CN108802368A - A kind of kit of enzyme linked immunosorbent detection ox miscarriage Chlamydia - Google Patents
A kind of kit of enzyme linked immunosorbent detection ox miscarriage Chlamydia Download PDFInfo
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56927—Chlamydia
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Abstract
The present invention relates to field of immunology, specifically disclose a kind of kit of enzyme linked immunosorbent detection ox miscarriage Chlamydia.The present invention is coated with solid phase carrier by the artificial synthesized polypeptide as shown in SEQ ID NO.1, and as antigen, to realize the MBP enzyme linked immuno-adsorbent assay of ox miscarriage Chlamydia.The present invention makes MBP enzyme linked immuno-adsorbent assay have the characteristics that specific height, sensitivity are good using the artificial synthesized polypeptide antigen as envelope antigen.The present invention provides the antigen protective agent of optimization also after antigen coat solid phase carrier, is protected to the antigen being coated with, and extends the holding time of antigen.
Description
Technical field
The present invention relates to field of immunology, specifically, being related to the kit of enzyme linked immunosorbent detection ox miscarriage Chlamydia.
Background technology
Ox miscarriage chlamydiosis (Bovine chlamydia abortus) is by miscarriage Chlamydia (Chlamydophila
Abortus the infecting both domestic animals and human communicable disease of the mammals such as a kind of cattle and sheep and pig miscarriage caused by).This disease of morbidity milk cattle infected
It is mainly shown as miscarriage, fecundity decline, production stillborn foetus, post-abortion retention of afterbirth；Decline simultaneously with the output of milk, appetite subtracts
It moves back, part infected cattle has the symptoms such as body temperature raising, apathetic.In addition experiment confirms inoculation miscarriage chlamydiosis in cow uteri
Original can cause mastadenitis of cow.Breeding oxen has infected miscarriage Chlamydia, and cause of disease, which enters in sperm, can cause under kind of ox fertility
Drop.The mankind are easy to cause to infect when contacting or handling aborted fetus or calf without taking proper safeguard measure, people's sense
Mainly occurs influenza-like symptom after dye, pregnant woman infects this cause of disease and is easy to cause to miscarry.
Include both at home and abroad at present two major classes i.e. antigen detection and Serologic detection to the means that predominantly detect of miscarriage Chlamydia.
Antigen detection method has：1) placenta tissue or the direct staining of vagina cotton swab smear, immunohistochemical staining.Direct staining disease
Former method method is easy to operate, detection is fast, but sensitivity and specificity are general.Cell immunohistochemical staining method operation is more multiple
The judgement of miscellaneous and above two methods and results is required for abundant technical expertise, and judgement result is larger by subjective impact,
It is extremely difficult when especially EB and RB of identification Chlamydia.2) cell culture of Antigen isolation and identification method detaches cause of disease chlamydia
Inclusion body carries out immunohistochemistry or immunology dyeing, needs fowl embryo or cell, separation process is more complicated and is grasped to experiment
Make, experimental situation and the more demanding general laboratory of base of Experimental Establishment are difficult to carry out.3) its advantage of Immunofluorescent Antibody technology
It is method simplicity, high specificity, non-specific fluorescence less coloration.The disadvantage is that sensibility is relatively low, fluorescence microscope is expensive not
It is suitable as common detection methods.4) although polymerase chain reaction has higher specificity and sensibility to prepare template more complicated
Required sample size is big, and needs the expensive instruments such as PCR instrument, and detection reagent is expensive.5) double antibodies sandwich enzyme-linked immunosorbent assay side
Method has simplicity, sensitive, it is efficient the features such as but the ELISA kit that can not detect miscarriage CHLA Casset domestic at present.
Relative to antigen detection method, detection antibody method is more simple and easy to do.For example immunofluorescent test, enzyme-linked exempt from
Epidemic disease adsorption test, indirect hemagglutination test, complement fixation test (CFT) and the detection experiment of colloid gold reagent item etc..1) immunofluorescent test
Specific antibody is detected with the known CHLA Casset of label, and the method advantage is simple and practicable, specific height.Disadvantage is sensitivity
It is relatively low, device therefor is expensive.2) enzyme-linked immunosorbent assay has easy to operate, specific and high sensitivity, to operating personnel's
Technology is of less demanding, experimental result is influenced the advantages that small by subjective factor and is widely used in clinical detection serum antibody technology
In, but there is no the enzyme-linked immunosorbent assay kit for miscarriage chlamydial antibody on domestic market.3) indirect hemagglutination test
Method operation sequence is simple, does not need special installation, but result judgement is affected by subjective factor, and it is more to couple antigen
For whole cell, may there are cross reactions with part Gram-negative bacteria, be as a result also easy to produce false positive.4) complement fixation test (CFT) side
Method is widely used in veterinary laboratories.But complement fixation test (CFT) is cumbersome, sensibility is low, detection time is long, it is higher to need
Professional knowledge and be easy with other Gram-negative bacteria serum generate cross reaction the defects of in clinical application by compared with
More limitations.5) operation is simple but detection sensitivity is relatively low for colloidal gold strip method.
