CN114660287A

CN114660287A - Kit for detecting I factor, detection method and application thereof

Info

Publication number: CN114660287A
Application number: CN202011531482.5A
Authority: CN
Inventors: 朱梅珍; 刘颖冰
Original assignee: Shanghai Yijue Biotechnology Co ltd
Current assignee: Shanghai Yijue Biotechnology Co ltd
Priority date: 2020-12-22
Filing date: 2020-12-22
Publication date: 2022-06-24

Abstract

The invention belongs to the field of immunodetection, and relates to a kit for detecting a factor I, a detection method and application thereof. In particular, the invention relates to a kit for detecting factor I, 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 a kit for detecting factor I. 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 I factor, detection method and application thereof

Technical Field

The invention belongs to the field of immunodetection, and particularly relates to a kit for detecting a factor I, a detection method and application thereof.

Background

The I factor (old called C3bINA) is heterodimer serum protein, has a double-sphere structure, and has a molecular full length of 13 nm. Wherein the globule (L chain) is 4.9nm, and has serine protease activity; the macrospheres (H chain) 5.4nm can be bound to C3 b. The molecular weight of factor I is 88kDa, the heavy chain is 50kDa, the light chain is 38kDa, and the chains are linked by disulfide bonds.

Infection is caused by invasion of bacteria and viruses into organisms, pathogens cannot be identified efficiently, abuse 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 I factor determination of all countries in the world including China continues to use the immunodiffusion method and the ELISA double-antibody sandwich technology, the method is time-consuming and labor-consuming, the accuracy is low, and 2 days are required for patients to take reports. The commercial complement factor I detection kit adopts a double-antibody sandwich ELISA method. Coating the enzyme label plate with anti-complement factor I antibody, combining the complement factor I in the sample or standard with the coated antibody during the experiment, and washing off the free components. Biotinylated anti-complement factor I antibody and horseradish peroxidase-labeled avidin were added in sequence. The anti-complement factor I antibody binds to complement factor I bound to the coating antibody, biotin specifically binds to avidin to form an immune complex, and the free components are washed away. Adding chromogenic substrate (TMB), wherein the TMB is blue under the catalysis of horseradish peroxidase, and becomes yellow after adding stop solution. Measuring OD value at 450nm wavelength with enzyme labeling instrument, and calculating the concentration of complement factor I in the sample by drawing standard curve, wherein the concentration of complement factor I is in direct proportion to OD450 value.

Complement factor I is only used for auxiliary detection of congenital factor I deficiency in practical application.

Due to the defects in the prior art, the kit for rapidly and accurately detecting the concentration of the factor I 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 factor I, wherein the kit comprises:

reagent I: a first buffer solution and a first PEG solution;

and (2) reagent II: the anti-factor I antibody comprises a monoclonal antibody and/or a polyclonal antibody, a second PEG solution and IgG; the pH value of the first buffer solution is 3-10.

(2) The kit of (1), wherein the anti-factor I antibody is one or a combination of more than two of rabbit anti-human factor I antibody, goat anti-human factor I antibody and mouse anti-human factor I monoclonal antibody; preferably, the rabbit anti-human factor I antibody, the sheep anti-human factor I antibody and the mouse anti-human factor I antibody are monoclonal antibodies or polyclonal antibodies; factor I antibody titers 1:16 to 1:128, the concentration of the antibody is 300ml/L-800 ml/L.

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

(4) The kit according to (1) or (2), wherein the factor I monoclonal antibody is modified by treatment with trace manganese dioxide or potassium permanganate, and the lipophilic group of the anti-factor I antibody is changed into hydrophilic.

(5) The kit according to any one of (1) to (4), wherein the anti-factor I antibody is covalently bound to a latex particle; preferably, the anti-factor I monoclonal antibody is bound first and then bound to the factor I polyclonal antibody covalently bound to the latex particles by 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 producing a diagnostic agent for detecting factor I 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 factor I polyclonal antibody is added to make the linearity wider, the monoclonal antibody contains mouse, rabbit, sheep and horse cell strains, and the polyclonal antibody contains 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 factor I concentration (mg/L) was calculated by the following formula:

(12) use of the kit according to any one of (1) to (5) for the preparation of a reagent or kit for detecting factor I.

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 accuracy reaches the absorbance value: 0.0001.

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 the diagnosis of the cancer, 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 the graphs of the standard substance for detecting six concentration gradients of the complement factor I kit of the present invention, each concentration gradient is detected 3 times, the average value is taken, the regression analysis is performed 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.

