CN114660292A - Kit for detecting C5 complement and detection method and application thereof - Google Patents

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

Kit for detecting C5 complement and its detection method and use

technical field

The invention belongs to the field of immune detection, and in particular relates to a kit for detecting C5 complement, a detection method and application thereof.

Background technique

Complement component 5 (The fifth component of complement, C5) is an important component of the complement system. At present, the C5 gene has been reported in several organisms such as human, mouse, rainbow trout and bovine. The human C5 gene is located in regions 32-34 of the long arm of chromosome 9. The C5 precursor protein is composed of two polymorphic chains of α and β through disulfide bonds, with a molecular weight of 190kDa. The 74-75 arginine-leucine bond near the N-terminus is the site of C5 convertase action. Under the action of C5 convertase, the inactive C5 precursor protein cleaves a small fragment C5a from the N-terminus of the C5 enzyme chain into the liquid phase, and the rest is C5b, which is still bound to the cell membrane surface.

Infection is caused by bacteria and viruses invading the body, and pathogens cannot be efficiently identified, resulting in more and more abuse of antibiotics. Bacterial infections lead to drug resistance, and antibiotic resistance continues to occur. Clinically, bacterial culture takes 3-5 days. The waiting time is too long and the treatment is delayed. The doctor cannot immediately give the patient's medication plan, and wait for the bacteria to be cultured before medication, which delays the pathogenesis.

C5 determination in countries around the world, including China, uses the immunodiffusion method and ELISA double-antibody sandwich technology. This method is time-consuming and labor-intensive, the accuracy rate is not high, and it takes 2 days for the patient to get the report. There is a complement C5 detection kit on the market that uses a double-antibody two-step sandwich enzyme-linked immunosorbent assay (ELISA). Add the standard and the sample to be tested to the transparent enzyme-labeled plate pre-coated with complement component 5 (C5), incubate for a sufficient time, wash to remove unbound components, then add the enzyme-labeled working solution and incubate for a sufficient time Afterwards, unbound components are removed by washing. Substrates A and B were added in sequence, and the substrate (TMB) was converted into a blue product under the catalysis of horseradish peroxidase (HRP), and turned yellow under the action of acid. The concentration of C5) was positively correlated, and the OD value was measured at a wavelength of 450 nm, and the content of complement component 5 (C5) in the sample was calculated according to the OD value of the standard and the sample.

Due to the defects in the prior art, there is an urgent need to provide a kit for rapidly and accurately detecting the concentration of C5 complement.

SUMMARY OF THE INVENTION

Invention to solve problem

Due to the above problems in the prior art, the purpose of the present invention is to provide a rapid, high-accuracy, high-precision, stable and linear detection kit for bacterial and/or viral infection.

solution to the problem

(1) a kit for detecting C5 complement, wherein the kit comprises:

Reagent I: the first buffer, the first PEG buffer;

Reagent II: anti-C5 complement antibody, comprising monoclonal antibody and/or polyclonal antibody, a second PEG buffer, and IgG; the pH of the first buffer is 3-10.

(2) The kit according to (1), wherein the anti-C5 complement antibody is selected from one or both of a rabbit anti-human C5 complement antibody, a goat anti-human C5 complement antibody, and a mouse anti-human C5 complement antibody A combination of the above; preferably, the rabbit anti-human C5 complement antibody, the goat anti-human C5 complement antibody, and the mouse anti-human C5 complement antibody are monoclonal antibodies or polyclonal antibodies; optionally, the anti-C5 The antibody titer of complement antibody is 1:64-1:128, and the concentration is 100ml/L～800ml/L.

(3) The kit according to (1) or (2), wherein after the polyclonal antibody is modified with a trace amount of manganese dioxide or potassium permanganate, the lipophilic group of the anti-C5 complement antibody is changed to hydrophilic.

(4) The kit according to (1) or (2), wherein after the C5 complement monoclonal antibody is modified with a trace amount of manganese dioxide or potassium permanganate, the lipophilic group of the anti-C5 complement antibody is changed to hydrophilic sex.

(5) The kit according to any one of (1) to (4), wherein the anti-C5 complement antibody is covalently bound to latex particles; preferably, the anti-C5 complement monoclonal antibody is bound first, Then, the C5 complement polyclonal antibody is covalently bound to the latex particles through a carbodiimide reaction.

(6) Use of the kit according to any one of (1) to (5) in the preparation of a diagnostic agent for detecting pathogen infection; optionally, the pathogen includes viruses, bacteria, fungi, mycoplasma, and chlamydia , one or more of rickettsite.

(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 tumor markers, wherein the tumor markers are selected from thyroid cancer, Liver cancer, stomach cancer, uterine cancer, cervical cancer, lung cancer, breast cancer, esophagus cancer, nasopharyngeal cancer, prostate cancer, colorectal cancer, kidney cancer, pancreatic cancer, bladder cancer, glioma.

(8) The use according to (6) or (7), wherein whole blood, peripheral blood, and serum are used for pre-detection.