The detection chlamydial antibody kit of domestic market circulation is all with the Chlamydia of Chlamydia whole cell and purifying
Lipopolysaccharides is as antigen.However, due to these antigens main component be Chlamydia Major Outer Membrane Protein (MOMP) and lipopolysaccharides,
It is that easily cause intersection anti-for the antigen of most common Chlamydia cross reaction, identification CHLA Casset can only be confined to and be unable to
The Chlamydia of respectively different kind, i.e., can not specific detection ox miscarriage Chlamydia.
Invention content
The object of the present invention is to provide a kind of kits of enzyme linked immunosorbent detection ox miscarriage Chlamydia, by with high specificity
Ox miscarriage chlamydial antibody is detected, realizes the indirect enzyme-linked immunosorbent detection for Chlamydia of miscarrying to ox.
In order to realize the object of the invention, technical scheme is as follows：
The present invention provides a kind of kit of enzyme linked immunosorbent detection ox miscarriage Chlamydia, the solid phase carrier in the kit
Upper coating is just like polypeptide shown in SEQ ID NO.1.
The polypeptide sequence comes from a segment polypeptide sequence of the POMP90 albumen of S26/3 plants of Chlamydia of miscarriage.
Polypeptide sequence can entrust commercial company to complete synthesis, and the polypeptide fragment used in the embodiment of the present invention is by Beijing
Matt bio tech ltd of section synthesizes.
Further, it after the solid phase carrier is coated with the polypeptide, is handled using antigen protective agent；The antigen is protected
Shield agent is the PBS containing 10% sucrose, 10% glycerine, preferable ph 7.2.
Further, the solid phase carrier is ELISA Plate, and such as 96 hole elisa Plates, the preparation method of the ELISA Plate are：
1) polypeptide is diluted to polypeptide solution using aseptic deionized water；
2) it utilizes carbonate buffer solution that the polypeptide solution obtained by step 1) is diluted 1000 times, each hole of ELISA Plate is added and incubates
It educates, after rinsing ELISA Plate with washing buffer PBST, is closed using confining liquid；
3) antigen protective agent is added per hole to the microwell plate closed to be incubated, outwells antigen protective agent, wash, dry in the air
It is dry to get；
The polypeptide is diluted to the polypeptide solution of final concentration of 1mg/mL preferably by aseptic deionized water；
The polypeptide solution obtained by step 1) is diluted to the more of final concentration of 1.0 μ g/mL preferably by carbonate buffer solution
Peptide solution；
It is preferred that the carbonate buffer solution is the carbonate buffer solution of pH9.6,50mM；
It is preferred that the confining liquid is the PBST solution containing 10% skimmed milk power.
Preferably, after each hole of ELISA Plate is added in the polypeptide solution of the final concentration of 1.0 μ g/mL, it is small that 16 are incubated at 4 DEG C
When；It is incubated 2 hours and is closed at 37 DEG C using the confining liquid；After the antigen protective agent is added, it is incubated 4 hours at 37 DEG C.
Above-mentioned preferred embodiment is combined, the preparation method of following ELISA Plate is obtained：
1) polypeptide is diluted to the polypeptide solution of final concentration of 1mg/mL using aseptic deionized water；
2) utilize the carbonate buffer solution of pH9.6,50mM that the polypeptide solution obtained by step 1) is diluted to final concentration of 1.0
Each hole of ELISA Plate is added in the polypeptide solution of μ g/mL, and 4 DEG C are incubated 16 hours, rinses ELISA Plate with washing buffer PBST, then use
Confining liquid (the PBST solution for containing 10% skimmed milk power) closing, is closed, 37 DEG C incubate 2 hours, outwell with 100 holes μ L/ of confining liquid
Confining liquid, PBST are washed 3 dryings and are closed with 200 holes μ L/ of confining liquid, and 37 DEG C incubate 2 hours, outwell confining liquid, PBST washings 3
Secondary drying；
3) antigen protective agent that 200 microlitres are added per hole for the microwell plate closed outwells antigen after 37 DEG C are incubated 2 hours
Protective agent washs, drying, ultraviolet irradiation 1 hour, and acquisition is coated with the polypeptide (miscarriage S26/3 plants of POMP90 eggs of Chlamydia
ELISA Plate in vain).
Further, the kit further includes enzyme labelled antibody, and the enzyme labelled antibody is horseradish peroxidase-labeled
Goat-anti ox IgG.