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

FIG. 3 shows a schematic diagram of the interpretation of the results of factor I detection using colloidal gold test strips.

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.

In the present specification, the numerical range represented by "numerical value a to numerical value B" means a range including the end point numerical value A, B.

In the present specification, "PEG" means polyethylene glycol; here, "PEG 2000" means PEG having a polymerization degree of 2000. In the present specification, reference to "some particular/preferred embodiments," "other particular/preferred embodiments," "embodiments," and the like, 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.

FIG. 1 is a graph showing the results of the six concentration gradient measurements performed by the complement factor I detection kit of the present invention, wherein each concentration gradient is measured 3 times, the 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.

< first aspect >

The invention provides a kit for detecting factor I, which comprises:

reagent I: a first buffer solution and a first PEG solution;

and (2) reagent II: anti-factor I antibody, second PEG solution, IgG; the pH value of the first buffer solution is 3-10.

The inventor finds that the I factor antigen can be adhered to the pathogen, and the concentration of the I factor is measured to obtain the amount of pathogen infection, so that whether the patient has the pathogen infection or not can be known.

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 filtration method or the chromatography method and the (Poct) immediate test are faster, and the report can be accurately reported within 2 minutes, thereby reducing the waiting time of patients. The method is expected to replace an ELISA double-antibody sandwich method for detecting the factor I, and the factor I is detected by using methods such as 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 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 genera staphylococcus, streptococcus, enterococcus, neisseria, moraxella, acinetobacter, pseudomonas, alcaligenes, brunella, bordetella, legionella, escherichia, citrobacter, salmonella, shigella, klebsiella, serratia, proteus, pluriphranilide, 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 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 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.

The reagent I comprises a first PEG solution (polyethylene glycol), when an anti-factor I antibody (antibody) is combined with a factor I (antigen) to form an immune complex with a certain structure, the immune complex becomes particles suspended in a reaction solution under the action of the PEG, so that the turbidity of a sample is changed.

In the present invention, the first PEG solution is different from the second PEG solution.

In some specific embodiments of the invention, the first PEG solution used in reagent I comprises: PEG 2000; the second PEG solution used in the reagent II is PEG 4000-20000, and the PEG with extremely large molecular weight and concentration difference between the reagent I and the reagent II is used for accelerating and strengthening the reaction. 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 PEG2000 is 400 g/L-600 g/L; in a preferred embodiment, the concentration of PEG2000 is 500g/L, and the concentration of PEG2000 reduces non-specific reaction in the sample.

When the anti-factor I antibody used in the invention is anti-factor I antiserum, other antibodies except the factor I antibody in the factor I antiserum are combined by the calcium chloride in the anti-factor I antiserum, so that useless antibodies can be removed, the affinity of the antibodies and the antigen is enhanced, the combination speed is accelerated, and the arrival of an 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 concentration of calcium chloride is 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-factor I antibody, a second PEG solution and IgG.

The reagent II comprises an anti-factor I antibody, and the anti-factor I antibody can be a polyclonal antibody or a monoclonal antibody. In some embodiments of the invention, the polyclonal antibody is an antiserum containing factor I antibodies. In a preferred embodiment, the anti-factor I antibody of the invention is a monoclonal antibody. In some embodiments of the invention, the anti-factor I antibody is selected from one or a combination of two or more of rabbit anti-human factor I antibody, goat anti-human factor I antibody, and mouse anti-human factor I antibody. In a preferred embodiment, the rabbit anti-human factor I antibody, the sheep anti-human factor I antibody, and the mouse anti-human factor I antibody are monoclonal antibodies. In some specific embodiments of the invention, the concentration of the anti-factor I antibody is from 100ml/L to 800 ml/L; in a preferred embodiment, the anti-factor I antibody is present at a concentration of 600 ml/L.

The reagent II comprises a second PEG solution, and the second PEG solution which can be used in the reagent II comprises: PEG 4000-20000. For example, the second PEG solution may be one or more of PEG with a molecular weight of 4000, 7000, 8000, 9000 or 20000. In the reagent II, the concentration of PEG 4000-20000 is 30 g/L-80 g/L; preferably, the concentration of the PEG 4000-20000 is 80 g/L. When the I factor antibody-antigen is combined 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. According to the invention, the PEG2000 is 400-600 g/L in the reagent I, the PEG 4000-20000 is 30-80 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, particularly the concentration difference of the first PEG solution and the second PEG solution. The absorbance at the end of the reaction is stable, and the affinity is stronger than that of the general reagent.