(9) Use of the kit according to any one of (1) to (8) in the preparation of a diagnostic agent for detecting C5 complement by the following method, wherein the method comprises: immunotransmission turbidimetry, immunoassay Nephelometric method, immune latex method, chemiluminescence method or colloidal gold diafiltration method or chromatography method, real-time detection; , The wavelength of immune latex method is 600nm-700nm. Especially in colloidal gold percolation or chromatography, Poct (point-of-care), chemiluminescence, microfluidic chip, magnetic particle method, the addition of mouse monoclonal antibody improves the sensitivity of the reagent, and C5 complement is added. Polyclonal antibodies will make the linearity wider. Monoclonal antibodies include mouse, rabbit, sheep, and horse cell lines. Polyclonal antibodies include 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 immunoturbidimetric method comprises the following steps:

Steps of adding sample reagent I: after adding the sample to reagent I, incubate, and read and measure the first sample A1;

Steps of adding sample reagent II: After adding step of sample reagent I, add reagent II, incubate, and read and measure the second sample A2.

Steps of adding calibrator reagent I: after adding the calibrator to reagent I, incubate, and read and measure the calibrator A1 at point 1;

Steps of adding calibrator reagent II: after the step of adding calibrator reagent I, add reagent II, incubate, and read and measure the second calibrator A2;

C5 complement concentration (mg/L) was calculated by the following formula:

(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.

effect of invention

The detection result of the kit provided by the invention has high accuracy, high precision, good stability and good linearity.

In one embodiment of the present invention, the use of the kit of the present invention can remove useless antibodies, so that the affinity of the antibody with the antigen is enhanced, the binding speed is accelerated, and the arrival of the end point is accelerated.

The immunoturbidimetric method of the present invention can produce a report in only 10 minutes, is convenient and fast, has high accuracy, shortens the waiting time of patients, and can see the report in 10 minutes.

The present invention is used to detect and diagnose thyroid cancer, liver cancer, stomach cancer, uterine cancer, cervical cancer, lung cancer, breast cancer, esophagus cancer, nasopharyngeal cancer, prostate cancer, colorectal cancer, kidney cancer, pancreatic cancer, bladder cancer The kit for cancer and glioma can improve the accuracy and specificity of the above-mentioned cancer diagnosis, and can be used for the preliminary screening and postoperative follow-up of the above-mentioned cancers, providing reliable information for the risk degree of tumor progression; it can also be used to detect viruses , bacteria, fungi, mycoplasma, chlamydia, rickettsia one or more pathogens, which has guiding significance for clinical practice.

Description of drawings

Fig. 1 shows that the kit in the kit 3 of the present invention detects the standard substance of six concentration gradients, each concentration gradient is detected 3 times, takes the average value, and performs regression analysis on the average value of the measured value and the theoretical concentration of the standard substance , the X-axis represents the theoretical concentration, and the Y-axis represents the mean of the measured values.

Figure 2 shows a schematic structural diagram of a colloidal gold test strip.

Figure 3 shows a schematic diagram of interpretation of the results of C5 complement detection with colloidal gold test strips.

Detailed ways

Other objects, features and advantages of the present application will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the present disclosure, are given for illustrative purposes only, since after reading this detailed description, Various changes and modifications will become apparent to those skilled in the art.

In addition, in order to better illustrate the present invention, numerous specific details are given in the following detailed description. It will be understood by those skilled in the art that the present invention may be practiced without certain specific details. In other instances, methods, means, devices and steps well known to those skilled in the art have not been described in detail so as to highlight the subject matter of the present invention.

It should be noted:

In the present specification, the numerical range represented by "numerical value A to numerical value B" means a range including numerical values A and B at the endpoints.

In this specification, "PEG" refers to polyethylene glycol; wherein, "PEG1000" refers to PEG having a degree of polymerization of 1000.

In this specification, references to "some specific/preferred embodiments", "other specific/preferred embodiments", "embodiments", etc. refer to the specific elements described in relation to the embodiment (eg, features, properties, and/or characteristics) are included in at least one embodiment described herein, and may or may not be present in other embodiments. Additionally, it should be understood that the described elements may be combined in any suitable manner in the various embodiments.

<Aspect 1>

The present invention provides a kit for detecting C5 complement, the kit comprising:

Reagent I: the first buffer solution, the first PEG solution;

Reagent II: anti-C5 complement antibody, second PEG solution, IgG; the pH of the first buffer solution is 3-10.

The inventors of the present invention found that C5 antigen can be leached with bacteria or adhered to virions, and by measuring the concentration of C5, the amount of bacterial or viral infection can be known, and whether the patient has bacterial infection or viral infection can be known.

The immunoturbidimetric method of the present invention can produce a report in only 10 minutes, is convenient and fast, has high accuracy, shortens the waiting time of patients, and can see the report in 10 minutes. Colloidal gold percolation method or chromatography method, (Poct) instant test is faster, and the report can be accurately issued in 2 minutes, reducing the waiting time of patients. The method of the present invention is expected to replace the ELISA double-antibody sandwich method for detecting C5 complement, and use immunotransmission turbidimetry, immunoscatter turbidimetry, immune latex method, chemiluminescence method, microfluidic chip method, colloidal gold percolation method or chromatography C5 complement was detected by methods such as method, Poct point-of-care test, etc.

Pathogens that can be detected by the kit of the present invention include: one or more of viruses, bacteria, fungi, mycoplasma, chlamydia, and rickettsia.

