor wavelength of the immunoturbidimetric assay is 700nm, the amount of the immunoturbidimetric assay sample or calibrator added is 3. mu.l to 5. mu.l, and the reaction time is 10 minutes.
The immunotransmission turbidimetry, immunoscattering turbidimetry, latex-enhanced immunoturbidimetry, chemiluminescence, and Poct instant detection latex-enhanced immunoturbidimetry further comprise C5 calibrator comprising 5g bovine albumin, 0.3ml Proclin, and water added to 100ml for extracting C5 complement antigen. The quality control product is composed of 700mg/L bovine albumin, 350mg/L Proclin, and C5 complement antigen extracted.
< third aspect >
In a third aspect of the invention there is provided the use of an anti-C5 complement antibody in the manufacture of a diagnostic for the detection of C5 complement.
The invention discovers that the thallus of bacteria and viruses is adhered to the antigen of C5 complement, and the content of the bacteria and the viruses can be known by detecting the concentration of the C5 complement antibody.
The invention can be used for bacterial infection and virus infection by detecting the C5 complement, and can be used for comparing before and after treatment to identify whether the disease is improved or not, and the diagnosis accuracy rate of the combined application of a plurality of projects can reach 100%. Pathogens include: one or more of viruses, bacteria, fungi, mycoplasma, chlamydia, rickettsia.
Further, viruses include RNA viruses, DNA viruses and protein viruses. In some specific embodiments, the virus detected is selected from one or more of hepatitis virus, enterovirus, measles virus, rubella virus, herpes virus, mumps virus, influenza virus, avian influenza virus, prion, coronavirus, adenovirus, respiratory syncytial virus. Illustratively, the hepatitis virus may be hepatitis a, b, c, d, e, etc.; the enterovirus can be norovirus, rotavirus, coxsackievirus, echovirus, astrovirus and the like; the herpesvirus may be herpes zoster virus, EB virus, etc.; the influenza virus can be influenza A virus, influenza B virus, parainfluenza virus, etc.;
further, bacteria include cocci, bacilli and spirochetes. In some embodiments, the viruses detected by the kits of the invention are selected from the group consisting of gram-positive aerobic cocci, gram-negative aerobic bacilli, gram-negative facultative anaerobes, gram-negative anaerobic bacilli, and the like. In some more specific embodiments, the bacteria detected are selected from one or more of staphylococcus, streptococcus, enterococcus, neisseria, moraxella, acinetobacter, pseudomonas, alcaligenes, brucella, bordetella, legionella, escherichia, citrobacter, salmonella, shigella, klebsiella, serratia, proteus, pluriphenylene, morganella, yersinia, haemophilus, vibrio, aeromonas, peptococcus, peptostreptococcus, micrococcus, bacteroides, clostridium, anthrax, bacillus, clostridium, listeria, erysipelothrix, corynebacterium, actinomyces, mycobacterium, shigella.
Illustratively, the genus Staphylococcus includes Staphylococcus aureus, Staphylococcus epidermidis, etc., the genus Streptococcus includes alpha-hemolytic streptococcus, beta-hemolytic streptococcus, non-hemolytic streptococcus, pneumococcus, etc., the genus enterococcus includes enterococcus, etc., the genus Neisseria includes meningococcus, gonococcus, Neisseria, etc., the genus Moraxella includes Moraxella catarrhalis, etc., the genus Acinetobacter includes Acinetobacter nitorum, Acinetobacter lofoerium, etc., the genus Pseudomonas includes Pseudomonas aeruginosa, etc., the genus Alcaligenes includes Alcaligenes faecalis, the genus Bordetella includes Bordetella, etc., the genus Escherichia includes Escherichia coli, etc., the genus Yersinia includes Yersinia pestis, etc., the genus Haemophilus includes Bacillus influenzae, etc., the genus Vibrio includes Vibrio cholerae, Vibrio Eltor, Vibrio parahemolytic vibrio, etc., the genus Aeromonas includes Aeromonas hydrophila, etc., the genus Pediococcus includes peptococcus, etc., the genus Peptostreptococcus includes Streptococcus digestions and the like, the genus Peptococcus includes Micrococcus freudenreichii and the like, the genus Clostridium includes Bacteroides fragilis, Clostridium and the like, the genus Bacillus includes Bacillus anthracis and the like, the genus Bacillus includes Bacillus cereus, Bacillus tetani and the like, the genus Clostridium includes Clostridium botulinum, Clostridium difficile and the like, the genus Corynebacterium includes Corynebacterium diphtheria and the like, the genus Mycobacterium includes Mycobacterium tuberculosis, Bacillus leprae and the like, the genus Shigella includes Bacillus dysenteriae and the like, the genus Actinomyces includes Nocardia stelleri and the like, the genus Klebsiella includes Bacillus pneumoniae and the like, and the genus Legionella includes Legionella pneumophila and the like.