Further, the kit further includes negative control sera and positive control serum；
The preparation method of the positive control serum is specific as follows：
1) taking S26/3 plants of POMP90 albumen KLH- couplings peptides of miscarriage Chlamydia, (polypeptide shown in the more NO.1 of SEQ ID is coupled
KLH carrier proteins form, and polypeptide coupling commission Shanghai Qiangyao Biotechnology Co., Ltd. completes), by antigen with 10mmol/L
PBS buffer solution (pH 7.4) is diluted to a concentration of 20mg/mL as immunizing antigen；
Wherein, PBS buffer solution is sodium chloride 8g, potassium chloride 0.2g, disodium hydrogen phosphate (12H2O) 3.63g, potassium dihydrogen phosphate
0.24g is dissolved in 900mL deionized waters, fully after dissolving, is adjusted pH value to 7.4 with 1mol/L hydrochloric acid solutions, is added deionization
Water is settled to obtained by 1L；
2) it is negative calf to select 24 monthly ages, chlamydial antibody, is immunized；
3) head exempts from：It is fully emulsified by Fu Shi Freund's complete adjuvants and immunizing antigen mixed in equal amounts, take the subcutaneous multiple spot note of 2mL necks
It penetrates；
4) two exempt from：Head exempts from the 14th day afterwards, takes Fu Shi Freund's incomplete adjuvants and immunizing antigen mixed in equal amounts, fully emulsified, takes 2mL
The subcutaneous multi-point injection of neck；
5) three exempt from：Two exempt from the 7th day afterwards, and it is primary to exempt from method booster immunization with two；
6) four exempt from：Three exempt from after the 7th day, the subcutaneous each injecting immune antigen 2mL of muscle and neck；
7) tail vein blood after 1 week detaches serum and dilutes, using the Chlamydia S26/3 that miscarries shown in SEQ ID NO.1
The strain coated ELISA Plate ELISA method of pomp90 polypeptides detects its immune serum antibody titer, chooses 1:When 200 dilution
OD450Calf of the value more than 1.0, carries out jugular vein blood collection, sterile separation serum, it is positive to obtain miscarriage Chlamydia for water-bath inactivation
Control serum (miscarriage Chlamydia S26/3 strain positive controls serum)；
The preparation method of the negative control sera is：
Bull calf (such as 2) is chosen, takes a blood sample respectively, detach serum, water-bath inactivation is examined by Chlamydia indirect hemagglutination
It surveys and ELISA detections, 2 kinds of results of collection is the cow's serum of feminine gender, mixing, (miscarriage clothing is former for the as described negative control sera
S26/3 plants of negative control seras of body).
Further, the kit further include washing buffer, enzyme labelled antibody dilute solution, Sample Dilution buffer solution,
Developing solution and terminate liquid.
Wherein, the washing buffer is the phosphate buffer (PBST) containing 1.0 ‰ Tween-20s, pH7.2；
The enzyme labelled antibody dilute solution be containing 0.1 ‰ merthiolates, 5% glycerine, 5% trehalose pH7.2 phosphorus
Phthalate buffer；
The Sample Dilution buffer solution is the PBST containing 0.3%Triton-100,0.1 ‰ merthiolates；
The terminate liquid is the sulfuric acid solution of 2N concentration, the H that preferably deionization is prepared2SO4.Wherein, 2N is that equivalent is dense
Degree, 2N are equal to 1M, the i.e. sulfuric acid solution of 1mol/L.
Further, the developing solution includes color developing agent A and color developing agent B：Color developing agent A be containing 1.46% disodium hydrogen phosphate,
0.93% citric acid, the aqueous solution of 0.045% hydrogen peroxide；Color developing agent B is containing 0.03% tetramethyl benzidine, 0.096% lemon
Lemon acid, 0.019% ethylenediamine tetraacetic ethanedioic acid sodium salt, 2%DMSO, the aqueous solution of 4% glycerine.
The present invention also provides the operating procedures of the kit：
Positive control serum, negative control sera and sample are diluted with 1: 10 times with Sample dilution, with 100 holes μ L/
It being added in polypeptide coating plate (wherein positive to set multiple holes with negative control), 37 DEG C of incubation 30min outwell liquid in reaction plate,
4 times are washed with washing buffer to pat dry；Then it is added with 100 holes μ L/ and is set with 1: 5000 times of diluted enzyme labelled antibody of enzyme labelled antibody
37 DEG C of incubation 30min；Taking-up reaction plate, which outwells enzyme labelled antibody, has washing buffer to wash 4 times；The isometric mixing of developing solution A, B
Afterwards, it is added with 100 holes μ L/, 37 DEG C of incubation 15min；100 holes μ L/ of terminate liquid are added, sets 450nm wave bands in microplate reader and measures respectively
Hole OD values, according to sample to be tested (ODS), positive control (ODPC) and negative control (ODNC) calculate positive control OD450Value and feminine gender
Compare OD450Value, if ODPCMean value >=0.45, and ODPC/ODNC>=3 are tested establishment.It is calculated on the basis of testing establishment
Each sample S/P percentages.S/P% values=(sample OD450/ positive control OD450) x100%.When sample S/P% values are less than
Be determined as when 26% it is negative, be determined as less than 31% more than 26% it is suspicious, be determined as the positive more than 31%.
The Enzyme-linked Immunosorbent Assay method of miscarriage chlamydial antibody is detected using kit of the present invention.Apply is in cows
In the analysis of no infective abortion Chlamydia cause of disease, sample to be tested is the serum for picking up from ox.
It should be noted that the present invention relates to raw material or reagent be ordinary commercial products, the operation being related to is such as
No specified otherwise is this field routine operation.
It will be appreciated by those skilled in the art that on the basis of common knowledge of the art, above-mentioned each optimum condition, Ke Yixiang
Mutually combination, obtains specific implementation mode.