Further, the viruses that can be detected 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 hepatitis virus, enterovirus, measles virus, rubella virus, herpes virus, mumps virus, influenza virus, avian influenza virus, prion, coronavirus One or more of virus, adenovirus, respiratory syncytial virus. Exemplarily, the hepatitis virus can be hepatitis A, B, C, D, E, etc.; the enterovirus can be norovirus, rotavirus, coxsackie virus, echo virus, stellate virus, etc.; herpes virus can be herpes zoster virus, EB virus, etc.; influenza virus can be influenza A virus, influenza B virus, parainfluenza virus, etc.;

Further, the bacteria that can be detected by the kit of the present invention include cocci, bacilli and spirochetes. In some specific embodiments, the virus detected by the kit of the present invention is selected from the group consisting of Gram-positive aerobic cocci, Gram-negative aerobic cocci, Gram-negative aerobic bacilli, Gram-negative facultative anaerobic bacteria Aerobic bacteria, Gram-negative anaerobic bacteria, etc. In some more specific embodiments, the bacteria detected by the kit of the present invention are selected from Staphylococcus, Streptococcus, Enterococcus, Neisseria, Moraxella, Acinetobacter, Pseudomonas Alcaligenes, Brucella, Bordetella, Legionella, Escherichia, Citrobacter, Salmonella, Shigella, Klebsiella , Serratia, Proteus, Prufidenella, Morganella, Yersinia, Haemophilus, Vibrio, Aeromonas, Peptococcus, Peptostreptococcus Genus, Micrococcus, Bacteroides, Fusobacterium, Bacillus anthracis, Bacillus, Clostridium, Listeria, Erysipelas, Corynebacterium, Actinomyces, Mycobacterium, One or more bacteria of the genus Shigella.

Illustratively, Staphylococcus includes Staphylococcus aureus, Staphylococcus epidermidis, etc., Streptococcus includes alpha-hemolytic streptococcus, beta-hemolytic streptococcus, non-hemolytic streptococcus, pneumococcus, etc., Enterococcus includes Enterococcus, etc. , Neisseria including meningococcus, Neisseria gonorrhoeae, Neisseria, etc., Moraxella including Moraxella catarrhalis, etc., Acinetobacter including Acinetobacter non-nitrogen, Acinetobacter loferia, etc., Pseudomonas includes Pseudomonas aeruginosa and other pseudomonas, Alcaligenes includes Alcaligenes faecalis, Bordetella includes Pertussis, etc. Escherichia includes Escherichia coli, etc., Yersinia The genus includes Yersinia pestis, etc., Haemophilus includes Influenza bacillus, etc., Vibrio includes Vibrio cholerae, Vibrio ElTor, Vibrio parahaemolyticus, etc., Aeromonas includes Aeromonas hydrophila, etc., Peptococcus Genus includes Peptococcus, etc., Peptostreptococcus includes Peptostreptococcus, etc., Micrococcus includes Micrococcus fischeri, etc., Fusobacterium includes Bacteroides fragilis, Fusobacterium, etc., Bacillus anthracis includes Bacillus anthracis, etc., Bacillus Including Bacillus cereus, Bacillus tetanus, etc., Clostridium including botulinum, Clostridium difficile, Clostridium difficile, etc., Corynebacterium including Diphtheria, etc., Mycobacterium including Mycobacterium tuberculosis, Leprosy, etc. , Shigella genus includes Shigella spp., Actinomyces genus includes Nocardia stellariformis, etc., Klebsiella genus includes Pneumococcus, etc., and Legionella genus includes Legionella pneumophila.

Further, the kit of the present invention can also detect fungi such as Candida, Aspergillus, Cryptococcus neoformans, Mucor, Mycoplasma such as Mycoplasma pneumoniae, Rickettsia such as Rickettsia Q fever, or pathogens such as Chlamydia.

The kit of the present invention can also detect tumor markers: thyroid cancer, liver cancer, stomach cancer, uterine cancer, cervical cancer, lung cancer, breast cancer, esophagus cancer, nasopharyngeal cancer, prostate cancer, colorectal cancer, kidney cancer, pancreatic cancer, Bladder cancer, glioma.

Reagent I

The reagent I of the present invention includes a first buffer solution and a first PEG solution; the pH value of the first buffer solution is 3-10.

In some specific embodiments of the invention, a tris buffer may be used. Tris(hydroxymethyl)aminomethane buffer solution is prepared by mixing tris(hydroxymethyl)aminomethane solution and hydrochloric acid solution in proportion. As a preferred embodiment, the pH of the tris buffer in the present invention is 7.5 to 9.5; as a further preferred embodiment, the pH of the tris buffer is 8.1.

In the present invention, the first PEG and the second PEG may be the same or different.

The reagent I of the present invention includes the first PEG solution (polyethylene glycol). When the anti-C5 complement antibody (antibody) is combined with the C5 complement (antigen) to form an immune complex with a certain structure, under the action of PEG, it becomes suspended in Microparticles in the reaction solution cause changes in sample turbidity.

In some specific embodiments of the present invention, the first PEG used in Reagent I includes: PEG1000; the second PEG used in Reagent II is PEG 6000-10000, and the molecular weights of Reagent I and Reagent II are greatly different. PEG, to accelerate and strengthen the reaction. The absorbance at the end of the reaction is stable, and the affinity is stronger than that of general reagents. In the reagent I of the present invention, the concentration of the PEG1000 is 400g/L～600g/L; as a preferred embodiment, the concentration of the PEG1000 is 500g/L, at the concentration of the PEG1000, Reduce non-specific reactions in samples.