Furthermore, the pathogen of Candida, Aspergillus, Cryptococcus neoformans, Mucor, mycoplasma such as Mycoplasma pneumoniae, rickettsia such as Q-fever, or Chlamydia can be detected.
Meanwhile, the screening reagent can also be used for screening tumor markers: thyroid cancer, liver cancer, gastric cancer, uterine cancer, cervical cancer, lung cancer, breast cancer, esophageal cancer, nasopharyngeal cancer, prostatic cancer, colorectal cancer, kidney cancer, pancreatic cancer, bladder cancer and glioma, and the accuracy rate can reach 90-98%. Can be popularized to the public, and can screen thyroid cancer, liver cancer, gastric cancer, uterine cancer, cervical cancer, lung cancer, breast cancer, esophageal cancer, nasopharyngeal cancer, prostatic cancer, colorectal cancer, renal cancer, pancreatic cancer, bladder cancer and glioma by simply one drop of blood.
Examples
Example 1: kit composition for detecting C5 concentration by latex immunoturbidimetry
The kit for detecting the concentration of C5 by using the enhanced immunoturbidimetry comprises two reagents: reagent I is prepared from tris buffer (pH8.1). 0.294g of calcium chloride is taken, 0.2mol/L of tris solution is added for dissolution, 1mol/L of hydrochloric acid solution is used for adjusting the pH value to 8.1, PEG 100050 g is added, water is added for dilution to 100ml, in a reagent I, the mass concentration of PEG1000 is 500g/L, a reagent II consists of suspension formed by cross-linking rabbit anti-human C5 antiserum, goat anti-human C5 antiserum, mouse anti-human C5 antiserum or mouse anti-human C5 monoclonal antibody on latex particles, and the reagent II is used for covalently bonding the rabbit anti-human C5 antibody on the latex particles through carbodiimide reaction. The titer of the rabbit anti-human C5 antiserum is 1:64-1:128, the content of the C5 antibody is 600ml/L, and the concentration of PEG6000 is 60 g/L.
Example 2: kit composition for detecting C5 concentration by using scattering immunoturbidimetry
Nephelometric immunoturbidimetryA kit for detecting the concentration of C5, comprising two reagents: the reagent I is prepared by taking 0.294g of calcium chloride from a tris buffer solution (pH8.1), adding 0.2mol/L tris solution to dissolve, adjusting the pH value to 8.1 by using 1mol/L hydrochloric acid solution, adding PEG 100030 g, adding water to dilute to 100ml, and in the reagent I, the mass percentage concentration of PEG1000 is 300 g/L. Reagent II is prepared by dissolving calcium chloride 0.294g in Tris buffer solution (pH8.1) with 0.2mol/L Tris solution, adjusting pH to 8.1 with 1mol/L hydrochloric acid solution, adding PEG6000 and rabbit anti-human C5 antiserum, and diluting with water to 100 ml. In the reagent II, the concentration of PEG6000 in percentage by mass is 60g/L, the titer of the rabbit anti-human C5 antiserum is 1:64-1:128, and the concentration of C5 antiserum is 600 ml/L.
