CN111458498A - Hand-foot-mouth EV71 antigen detection kit - Google Patents
Hand-foot-mouth EV71 antigen detection kit Download PDFInfo
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- CN111458498A CN111458498A CN201910056766.4A CN201910056766A CN111458498A CN 111458498 A CN111458498 A CN 111458498A CN 201910056766 A CN201910056766 A CN 201910056766A CN 111458498 A CN111458498 A CN 111458498A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
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Abstract
The invention is suitable for the technical field of in-vitro diagnostic reagents, and provides a hand-foot-mouth EV71 antigen detection kit. The hand-foot-mouth EV71 antigen detection kit comprises an ELISA plate coated with an anti-EV 71-VP1 monoclonal antibody, an HRP-labeled anti-EV 71 monoclonal antibody for detection, an enterprise reference product, a sample diluent, a concentrated washing solution, a developing solution A, a developing solution B and a stop solution. The hand-foot-mouth EV71 antigen detection kit adopts a specific antigen expression sequence SEQ ID NO: 1, the kit is used for detecting EV71 antigen in serum, can effectively improve detection sensitivity, and is used for early auxiliary diagnosis of clinical patients infected with hand-foot-and-mouth disease EV 71; the enterprise reference materials contained in the kit can be used for quality control of EV71 antigen detection kits produced by enterprises, and can well control the product quality of the kits.
Description
Technical Field
The invention belongs to the technical field of in-vitro diagnostic reagents, and particularly relates to a hand-foot-mouth EV71 antigen detection kit.
Background
Hand-foot-and-mouth disease (HFMD) is an infectious disease with herpes of the skin of hands, feet and oral mucosa as the main symptom caused by enterovirus infection. Enteroviruses causing hand-foot-and-mouth disease include enterovirus type 71 (EV71) and some serotypes of coxsackie a group virus (CoxA), echovirus (Echo). EV71 infection causes a large proportion of severe cases. The enterovirus has strong infectivity and is easy to cause outbreak or epidemic.
Currently, the related diagnosis of the hand-foot-and-mouth disease mainly comprises two main categories of clinical diagnosis and laboratory diagnosis. Wherein the clinical diagnosis related to the hand-foot-and-mouth disease is as follows: acute onset, fever, maculopapular and herpes in the palms or soles, and rashes in the buttocks or knees. Inflammatory halos are formed around the rash, and fluid in the blisters is less; the oral mucosa has dispersed herpes and obvious pain. Some children may be accompanied by cough, runny nose, loss of appetite, nausea, vomiting and headache. Severe cases manifest as: 1. patients with clinical manifestations of hand-foot-and-mouth disease are accompanied by myoclonus, or encephalitis, acute delayed paralysis, cardiopulmonary failure, pulmonary edema, etc. 2. Infants in the area with the epidemic hand-foot-mouth disease have no typical hand-foot-mouth disease, but have fever with myoclonus, encephalitis, acute delayed paralysis, cardiopulmonary failure, pulmonary edema and the like. At present, laboratory diagnosis of hand-foot-and-mouth disease mainly comprises methods such as virus separation, nucleic acid detection, serology detection and the like; (1) and (3) separating the virus, namely separating the enterovirus from tissue specimens such as throat swabs or throat washing liquid, excrement or anus swabs, cerebrospinal fluid or herpes fluid, brain, lung, spleen, lymph nodes and the like. (2) And (3) nucleic acid detection: pathogenic nucleic acids are detected from samples such as patient serum, throat swab or throat wash, feces or anal swab, cerebrospinal fluid or herpes fluid, and tissue samples such as brain, lung, spleen, and lymph node. (3) Serological detection: the specific IgM antibody in the patient serum is positive, or the acute phase serum IgG antibody and the convalescent serum IgG antibody are increased by more than 4 times. There is currently no approved antigen detection reagent.
Human enteroviruses causing hand-foot-and-mouth disease can be divided into 4 groups (enteroviruses of group A, group B, group C and group D) according to the genetic affinity relationship, wherein the enteroviruses of group A currently known comprise 17 serotypes, namely novel enteroviruses 71, 76 and 89-92 of Coxsackie group A viruses 2-8, 10, 12, 14 and 16 respectively. The most major pathogens causing HFMD are EV71 in group a enteroviruses and coxsackievirus group a 16. Enterovirus 71 (Enterovirus 71, EV71) infection clinically the main infection subjects were young children under the age of 5. Adults can also become infected and spread within the same family member, but clinical symptoms are not evident. This subclinical or occult infection is also another important pathway for rapid transmission of EV 71.
EV71 virus is enterovirus of picornaviridae, the viral genome is single-strand positive-strand RNA, encodes four virus capsid proteins of VP1, VP2, VP3 and VP4, except that VP4 is embedded inside the shell of the virus particle and is tightly connected with the virus core, other 3 structural proteins are exposed on the surface of the virus particle, the antigenic determinant is basically located on VP 1-VP 3, Shih and the like use a prokaryotic expression product of VP1 gene of EV71 as antigen, and establish a corresponding EV71-IgM detection reagent by an indirect enzyme-linked immunosorbent assay, thereby preliminarily proving the important position of the recombinant protein VP1 antigen in preparing EV71-E L ISA diagnostic reagents.
Early diagnosis of EV71 is of great significance to prevention and treatment of later-stage diseases. Although the RNA detection has been successfully developed, the method is complex to operate and expensive, and is difficult to popularize and apply in the basic level. Meanwhile, due to the existence of the EV71 recessive infection, whether a patient has a recent disease or not can not be determined only by RNA positivity. In contrast, enzyme-linked detection is simple and cheap, and EV71IgM is an important marker of acute infection, and has a wider application value. At present, antigens adopted in commercial EV71IgM detection reagents are all inactivated EV71 whole virus particles, and the existence of IgM antibodies can be detected on the next day of morbidity. However, the reagent is developed on the basis of the recombinant antigen, the performance of inactivating the EV71 whole virus particle antigen can be completely achieved through the optimization of the process and the methodology, and the reagent has obvious advantages in the aspects of production, operation and the like.