The reagent I of the present invention may further comprise calcium chloride. When the anti-C5 complement antibody used in the present invention is an anti-C5 complement antiserum, other antibodies except the C5 complement antibody in the C5 complement antiserum are bound by the calcium chloride therein. , which can remove useless antibodies, enhance the affinity of antibodies and antigens, accelerate the binding speed, and accelerate the arrival of the end point. As a preferred embodiment, the concentration of the calcium chloride is 0.25 g/mL to 0.35 g/mL; as a further preferred embodiment, the concentration of the calcium chloride is 0.294 g/mL. The addition of CaCl ₂ in the present invention enhances the affinity between the antibody and the antigen and accelerates the reaction binding speed. More importantly, the response linearity of immunoturbidimetry was greatly improved.

Reagent II

Reagent II of the present invention includes anti-C5 complement antibody, second PEG solution, and IgG.

The reagent II of the present invention includes an anti-C5 complement antibody, and the anti-C5 complement antibody can be a polyclonal antibody or a monoclonal antibody. In some specific embodiments of the present invention, the polyclonal antibody is an antiserum containing anti-C5 complement antibody. As a preferred embodiment, the anti-C5 complement antibody in the present invention is a monoclonal antibody. In some specific embodiments of the present invention, the anti-C5 complement antibody is selected from one or more combinations of rabbit anti-human C5 complement antibody, goat anti-human C5 complement antibody, and mouse anti-human C5 complement antibody. As a preferred embodiment, the rabbit anti-human C5 complement antibody, the goat anti-human C5 complement antibody, and the mouse anti-human C5 complement antibody are monoclonal antibodies. In some specific embodiments of the present invention, the concentration of the anti-C5 complement antibody is 100ml/L-800ml/L. As a preferred embodiment, the concentration of the anti-C5 complement antibody is 600ml/L.

The reagent II of the present invention includes the second PEG, and the second PEG that can be used in the reagent II includes: PEG6000-10000. For example, the second PEG solution can be one or more of PEGs with molecular weights of 6000, 7000, 8000, 9000 or 10000. The concentration of the PEG 6000-10000 in the reagent II of the present invention 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) combines with the C5 complement (antigen) to form an immune complex with a certain structure, under the action of PEG, it becomes a particle suspended in the reaction solution, which changes the turbidity of the sample. In the present invention, PEG 1000 is 400g/L～600g/L in Reagent I, and PEG 6000～10000 is 50～70g/L in Reagent II, and the ratio of Reagent I and Reagent II is greatly different to accelerate the reaction, strengthen. The absorbance at the end of the reaction is stable, and the affinity is stronger than that of general reagents.

In some specific embodiments of the present invention, the reagent II further includes a second buffer. The pH value of the second buffer solution is 3-10; preferably, it is 8.1. As a preferred embodiment, the second buffer is tris buffer. The pH of the buffer is 8.1, the reaction is accelerated, and the end point is reached quickly.

In some specific embodiments of the present invention, before the antibody is treated with PEG, a trace amount of manganese dioxide or potassium permanganate, or manganese dioxide and potassium permanganate is added for modification, and a small amount of manganese dioxide or permanganate is added to the antibody. Potassium, or manganese dioxide and potassium permanganate have a certain effect before reaching saturation, and in practical applications, manganese dioxide and potassium permanganate have the best effect when the concentration is 5-15mg/L. After the polyclonal antibody is modified with a trace amount of manganese dioxide or potassium permanganate, the lipophilic group of the anti-C5 complement antibody is changed to hydrophilic, so that the efficacy of the antibody is maximized, the efficacy is stronger, and the stability of the reagent is better. , the shelf life is longer, it can be stored for 24 months, the linearity is better, and the anti-interference is stronger.

In some specific embodiments of the present invention, before the antibody is treated with PEG, a trace amount of manganese dioxide or potassium permanganate, or manganese dioxide and potassium permanganate is added for modification, and a small amount of manganese dioxide or permanganate is added to the antibody. Potassium, or manganese dioxide and potassium permanganate have a certain effect before reaching saturation, and in practical applications, manganese dioxide and potassium permanganate have the best effect when the concentration is 5-15mg/L. After the C5 complement monoclonal antibody was modified with a trace amount of manganese dioxide or potassium permanganate, the lipophilic group of the anti-C5 complement antibody was changed to hydrophilic. It can improve the sensitivity of the reagent, make it fast and stable, with small error, and can correspondingly drive the polyclonal C5 complement antibody to have better linearity and better response.

In some specific embodiments of the invention, the anti-C5 complement antibody is covalently bound to latex particles. As a preferred embodiment, the anti-C5 complement antibody is covalently bound to latex particles through a carbodiimide reaction. In the present invention, the type and size of the latex particles are not particularly limited, and latex particles with a diameter of 15 to 120 nm can be used.

<Second aspect>

In a second aspect of the present invention, there is provided a use of the kit according to the first aspect in the preparation of a diagnostic agent for detecting C5 complement by the following method, the method comprising: immunoturbidimetry and/or chemiluminescence Law. The immunoturbidimetric method in the present invention is selected from the group consisting of immunotransmission turbidimetry, immune scattering turbidimetry, and immune latex method.