In some specific embodiments of the present invention, the reagent II further comprises a second buffer solution, wherein the pH value of the second buffer solution is 3 to 10; preferably 8.1. In a preferred embodiment, the second buffer is tris buffer. The pH of the buffer solution is 8.1, the reaction is accelerated, and the end point reaching speed is high.

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 factor I antibody is changed into hydrophilic, so that the efficiency of the antibody is higher, the efficiency is higher, the stability of the reagent is better, the storage period is longer, the reagent can be stored for 24 months, the linearity is better, and the anti-interference performance is higher.

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 factor I monoclonal antibody is treated and modified by trace manganese dioxide or potassium permanganate, the lipophilic group of the C5 antibody is changed into hydrophilic. The sensitivity of the reagent can be improved, the reagent is quick and stable, the error is small, and the polyclonal factor I antibody can be correspondingly driven to have better linear reaction.

In some embodiments of the invention, the anti-factor I antibody is covalently bound to a latex particle. As a preferred embodiment, the anti-factor I 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 may be used.

< second aspect >

In a second aspect the present invention provides the use of a kit according to the first aspect in the manufacture of a diagnostic for the detection of factor I 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:

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, factor I concentration (mg/L) was calculated by the following formula:

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 main wavelength measured by the immunoturbidimetry is 340nm, and the sub-wavelength measured by the immunoturbidimetry is 700 nm. The above immunoturbidimetric assay sample or calibrator is added in an amount of 3. mu.l to 5. mu.l, and the reaction time is 10 minutes.

The immune transmission turbidimetry, the immune scattering turbidimetry, the latex enhanced immune turbidimetry, the chemiluminescence method and the Poct instant detection also comprise a factor I calibrator: is prepared from bovine albumin (5 g) and Proclin (0.3 ml) through adding water to 100ml, and extracting factor I antigen. The quality control product consists of 700mg/L bovine albumin, 350ml/L Proclin and I factor antigen.

< third aspect >

In a third aspect the invention provides the use of an anti-factor I antibody for the manufacture of a diagnostic for the detection of a factor I antibody.

The invention finds that the thallus of bacteria and virus is adhered to the antigen of the factor I, and the content of the bacteria and the virus can be obtained by detecting the concentration of the antibody of the factor I.

The invention can be used for pathogen infection by detecting the factor I, and can be used for identifying whether the pathogen infection is improved or not by comparing before and after treatment, and the accuracy 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 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 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.

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 method can also be used for screening tumor markers: 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, 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: composition of latex immunoturbidimetric kit

The kit for detecting the content of the factor I by using the latex immunoturbidimetry comprises two reagents: reagent I contains Tris-buffered saline (pH 8.1). Taking 0.294g of calcium chloride, adding 0.2mol/L of tris solution to dissolve the calcium chloride, adjusting the pH value to 8.1 by using 1mol/L of hydrochloric acid solution, adding 50g of PEG1000 solution, adding water to dilute to 100ml, wherein in a reagent I, the mass concentration of the PEG1000 solution is 500g/L, a reagent II consists of suspension formed by crosslinking rabbit anti-human factor I antiserum on latex particles, and a reagent II is formed by covalently bonding rabbit anti-human factor I antibodies and IgG on the latex particles through carbodiimide reaction. The titer of the rabbit anti-human factor I antiserum is 1:16 to 1:128, the content of the factor I antibody is 600ml/L, and the concentration of PEG6000 is 80 g/L.

Example 2: kit composition for detecting I factor concentration by using scattering immunoturbidimetry

A kit for detecting the concentration of factor I by a scattering immunoturbidimetry method comprises two reagents, wherein the reagent I contains a tris buffer solution (pH 8.1). 0.294g of calcium chloride is taken and added with 0.2mol/L of tris solution for dissolution, 1mol/L of hydrochloric acid solution is used for adjusting the pH value to 8.1, PEG200050g is added, water is added for dilution to 100ml, and in the reagent I, the PEG2000 is 500g/L in mass percentage concentration. Reagent II contained a tris buffer solution (pH 8.1). 0.294g of calcium chloride is taken and added with 40mg/L of 0.2mol/L of tris solution to dissolve, 1mol/L of hydrochloric acid solution is used for adjusting the pH value to 8.1, PEG6000 and rabbit anti-human factor I antiserum are added with water and diluted to 100ml, in the reagent II, the mass percentage concentration of the PEG6000 is 50g/L, the volume concentration of the rabbit anti-human factor I antiserum is 600ml/L, the titer of the rabbit anti-human factor I antiserum is 1:16, and the concentration of the factor I antiserum is 600 ml/L.