The research shows that the epitope of EV71 is mainly located in VP1, VP2, VP3, Shih and the like prove that the recombinant VP1 antigen has important function in preparing EV71-E L ISA diagnostic reagent, therefore, the invention solves the problem of VP1 antigen expression, and then uses EV71-VP1 antigen to prepare monoclonal antibody, and develops a double-antibody sandwich enzyme-linked immunoassay kit with higher sensitivity for detecting EV71 antigen.
Disclosure of Invention
The embodiment of the invention provides a hand-foot-mouth EV71 antigen detection kit, and aims to solve the problems that the existing RNA detection operation is complex, the price is high, and the basic popularization and application are difficult. The detection kit provided by the invention is an in-vitro diagnosis kit for determining enterovirus (EV71) antigen by adopting an enzyme-linked immunosorbent assay, has the characteristics of simple operation and low cost, and EV71IgM is an important marker of acute infection, so that the detection kit has wider application value.
The embodiment of the invention is realized in such a way that a hand-foot-mouth EV71 antigen detection kit comprises an ELISA plate coated with an anti-EV 71-VP1 monoclonal antibody, an HRP-labeled anti-EV 71 monoclonal antibody for detection, an enterprise reference product, a sample diluent, a concentrated washing solution, a developing solution A, a developing solution B and a stop solution.
The nucleotide sequence of the anti-EV 71 virus antigen used in the preparation process of the anti-EV 71-VP1 monoclonal antibody is shown as SEQ ID NO: 1 is shown.
The preparation method of the ELISA plate for resisting the EV71-VP1 monoclonal antibody comprises the following steps:
1) diluting the anti-EV 71-VP1 monoclonal antibody with a pH9.6 carbonate buffer solution, wherein the final concentration of the antibody is 5 mu g/m L, adding the antibody into a microporous plate, keeping the antibody at 100 mu L/hole, and standing at 2-8 ℃ for not less than 16 hours;
2) washing the plate 2 times with 0.01 mol/L PBS pH 7.2;
3) adding sealing liquid, sealing at 120 mu L per hole at 2-8 ℃ for not less than 16 hours;
4) vacuum drying for 3 hours;
5) packaging the coated antibody plate with aluminum foil bag, and storing at 2-8 deg.C.
The preparation method of the anti-EV 71-VP1 monoclonal antibody comprises the following steps:
using anti-EV 71 monoclonal antibody prepared by EV71-VP1 antigen immune mouse, making ascites pass through 200-mesh stainless steel sieve, removing fat layer, measuring supernatant volume, adding PBS (pH7.20.01mol/L) according to volume ratio of 1:1, adding saturated ammonium sulfate under stirring to 50% saturation, acting at 25 + -2 deg.C for 30 min, centrifuging at 4 deg.C 10000rpm for 15 min, discarding supernatant, dissolving precipitate with PBS (pH7.20.01mol/L), measuring volume, adding saturated ammonium sulfate to 33% saturation, acting at 25 + -2 deg.C for 30 min, centrifuging at 4 deg.C 10000rpm for 15 min, discarding supernatant, dissolving precipitate with PBS (pH7.20.01mol/L), placing into dialysis bag, dialyzing with PBS (pH7.20.01mol/L) for 48 hr until no SO is produced4 2－Until the end; and (3) passing through a Protein-A affinity chromatography column to obtain the anti-EV 71 monoclonal antibody.
The preparation method of the EV71-VP1 antigen comprises the following specific steps:
(1) culturing the expression strain, namely inoculating 20 mu L of EV71-VP1 expression strain preserved at the temperature of-70 ℃ into L B culture medium (100m L L B/500m L triangular flask), culturing overnight by an air shaking table at the temperature of 30 ℃, transferring the strain into L B culture medium (100m L L B/500m L triangular flask) according to the proportion of 5 percent on the next day, culturing for about 3 hours by the air shaking table at the temperature of 37 ℃, immediately adding IPTG (isopropyl-beta-D-thiogalactoside) when the OD600nm value reaches 0.7, transferring the culture bottle into a shaking table at the temperature of 25 ℃, inducing and culturing for 4 hours, combining bacterial liquid, centrifuging for 20 minutes at the rpm of 6000, abandoning supernatant, and collecting a precipitation part;
(2) and (2) purifying, namely suspending and precipitating by using 50 mmol/L Tris-HCl buffer solution with the pH value of 8.3, performing ultrasonic bacteria crushing method in ice bath, performing ultrasonic bacteria crushing method for 10 times at the interval of 30 seconds every time, performing centrifugation for 20 minutes at the temperature of 8 ℃, passing through GST agarose gel FF column, eluting by using buffer solution prepared by reduced glutathione, collecting elution peak, and identifying various purified recombinant epitope antigens by SDS-PAGE to obtain the EV71-VP1 antigen.
The EV71-VP1 expression strain is constructed by the following method:
carrying out annealing extension PCR on a VP1 sequence shown as SEQ ID NO. 1, introducing BamHI and EcoRI restriction sites at two ends of a connecting arm to be suitable for an expression vector pBET-28a in order to facilitate gene cloning to an expression vector, carrying out double restriction, inserting the double restriction sites into the vector pBET-28a to construct a corresponding expression plasmid, carrying out EV71-VP1 antigen expression plasmid, transforming E.coli B L21, inducing by IPTG, taking a whole bacteria solution to carry out SDS-PAGE identification, and proving that the plasmids all obtain high-efficiency expression to obtain an EV71-VP1 expression strain.