The immunoturbidimetric method in the present invention comprises the following steps:

Steps of adding sample reagent I: after adding the sample to reagent I, incubate, and read and measure the first sample A1;

Steps of adding sample reagent II: After adding step of sample reagent I, add reagent II, incubate, and read and measure the second sample A2.

Steps of adding calibrator reagent I: after adding the calibrator to reagent I, incubate, and read and measure the calibrator A1 at point 1;

Steps of adding calibrator reagent II: after the step of adding calibrator reagent I, add reagent II, incubate, and read and measure calibrator A2 at point 2.

After immunoturbidimetric determination, the C5 complement concentration (mg/L) was calculated by the following formula:

Specifically, the immunoturbidimetric method in the present invention includes: the step of using a biochemical analyzer to measure the sample, adding 160 ul of reagent I to the sample, incubating at 37°C for 5 min, reading and measuring the first sample A1, adding 40 ul of reagent II, and incubating at 37°C for 5 min, Read and measure the second point sample A2, sample A = sample A2 - sample A1; also includes a step of using the biochemical analyzer to measure the calibrator, add the calibrator to reagent I 160ul, incubate at 37°C for 5 minutes, read and measure the first point of calibration A1 , add 40ul of Reagent II, incubate at 37°C for 5min, read and measure the second calibration point A2, calibration A=calibration A2-calibration A1;

In one embodiment, the ratio of reagent I and reagent II added in the above method can be appropriately adjusted according to the ratio of 4:1.

The temperature measured by the above-mentioned immunoturbidimetric method is 36.8-37.2; as a preferred embodiment, the temperature measured by the above-mentioned immunoturbidimetric method is 37° C., the dominant wavelength measured by the above-mentioned immunoturbidimetric method is 340 nm, and the temperature measured by the above-mentioned immunoturbidimetric method is 340 nm. The secondary wavelength is 700 nm, the amount of the sample or calibrator measured by the above-mentioned immunoturbidimetric method is 3 μl-5 μl, and the reaction time is 10 minutes.

Immunotransmission turbidimetry, immune nephelometry, latex-enhanced immunoturbidimetry, chemiluminescence, Poct point-of-care latex-enhanced immunoturbidimetry also includes C5 calibrator, which is composed of 5g bovine albumin, 0.3ml Proclin, and water to 100ml, extract the C5 complement antigen composition. The quality control material is composed of 700mg/L bovine albumin, 350mg/L Proclin, and C5 complement antigen extracted.

<The third aspect>

The third aspect of the present invention provides the use of an anti-C5 complement antibody for preparing a diagnostic agent for detecting C5 complement.

The present invention finds that bacteria and viruses adhere to the antigen of C5 complement, and the content of bacteria and viruses can be known by detecting the concentration of C5 complement antibody.

The invention can be used for bacterial infection and viral infection by detecting C5 complement, comparing before and after treatment to identify whether it improves, and the diagnostic accuracy rate of combined application of multiple items can reach 100%. Pathogens include: one or more of viruses, bacteria, fungi, mycoplasma, chlamydia, and rickettsia.

Further, viruses include RNA viruses, DNA viruses and protein viruses. In some specific embodiments, the detected virus is selected from the group consisting of hepatitis virus, enterovirus, measles virus, rubella virus, herpes virus, mumps virus, influenza virus, avian influenza virus, prion, coronavirus, adenovirus, One or more of the respiratory syncytial viruses. Exemplarily, the hepatitis virus can be hepatitis A, B, C, D, E, etc.; the enterovirus can be norovirus, rotavirus, coxsackie virus, echo virus, stellate virus, etc.; herpes virus can be herpes zoster virus, EB virus, etc.; influenza virus can be influenza A virus, influenza B virus, parainfluenza virus, etc.;

Further, bacteria include cocci, bacilli and spirochetes. In some specific embodiments, the virus detected by the kit of the present invention is selected from the group consisting of Gram-positive aerobic cocci, Gram-negative aerobic cocci, Gram-negative aerobic bacilli, Gram-negative facultative anaerobic bacteria Aerobic bacteria, Gram-negative anaerobic bacteria, etc. In some more specific embodiments, the detected bacteria are selected from the group consisting of Staphylococcus, Streptococcus, Enterococcus, Neisseria, Moraxella, Acinetobacter, Pseudomonas, Alcalibacterium, Brucella, Bordetella, Legionella, Escherichia, Citrobacter, Salmonella, Shigella, Klebsiella, Serratia , Proteus, Prufidenella, Morganella, Yersinia, Haemophilus, Vibrio, Aeromonas, Peptococcus, Peptostreptococcus, Micrococcus , Bacteroides, Fusobacterium, Bacillus anthracis, Bacillus, Clostridium, Listeria, Erysipelas, Corynebacterium, Actinomyces, Mycobacterium, Shigella one or more bacteria.

