CN104865243B - A kind of bombyx mori egg microsporidiosis light activating chemical luminescence luminous immune detecting method - Google Patents
A kind of bombyx mori egg microsporidiosis light activating chemical luminescence luminous immune detecting method Download PDFInfo
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Abstract
The present invention provides a kind of bombyx mori egg microsporidiosis light activating chemical luminescence luminous immune detecting method, include the preparation of cultivated silkworm microsporidian sporoderm protein antigen, the foundation of mouse resisting silkworm microsporidian sporoderm protein monoclonal antibody hybridoma cell strain, the ascites of cultivated silkworm microsporidian sporoderm protein monoclonal antibody is prepared and purified, and nosema bombycis AlphaLISA detection methods foundation.When being detected using the inventive method, when special interaction is not present in biomolecule, free oxygen can not be diffused into Acceptor beads, the generation of signal is not had then, and then higher sensitiveness and accuracy is brought, testing result is more accurate, with higher homogeneity, and ambient interferences are few, background is low, with extensive dynamic detection range；Sample requirement amount is few when being detected using kit of the present invention, and testing process is simplified, with good market prospects without washing in detection process.
Description
Technical field
The invention belongs to agricultural biological technical field, and in particular to a kind of bombyx mori egg microsporidiosis light activation hair
Light immunologic detection method.
Background technology
The granulosis that nosema bombycis (Nosema bombycis, Nb) infects silkworm initiation is the destructiveness of silkworm
Infectious disease, is confirmed as the only quarantine disease in world sericulture area.Inspection of the recent domestic scholar in pebrine disease
Carry out numerous studies work in terms of survey, obtain greater advance, but due to sides such as cost, sensibility in practice, the complexity of operation
The influence in face, fails have a kind of technology that existing light microscope can be replaced to detect method so far.AlphaLISA technologies are a kind of
Chemiluminescent novel homogeneous detection technique based on microballon, AlphaLISA Cleaning Principle is similar to double-antibody method, mainly
Dependent on the interaction of Alpha donors microballon and Acceptor beads, when biological respinse makes donor microballon and Acceptor beads phase mutual connection
When near, laser excitation cascade reaction so as to produce the signal greatly amplified, and then is detected.
In recent years, the technology is widely used in medical science and biology field, but for the micro- spore of silkworm
The AlphaLISA detection kit of sub- worm, is not reported both at home and abroad at present.Compared with traditional elisa technique,
AlphaLISA detection kit has that higher sensitiveness, accuracy, homogeneity, background is low, extensive dynamic detection range,
And without washing and sample requirement amount it is few the features such as.The technology has been obtained widely in medical science and biology field
Using.But, it is not reported both at home and abroad for nosema bombycis AlphaLISA detections.
The content of the invention
For deficiencies of the prior art, it is an object of the invention to provide a kind of bombyx mori egg microsporidiosis
Light activating chemical luminescence luminous immune detecting method.