The preparation method of the anti-EV 71 monoclonal antibody for HRP labeling detection comprises the following steps:
(1) measuring 1mg protein and 1mg HRP, dissolving 5mg horse radish peroxidase in 1.0m L double distilled water to fully dissolve HRP;
(2) 0.5m L0.06 mol/L NaIO was added4Stirring for 30 minutes at room temperature in dark place;
(3) taking out, adding 0.16 mol/L ethylene glycol aqueous solution 0.5m L, and keeping out of the sun for 30 minutes at room temperature;
(4) taking a proper amount of purified antibody, fully dissolving the antibody by using double distilled water, mixing the antibody with enzyme solution, filling the mixture into a dialysis bag, dialyzing the mixture for 20 hours at 2-8 ℃ in 0.01 mol/L carbonate buffer solution with pH value of 9.6 in a dark place, and changing the solution for 3 times in the middle;
(5) collecting the dialyzed combined solution, adding 0.2m L NaBH according to 5mg HRP4Metering in solution, adding NaBH4After the stirring is carried out for 30 minutes,standing for 4 hours at the temperature of 2-8 ℃;
(6) loading the enzyme conjugate into a dialysis bag, and dialyzing with PBS (pH7.20.01mol/L) for 48 hours;
(7) measuring the volume of the concentrated solution, taking a small sample of 0.1m L, and inspecting;
(8) adding glycerol of the same volume to the other concentrated solution to obtain the HRP-labeled detection anti-EV 71 monoclonal antibody, and storing the monoclonal antibody below-20 ℃ for later use.
The enterprise reference product is used for quality control of EV71 antigen detection kits produced by enterprises; the enterprise reference comprises 10 parts of a positive reference P01-P10, 10 parts of a negative reference N01-N10, 1 part of a minimum detection limit reference S1-S5 and 1 part of a repeatability reference CV.
The positive reference substance is a patient serum sample with a positive detection result of EV71 antigen;
the negative reference substance is a healthy human serum sample with negative detection result;
the lowest detection limit reference substance is a serum sample obtained by diluting the serum of a patient with strong positive detection antibodies by a fold ratio with the serum of a healthy person;
the repeated reference substance is a patient serum sample with a positive detection result of EV71 antigen.
The technical requirements of the enterprise reference product comprise the following contents:
(1) appearance: all reference liquid should be clear and transparent without visible precipitation;
(2) negative reference product compliance rate: the positive reference products P01-P10 are all positive when the kit is used for detecting, and the coincidence rate of the positive reference products is 100%;
(3) positive reference compliance rate: the kit is used for detecting that the negative reference products N01-N10 are all negative, and the coincidence rate of the negative reference products is 100%;
(4) the lowest detection limit is: detecting a minimum detection limit reference substance S1-S5, wherein S1, S2 and S3 are all positive, S5 is negative, and S4 is not required;
(5) repeatability: detecting a repetitive reference product CV by using the kit, and simultaneously detecting 10 holes, wherein the repeatability (CV%) of the determined OD value is less than or equal to 15.0%;
(6) inter-batch difference: and 3 batch number kits are used for detecting the repetitive reference substance, and the batch-to-batch variation coefficient (CV%) among the 3 batch number kits is less than or equal to 15.0%.
Operation description of the enterprise reference:
(1) storage conditions were as follows: the long-term storage temperature should be below-20 deg.C.
(2) And adding the sample after the reference substance is completely melted and uniformly mixed.
(3) The product should be stored and transported at-20 deg.C or below to avoid repeated freezing and thawing.
(4) Duplicate reference (CV) was applied directly to each well at 100. mu. L, and 10 wells were tested in parallel.
(5) The reference substance should be treated with infectious substances during use.
(6) The reference product has an expiration date of 2 years below-20 ℃.
Second, the main research results and evaluation of related products
1. Results of Performance Studies of the finished product
Negative reference product compliance rate: and (5) detecting by using 10 enterprise negative reference products, and detecting all negative products.
Positive reference compliance rate: the detection is carried out by 10 enterprise positive reference products, and the coincidence rate is 10/10.
Precision reference product: and (4) carrying out detection by using an enterprise precision reference product, wherein CV (%) is less than or equal to 15.0% (n is 10).
Three batches of reagents produced continuously, 3% were sampled according to the above criteria. All meet the standard requirements.
And (3) stability verification results: stability tests are carried out on the three batches of reagents, and the results prove that the product reaches the quality inspection standard after 5 days of accelerated tests at 37 ℃; the kit can reach the quality inspection standard after being stored for 12 months at the temperature of 2-8 ℃.
2. Product evaluation
The developed reagent is subjected to analysis performance evaluation, anti-interference capability evaluation and stability (long-term storage stability and thermal stability) evaluation, and clinical tests are compared with the kit on the market, and the results show that the reagent meets the technical performance requirements, can meet the clinical use requirements of basic medical institutions, fills the blank of the in-vitro diagnostic reagent for directly measuring the enterovirus EV71 antigen by using the domestic enzyme-linked immunosorbent assay, and has higher sensitivity than the similar kit in the market.
The sample diluent is 0.24% Tris, 1.78% NaCl, 0.5% sodium caseinate, 0.2% bovine serum albumin, 0.1% Tween-20, 0.1% Triton X-100, 5% goat serum and 0.1% thimerosal;
the concentrated washing solution is 0.9% NaCl-0.1% Triton X-100;
the color development liquid A is boric acid buffer solution, L mininol;
the color developing solution B is citric acid buffer solution H2O2；
The stop solution is 2M H2SO4。
The detection principle of the detection kit of the invention is as follows: the method comprises the steps of adopting a double-antibody sandwich enzyme-linked immunoassay principle, coating a microporous plate with an anti-EV 71 monoclonal antibody, adding serum to be detected, specifically binding an antigen antibody when an EV71 antigen exists in a sample to be detected, adding another anti-EV 71 monoclonal antibody marked by an enzyme, reacting the enzyme conjugate with the antigen-antibody conjugate, fully washing, adding a TMB substrate for color development, stopping the reaction by using a stop solution, measuring absorbance by using an enzyme-linked immunosorbent assay, and judging whether the EV71 antigen exists in the sample to be detected according to a judgment mode provided by a product. The kit is used for early auxiliary diagnosis and epidemiological investigation of clinical patients infected with the hand-foot-and-mouth disease.