Illustratively, Staphylococcus includes Staphylococcus aureus, Staphylococcus epidermidis, etc., Streptococcus includes alpha-hemolytic streptococcus, beta-hemolytic streptococcus, non-hemolytic streptococcus, pneumococcus, etc., Enterococcus includes Enterococcus, etc. , Neisseria including meningococcus, Neisseria gonorrhoeae, Neisseria, etc., Moraxella including Moraxella catarrhalis, etc., Acinetobacter including Acinetobacter non-nitre, Acinetobacter lofelii, etc., Pseudomonas includes Pseudomonas aeruginosa and other pseudomonas, Alcaligenes includes Alcaligenes faecalis, Bordetella includes Pertussis, etc. Escherichia includes Escherichia coli, etc., Yersinia The genus includes Yersinia pestis, etc., Haemophilus includes Influenza bacillus, etc., Vibrio includes Vibrio cholerae, Vibrio ElTor, Vibrio parahaemolyticus, etc., Aeromonas includes Aeromonas hydrophila, etc., Peptococcus Genus includes Peptococcus, etc., Peptostreptococcus includes Peptostreptococcus, etc., Micrococcus includes Micrococcus fischeri, etc., Fusobacterium includes Bacteroides fragilis, Fusobacterium, etc., Bacillus anthracis includes Bacillus anthracis, etc., Bacillus Including Bacillus cereus, Bacillus tetanus, etc., Clostridium including botulinum, Clostridium difficile, Clostridium difficile, etc., Corynebacterium including Diphtheria, etc., Mycobacterium including Mycobacterium tuberculosis, Leprosy, etc. , Shigella genus includes Shigella spp., Actinomyces genus includes Nocardia stellariformis, etc., Klebsiella genus includes Pneumococcus, etc., and Legionella genus includes Legionella pneumophila.

Further, fungi such as Candida, Aspergillus, Cryptococcus neoformans, Mucor, Mycoplasma such as Mycoplasma pneumoniae, Rickettsia such as Rickettsia Q fever, or pathogens such as Chlamydia can also be detected.

At the same time, the discovery can also be used for tumor marker screening: thyroid cancer, liver cancer, stomach cancer, uterine cancer, cervical cancer, lung cancer, breast cancer, esophagus cancer, nasopharyngeal cancer, prostate cancer, colorectal cancer, kidney cancer, pancreas cancer Cancer, bladder cancer, glioma, the accuracy rate can reach 90%-98%. It can be popularized to the general public, and a simple drop of blood can screen thyroid cancer, liver cancer, stomach cancer, uterine cancer, cervical cancer, lung cancer, breast cancer, esophagus cancer, nasopharyngeal cancer, prostate cancer, colorectal cancer, kidney cancer, pancreatic cancer , bladder cancer, glioma.

Example

Example 1: The composition of the kit for detecting C5 concentration by latex immune turbidimetry

The kit for the detection of C5 concentration by enhanced immunoturbidimetry includes two reagents: Reagent I consists of tris buffer (pH 8.1). Get calcium chloride 0.294g, add 0.2mol/L tris solution to dissolve, adjust pH value to 8.1 with 1mol/L hydrochloric acid solution, add PEG1000 50g, add water and dilute to 100ml, in reagent I, the described The mass concentration of PEG1000 is 500g/L, and reagent II is a suspension formed by cross-linking 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 latex particles. The reagent II is composed of a turbid liquid, and the reagent II is to covalently bind the rabbit anti-human C5 antibody to the latex particles through the carbodiimide reaction. The titer of the rabbit anti-human C5 antiserum was 1:64-1:128, the C5 antibody content was 600ml/L, and the PEG 6000 concentration was 60g/L.

Example 2: The composition of the kit for detecting C5 concentration by scattering immunoturbidimetry

The kit for the detection of C5 concentration by scattering immunoturbidimetry , including two reagents: Reagent I, 0.294 g of calcium chloride from tris buffer (pH 8.1), plus 0.2 mol/L tris of tris. Methane solution was dissolved, adjusted pH value to 8.1 with 1mol/L hydrochloric acid solution, added PEG1000 30g, added water and diluted to 100ml, in reagent I, the mass percent concentration of described PEG1000 was 300g/L. Reagent II: Take 0.294 g of calcium chloride from tris buffer (pH 8.1), add 0.2 mol/L tris solution to dissolve, and adjust the pH to 8.1 with 1 mol/L hydrochloric acid solution, Add PEG6000 and rabbit anti-human C5 antiserum and dilute to 100ml with water. In the reagent II, the mass percentage concentration of the PEG6000 is 60g/L, the titer of the rabbit anti-human C5 antiserum is 1:64-1:128, and the C5 antiserum concentration is 600ml/L.

Example 3: The composition of the kit for the detection of C5 concentration by enhanced nanolatex immunoturbidimetry

A kit for the detection of C5 concentration by enhanced nanolatex immunoturbidimetry, including two reagents: Reagent I takes 0.294 g of calcium chloride from tris buffer (pH 8.1), and adds 0.2 mol/L tris. The base aminomethane solution was dissolved, and the pH value was adjusted to 8.1 with 1mol/L hydrochloric acid solution, PEG1000 50g was added, and water was added to dilute to 100ml. In reagent I, the mass concentration of the PEG1000 was 500g/L. Reagent II is composed of rabbit anti-human C5 antiserum or goat anti-human C5 antiserum or mouse anti-human C5 antiserum or mouse anti-human C5 monoclonal antibody cross-linked on latex particles. Imine reaction Rabbit anti-human C5 antibody, or goat anti-human C5 antibody, or mouse anti-human C5 antibody, or mouse anti-human C5 monoclonal antibody, IgG is covalently bound to latex particles. The titer of the rabbit anti-human C5 antiserum is 1:64-1:128, the concentration of C5 antiserum is 600ml/L, and the concentration of PEG6000 is 60g/L.