Above-mentioned purpose is realized, the present invention is adopted the following technical scheme that：A kind of bombyx mori egg microsporidiosis light activation
Electrochemiluminescent immunoassay detection method, comprises the following steps：
1) preparation of cultivated silkworm microsporidian sporoderm protein antigen：First by the spore of microsporidian bead broken 12~13
After hour, DAPI dyeing observations are carried out, and are screened, the spore core for sending blue light is not seen in the visual field, it is many by observing
Individual visual field statistics, the purity of prepared spore shell is more than 99%, obtains microsporidian spore shell；Then, by the microsporidian spore
Shell is broken 12~13 hours with bead again, obtains spore shell fragmin, i.e. cultivated silkworm microsporidian sporoderm protein antigen；
2) foundation of mouse resisting silkworm microsporidian sporoderm protein monoclonal antibody hybridoma cell strain：With step 1) it is obtained
Spore shell fragmin is antigen, and immune balb/c mice using PEG1500 cell-fusion techniques, and by subclone screening, is obtained
To the hybridoma cell strain of secrete monoclonal antibody；
3) ascites of cultivated silkworm microsporidian sporoderm protein monoclonal antibody is prepared and purified：Recovery step 2) made from silkworm
Microsporidian sporoderm protein monoclonal antibody hybridoma cell, when cultivating to exponential phase, is injected intraperitoneally BALB/C mice, receives
Collect ascites, and using the purifying of caprylic acid-ammonium progress antibody；The cultivated silkworm microsporidian sporoderm protein monoclonal of purifying is resisted
Body is divided into two parts, portion biotin labeling, and another and acceptor microballoon are coupled；
4) foundation of nosema bombycis AlphaLISA detection methods：Microsporidian is detected using double antibody sandwich method；
1. cultivated silkworm microsporidian sporoderm protein monoclonal antibody is connected with biotin：Take step 3) prepare antibody purification
Lmg is added in the centrifuge tube with filter membrane, and 8min is centrifuged with 9000r/min；With mark buffer solution (0.1mol/L Na2CO3/
NaHCO3, pH9.5) and repeated washing 6 times；Antibody is collected, 50 μ L antibody-solutions are configured to, the antibody-solutions include silkworm
The biotin (being prepared with DMSO) of microsporidian sporoderm protein monoclonal antibody, mark buffer solution and 5 μ L 22mg/mL；Will be described
Antibody-solutions are incubated 4h in shaking at room temperature, remove unnecessary biotin using the centrifuge tube with filter membrane, obtain biotinylated antibody,
And it is 0.5mg/mL that biotinylated antibody is adjusted into concentration with 1xAphaLISA cushioning liquid；
2. cultivated silkworm microsporidian sporoderm protein monoclonal antibody is coupled with acceptor microballoon：Take step 3) prepare antibody purification
0.2mg is added in the centrifuge tube with filter membrane, 8min is centrifuged with 9000r/min, with buffer solution (0.13mol/L, pH 8.0PBS)
Antibody is collected after repeated washing 6 times, 1mg acceptors microballoon, 10 μ L 25mg/mLNaBH are added in antibody-solutions3CN is (slow with PBS
Fliud flushing prepare, it is now with the current), 1.25 μ L mass concentrations be 10% Tween-20, then cumulative volume is added to buffer solution
200 μ L, in 37 DEG C of lucifuge oscillating reactions 48h；It is eventually adding 10 μ L 65mg/mL carboxymethyl hydroxylamine hydrochlorides solution (use
0.8mol/L NaOH are prepared, now with the current), the uncombined site of 1h closings is incubated in 37 DEG C of lucifuges, centrifuge washing has been connected
The acceptor microballoon of antibody is connect, it is 5mg/mL to adjust acceptor microballoon concentration with 1xAphaLISA buffer solutions；
3. the foundation of method：By step 2. the obtained acceptor microballoon for having connected antibody and 1xAphaLISA buffer solutions according to
1:50~1:100 volume ratio is diluted, step 1. obtained biotinylated antibody and 1xAphaLISA buffer solutions according to 1:
200~1:400 volume ratio is diluted；Be separately added into microwell plate standard items or detected sample, connection antibody by
Body microballoon and each 25 μ L of biotinylated antibody, are incubated 20min in 37 DEG C of vibrations, then add streptavidin under the conditions of lucifuge
Donor microballoon l75 μ L, in 37 DEG C vibration be incubated 20min after, the detected signal value on AlphaScreen/Lisa detectors.