The detection method of the hand-foot-mouth EV71 antigen detection kit comprises the following steps:
1. experimental preparation the kit was removed from the refrigerated environment and allowed to equilibrate at room temperature (18 ℃ C. -25 ℃ C.) for 30 minutes while diluting the 20 × concentrated wash solution at 1: 19;
2. adding the sample to be tested, namely setting two holes of a negative control and a positive control in each experiment, respectively adding 100 mu L of the negative control and the positive control, setting a hole blank control (without adding any reagent), adding 50 mu L of sample diluent into the other holes, adding 50 mu L of the sample to be tested, fully mixing uniformly, incubating at 37 ℃ for 60 minutes, removing the sample in the holes, and drying;
3. washing the plate: discarding liquid in the holes of the enzyme label plate strip, and patting the liquid on absorbent paper to be dry; filling each hole with washing liquid, standing for 5-10 seconds, removing the washing liquid in the hole, and drying by beating for 5 times;
4. adding enzyme conjugate, adding 100 μ L to each well, adding no blank control well, mixing well, and incubating at 37 deg.C for 30 min;
5. washing the plate: the operation steps are the same as 3;
6. adding color development liquid, namely 50 mu L of color development liquid A and 50 mu L of color development liquid B, fully and uniformly mixing, and incubating for 10 minutes at 37 ℃ in a dark place;
7. adding 50 mu L of stop solution, fully oscillating and uniformly mixing, setting the detection wavelength of 450nm or dual-wavelength 450nm/630nm of an enzyme-labeling instrument, measuring the light absorption value of each hole, and reading within 10 minutes after stopping.
The invention has the beneficial effects that the hand-foot-mouth EV71 antigen detection kit is a double-antibody sandwich enzyme-linked immunoassay kit, and adopts a specific antigen expression sequence SEQ ID NO: 1, the kit is used for detecting EV71 antigen in serum, can effectively improve detection sensitivity, and is used for early auxiliary diagnosis of clinical patients with hand-foot-and-mouth disease EV71 infection.
The hand-foot-mouth EV71 antigen detection kit provided by the invention contains enterprise reference products which can be used for quality control of EV71 antigen detection kits produced by enterprises, and can well control the product quality of the kits.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The sample diluent is 0.24% Tris, 1.78% NaCl, 0.5% sodium caseinate, 0.2% bovine serum albumin, 0.1% Tween-20, 0.1% Triton X-100, 5% goat serum and 0.1% thimerosal;
the concentrated washing solution is 0.9% NaCl-0.1% Triton X-100;
the color development liquid A is boric acid buffer solution, L mininol;
the color developing liquid B is citric acidBuffer solution H2O2；
The stop solution is 2M H2SO4。
Example one
The embodiment of the invention is realized in such a way that a hand-foot-mouth EV71 antigen detection kit comprises an ELISA plate coated with an anti-EV 71-VP1 monoclonal antibody, an HRP-labeled anti-EV 71 monoclonal antibody for detection, an enterprise reference product, a sample diluent, a concentrated washing solution, a developing solution A, a developing solution B and a stop solution.
The nucleotide sequence of the anti-EV 71 virus antigen used in the preparation process of the anti-EV 71-VP1 monoclonal antibody is shown as SEQ ID NO: 1 is shown.
Preparation of ELISA plate for anti-EV 71-VP1 monoclonal antibody
The preparation method of the ELISA plate for resisting the EV71-VP1 monoclonal antibody comprises the following steps:
1) diluting the anti-EV 71-VP1 monoclonal antibody with a pH9.6 carbonate buffer solution, wherein the final concentration of the antibody is 5 mu g/m L, adding the antibody into a microporous plate, keeping the antibody at 100 mu L/hole, and standing at 2-8 ℃ for not less than 16 hours;
2) washing the plate 2 times with 0.01 mol/L PBS pH 7.2;
3) adding sealing liquid, sealing at 120 mu L per hole at 2-8 ℃ for not less than 16 hours;
4) vacuum drying for 3 hours;
5) packaging the coated antibody plate with aluminum foil bag, and storing at 2-8 deg.C.
The preparation method of the anti-EV 71-VP1 monoclonal antibody comprises the following steps:
using an anti-EV 71 monoclonal antibody prepared by an EV71-VP1 antigen immunized mouse, enabling ascites to pass through a 200-mesh stainless steel sieve, removing a fat layer, measuring the volume of a supernatant, adding PBS (pH7.20.01mol/L) according to the volume ratio of 1:1, adding saturated ammonium sulfate under stirring to enable the saturated ammonium sulfate to reach 50% saturation, acting for 30 minutes at 25 +/-2 ℃, centrifuging for 15 minutes at 10000rpm at 4 ℃, discarding the supernatant, dissolving a precipitate with PBS (pH7.20.01mol/L), measuring the volume, adding saturated ammonium sulfate to enable the saturated ammonium sulfate to reach 33% saturation, acting for 30 minutes at 25 +/-2 ℃, centrifuging for 15 minutes at 10000rpm at 4 ℃, discarding the supernatant, dissolving the precipitate with a proper amount of PBS (pH7.20.01mol/L), and filling the precipitate into a dialysis bag;dialyzing with PBS (pH7.20.01mol/L) for 48 hours until free of SO4 2－Until the end; passing through Protein-A affinity chromatography column to obtain anti-EV 71 monoclonal antibody; measuring concentration and purity, diluting the finally obtained concentrated solution by 10 times, measuring OD280nm value, and calculating the concentration of the purified antibody; and (3) purity determination: the identification is carried out by an SDS-PAGE method; the loading amount is 10. mu.g, and the purified monoclonal antibody should present two bands (one heavy chain; one light chain) on SDS-PAGE; subpackaging: freeze-drying 5mg per bottle at-20 deg.C or below.