The beneficial effects of the present invention are：
The present invention is carried by synthesis miscarriage S26/3 plants of POMP90 polypeptides of Chlamydia as antigen coat solid phase
Body carries out MBP enzyme linked immuno-adsorbent assay.Kit of the present invention is that coating is anti-using the polypeptid specificity segment of artificial synthesized high-purity
Original, specificity is high, and sensitivity is good.And the antigen being coated with is protected with antigen protective agent after antigen coat solid phase carrier
Shield, extends the holding time of antigen.In addition, the enzyme-linked antibody that the present invention uses is the goat-anti ox of horseradish peroxidase-labeled
IgG, can specificity in cow's serum miscarry S26/3 plants of serum antibodies of Chlamydia detection.
Kit provided by the invention can improve detection ox using Chlamydia POMP90 protein-specifics polypeptide of miscarrying as antigen
The specificity and sensibility for Chlamydia specific antibody ELISA kit of miscarrying.At present both at home and abroad not individually for detection ox
The kit that chlamydial antibody of miscarrying detects, the present invention can make up specific detection ox miscarriage Chlamydia ELISA kit
Vacancy.Subjective factor that is short the time required to kit of the present invention, reducing operating personnel influences.And it is economical and practical, to instrument
It is of less demanding, it is only necessary to common constant water bath box, microplate reader etc..The present invention is suitable for the popularization of large-scale commercial applications detection method,
With good market prospects.The kit is alternatively milk cow and plants the epidemiological survey and diagnosis of ox miscarriage chlamydiosis
New technical foundation has been established, the demand of clinical examination is met.
Description of the drawings
Fig. 1 is the present invention positive and negative serum ROC curve figure.
Fig. 2 is the coordinate points of ROC curve of the present invention and corresponding sensibility and specificity, and testing result variable is S/ in figure
P% values.
Specific implementation mode
The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real
Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field
Art personnel can carry out various modifications and replace to the present invention without departing substantially from spirit of the invention and spirit.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The synthesis of the miscarriage S26/3 plants of POMP90 polypeptides of Chlamydia of embodiment 1
According to including miscarriage Chlamydia S26/3 plants of POMP90 protein sequences (Genbank Accession on Genbank：
U65943), compare what one section of selection after analyzing different chlamydia trachomatis gene sequences was made of 25 amino acid using DNAstar softwares
Specific peptide sequences carry out artificial synthesized.Quantitative polypeptide dispenses, and is saved backup for -20 DEG C after freezing is drained.
The selection of 2 antigen protective agent of embodiment
Selection-sucrose, glucan, Macrogol 6000, glycerine, is configured to antigen protection according to the prescription of table 1 at trehalose
Agent is coated with miscarriage Chlamydia POMP90 albumen ELISA Plates with optimum optimizing condition, 200 holes μ L/ is separately added into after closing, washing
Antigen stabilizer (prescription 1, prescription 2, prescription 3, prescription 4), 4 DEG C overnight, and next day, which takes out, discards liquid in hole, and PBST solution is added
300 holes μ L/ are washed 3 times, and each 3min is used in combination blotting paper to pat dry, and ultraviolet light irradiates 1h, and vacuum bag is packed into after drying and is placed in 37
DEG C environment continuously preserves, and the 14th day and 21 days when takes out ELISA Plate respectively, according to determining ELISA Programmable detection positive controls
Product repeat 4 holes, calculate its average value, compare its OD450Decline situation.The existing coated nothing of selection adds any protectant enzyme mark
Plate is as a control group.
Table 1 is coated with the prescription of stabilizer
| Sucrose | Glucan | Trehalose | PEG | Glycerine | PBS | |
| Prescription 1 | 5% | 4% | 5% | To 100% | ||
| Prescription 2 | 2%- | 4% | 10% | To 100% | ||
| Prescription 3 | - | 2% | 2% | 4% | 10% | To 100% |
| Prescription 4 | 10% | - | - | 10% | To 100% | |
| Control group | - | - | - | - | - | - |
Measurement OD value of the different coating stabilizers of table 2 at 37 DEG C
| 0 day | 7 days | 14 days | 21 days | |
| Prescription 1 | 0.96 | 0.85 | 0.71 | 0.60 |
| Prescription 2 | 0..92 | 0.78 | 0.68 | 0.55 |
| Prescription 3 | 0.90 | 0.76 | 0.65 | 0.49 |
| Prescription 4 | 0.95 | 0.91 | 0.85 | 0.72 |
| Control group | 0.96 | 0.97 | 1.02 | 1.06 |
37 DEG C preserve 7,14 days and 21 after testing result show envelope antigen protect the 4th group of liquid compared with control group result
OD450It is worth reduced rate difference 6.18%, 16.7% and 32%, remaining prescription group OD450Value at 7 days rate of descent more than 10%,
OD at 14 days450It is worth rate of descent more than 30%.Therefore it is antigen protective agent to select coating prescription 4, and the antigen of screening protects prescription
Group, which becomes in 100ml, to be contained：10% sucrose, 10% glycerine.
Embodiment 3 is coated with the preparation of the ELISA Plate of miscarriage Chlamydia POMP90 polypeptides
1, synthetic miscarriage Chlamydia POMP90 polypeptides are diluted to final concentration 1mg/mL with aseptic deionized water.