Further, the specific steps of setting up of the step 3. method include：
(1) by the acceptor microballoon for having connected antibody and 1xAphaLISA buffer solutions according to 1:50~1:100 volume
Than being diluted, reagent 1 is used as；
(2) by the biotinylated antibody and 1xAphaLISA buffer solutions according to 1:200~1:400 volume ratio carries out dilute
Release, be used as reagent 2；
(3) 10 × AlphaLISA assay Buffer are diluted to 1 × AlphaLISA assay with ultra-pure water
Buffer, the donor microballoon of the streptavidin is diluted to 1 × AlphaLISA assay Buffer final concentration of
40 μ g/mL, are used as reagent 3；
(4) after Eggs of Silkworm is ground with PBS (0.01mol/L, pH7.4), with filtered through gauze, take filtrate in
500~600r/min centrifuge 2~3min, collect supernatant, and by supernatant then at 3000~3500r/min centrifugation 10~
11min, collects precipitation, and precipitation is diluted to 5mL with PBS (0.01mol/L, pH7.4), is used as silkworm seed lapping liquid；
(5) nosema bombycis is added in the silkworm seed lapping liquid prepared to step (4), nosema bombycis is ground in silkworm seed
Concentration in grinding fluid is followed successively by 107spores/mL、106spores/mL、105spores/mL、104Spores/mL and
103spores/mL；
(6) foundation of standard curve：Take silkworm seed lapping liquid, step (1) that step (5) each concentration contains nosema bombycis
Each 25 μ L of reagent 2 prepared by the reagent 1 and step (2) of preparation, are well mixed, and constitute standard items experimental group；Step (4) is taken to prepare
Silkworm seed lapping liquid, step (1) prepare reagent 1 and step (2) prepare each 25 μ L of reagent 2, be well mixed, as feminine gender it is right
According to；The sample of the standard items experimental group and negative control is incubated 20min in 37 DEG C of vibrations, then added under the conditions of lucifuge
Enter the reagent 3 of 175 μ L steps (3) preparation, be incubated then at 37 DEG C of vibrations after 20min, on AlphaScreen/Lisa detectors
At 615nm, each signal value of examination criteria product experimental group；Logarithm value using each concentration of standard item group is abscissa, each concentration mark
The ratio of the signal value of quasi- product and the signal value of negative control is ordinate, draws standard curve；
(7) detection of testing sample：Testing sample obtains treating test sample using being handled with step (4) identical method
Savor lapping liquid；Reagent 1 prepared by testing sample lapping liquid, step (1) and each 25 μ L of reagent 2 prepared by step (2) are taken, mixing is equal
It is even, in 37 DEG C vibration be incubated 20min, 175 μ L steps 3 are then added under the conditions of lucifuge) prepare reagent 3, shaken then at 37 DEG C
It is dynamic to be incubated after 20min, on AlphaScreen/Lisa detectors at 615nm, the signal value of testing sample is detected, and according to step
Suddenly the standard curve that (6) are set up, is calculated, and obtains the concentration of microsporidian in testing sample lapping liquid, and then detection is become a Buddhist monk or nun
Microsporidian content in silkworm silkworm seed.
Further, the acceptor microballoon and donor microballoon are nano-scale particle；Being coated with of acceptor microsphere surface
Learn luminous agent and lanthanide series europium；The donor microsphere surface is coated with streptavidin and sensitising agent.
Compared with prior art, the present invention has the advantages that：
1st, first using hybridoma technology, to set up mouse resisting silkworm microsporidian sporoderm protein monoclonal antibody miscellaneous for the inventive method
Hand over tumor cell strain；Mouse resisting silkworm microsporidian sporoderm protein monoclonal antibody is prepared by ascites purifying；And pass through verification experimental verification
Obtained monoclonal antibody has good stability, and has specificity to nosema bombycis, available for nosema bombycis cause of disease
Detection.
2nd, obtained mouse resisting silkworm microsporidian sporoderm protein 1G5 monoclonal antibodies are coated in AlphaLISA by the present invention
On the acceptor microballoon of method, and with mouse resisting silkworm microsporidian sporoderm protein 2D9 monoclonal antibodies made from biotin labeling, with confession
Body microballoon constitutes the detection kit for detecting microsporidian in the lump；When being detected using the inventive method, Alpha donors
Microballon and Acceptor beads interaction, under 680nm laser irradiation, sensitising agent on donor microballon is by the oxygen in surrounding environment
Gas is converted into more active free oxygen, and free oxygen diffuses to Acceptor beads, produces a series of chemiluminescence reaction, finally will
On europium in energy transmission to Acceptor beads, launch wavelength is 615nm, and then is detected.