Example 3: kit composition for detecting C5 concentration by enhanced nano latex immunoturbidimetry
The kit for detecting the concentration of C5 by using the enhanced nano latex immunoturbidimetry comprises two reagents: reagent I is prepared by taking 0.294g of calcium chloride from a tris buffer solution (pH8.1), adding 0.2mol/L tris solution to dissolve, adjusting the pH value to 8.1 by using 1mol/L hydrochloric acid solution, adding PEG 100050 g, adding water to dilute to 100ml, and in reagent I, the mass concentration of PEG1000 is 500 g/L. And the reagent II is formed by crosslinking rabbit anti-human C5 antiserum or goat anti-human C5 antiserum or mouse anti-human C5 antiserum or mouse anti-human C5 monoclonal antibody on a suspension on latex particles, and the rabbit anti-human C5 antibody, goat anti-human C5 antibody, mouse anti-human C5 antibody, mouse anti-human C5 monoclonal antibody and IgG are covalently bound on the latex particles through carbodiimide reaction. The titer of the rabbit anti-human C5 antiserum is 1:64-1:128, the concentration of the C5 antiserum is 600ml/L, and the concentration of the PEG6000 antiserum is 60 g/L.
Example 4: method for detecting concentration of C5 in human serum
A method for detecting the concentration of C5 in human serum comprises the steps of measuring a sample by a Hitachi 7100 biochemical analyzer, adding a reagent I R1 into the sample, incubating for 5min at 37 ℃, reading a sample A1 at the 1 st point, adding a reagent II R2, incubating for 5min at 37 ℃, reading a sample A2 at the 2 nd point, wherein the sample A is sample A2-sample A1; the method also comprises a step of measuring the calibrator by using a Hitachi 7100 biochemical analyzer, wherein in the step of measuring the calibrator by using the biochemical analyzer, the calibrator is added into a reagent I R1, then the mixture is incubated at 37 ℃ for 5min, the calibrator A1 at the 1 st point is read, the reagent II R2 is added, the mixture is incubated at 37 ℃ for 5min, and the calibrator A2 at the 2 nd point is read, wherein the calibrator A is calibrator A2-calibrator A1;
the parameters measured above were: temperature 37 ℃, dominant wavelength 340nm, secondary wavelength 700nm, sample or calibrator 3 μ l, R1: 240 μ l, R2: 60 μ l, reaction time 10 min.
Example 5: method for detecting C5 concentration in human serum by using 50 healthy human normal value samples (immunoturbidimetry measurement)
In the step of measuring the sample by using the biochemical analyzer, adding a reagent I R1 into the sample, incubating at 37 ℃ for 5min, reading and measuring a sample A1 at a1 st point, adding a reagent II R2, incubating at 37 ℃ for 5min, reading and measuring a sample A2 at a2 nd point, wherein the sample A is sample A2-sample A1; the method also comprises a step of measuring the calibrator by using a Hitachi 7100 biochemical analyzer, wherein in the step of measuring the calibrator by using the biochemical analyzer, the calibrator is added into a reagent I R1, then the mixture is incubated at 37 ℃ for 5min, the calibrator A1 at the 1 st point is read, the reagent II R2 is added, the mixture is incubated at 37 ℃ for 5min, and the calibrator A2 at the 2 nd point is read, wherein the calibrator A is calibrator A2-calibrator A1;
the parameters measured above were: temperature 37 ℃, dominant wavelength 600nm, secondary wavelength 700nm, sample or calibrator 3 μ l, R1: 240 μ l, R2: 60 μ l, reaction time 10 min.
TABLE 150C 5 content of healthy people
Healthy person value: liver function, renal function, biochemical index and immunologic function index are all normal
60 cases of tumor patients (thyroid cancer, liver cancer, stomach cancer, uterine cancer, cervical cancer, lung cancer, breast cancer, esophageal cancer, nasopharyngeal cancer, prostate cancer, colorectal cancer, kidney cancer, pancreatic cancer, bladder cancer, glioma)
Concentrations between 36.7mg/L and 72mg/L are normal.
The results of 60 cases of clinical diagnosis are more than 72mg/L, are positive and accord with the clinical diagnosis standard.