The preparation method of the EV71-VP1 antigen comprises the following specific steps:
(1) culturing the expression strain, namely inoculating 20 mu L of EV71-VP1 expression strain preserved at the temperature of-70 ℃ into L B culture medium (100m L L B/500m L triangular flask), culturing overnight by an air shaking table at the temperature of 30 ℃, transferring the strain into L B culture medium (100m L L B/500m L triangular flask) according to the proportion of 5 percent on the next day, culturing for about 3 hours by the air shaking table at the temperature of 37 ℃, immediately adding IPTG (isopropyl-beta-D-thiogalactoside) when the OD600nm value reaches 0.7, transferring the culture bottle into a shaking table at the temperature of 25 ℃, inducing and culturing for 4 hours, combining bacterial liquid, centrifuging for 20 minutes at the rpm of 6000, abandoning supernatant, and collecting a precipitation part;
(2) purifying, namely suspending and precipitating by using 50 mmol/L Tris-HCl buffer solution with the pH value of 8.3, performing ultrasonic bacteria crushing method in ice bath, exceeding 30 seconds each time, exceeding 10 times at intervals of 30 seconds, centrifuging at 12000rpm at the temperature of 8 ℃ for 20 minutes, taking supernatant, passing through a GST agarose gel FF column, eluting by using buffer solution prepared by reduced glutathione, collecting elution peaks, and identifying various purified recombinant epitope antigens by SDS-PAGE;
measuring the absorbance of the protein sample at 280nm and 260nm by ultraviolet absorption method, and calculating the concentration of C (mg/L) ═ A280nm × 1.45.45 to A260nm × 0.74.74;
(3) and (3) antigen activity identification, namely diluting the purified antigen to 2.5 mu g/m L by using a carbonic acid buffer solution respectively, coating an E L ISA micropore plate, discarding the solution after the overnight at 2-8 ℃ in each hole of 100L, washing the plate for 3 times by using a washing solution, draining the plate, adding a confining solution 100L into each hole, discarding the solution after the overnight at 2-8 ℃, adding 10L of positive serum from different regions in China, which is confirmed by detection, into each hole after 90L sample diluted solution is added, and using 10 of normal human serum as a control.
The EV71-VP1 expression strain is constructed by the following method:
annealing extension PCR is carried out on a VP1 sequence shown as SEQ ID NO. 1, in order to facilitate gene cloning to an expression vector, BamHI and EcoRI two enzyme cutting sites are introduced at two ends of a connecting arm to be suitable for the expression vector pBET-28a, the double enzyme cutting is carried out and then inserted into the vector pBET-28a to construct corresponding expression plasmids, a sequencing method proves that each gene fragment is correctly inserted, EV71-VP1 antigen expression plasmids are transformed into E.coliB L21, the whole bacteria liquid is taken for SDS-PAGE identification through IPTG induction to prove that the plasmids have all obtained high-efficiency expression, obtain 71-VP1 expression strains EV, and-70 is stored for later use.
Preparation of anti-EV 71 monoclonal antibody for HRP-labeled detection
The preparation method of the anti-EV 71 monoclonal antibody for HRP labeling detection comprises the following steps:
(1) dissolving 5mg of horseradish peroxidase (1 mg of protein and 1mg of HRP) in 1.0m L double distilled water to fully dissolve the HRP;
(2) adding 0.5m L0.06 mol/L NaIO4, and stirring for 30 minutes at room temperature in the dark;
(3) taking out, adding 0.16 mol/L ethylene glycol aqueous solution 0.5m L, and keeping out of the sun for 30 minutes at room temperature;
(4) taking a proper amount of purified antibody, fully dissolving the antibody by using double distilled water, mixing the antibody with enzyme solution, filling the mixture into a dialysis bag, dialyzing the mixture for 20 hours at 2-8 ℃ in 0.01 mol/L carbonate buffer solution with pH value of 9.6 in a dark place, and changing the solution for 3 times in the middle;
(5) adding NaBH4 (calculated by adding 0.2m L NaBH4 solution into 5mg HRP) into the dialyzed binding solution, stirring for 30 minutes, and standing for 4 hours at the temperature of 2-8 ℃;
(6) loading the enzyme conjugate into a dialysis bag, and dialyzing with PBS (pH7.20.01mol/L) for 48 hours;
(7) measuring the volume of the concentrated solution, taking a small sample of 0.1m L, and inspecting;
(8) adding equal volume of glycerol into the rest concentrated solution, and storing at-20 deg.C below;
(9) selecting the concentration of the coating antibody and the concentration of the enzyme-labeled antibody;
and selecting the optimal concentration of the coating antibody and the working concentration of the enzyme-labeled antibody by a matrix titration method.
(III) preparation of reference products of enterprises
1. Origin of sample
Collecting the serum of patients with hand-foot-and-mouth disease and healthy people, and preparing enterprise reference products: the method comprises the following steps of 10 patient serum samples with positive detection results of EV71 antigens, wherein 1 patient serum with strong positive detection antibodies is subjected to multiple dilution by using healthy human serum to serve as a sensitivity reference, and 1 patient serum with positive detection results of EV71 antigens serves as precision control; and 10 parts of healthy human serum with negative detection results. The enterprise reference product is used for quality control of EV71 antigen detection kits produced by enterprises.