2, the 1mg/mL polypeptides diluted are diluted to 1.0 μ g/mL of final concentration with pH9.6,50mM carbonate buffer solution, added
Enter each hole of ELISA Plate, 4 DEG C are incubated 16 hours, rinse ELISA Plate with washing buffer PBST, then (contain 10% defatted milk with confining liquid
The PBST solution of powder) closing, it is closed with 100 holes μ L/ of confining liquid, 37 DEG C incubate 2 hours, outwell confining liquid, and PBST is washed 3 times and got rid of
Dry to be closed with 200 holes μ L/ of confining liquid, 37 DEG C incubate 2 hours, outwell confining liquid, and PBST washs 3 dryings；
3,200 microlitres of antigen protective agent is added per hole for the microwell plate closed, and 37 degree of pH7.2PBS solution is incubated 2 hours
Afterwards, antigen protection solution is outwelled, is washed, drying, purple light irradiation 1 hour, that is, obtains and is coated with S26/3 plants of Chlamydia of miscarriage
The ELISA Plate of POMP90 polypeptides.The preparation of the miscarriage Chlamydia positive control serum of embodiment 4
1, taking S26/3 plants of pomp90 albumen KLH- couplings peptides of miscarriage Chlamydia, (shine by force biological section in polypeptide coupling commission Shanghai
Skill Co., Ltd complete), by the coupling peptide using 10mmol/L PBS buffer solution (pH 7.4) be diluted to a concentration of 20mg/mL as
Immunizing antigen.- 20 DEG C save backup, S26/3 plants of immunizing antigens of Chlamydia of as miscarrying.
2,4 monthly age calves of selection detect through Chlamydia IHA kits, and chlamydial antibody is negative calf 2, is exempted from
Epidemic disease, blood sampling, separation serum.
3, head exempts from：It is fully emulsified by Fu Shi Freund's complete adjuvants and immunizing antigen mixed in equal amounts, take the subcutaneous multiple spot note of 1mL necks
It penetrates.
4, two exempt from：Head exempts from the 14th day afterwards, takes Fu Shi Freund's incomplete adjuvants and immunizing antigen mixed in equal amounts, fully emulsified, takes 1mL
The subcutaneous multi-point injection of neck.
5, three exempt from：Two exempt from the 7th day afterwards, and it is primary to exempt from method booster immunization with two.
6, booster immunization：Three exempt from after the 7th day, the subcutaneous each injecting immune antigen 1 mL of muscle and neck.
7, serum antibody titer detects：Tail vein is respectively taken a blood sample 1mL after booster immunization 7 days, until in sterile eppendorf tubes, in
37 DEG C stand 2 hours, then stay overnight for 4 DEG C.Next day 8000g centrifuges 2 minutes, detaches serum and dilutes, prepared by embodiment 3
ELISA Plate ELISA method detect its immune serum antibody titer, choose 1:OD when 200 dilution450Calf of the value more than 1.0, into
Row jugular vein blood collection, sterile separation serum, 56 DEG C of water-baths inactivate 30min, that is, obtain miscarriage S26/3 plants of ox positive controls of Chlamydia
Serum, -20 DEG C save backup.
The preparation of 5 Ns of miscarriage Chlamydia negative control seras of embodiment
1,2 3-4 monthly age calves to be chosen, takes a blood sample respectively, detach serum, 56 DEG C of water-baths inactivate 30min,
2, serum is detected by Chlamydia indirect hemagglutination (IHA) kit and ELISA kit, is collected 2 kinds of results and is
Negative cow's serum, mixing, S26/3 plants of negative control seras of Chlamydia of as miscarrying, -20 DEG C save backup.
6 Ns of miscarriage chlamydiosis enzyme-linked immunologic detecting kits of embodiment
1) ELISA Plate that embodiment 3 is prepared；
2) positive control serum and negative control sera prepared by embodiment 4-5；
3) washing buffer is the phosphate buffer (PBST) containing 1.0 ‰ Tween-20s, pH7.2；
4) Sample Dilution buffer solution is the phosphate containing 0.3%Triton-100, the pH 7.2 of 0.1 ‰ merthiolates
Buffer solution
5) enzyme labelled antibody dilute solution is to contain 1.0% bovine serum albumin(BSA), 0.1 ‰ merthiolates, 5% glycerine, pH7.2
Phosphate buffer；
6) color developing agent A is containing 1.46% disodium hydrogen phosphate, 0.93% citric acid, the aqueous solution of 0.045% hydrogen peroxide.
7) color developing agent B is containing 0.03% tetramethyl benzidine, 0.096% citric acid, 0.019% ethylenediamine tetraacetic sodium oxalate
Salt, 2%DMSO, the aqueous solution of 4% glycerine.
The application of 7 Ns of miscarriage chlamydiosis enzyme-linked immunologic detecting kits of embodiment
The specific implementation step of ox miscarriage chlamydiosis enzyme linked immunological antibody testing method is as follows：
1) sample dilutes：Test serum, feminine gender and positive control serum 1: 10 are diluted with Sample Dilution buffer solution.