A. And (3) detecting the precision: the samples were continuously extracted 20 times from the same sample, and the sample was measured using a hitachi 7100 biochemical analyzer according to the method for detecting the concentration of C5 in human serum, and the average value, Standard Deviation (SD) and Coefficient of Variation (CV) of the measured values were calculated, and CV (standard deviation/average value) was 100%, and the results are shown in table 2. For clinical chemistry testing projects, a method precision of less than 5% CV is well recognized as acceptable.
Table 2: results of precision measurement
| Reagent kit | pH value | Mean value (mg/L) | Standard Deviation (SD) | Coefficient of Variation (CV) |
| Kit 1 | 4.0-6.0 | 47.81 | 1.79 | 3.74％ |
| Kit 2 | 6.0-8.0 | 48.87 | 1.49 | 3.05％ |
| Kit 3 | 8.0-11.0 | 48.16 | 0.75 | 1.57％ |
The CV value in the table 2 is less than or equal to 3.74%, which shows that the precision of the kit provided by the invention is higher, wherein the precision of the kit 3 is the highest.
B. And (3) accuracy detection: the measurement is carried out by using a quality control product with the serum anti-C5 complement antibody content of 60mg/L (the quality control product is C5 complement antigen formed by 5g of bovine albumin, 0.3ml of Proclin and adding water to 100ml and extracting the antigen), the operation is repeated for 5 times, the average value is taken, the relative deviation (CB) is calculated, and the detection result is shown in Table 3. For clinical chemistry test items, the relative deviation was not more than ± 15%, which was considered to have excellent accuracy.
Table 3: accuracy detection result
The relative deviation in table 3 does not exceed +/-2.27%, which indicates that the kit provided by the invention has high accuracy, wherein the kit accuracy of the kit 3 is the highest.
C. And (3) stability detection: the same standard sample with serum anti-C5 complement antibody content of 60mg/L was assayed 10 times per sample at 0 month, 6 months and 12 months respectively under storage conditions of 2-8 deg.C, and the results are shown in Table 4.
Table 4: stability test results
| Reagent kit | pH value | For 0 month | 6 months old | 12 months old |
| Kit 1 | 4.0-6.0 | 66.41 | 65.79 | 65.97 |
| Kit 2 | 6.0-8.0 | 66.33 | 65.53 | 65.57 |
| Kit 3 | 8.0-11.0 | 66.53 | 66.24 | 66.45 |
As can be seen from Table 4, the detection values have small differences, which indicates that the kit provided by the invention has good stability, and can be stored at 2-8 ℃ for at least more than 12 months, wherein the kit described in kit 3 has the best stability.
D. Linear detection: the kit of the kit 3 is used for detecting the standard substance with six concentration gradients, the concentration of each gradient is detected for 3 times, the average value is taken, the regression analysis is carried out on the average value of the measured values and the theoretical concentration of the standard substance, the X axis represents the theoretical concentration, and the Y axis represents the average value of the measured values. In general R2≧ 0.9900 was considered to have good linearity, the results are shown in FIG. 1.
The linear regression equation obtained was: y 0.9798x +1.5804, correlation coefficient: r20.9994. The result shows that the kit provided by the invention has good linear correlation relationship.
Interference test
Selecting four potential interferents, namely bilirubin, hemoglobin, triglyceride and ascorbic acid, and adding 250mg/L, 300mg/L and 350mg/L bilirubin into a quality control product respectively; 3.5g/L, 4.0g/L, 4.5g/L of hemoglobin; triglycerides at 7.5g/L, 8.0g/L, 8.5 g/L; ascorbic acid of 450mg/L, 500mg/L and 550mg/L and a control group are quality control products without any interferents, the detection method of the kit described in the kit 3 is utilized for determination, each sample is determined for 5 times, and the results are shown in Table 5.
Table 5: interferent test results
As can be seen from Table 5, bilirubin is less than or equal to 300mg/L, hemoglobin is less than or equal to 4.0g/L, triglyceride is less than or equal to 8.0g/L, and ascorbic acid is less than or equal to 500mg/L, and no obvious interference is caused to the detection result.
In conclusion, the kit provided by the invention has the advantages of high accuracy of the detectable result, high precision, good stability and good linearity. Wherein, the detection method of the kit 3 is used for detection, and the detection result is optimal.