2. Preparation of
(1) 10 parts of a positive reference substance: collecting clinical patient serum samples with confirmed hand-foot-mouth clinical diagnosis, detecting positive enterovirus (EV71) antigens, wherein the positive enterovirus antigens are strong positive, medium positive and weak positive, and subpackaging at the temperature of below-20 ℃ for later use. Subpackaging at-20 deg.C or below for use.
(2) 10 parts of a negative reference product: and (3) packaging the healthy human serum with the negative EV71 antigen detection result at the temperature of below-20 ℃ for later use.
(3) Sensitivity reference product: 1 part of EV71 antigen is detected to be positive patient serum, 5 diluted concentrations are formed by diluting healthy human serum in a multiple ratio, and the serum is subpackaged below 20 ℃ for later use.
(4) Precision reference product: 1 sample positive for the EV71 antigen was selected for control of reagent precision.
3. Composition of enterprise reference
Composition of enterprise reference
Number of reference name number specification
Positive reference P01-P10500 mu L/10 branches
Negative reference N01-N10500 mu L/10
Minimum detection limit reference substance S1-S5500 mu L/5 counts
Repetitive reference CV 2m L/5
4. Technical requirements
(1) Appearance: all reference liquids should be clear, transparent, without visible precipitation to the naked eye.
(2) Negative reference product compliance rate: the positive reference products P01-P10 are all positive when the kit is used for detecting, and the coincidence rate of the positive reference products is 100%.
(3) Positive reference compliance rate: the kit is used for detecting that the negative reference products N01-N10 are all negative, and the coincidence rate of the negative reference products is 100%.
(4) The lowest detection limit is: when the minimum detection limit reference substance S1-S5 is detected, S1, S2 and S3 are all positive, S5 is negative, and S4 is not required.
(5) Repeatability: and (3) detecting the repetitive reference CV by using the kit, and simultaneously detecting 10 holes, wherein the repeatability (CV%) of the determined OD value is less than or equal to 15.0%.
(6) Inter-batch difference: and 3 batch number kits are used for detecting the repetitive reference substance, and the batch-to-batch variation coefficient (CV%) among the 3 batch number kits is less than or equal to 15.0%.
5. Description of the operation
(1) Storage conditions were as follows: the long-term storage temperature should be below-20 deg.C.
(2) And adding the sample after the reference substance is completely melted and uniformly mixed.
(3) The product should be stored and transported at-20 deg.C or below to avoid repeated freezing and thawing.
(4) Duplicate reference (CV) was applied directly to each well at 100. mu. L, and 10 wells were tested in parallel.
(5) The reference substance should be treated with infectious substances during use.
(6) The reference product has an expiration date of 2 years below-20 ℃.
First, biological safety description
The main raw materials for production are anti-EV 71 monoclonal antibody, coated enzyme linked plate and auxiliary components which are nontoxic and infectious. Inactivation method of positive control sample: 56 ℃ for 60 minutes.
Second, the main research results and evaluation of related products
1. Results of Performance Studies of the finished product
Negative reference product compliance rate: and (5) detecting by using 10 enterprise negative reference products, and detecting all negative products.
Positive reference compliance rate: the detection is carried out by 10 enterprise positive reference products, and the coincidence rate is 10/10.
Precision reference product: and (4) carrying out detection by using an enterprise precision reference product, wherein CV (%) is less than or equal to 15.0% (n is 10).
Three batches of reagents produced continuously, 3% were sampled according to the above criteria. All meet the standard requirements.
And (3) stability verification results: stability tests are carried out on the three batches of reagents, and the results prove that the product reaches the quality inspection standard after 5 days of accelerated tests at 37 ℃; the kit can reach the quality inspection standard after being stored for 12 months at the temperature of 2-8 ℃.
2. Product evaluation
The developed reagent is subjected to analysis performance evaluation, anti-interference capability evaluation and stability (long-term storage stability and thermal stability) evaluation, and clinical tests are compared with the kit on the market, and the results show that the reagent meets the technical performance requirements, can meet the clinical use requirements of basic medical institutions, fills the blank of the in-vitro diagnostic reagent for directly measuring the enterovirus EV71 antigen by using the domestic enzyme-linked immunosorbent assay, and has higher sensitivity than the similar kit in the market.
The detection method of the hand-foot-mouth EV71 antigen detection kit comprises the following steps:
1. experimental preparation the kit was removed from the refrigerated environment and allowed to equilibrate at room temperature (18 ℃ C. -25 ℃ C.) for 30 minutes while diluting the 20 × concentrated wash solution at 1: 19;
2. adding the sample to be tested, namely setting two holes of a negative control and a positive control in each experiment, respectively adding 100 mu L of the negative control and the positive control, setting a hole blank control (without adding any reagent), adding 50 mu L of sample diluent into the other holes, adding 50 mu L of the sample to be tested, fully mixing uniformly, incubating at 37 ℃ for 60 minutes, removing the sample in the holes, and drying;
3. washing the plate: discarding liquid in the holes of the enzyme label plate strip, and patting the liquid on absorbent paper to be dry; filling each hole with washing liquid, standing for 5-10 seconds, removing the washing liquid in the hole, and drying by beating for 5 times;
4. adding enzyme conjugate, adding 100 μ L to each well, adding no blank control well, mixing well, and incubating at 37 deg.C for 30 min;
5. washing the plate: the operation steps are the same as 3;
6. adding color development liquid, namely 50 mu L of color development liquid A and 50 mu L of color development liquid B, fully and uniformly mixing, and incubating for 10 minutes at 37 ℃ in a dark place;
7. adding 50 mu L of stop solution, fully oscillating and uniformly mixing, setting the detection wavelength of 450nm or dual-wavelength 450nm/630nm of an enzyme-labeling instrument, measuring the light absorption value of each hole, and reading within 10 minutes after stopping.