2) it is loaded：It is added in the ELISA Plate hole after closing with 100 holes μ L/ samples to be tested, while multiple holes is set with 100 holes μ L/
Negative control and positive control, 37 DEG C incubate 30 minutes.PBST is rinsed 4 times, drying.
3) enzyme labeling antibody：Enzyme labelled antibody is diluted to 1: 5000 enzyme working solution with enzyme table antibodies buffer, is added per hole
Enter 100 μ L, 37 DEG C incubate 30 minutes.PBST is rinsed 4 times, drying.
4) it develops the color：Successively it is added color developing agent A, B each 50 μ L per hole, 37 DEG C of Incubation in dark 15 minutes.
5) it terminates：100 μ L terminate liquids are added per hole.
6) it detects：In detection 450nm absorption values in microplate reader.
The sensitivity and specificity of 8 Ns of miscarriage chlamydiosis enzyme-linked immunologic detecting kits of embodiment
1, sensitivity detects：Miscarriage S26/3 plants of POMP90 eggs of Chlamydia are detected with kit of the present invention (embodiment 6)
White specific primer PCR detects infective abortion Chlamydia milk cow vagina cotton swab, and assay certificate is 32 parts of infective abortions of the positive
The cow's serum of Chlamydia judges.Sensitivity indicates with true positive rate, i.e. sensitivity (%)=[number positive/(true positives number+vacation is cloudy
Property number)] x 100%.
2, specific detection：
1) known serum sample is detected：S26/3 plants of Chlamydia of miscarriage is detected with kit of the present invention (embodiment 6)
POMP90 albumen specific primer PCRs detect milk cow vagina cotton swab, 107 parts of cow serums of assay certificate feminine gender and positive 32
Part serum, judges.Specificity indicates with true negative rate, i.e., specific (%)=[negative number/(true negative number+false positive number)] x
100%.
3 testing result of table
Note "+" represents the positive；"-" represents feminine gender.
2) cross reaction of different Chlamydia positive serums is detected：Chicken is detected with kit of the present invention (embodiment 6)
It is anti-that anti-chlamydia psittaci 6BC pearls positive serum, chlamydia pneumoniae positive serum and chlamydia trachomatis positive serum set chicken simultaneously
Miscarriage Chlamydia positive serum does positive control and to do negative control result as follows for SPF chicken serums rather：
The different Chlamydia positive serum ELISA of table 4 detect OD450Value
Testing result shows kit detection chlamydia psittaci 6BC pearls and chlamydia pneumoniae, trachoma clothing described in this patent
The OD of substance positive serum450Respectively less than kit feminine gender judgement critical value (i.e. S/P% ﹤ 26%) belongs to negative, illustrates this specially
The profit ox miscarriage Chlamydia ELISA detection kit does not generate cross reaction with other Chlamydia serum.
3,32 parts of samples by the PCR detection positives, ELISA kit detect 29 parts of positives, 3 parts of feminine genders；107 parts by PCR
Negative sample is detected, ELISA kit detects 0 part of positive, 107 parts of feminine genders.X2 inspections are done to two methods with SPSS softwares
(McNemar Test), P > 0.05 show that two methods do not have marked difference, while tentatively obtaining the sensitivity of this kit
It is 90.6%, specificity is 100%.
9 repeatability detection of embodiment
According to ELISA operating methods, with each replication of 4 parts of cow's serums of same reagent box pair (2 parts of feminine genders, 2 parts of positives) 3
It is secondary；Meanwhile respectively 4 parts of cow's serums are carried out repeating to detect each 3 times with the different kits of same a batch and the kit of different batches,
Calculate its respectively OD450Average value, according to CV (%)=(SD/AV OD450) x100% calculate the ELISA kit batch in
With interassay coefficient of variation CV values, the repeatability of the ELISA kit is verified.Testing result is as follows：
Table 5 repeats to test
ELISA kit variation within batch coefficient is respectively less than 10%, and interassay coefficient of variation is respectively less than 15%.
The foundation of 10 Ns of miscarriage chlamydiosis enzyme linked immunological antibody assay kit standards systems of embodiment
1, the foundation of critical value：According to the ELISA experimental conditions of optimization establishment, 32 parts are detected by miscarriage Chlamydia S26/
3 plants of POMP90 albumen specific primer PCRs detections identify that positive miscarriage Chlamydia positive serum and 107 parts of process PCR detect,
Identification negative serum is detected, and calculates the OD of each serum450With positive control OD450Ratio S/P% and calculate separately out sun
Property serum S/P% ± 2SD and negative serum S/P% ± 2SD values.Result of calculation is as follows：
The 6 PCR positives of table and negative serum ELISA detected value statistical values
Using 1- specificity as abscissa, susceptibility is ordinate, and with 20.0 Software on Drawing ROC curves of SPSS, statistics is outstanding
Mounting index (Youden ' s Index, YI), YI=sensibility+specificity -1 find out positive S/P% ± 2SD and feminine gender S/ respectively
P% ± 2SD values correspond to sensitivity and special angle value.Because it is very low that 69.72 and 14.41 critical values correspond to sensitivity and specificity
So only from critical value in the best positive and negative critical value of 21.88 and 34.99 interval selections.Different critical value is corresponding
ELISA sensitivitys and special as follows：
Table 7
| Critical value | Sensitivity (%) | Specificity (%) |
| 21.9 | 100 | 56.1 |
| 25.8 | 100 | 79.4 |
| 26.4 | 100 | 81.3 |
| 29.8 | 96.9 | 90.7 |
| 30.8 | 96.9 | 92.5 |
| 31.2 | 90.6 | 92.5 |
| 33.6 | 78.1 | 94.4 |
It is best cut point to select the outstanding mounting index maximum in this section, this puts corresponding sensibility and specificity most
Height determines the S/P% values at this as kit positive critical value (CUT-OFF values), while it is big to choose another sensitivity
In 0.9, specificity is more than 0.8 respective value as kit feminine gender critical value.It is considered as suspicious sample among detected value the two.