Example 6Poct method for detecting the content of C5 in a sample to be tested (gold-labeled):
example 6 gold-labeling was performed using a FT-J5 gold-labeled reader, manufactured by Shanghai North China Biochemical Co., Ltd. The colloidal gold test strip for detection is shown in FIG. 2, wherein colloidal gold, Staphylococcal Protein (SPA) and goat anti-human are coated on the colloidal gold padC5 complementAntibody conjugate, detection line coated rabbit anti-humanC5 complementAntibody, quality testing line coated antigen.
The detecting the sample comprises: 24 positive human serum samples infected with gram-positive bacteria, 8 positive human serum samples infected with staphylococcus aureus, 8 positive human serum samples infected with candida glabrata, 8 positive human serum samples infected with pneumococcus, 8 positive human serum samples infected with hepatitis c virus, and 20 healthy human serum samples.
The detection results are interpreted as shown in fig. 3: if the quality detection line and the detection line are both colored, the result is positive; if the quality detection line is colored and the detection line is colorless, the result is negative; if the detection line is colored and the quality detection line is colorless, or the detection line and the quality detection line are both colorless, the result is invalid.
The detection steps are as follows:
1. all reagents and samples were removed before use and allowed to stand at room temperature for 15-30 minutes to allow them to return to room temperature.
2. And taking out the detection plate.
3. And adding 2 drops of sealing liquid into the center of the reaction hole of the detection plate until the sealing liquid completely permeates.
4. And adding 40 microliter of the sample to be detected into the reaction hole center of the detection plate by using a pipette until the sample completely permeates.
5. 4 drops of washing solution are added to the center of the reaction hole of the detection plate until the washing solution is completely infiltrated.
6. Adding colloidal gold, Staphylococcal Protein (SPA) and goat anti-human in the center of the reaction well of the detection plateC5 complementAntibody conjugate 3 drops, and the colloidal gold conjugate is allowed to penetrate completely.
7. 4 drops of washing liquid are added to the center of the reaction hole of the detection plate, and the result is visually observed after the washing liquid completely permeates.
TABLE 6 results of C5 complement detection by Poct method
Bacterial infection:
gram-positive bacteria, gram-negative bacteria, streptococci glabrata, staphylococcus aureus, pneumococcus and pyogenes infections.
Viral infection: hepatitis C
Example 7: method for detecting C5 content in sample to be detected by using immune transmission turbidimetry
Measuring a sample by using a Hitachi 7100 biochemical analyzer, adding a reagent R1 into the sample, incubating for 5min at 37 ℃, reading a sample A1 at the 1 st point, adding a reagent R2, incubating for 5min at 37 ℃, reading a sample A2 at the 2 nd point, wherein the sample A is a sample A2-a sample A1; the method also comprises a step of measuring the calibrator by using a Hitachi 7100 biochemical analyzer, wherein in the step of measuring the calibrator by using the biochemical analyzer, the calibrator is added with a reagent R1I, then the mixture is incubated at 37 ℃ for 5min, a1 st calibration A1 is read, a reagent R2 II is added, the mixture is incubated at 37 ℃ for 5min, a2 nd calibration A2 is read, and the calibration A is calibration A2-calibration A1;
the parameters measured above were: temperature 37 ℃, dominant wavelength 340nm, secondary wavelength 700nm, sample or calibrator 3 μ l, R1: 240 μ l, R2: 60 μ l, reaction time 10 min. The kit comprises the following components: r1 Tris buffer (pH8.1), PEG 2000, R2 Tris buffer (pH8.1), PEG6000, C5 complement.
TABLE 7 results of C5 content determination by immunoturbidimetry
Note: the value was positive at 72mg/L or more, and negative at below.
The above examples of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (12)
1. A kit for detecting C5 complement, wherein the kit comprises:
reagent I: a first buffer solution and a first PEG buffer solution;
and (2) reagent II: the anti-C5 complement antibody comprises a monoclonal antibody and/or a polyclonal antibody, a second PEG buffer solution and IgG; the pH value of the first buffer solution is 3-10.