Interpretation of the test results:
1. critical value (Cutoff) calculation: cutoff ═ negative control mean OD value +0.13
2. The OD value of the sample to be tested is greater than or equal to the critical value (Cutoff) and is positive; the sample to be tested is negative when the OD value is less than the critical value (Cutoff).
3. The negative control OD value is less than or equal to 0.12, the positive control OD value is more than or equal to 1.0, otherwise, the experiment is invalid. The OD value of the negative control is calculated as 0.05 when the OD value is lower than 0.05, and is calculated as the actual OD value when the OD value is higher than 0.05.
4. The positive samples are repeated, and all the positive samples should be confirmed by proper means.
(determination of the cut-off value: detection of OD value average +3SD in 270 negative samples).
The invention has the beneficial effects that the hand-foot-mouth EV71 antigen detection kit is a double-antibody sandwich enzyme-linked immunoassay kit, and adopts a specific antigen expression sequence SEQ ID NO: 1, the kit is used for detecting EV71 antigen in serum, can effectively improve detection sensitivity, and is used for early auxiliary diagnosis of clinical patients with hand-foot-and-mouth disease EV71 infection.
The hand-foot-mouth EV71 antigen detection kit provided by the invention contains enterprise reference products which can be used for quality control of EV71 antigen detection kits produced by enterprises, and can well control the product quality of the kits.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> Avicel Biotechnology Ltd
SHANDONG ORIENTAL OCEAN SCI-TECH Co.,Ltd.
Eastern ocean (Beijing) medical research institute Co., Ltd
<120> hand-foot-mouth EV71 antigen detection kit
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<213> Artificial Sequence (Artificial Sequence)
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attcaa 726
Claims (10)
1. A hand-foot-mouth EV71 antigen detection kit is characterized by comprising an ELISA plate coated with an anti-EV 71-VP1 monoclonal antibody, an HRP-labeled detection anti-EV 71 monoclonal antibody, an enterprise reference product, a sample diluent, a concentrated washing solution, a developing solution A, a developing solution B and a stop solution.
2. The hand-foot-mouth EV71 antigen detection kit of claim 1, wherein the nucleotide sequence of the anti-EV 71 virus antigen used in the preparation process of the anti-EV 71-VP1 monoclonal antibody is shown as SEQ ID NO: 1 is shown.
3. The hand-foot-mouth EV71 antigen detection kit of claim 1, wherein the preparation method of the enzyme label plate for resisting the EV71-VP1 monoclonal antibody comprises the following steps:
1) diluting the anti-EV 71-VP1 monoclonal antibody with a pH9.6 carbonate buffer solution, wherein the final concentration of the antibody is 5 mu g/m L, adding the antibody into a microporous plate, keeping the antibody at 100 mu L/hole, and standing at 2-8 ℃ for not less than 16 hours;
2) washing the plate 2 times with 0.01 mol/L PBS pH 7.2;
3) adding sealing liquid, sealing at 120 mu L per hole at 2-8 ℃ for not less than 16 hours;
4) vacuum drying for 3 hours;
5) packaging the coated antibody plate with aluminum foil bag, and storing at 2-8 deg.C.
4. The hand-foot-mouth EV71 antigen detection kit of claim 1, wherein the preparation method of the anti-EV 71-VP1 monoclonal antibody comprises the following steps:
using anti-EV 71 monoclonal antibody prepared by EV71-VP1 antigen immune mouse, making ascites pass through 200-mesh stainless steel sieve, removing fat layer, measuring supernatant volume, adding PBS (pH7.20.01mol/L) according to volume ratio of 1:1, stirring and adding saturated ammonium sulfate to make 50% saturation, acting at 25 + -2 deg.C for 30 min, centrifuging at 4 deg.C 10000rpm for 15 min, removing supernatant, dissolving precipitate with PBS (pH7.20.01mol/L), measuring volume, addingSaturating with ammonium sulfate to 33% saturation, treating at 25 + -2 deg.C for 30 min, centrifuging at 4 deg.C at 10000rpm for 15 min, discarding supernatant, dissolving precipitate with appropriate amount of PBS (pH7.20.01mol/L), packaging into dialysis bag, dialyzing with PBS (pH7.20.01mol/L) for 48 hr until no SO is present4 2－Until the end; and (3) passing through a Protein-A affinity chromatography column to obtain the anti-EV 71 monoclonal antibody.
5. The hand-foot-mouth EV71 antigen detection kit of claim 4, wherein the EV71-VP1 antigen preparation method specifically comprises the following steps:
(1) culturing the expression strain, namely inoculating 20 mu L of EV71-VP1 expression strain preserved at the temperature of-70 ℃ into L B culture medium, culturing overnight at the temperature of 30 ℃ by using an air shaking table, transferring the EV71-VP1 expression strain into L B culture medium according to the proportion of 5% the next day, culturing for about 3 hours at the temperature of 37 ℃ by using the air shaking table, immediately adding IPTG (isopropyl-beta-thiogalactoside) when the OD600nm value reaches 0.7, transferring a culture bottle into a shaking table at the temperature of 25 ℃ for induced culture for 4 hours, combining bacterial liquid, centrifuging at 6000rpm for 20 minutes, removing supernatant, and collecting a precipitate;
(2) and (2) purifying, namely suspending and precipitating by using 50 mmol/L Tris-HCl buffer solution with the pH value of 8.3, performing ultrasonic bacteria crushing method in ice bath, performing ultrasonic bacteria crushing method for 10 times at the interval of 30 seconds every time, performing centrifugation for 20 minutes at the temperature of 8 ℃, passing through GST agarose gel FF column, eluting by using buffer solution prepared by reduced glutathione, collecting elution peak, and identifying various purified recombinant epitope antigens by SDS-PAGE to obtain the EV71-VP1 antigen.