As depicted in figs. 1 and 2, ROC curve is obtained through SPSS20.0 Software on Drawing, finger is stepped in area under the curve=0.976 especially
Number maximum value=0.894, it is 0.969 that this, which puts corresponding sensitivity, and specificity is 0.925, and respective coordinates point is S/P%=
30.81.Negative sample critical value corresponds to sensitivity 1, specificity 0.813, and respective coordinates point is S/P%=26.33, is rounded up
This kit positive critical S/P%=31%, negative critical value S/P%=26% afterwards, then judgement can among 26% and 31%
Doubt sample.
2, testing result criterion：Sample to be tested (S), the OD of positive control (P) and negative control (N)450And S/P%
Value.If ODPCMean value >=0.45 and ODPC/ODNC>=3.0 kits are effective.S/P% >=31% item is determined as on this basis
Chlamydia is positive.S/P% < 26% are then determined as Chlamydia feminine gender, and 31% >=S/P% >=26% item judges suspicious.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>China Agricultural University
<120>A kind of kit of enzyme linked immunosorbent detection ox miscarriage Chlamydia
<141> 2018-02-28
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Ser Thr Ser Lys Gly Gly Ala Ile Tyr Ala Asp Lys Leu Thr Ile Val
1 5 10 15
Ser Gly Gly Pro Thr Leu Phe Ser Asn
20 25
Claims (10)
1. a kind of kit of enzyme linked immunosorbent detection ox miscarriage Chlamydia, which is characterized in that the solid phase carrier in the kit
Upper coating is just like polypeptide shown in SEQ ID NO.1.
2. kit according to claim 1, which is characterized in that after the solid phase carrier is coated with the polypeptide, using anti-
Former protective agent is handled；The antigen protective agent is the PBS containing 10% sucrose, 10% glycerine, and preferred antigens are protectant
PH value is 7.2.
3. kit according to claim 2, which is characterized in that the solid phase carrier is ELISA Plate, the ELISA Plate
Preparation method is：
1) polypeptide is diluted to polypeptide solution using aseptic deionized water；
2) it utilizes carbonate buffer solution that the polypeptide solution obtained by step 1) is diluted 1000 times, each hole of ELISA Plate is added and is incubated, uses
After washing buffer PBST rinses ELISA Plate, closed using confining liquid；
3) antigen protective agent is added per hole to the microwell plate closed to be incubated, outwells antigen protective agent, wash, dry, i.e.,
?；
The polypeptide is diluted to the polypeptide solution of final concentration of 1mg/mL preferably by aseptic deionized water；
The polypeptide that polypeptide solution obtained by step 1) is diluted to final concentration of 1.0 μ g/mL preferably by carbonate buffer solution is molten
Liquid；
It is preferred that the carbonate buffer solution is the carbonate buffer solution of pH9.6,50mM；
It is preferred that the confining liquid is the PBST solution containing 10% skimmed milk power.
4. kit according to claim 3, which is characterized in that the preparation method of the ELISA Plate is：
After each hole of ELISA Plate is added in the polypeptide solution of the final concentration of 1.0 μ g/mL, it is incubated 16 hours at 4 DEG C；
It is incubated 2 hours and is closed at 37 DEG C using the confining liquid；
After the antigen protective agent is added, it is incubated 4 hours at 37 DEG C.
5. according to Claims 1 to 4 any one of them kit, which is characterized in that the kit further includes enzyme labelled antibody,
The enzyme labelled antibody is the goat-anti ox IgG of horseradish peroxidase-labeled.