2. The kit of claim 1, wherein the anti-C5 complement antibody is selected from one or a combination of two or more of a rabbit anti-human C5 complement antibody, a goat anti-human C5 complement antibody, a mouse anti-human C5 complement antibody; preferably, the rabbit anti-human C5 complement antibody, the sheep anti-human C5 complement antibody and the mouse anti-human C5 complement antibody are monoclonal antibodies or polyclonal antibodies; optionally, the anti-C5 complement antibody has an antibody titer of 1:64 to 1:128 and a concentration of 100ml/L to 800 ml/L.
3. The kit of claim 1 or 2, wherein the lipophilic group of the anti-C5 complement antibody is changed to hydrophilic after modification of the polyclonal antibody by treatment with trace amounts of manganese dioxide or potassium permanganate.
4. The kit of claim 1 or 2, wherein the lipophilic group of the anti-C5 complement antibody is changed into hydrophilic after the C5 complement monoclonal antibody is modified by treating with trace manganese dioxide or potassium permanganate.
5. A kit according to any one of claims 1 to 4 wherein the anti-C5 complement antibody is covalently bound to a latex particle; preferably, the anti-C5 complement monoclonal antibody is bound first and then bound to the C5 complement polyclonal antibody by covalent binding to latex particles via a carbodiimide reaction.
6. Use of a kit according to any one of claims 1 to 5 in the preparation of a diagnostic agent for the detection of pathogen infection; optionally, the pathogen comprises one or more of a virus, a bacterium, a fungus, a mycoplasma, a chlamydia, a rickettsia.
7. Use of the kit according to any one of claims 1 to 6 in the preparation of a diagnostic agent for early diagnosis screening of tumor markers selected from thyroid cancer, liver cancer, stomach cancer, uterine cancer, cervical cancer, lung cancer, breast cancer, esophageal cancer, nasopharyngeal cancer, prostate cancer, colorectal cancer, renal cancer, pancreatic cancer, bladder cancer, glioma.
8. Use according to claim 6 or 7, wherein whole blood, peripheral blood, serum is used for the detection.
9. Use of a kit according to any one of claims 1 to 8 in the preparation of a diagnostic for the detection of C5 complement by a method comprising: immunotransmission turbidimetry, immunoscattering turbidimetry, immunolatex, chemiluminescence, or colloidal gold filtration or chromatography, and real-time detection; preferably, the immunoturbidimetry comprises an immunotransmission turbidimetry, an immunoscattering turbidimetry wavelength of 340nm, and an immunolatex method wavelength of 600nm-700 nm. Especially in the colloidal gold percolation method or chromatography, Poct (point of care), chemiluminescence method, microfluidic chip and magnetic particle method, the mouse monoclonal antibody is added to improve the sensitivity of the reagent, and the C5 complement polyclonal antibody is added to improve the linearity, wherein the monoclonal antibody comprises mouse, rabbit, sheep and horse cell strains, and the polyclonal antibody comprises mouse, rabbit, sheep and horse antibodies.
10. Use according to claim 9, wherein human peripheral serum, whole blood and peripheral blood are tested.
11. Use according to claim 9 or 10, wherein the immunoturbidimetry comprises the following steps:
sample reagent I addition step: adding the sample into the reagent I, incubating, and reading the sample A1 at the 1 st point;
sample reagent II addition step: after the sample reagent I addition step, reagent II is added, incubated, and sample A2 at point 2 is read.
Adding a calibrator reagent I: adding the calibrator into the reagent I, incubating, and reading the calibrator A1 at the 1 st point;
adding a calibrator reagent II: after the step of adding the calibrator reagent I, adding a reagent II, incubating, and reading and measuring a2 nd point calibrator A2; wherein,
the C5 complement concentration (mg/L) was calculated by the following equation:
12. use of a kit according to any one of claims 1 to 5 in the preparation of a reagent or kit for the detection of C5 complement.
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| CN117471108B (en) * | 2023-12-28 | 2024-03-01 | 北京万泰德瑞诊断技术有限公司 | Complement C1q reference substance, preparation method and application thereof |
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