During the 3rd, using the present invention for detecting that the kit of microsporidian is detected, it is not present specifically in biomolecule
During interaction, free oxygen can not be diffused into Acceptor beads, then do not have the generation of signal, and then bring higher sensitiveness
And accuracy, testing result is more accurate.
4th, the present invention is used to detect that the kit of microsporidian to have higher homogeneity, and ambient interferences are few, background is low,
With extensive dynamic detection range；Sample requirement amount is few when being detected using kit of the present invention, and in detection process
In without washing, testing process is simplified, with good market prospects.
Brief description of the drawings
Fig. 1 is broken spore total protein, broken spore shell fragment, germinating spore total protein and the SDS- for sending out spore coat fragment
PAGE schemes；
Fig. 2 is the growing state that mouse resisting silkworm microsporidian sporoderm protein monoclonal antibody is carried out the 8th day after cell fusion
(10×10)；
Fig. 3 is nosema bombycis protein in cell wall 1G5 monoclonal antibodies ascites rear SDS-PAGE electroresis appraisals before purification；
Fig. 4 is self-control reagent dosage-response curve.
Embodiment
The present invention is described in further detail with specific embodiment below in conjunction with the accompanying drawings, it is raw materials used such as nothing in embodiment
Specified otherwise, is common commercially available；The biological test used unless otherwise specified, as conventional method.
First, a kind of bombyx mori egg microsporidiosis light activating chemical luminescence luminous immune detecting method, comprises the following steps：
1st, the strain of mouse resisting silkworm microsporidian sporoderm protein monoclonal antibody hybridoma cell 1G5,2D9 foundation；
2nd, the ascites of nosema bombycis protein in cell wall 1G5,2D9 monoclonal antibody is prepared and purified；
3rd, nosema bombycis AlphaLISA detection；
4th, the evaluation of AlphaLISA detection methods.
2nd, specific steps include：
1st, the foundation of mouse resisting silkworm microsporidian sporoderm protein monoclonal antibody hybridoma cell strain；
The preparation of 1.1 cultivated silkworm microsporidian sporoderm protein antigens：
Based on the principle of " the natural space conformation for retaining antigen protein as far as possible ", silkworm is prepared using " two step crush methods "
" the spore shell fragment " of microsporidian；
After the spore and germinating spore of microsporidian are crushed 12 hours with bead, carry out DAPI dyeing observations and simultaneously carry out
Do not see the spore core for sending blue light in screening, obtained purer spore shell, the visual field, counted by observing multiple visuals field, institute
The purity for preparing spore shell is more than 99%, and spore shell fragment is prepared for further crushing；Spore shell after above-mentioned primary fragmentation is entered
Row second is broken, and by prepared " broken spore total protein ", " broken spore shell fragment ", " germinating spore total protein " and
" hair spore coat fragment " carries out SDS-PAGE detections, as a result such as Fig. 1；M swimming lanes are Marker in Fig. 1；1：Germinating spore total protein；
2：Broken spore total protein；3：Spore suspension be broken 4 hours after supernatant；4：Spore suspension be broken 2 hours after supernatant；
5：Send out spore coat fragment；6：Broken spore shell fragment.As can be drawn from Figure 1：Two kinds of spore shell micrinites have between tying up to 25-35KDa
Three obvious bands, also have band in below 25KDa, and these bands are included in the band of total protein, illustrate institute
The spore shell fragment band of preparation relatively enriches.