6. kit according to claim 5, which is characterized in that the kit further includes negative control sera and the positive
Control serum；
The preparation method of the positive control serum is：
Polypeptide coupling KLH carrier proteins shown in SEQ ID NO.1 are obtained into coupling peptide, with 10mmol/L PBS buffer solution (pH
7.4) a concentration of 20mg/mL is diluted to as immunizing antigen；
The calf that selection miscarriage chlamydial antibody is negative, is immunized；
Head exempts from：It is fully emulsified by Fu Shi Freund's complete adjuvants and immunizing antigen mixed in equal amounts, take 1mL musculi collis to inject；
Two exempt from：Head exempts from the 14th day afterwards, takes Fu Shi Freund's incomplete adjuvants and immunizing antigen mixed in equal amounts, fully emulsified, takes 1mL neck skins
Lower multi-point injection；
Three exempt from：Two exempt from the 7th day afterwards, and it is primary to exempt from method booster immunization with two；
Four exempt from：Three exempt from after the 7th day, the subcutaneous each injecting immune antigen 1 mL of muscle and neck；
It takes a blood sample, detach serum and dilutes after 1 week, using the coated ELISA Plate of polypeptide shown in SEQ ID NO.1, it is immune to detect it
Serum antibody titer chooses 1:OD when 200 dilution450Calf more than 1.0 carries out jugular vein blood collection, sterile separation serum, water
Bath inactivation obtains miscarriage Chlamydia positive control serum；
The preparation method of the negative control sera is：
Bull calf is chosen, takes a blood sample respectively, detach serum, water-bath inactivation is examined by the detection of Chlamydia indirect hemagglutination and ELISA
It surveys, collects the cow's serum that result is negative, mixing, Chlamydia negative control sera of as miscarrying.
7. kit according to claim 5, which is characterized in that the kit further includes that washing buffer, enzyme mark are anti-
Body dilute solution, Sample Dilution buffer solution, developing solution and terminate liquid.
8. kit according to claim 7, which is characterized in that the washing buffer be containing 0.5 ‰ Tween-20s,
The phosphate buffer (PBST) of pH7.2；
The enzyme labelled antibody dilute solution is the phosphate containing, the pH7.2 of 0.1 ‰ merthiolates, 5% glycerine, 5% trehalose
Buffer solution；
The Sample Dilution buffer solution is the phosphate-buffered containing, the pH7.2 of 0.3%Triton-100,0.1 ‰ merthiolates
Liquid；
The terminate liquid is strong acid.
9. kit according to claim 8, which is characterized in that the terminate liquid is the H that deionized water is prepared2SO4。
10. kit according to claim 8 or claim 9, which is characterized in that the developing solution includes color developing agent A and color developing agent
B：Color developing agent A is containing 1.46% disodium hydrogen phosphate, 0.93% citric acid, the aqueous solution of 0.045% hydrogen peroxide；Color developing agent B is
Containing 0.03% tetramethyl benzidine, 0.096% citric acid, 0.019% ethylenediamine tetraacetic ethanedioic acid sodium salt, 2%DMSO, 4% glycerine
Aqueous solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810259741.XA CN108802368B (en) | 2018-03-27 | 2018-03-27 | Kit for enzyme-linked immunosorbent assay of bovine chlamydia abortus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810259741.XA CN108802368B (en) | 2018-03-27 | 2018-03-27 | Kit for enzyme-linked immunosorbent assay of bovine chlamydia abortus |
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| CN108802368A true CN108802368A (en) | 2018-11-13 |
| CN108802368B CN108802368B (en) | 2021-05-04 |
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ID=64095460
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810259741.XA Active CN108802368B (en) | 2018-03-27 | 2018-03-27 | Kit for enzyme-linked immunosorbent assay of bovine chlamydia abortus |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111024947A (en) * | 2019-11-19 | 2020-04-17 | 江苏美克医学技术有限公司 | Candida albicans fluorescence immunochromatography assay kit and preparation method thereof |
| CN111239417A (en) * | 2020-02-21 | 2020-06-05 | 深圳市伯劳特生物制品有限公司 | Coating buffer solution, kit and application |
| CN113567665A (en) * | 2021-08-16 | 2021-10-29 | 固安林科特生物工程有限公司 | Lysate for detecting chlamydia trachomatis antigen and detection method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102180953A (en) * | 2011-02-28 | 2011-09-14 | 中国农业大学 | Chlamydophila abortus outer membrane recombinant protein, and coding gene and application thereof |
| CN105606826A (en) * | 2016-02-05 | 2016-05-25 | 中国农业大学 | Kit for detecting avian chlamydia psittaci by enzyme linked immunosorbent assay |
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2018
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102180953A (en) * | 2011-02-28 | 2011-09-14 | 中国农业大学 | Chlamydophila abortus outer membrane recombinant protein, and coding gene and application thereof |
| CN105606826A (en) * | 2016-02-05 | 2016-05-25 | 中国农业大学 | Kit for detecting avian chlamydia psittaci by enzyme linked immunosorbent assay |
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| EVANGELIA VRETOU ET AL.: "Antigenic Organization of the N-Terminal Part of the Polymorphic Outer Membrane Proteins 90, 91A, and 91B of Chlamydophila abortus", 《INFECTION AND IMMUNITY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111024947A (en) * | 2019-11-19 | 2020-04-17 | 江苏美克医学技术有限公司 | Candida albicans fluorescence immunochromatography assay kit and preparation method thereof |
| CN111239417A (en) * | 2020-02-21 | 2020-06-05 | 深圳市伯劳特生物制品有限公司 | Coating buffer solution, kit and application |
| CN113567665A (en) * | 2021-08-16 | 2021-10-29 | 固安林科特生物工程有限公司 | Lysate for detecting chlamydia trachomatis antigen and detection method |
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| CN108802368B (en) | 2021-05-04 |
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