Example 3: kit for detecting concentration of factor I by enhanced nano latex immunoturbidimetry

The kit for detecting the concentration of the factor I by the enhanced nano latex immunoturbidimetry comprises two reagents, wherein the reagent I contains a tris buffer solution (pH 8.1). 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, PEG200050g is added, water is added for dilution to 100ml, in a reagent I, the mass concentration of PEG2000 is 500g/L, a reagent II is formed by cross-linking rabbit anti-human factor I antiserum on latex particles, and the reagent II is formed by covalently bonding rabbit anti-human factor I antibodies and IgG on the latex particles through carbodiimide reaction. The titer of the rabbit anti-human factor I antiserum is 1:64, the concentration of the factor I antiserum is 600ml/L, and the concentration of PEG6000 is 50 g/L.

Example 4: detecting the concentration of factor I in human serum

A method for detecting concentration of factor I in human serum utilizes the kit of the invention, sample adopts Hitachi 7100 biochemical analyzer to measure, add sample into reagent IR 1, incubate for 5min at 37 ℃, read and measure sample A1 at 1 st point, add reagent IIR 2, incubate for 5min at 37 ℃, read and measure sample A2 at 2 nd point, sample A is 2-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, sample or calibrator 3 μ l, R1: 240 μ l, R2: 60 μ l, reaction time 10 min.

Example 5: average value (determined by immune transmission turbidimetry) of 50 healthy human normal value samples for detecting factor I in human serum Concentration of

Measuring a sample by using a Hitachi 7100 biochemical analyzer, adding a reagent I R1 into the sample, incubating the sample at 37 ℃ for 5min, reading a sample A1 at the 1 st point, adding a reagent II R2, incubating the sample 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 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, sample or calibrator 3 μ l, R1: 240 μ l, R2: 60 μ l, reaction time 10 min.

TABLE 150 contents of factor I in healthy people

Wherein, the definition for healthy people is: liver function, renal function, biochemical index and immune 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 20.5mg/L and 52.3mg/L are normal.

The results of 60 cases of clinical diagnosis are more than 52.3mg/L, are positive and accord with the clinical diagnosis standard.

A. And (3) detecting the precision: continuously extracting the same sample for 20 times, determining the sample by Hitachi 7100 biochemical analyzer according to the method for detecting the concentration of the factor I in human serum,

adding a reagent I R1 into the sample, incubating at 37 ℃ for 5min, reading and measuring a sample A1 at the 1 st point, adding a reagent II R2, incubating at 37 ℃ for 5min, reading and measuring 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 Hitachi 7100 biochemical analyzer, wherein in the step of measuring the calibrator by using the biochemical analyzer, the calibrator is added with a reagent I R1, then the mixture is incubated at 37 ℃ for 5min, the 1 st calibration A1 is read, the reagent II R2 is added, the mixture is incubated at 37 ℃ for 5min, and the 2 nd calibration A2 is read, wherein the calibration A is calibration A2-calibration A1;

The average, Standard Deviation (SD), and Coefficient of Variation (CV) of the measured values were calculated, and CV (standard deviation/average) × 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

Composition of kits 1, 3: r1 Tris buffer (pH8.1), PEG2000, R2 Tris buffer (pH8.1), PEG6000, factor I antibody.

The CV value in the table 2 is less than or equal to 3.41%, 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: a quality control product with the antigen content of the serum complement I factor of 40mg/L is used (the quality control product is the I factor antigen formed by adding water into 5g of bovine albumin and 0.3ml of Proclin to 100ml and extracting the antigen). Repeat 5 times, take the mean value, calculate the relative deviation (CB), the test result is shown in Table 3. For clinical chemistry test items, the relative deviation is not more than ± 15%, which is considered to have excellent accuracy.

Table 3: accuracy detection result

The relative deviation in table 3 does not exceed +/-1.70%, 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 40mg/L serum complement I factor antigen content was assayed 10 times per sample at 0 month, 6 months and 12 months respectively under 2-8 deg.C storage condition, and the results are shown in Table 4.