The foundation of 1.2 mouse resisting silkworm microsporidian sporoderm protein monoclonal antibody hybridoma cell strains：
The nosema bombycis that 1.1 are prepared crushes the spore shell fragmin immune balb/c mice (Nosema bombycis
The amino acid sequence of worm spore shell fragmin is as shown in SEQ ID NO.1), using PEG1500 cell-fusion techniques, prepare Dan Ke
Grand antibody, as a result shows that Single cell fusion is divided into and fills 384 holes (being divided into the orifice plate of 4 plate 96 to dispense 384 samples), merges successfully 370
Hole, fusion rate is up to 96.3%, ELISA initial surveies positive rate up to 17.3%；The higher 38 holes subclone of the positive value of selection, by 2
Secondary subclone, continuous repeated screening has finally given 2 plants of hybridoma cell strains compared with stably excreting antibody, be named as 1G5,
2D9.The selection result of cell fusion is as shown in table 1, Fig. 2；Shown in table 1 and hole count is merged in 4 96 orifice plates, ELISA is positive
Hole count and ELISA positive rates.Fig. 2 shows that mouse resisting silkworm microsporidian sporoderm protein monoclonal antibody is carried out the 8th after cell fusion
Its growth conditions is good.
The cell fusion of table 1 and positive rate list
The biological nature identification of 1.3 monoclonal antibodies
1.3.1 positive hybridoma cell stability test
1G5 the and 2D9 cell lines squamous subculture that 1.2 are obtained more than 6 months, takes culture supernatant to carry out ELISA potency
(it is existing known technology that ELISIA methods used, which carry out titration) is determined, as a result shows that its culture supernatant ELISA potency is still protected
Hold 1:3200-6400；1G5 and 2D9 cell lines are frozen respectively and recovered behind 1,2,4 and 6 months, titration are carried out, as a result
Show that its culture supernatant ELISA potency remains at 1:3200-6400；It can to sum up draw, 1G5 and 2D9 positive hybridomas are thin
Born of the same parents' strain has good stability.
1.3.2 the specificity analysis of monoclonal antibody
Identify that the specificity of 1G5 and 2D9 secrete monoclonal antibodies is (used with tri-antibody sandwich ELSIA (TAS-ELISA) method
ELISIA methods identify that the specificity of monoclonal antibody is existing known technology), it is used as coating with the rabbit-anti sporozoite polyclonal antibody of preparation
Antigen, the monoclonal antibody of 1G5 or 2D9 cell lines secretion are antibody to be detected, and enzyme labelled antibody is goat anti-mouse igg-HRP,
Selection deformation microsporidian (Shandong strain), bombyx mori nuclear polyhydrosis virus (NPV), bacillus thuringiensis, Escherichia coli, black chest
Sepsis bacillus, Serratieae, nosema bombycis and healthy Midgut of Silkworm, Bombyx Mori liquid carry out specific analysis, and list is determined with this
Anti- specificity, as a result as shown in table 2 and table 3；As shown in Table 2, two kinds of each antigen in addition to positive controls it is dense
Degree shows that 1G5 monoclonal antibodies have specificity for the nosema bombycis of positive control all shown as feminine gender；As shown in Table 3, except
Two kinds of concentration of each antigen beyond positive controls show silkworm of the 2D9 monoclonal antibodies for positive control all shown as feminine gender
Microsporidian has specificity.
The specificity analysis of the 1G5 monoclonal antibodies of table 2
Remarks：The positive is judged as more than 2.0 than negative OD values with the OD values of sample.