6. The hand-foot-mouth EV71 antigen detection kit of claim 5, wherein the EV71-VP1 expression strain is constructed by the following method:
carrying out annealing extension PCR on a VP1 sequence shown as SEQ ID NO. 1, introducing BamHI and EcoRI restriction sites at two ends of a connecting arm to be suitable for an expression vector pBET-28a in order to facilitate gene cloning to an expression vector, carrying out double restriction, inserting the double restriction sites into the vector pBET-28a to construct a corresponding expression plasmid, carrying out EV71-VP1 antigen expression plasmid, transforming E.coli B L21, inducing by IPTG, taking a whole bacteria solution to carry out SDS-PAGE identification, and proving that the plasmids all obtain high-efficiency expression to obtain an EV71-VP1 expression strain.
7. The hand-foot-and-mouth EV71 antigen detection kit of claim 1, wherein the preparation method of the anti-EV 71 monoclonal antibody for HRP labeling detection comprises the following steps:
(1) measuring 1mg protein and 1mg HRP, dissolving 5mg horse radish peroxidase in 1.0m L double distilled water to fully dissolve HRP;
(2) 0.5m L0.06 mol/L NaIO was added4Stirring for 30 minutes at room temperature in dark place;
(3) taking out, adding 0.16 mol/L ethylene glycol aqueous solution 0.5m L, and keeping out of the sun for 30 minutes at room temperature;
(4) taking a proper amount of purified antibody, fully dissolving the antibody by using double distilled water, mixing the antibody with enzyme solution, filling the mixture into a dialysis bag, dialyzing the mixture for 20 hours at 2-8 ℃ in 0.01 mol/L carbonate buffer solution with pH value of 9.6 in a dark place, and changing the solution for 3 times in the middle;
(5) collecting the dialyzed combined solution, adding 0.2m L NaBH according to 5mg HRP4Metering in solution, adding NaBH4Stirring for 30 minutes, and standing for 4 hours at the temperature of 2-8 ℃;
(6) loading the enzyme conjugate into a dialysis bag, and dialyzing with PBS (pH7.20.01mol/L) for 48 hours;
(7) measuring the volume of the concentrated solution, taking a small sample of 0.1m L, and inspecting;
(8) adding equal volume of glycerol into the rest concentrated solution to obtain the HRP-labeled anti-EV 71 monoclonal antibody for detection.
8. The hand-foot-mouth EV71 antigen testing kit of claim 1, wherein the enterprise references comprise 10 parts of positive references P01-P10, 10 parts of negative references N01-N10, 1 part of minimum detection limit references S1-S5, and 1 part of repetitive references CV.
9. The hand-foot-mouth EV71 antigen detection kit of claim 8, wherein the positive reference substance is a serum sample of a patient with a positive detection result of EV71 antigen;
the negative reference substance is a healthy human serum sample with negative detection result;
the lowest detection limit reference substance is a serum sample obtained by diluting the serum of a patient with strong positive detection antibodies by a fold ratio with the serum of a healthy person;
the repeated reference substance is a patient serum sample with a positive detection result of EV71 antigen.
10. The hand-foot-and-mouth EV71 antigen detection kit of claim 1, wherein the sample diluent is 0.24% Tris, 1.78% NaCl, 0.5% sodium caseinate, 0.2% bovine serum albumin, 0.1% Tween-20, 0.1% Triton X-100, 5% goat serum, 0.1% thimerosal;
the concentrated washing solution is 0.9% NaCl-0.1% Triton X-100;
the color development liquid A is boric acid buffer solution, L mininol;
the color developing solution B is citric acid buffer solution H2O2；
The stop solution is 2M H2SO4。
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| CN201910056766.4A CN111458498A (en) | 2019-01-19 | 2019-01-19 | Hand-foot-mouth EV71 antigen detection kit |
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| CN201910056766.4A CN111458498A (en) | 2019-01-19 | 2019-01-19 | Hand-foot-mouth EV71 antigen detection kit |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113049819A (en) * | 2021-04-13 | 2021-06-29 | 江苏泽成生物技术有限公司 | HSV1+2 type IgM antibody detection kit reference |
| CN115541895A (en) * | 2022-11-29 | 2022-12-30 | 天津德祥生物技术股份有限公司 | Formula liquid for improving sensitivity of micro-fluidic inverse detection card and application |
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| CN102243232A (en) * | 2010-05-14 | 2011-11-16 | 北京贝尔生物工程有限公司 | Diagnostic reagent kit (enzyme-linked immunosorbent assay (ELISA)) for enterovirus (EV) 71-type antibody (immune globulin M (IgM)) |
| CN104569428A (en) * | 2014-12-30 | 2015-04-29 | 浙江普康生物技术股份有限公司 | ELISA (enzyme-linked immunosorbent assay) kit for EV (enterovirus) 71 inactivated vaccine antigen |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102243232A (en) * | 2010-05-14 | 2011-11-16 | 北京贝尔生物工程有限公司 | Diagnostic reagent kit (enzyme-linked immunosorbent assay (ELISA)) for enterovirus (EV) 71-type antibody (immune globulin M (IgM)) |
| CN104569428A (en) * | 2014-12-30 | 2015-04-29 | 浙江普康生物技术股份有限公司 | ELISA (enzyme-linked immunosorbent assay) kit for EV (enterovirus) 71 inactivated vaccine antigen |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113049819A (en) * | 2021-04-13 | 2021-06-29 | 江苏泽成生物技术有限公司 | HSV1+2 type IgM antibody detection kit reference |
| CN115541895A (en) * | 2022-11-29 | 2022-12-30 | 天津德祥生物技术股份有限公司 | Formula liquid for improving sensitivity of micro-fluidic inverse detection card and application |
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