Table 4: stability test results

Reagent kit	pH value	0 month	6 months old	12 months old
					Kit 1	4.0-6.0	39.38	38.66	38.91
Kit 2	6.0-7.0	40.35	39.21	39.03
					Kit 3	7.0-11.0	40.27	40.36	40.30

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 is 0.9963x +0.895, correlation coefficient: r²0.9992. 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 factor I in a sample to be tested (gold-labeled):

example 6 was conducted by using a gold-labeled qualitative method using a FT-J5 gold-labeled reader manufactured by Shanghai North China Biochemical industries, Ltd. The colloidal gold test strip for detection is shown in figure 2, wherein the colloidal gold pad is coated with a conjugate of colloidal gold, Staphylococcal Protein (SPA) and goat anti-human factor I antibody, the detection line is coated with a rabbit anti-human factor I antibody, and the quality detection line is coated with an 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 result is 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; and 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 pipettor until the sample completely permeates into the reaction hole center.

5.4 drops of washing liquid are added to the center of the reaction hole of the detection plate until the washing liquid completely permeates.

6. 3 drops of conjugate of colloidal gold, Staphylococcal Protein (SPA) and goat anti-human factor I antibody are added into the reaction hole center of the detection plate until the colloidal gold conjugate is completely infiltrated.

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 detecting factor I by Poct method

Bacterial infection:

gram-positive bacteria, gram-negative bacteria, Candida glabrata, Staphylococcus aureus, and pneumococcus.

Viral infection:

hepatitis C Virus (HCV)

Example 7: immune transmission turbidimetry method for detecting I factor content in sample to be detected

A method for detecting the concentration of a factor I in human serum comprises the steps of measuring a sample by a Hitachi 7100 biochemical analyzer, adding a reagent IR 1 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 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 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, 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), PEG2000, R2 Tris buffer (pH8.1), PEG6000, factor I antibody

The results are shown in Table 7.

TABLE 7 results of detection of factor I content by immunotransmission turbidimetry

Note: the value was positive at 53mg/L or more, and negative at below.

Example 8Poct method for detecting the content of factor I in a sample to be tested (gold-labeled):

example 8 was conducted by using a gold-labeled qualitative method using a FT-J5 gold-labeled reader manufactured by Shanghai North China Biochemical industries, Ltd. The colloidal gold test strip for detection is shown in figure 2, wherein the colloidal gold pad is coated with a conjugate of colloidal gold, Staphylococcal Protein (SPA) and goat anti-human factor I antibody, the detection line is coated with a rabbit anti-human factor I antibody, and the quality detection line is coated with an antigen.

The detecting the sample comprises: 30 positive human serum samples infected with legionella pneumophila, 30 positive human serum samples infected with mycoplasma pneumoniae, 30 positive human serum samples infected with Q-fever rickettsia, 30 positive human serum samples infected with chlamydia pneumoniae, 30 positive human serum samples infected with adenovirus, 30 positive human serum samples infected with respiratory syncytial virus, 30 positive human serum samples infected with influenza A virus, 30 positive human serum samples infected with influenza B virus, and 30 positive human serum samples infected with parainfluenza virus. Healthy human serum samples 19 x 9 (total 171).

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:

2. And taking out the detection plate.

The results are shown in Table 8.

TABLE 8 detection by Poct methodFactor IResults of content

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

1. A kit for detecting factor I, wherein the kit comprises:

reagent I: a first buffer solution and a first PEG solution;

2. The kit according to claim 1, wherein the anti-factor I antibody is selected from one or a combination of more than two of rabbit anti-human factor I antibody, goat anti-human factor I antibody and mouse anti-human factor I monoclonal antibody; preferably, the rabbit anti-human factor I antibody, the sheep anti-human factor I antibody and the mouse anti-human factor I antibody are monoclonal antibodies or polyclonal antibodies; factor I antibody titers 1:16 to 1:128, the concentration of the antibody is 300ml/L-800 ml/L.

3. The kit according to claim 1 or 2, wherein the lipophilic group of the anti-factor I 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 according to claim 1 or 2, wherein the factor I monoclonal antibody is modified by treatment with trace amounts of manganese dioxide or potassium permanganate to change the lipophilic group of the anti-factor I antibody to hydrophilic.

5. A kit according to any one of claims 1 to 4, wherein the anti-factor I antibody is covalently bound to latex particles; preferably, the anti-factor I monoclonal antibody is bound first and then bound to the factor I polyclonal antibody covalently bound to the latex particles by 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 factor I by a method comprising: immunotransmission turbidimetry, immunoscattering turbidimetry, immunolatex, chemiluminescence, or 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 factor I polyclonal antibody is added to make the linearity wider, 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:

the factor I concentration (mg/L) was calculated by the following formula:

12. use of a kit according to any one of claims 1 to 5 in the manufacture of a reagent or kit for the detection of factor I.