Example 4: Method for detecting C5 concentration in human serum

The method for detecting the concentration of C5 in human serum is to use the Hitachi 7100 biochemical analyzer to measure the sample. After adding the reagent IR1 to the sample, incubate at 37 °C for 5 minutes, read the first point sample A1, add reagent IIR2, incubate at 37 °C for 5 minutes, read Measure the second point sample A2, sample A=sample A2-sample A1; also includes a step of using the Hitachi 7100 biochemical analyzer to measure the calibrator, and in the step of using the biochemical analyzer to measure the calibrator, the After the calibrator is added with reagent IR1, incubate at 37°C for 5 minutes, read and measure the first point of calibration A1, add reagent IIR2, incubate at 37°C for 5 minutes, read and measure the second point of calibration A2, calibration A=calibration A2-calibration A1;

The parameters of the above determination are: temperature 37°C, main wavelength 340nm, secondary wavelength 700nm, sample or calibrator 3μl, R1: 240μl, R2: 60μl, and the reaction time is 10 minutes.

Example 5: The method for detecting the concentration of C5 in human serum in 50 healthy samples with normal values (measured by immunoturbidimetry)

In the method for detecting the concentration of C5 in human serum, the sample is measured by Hitachi 7100 biochemical analyzer. In the step of using the biochemical analyzer to measure the sample, the sample is added to the reagent IR1, incubated at 37°C for 5 minutes, and then read and measured. Sample A1 at point 1, add reagent IIR2, incubate at 37°C for 5 minutes, read and measure sample A2 at point 2, sample A = sample A2 - sample A1; it also includes a step of measuring the calibrator with Hitachi 7100 biochemical analyzer, in all In the above-mentioned step of using the biochemical analyzer to measure the calibrator, after adding the calibrator to reagent IR1, incubate at 37°C for 5 minutes, read and measure the first point of calibration A1, add reagent IIR2, incubate at 37°C for 5 minutes, read and measure the second point calibration A2, calibration A = calibration A2 - calibration A1;

The parameters of the above determination are: temperature 37°C, main wavelength 600nm, secondary wavelength 700nm, sample or calibrator 3 μl, R1: 240 μl, R2: 60 μl, and the reaction time is 10 minutes.

Table 1 Content of C5 in 50 healthy people

Healthy people: liver function, kidney function, biochemical indicators, immune function indicators are normal

Detection of 60 tumor patients (thyroid cancer, liver cancer, stomach cancer, uterine cancer, cervical cancer, lung cancer, breast cancer, esophagus cancer, nasopharyngeal cancer, prostate cancer, colorectal cancer, kidney cancer, pancreatic cancer, bladder cancer, glial cancer tumor)

Concentrations between 36.7mg/L-72mg/L are normal.

The clinical diagnosis results of 60 cases were more than 72 mg/L, which were positive, which met the clinical diagnostic criteria.

A. Precision detection: 20 consecutive samples were drawn from the same sample, and the samples were measured by Hitachi 7100 biochemical analyzer according to the method for detecting the concentration of C5 in human serum, and the mean value, standard deviation (SD) and coefficient of variation of the measured values were calculated. (CV), CV=(standard deviation/average)*100%, the results are shown in Table 2, CV is usually used to measure the precision of a measurement method, the smaller the CV value, the better the precision of the measurement method. . Method precision with a CV of less than 5% is generally accepted as acceptable for clinical chemistry testing programs.

Table 2: Precision Test Results

Reagent test kit pH value Average (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 Table 2 is less than or equal to 3.74%, indicating that the kit provided by the present invention has high precision, and the kit 3 has the highest precision.

B. Accuracy test: use the quality control product with serum anti-C5 complement antibody content of 60mg/L (the quality control product is composed of 5g bovine albumin, 0.3ml Proclin, add water to 100ml, and extract the antigen to form C5 complement antigen) for determination, Repeat 5 times, take the mean value, and calculate the relative deviation (CB). The test results are shown in Table 3. For clinical chemistry test items, the relative deviation does not exceed ±15%, which is considered to have excellent accuracy.

Table 3: Accuracy Test Results

The relative deviations in Table 3 all do not exceed ±2.27%, indicating that the kit provided by the present invention has high accuracy, and the kit described in Kit 3 has the highest accuracy.

C. Stability test: The same standard sample with serum anti-C5 complement antibody content of 60 mg/L was measured at 0 months, 6 months and 12 months under the storage conditions of 2-8 ℃, and each sample was tested. The measurement was performed 10 times, and the mean value was taken. The results are shown in Table 4.

Table 4: Stability Test Results

Reagent test kit pH value 0 months 6 months 12 months 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

It can be seen from Table 4 that the difference in the detected values is small, indicating that the kit provided by the present invention has better stability, and can be stored at 2-8 ° C for at least 12 months or more, and the kit described in Kit 3 is stable. Sex is the best.

D. Linear detection: use the kit described in Kit 3 to detect the standard substance of six concentration gradients, each gradient concentration is detected 3 times, take the average value, and perform regression analysis on the average value of the measured value and the theoretical concentration of the standard substance , the X-axis represents the theoretical concentration, and the Y-axis represents the mean of the measured values. Generally, R ² ≥ 0.9900 is considered to have good linearity, and the results are shown in Figure 1.

The obtained linear regression equation is: y=0.9798x+1.5804, and the correlation coefficient: R ² =0.9994. The results show that the kit provided by the present invention has a good linear correlation.

interference test

Select four potential interfering substances, bilirubin, hemoglobin, triglyceride and ascorbic acid, and add 250mg/L, 300mg/L, 350mg/L bilirubin to the quality control substance; 3.5g/L, 4.0g/L L, 4.5g/L hemoglobin; 7.5g/L, 8.0g/L, 8.5g/L triglyceride; 450mg/L, 500mg/L, 550mg/L ascorbic acid, the control group is without any interfering substances The quality control products were measured using the detection method of the kit described in kit 3. Each sample was measured 5 times, and the average value was taken. The results are shown in Table 5.

Table 5: Interfering substance test results

It can be seen from Table 5 that bilirubin≤300mg/L, hemoglobin≤4.0g/L, triglyceride≤8.0g/L, and ascorbic acid≤500mg/L, which have no obvious interference to the test results.

In conclusion, the test kit provided by the present invention has high accuracy, high precision, good stability and good linearity of the detectable results. Among them, the detection method of the kit 3 is used for detection, and the detection result is the best.

Embodiment 6Poct method detects the content of C5 in the sample to be tested (gold calibration):

In Example 6, the gold standardization method was adopted, and the FT-J5 gold standard reader produced by Shanghai Beijia Biochemical Reagent Co., Ltd. was used for determination. The colloidal gold test strip used for the detection is shown in Figure 2, wherein the colloidal gold pad is coated with a combination of colloidal gold, staphylococcal protein (SPA) and goat anti-human C5 complement antibody, and the detection line is coated with rabbit anti-human C5 complement antibody. The quality line is coated with antigen.

The samples tested included: 24 positive human serum samples infected with Gram-positive bacteria, 8 positive human serum samples infected with Gluconoccus aureus, 8 positive human serum samples infected with Candida glabrata, and 8 positive human serum samples infected with Pneumococcus Human serum samples, 8 positive human serum samples infected with hepatitis C virus, and 20 healthy human serum samples.

The interpretation of the test results is shown in Figure 3: if the quality inspection line and the test line are both colored, it is a positive result; if the quality inspection line is colored and the test line is colorless, it is a negative result; if the test line is colored and the quality inspection line has no color color, or the detection line and the quality inspection line are colorless, it is an invalid result.

The detection steps are as follows:

1. Take out all the reagents and samples before use, and place them at room temperature for 15-30 minutes to return to room temperature.

2. Take out the detection board.

3. Add 2 drops of blocking solution to the center of the reaction well of the detection plate, and wait for the blocking solution to penetrate completely.

4. Use a pipette to add 40 microliters of the sample to be tested in the center of the reaction well of the detection plate, and wait until the sample is completely infiltrated.

5. Add 4 drops of washing solution to the center of the reaction well of the detection plate, and wait for the washing solution to penetrate completely.

6. Add 3 drops of the conjugate of colloidal gold, staphylococcal protein (SPA) and goat anti-human C5 complement antibody to the center of the reaction well of the detection plate, and wait until the colloidal gold conjugate is completely infiltrated.

7. Add 4 drops of washing solution to the center of the reaction well of the detection plate. After the washing solution is completely infiltrated, check the result visually.

Table 6 The results of detecting C5 complement by Poct method

Bacterial infections:

Gram-positive bacteria, Gram-negative bacteria, Streptococcus glabrata, Gluconococcus aureus, Pneumococcus, Pseudomonas infection.

Viral infection: Hepatitis C

Embodiment 7: Immunotransmission turbidimetry detects C5 content in the sample to be tested

The sample was measured by Hitachi 7100 biochemical analyzer. In the step of using the biochemical analyzer to measure the sample, after adding the sample I reagent R1, incubate at 37 °C for 5 minutes, read the first point sample A1, add II reagent R2, 37 °C Incubate for 5min, read and measure the second sample A2, sample A=sample A2-sample A1; also includes a step of measuring the calibrator with Hitachi 7100 biochemical analyzer, in the step of measuring the calibrator with the biochemical analyzer, the After adding the calibrator I reagent R1, incubate at 37°C for 5 minutes, read and measure the first point of calibration A1, add II reagent R2, incubate at 37°C for 5 minutes, read and measure the second point of calibration A2, calibration A=calibration A2-calibration A1;

The parameters of the above determination are: temperature 37°C, main wavelength 340nm, secondary wavelength 700nm, sample or calibrator 3 μl, R1: 240 μl, R2: 60 μl, and the reaction time is 10 minutes. Kit composition: R1 Tris buffer (PH 8.1), PEG 2000, R2Tris buffer (PH 8.1), PEG 6000, C5 complement.

Table 7 utilizes immunotransmittance turbidimetry to detect the results of C5 content

Note: The value above 72mg/L is positive, and the following is negative.

The above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those of ordinary skill in the art, changes or modifications in other different forms can also be made on the basis of the above description. There is no need and cannot be exhaustive of all implementations here. Any modifications, equivalent replacements and improvements made within the spirit and principle of the present invention shall be included within 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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