The specificity analysis of the 2D9 monoclonal antibodies of table 3
Remarks：The positive is judged as more than 2.0 than negative OD values with the OD values of sample
2nd, the preparation and purifying of nosema bombycis protein in cell wall 1G5,2D9 monoclonal antibody ascites：
The preparation and purifying of 2.1 nosema bombycis protein in cell wall 1G5,2D9 monoclonal antibodies
Recovery nosema bombycis protein in cell wall 1G5,2D9 hybridoma, when cultivating to exponential phase, takes 12 week old
BALB/c mouse 20, be injected intraperitoneally incomplete Freund's adjuvant, 0.2-0.3mL/ only；Pneumoretroperitoneum is inoculated with without blood respectively within 3 days
1G5,2D9 hybridoma of clear culture medium dilution, every mouse 3 × 106/ 0.3mL, each 10；Interval is after 5-7 days, collection
Ascites, is continuously adopted 2-3 times, every mouse gathers 5-10mL ascites, and 1G5 ascites 75mL, 2D9 ascites 60mL is collected altogether, is adopted respectively
The purifying of antibody is carried out with caprylic acid-ammonium.Ascites after before purification is subjected to SDS-PAGE electroresis appraisals, as a result such as Fig. 3,
Show that clearly heavy chain and light chain mainly occur in purification ascites nosema bombycis protein in cell wall 1G5 monoclonal antibodies, purity compared with
It is good.It is 8.6mg/mL, 6.7mg/mL that 1G5,2D9 antibody concentration, which are respectively, after measuring purification through ultraviolet specrophotometer；Between
Connect the potency after ELISA determines mouse 1G5,2D9 ascites antibody before purification, mouse 1G5,2D9 ascites antibody potency point before purification
Wei 1：128000、1:64000,1G5,2D9 antibody titer are 1 after purification：64000、1:16000, show that antibody purifies aftereffect
Valency loss is small.Ascites nosema bombycis protein in cell wall 1G5, the 2D9 monoclonal antibody of preparation has provided for follow-up research
The reagent of effect.
The biotin labeling of 2.2 antibody：
Take nosema bombycis protein in cell wall 1G5 monoclonal antibodies lmg after purification add Millipore companies with filter
In the centrifuge tube of film, 8min is centrifuged with 9000r/min；With mark buffer solution (0.1mol/LNa2CO3/NaHCO3,PH9.5)) weight
After backwashing is washed 6 times；Antibody is collected, 50 μ L antibody-solutions are configured to, the antibody-solutions include cultivated silkworm microsporidian sporoderm egg
The biotin (being prepared with DMSO) of white 1G5 monoclonal antibodies, mark buffer solution and 5 μ L 22mg/mL；By the antibody-solutions in
Shaking at room temperature is incubated 4h, removes unnecessary biotin using the centrifuge tube with filter membrane of Millipore companies, obtains biotinylation
Antibody is simultaneously；It is 0.5mg/mL that biotinylated antibody is adjusted into concentration with 1xAphaLISA cushioning liquid.
2.3 antibody are coupled with acceptor microballoon
Nosema bombycis protein in cell wall 2D9 monoclonal antibodies 0.2mg after purification is taken to add carrying for Millipore companies
In the centrifuge tube of filter membrane, 8min is centrifuged with 9000r/min, after buffer solution (0.13mol/L, pH 8.0PBS) repeated washing 6 times
Antibody is collected, 1mg acceptors microballoon (Perkinelmer Products, article No. are added in antibody-solutions：AL105C)、10μL
25mg/mL NaBH3CN (using buffer, now with the current), 1.25 μ L mass concentrations are 10% Tween-20, are then used
Cumulative volume is added to 200 μ L by buffer solution, in 37 DEG C of lucifuge oscillating reactions 48h；It is eventually adding 10 μ L 65mg/mL CMO (carboxylics
Methyl hydroxylamine hydrochloride solution) after (being prepared with 0.8mol/L NaOH, now with the current), it is incubated 1h closings in 37 DEG C of lucifuges and does not tie
Site is closed, centrifuge washing has been connected the acceptor microballoon of mouse resisting silkworm microsporidian sporoderm protein monoclonal antibody, used 1xAphaLISA
Buffer solution adjustment acceptor microballoon concentration is 5mg/mL.
Wherein, used acceptor microballoon (Perkinelmer Products, article No.：AL105C), donor microballoon
(Perkinelmer Products, article No.：6760002s)、AlphaLISA Immunoassay Buffer(10x)
(Perkinelmer Products, article No.：AL000F).
3rd, the foundation of nosema bombycis AlphaLISA detection methods：
By the acceptor microballoon for having connected cultivated silkworm microsporidian sporoderm protein 2D9 monoclonal antibodies obtained in step 2.3 with
1xAphaLISA buffer solutions are according to 1:50~1:100 volume ratio is diluted；The micro- spore for the biotin labeling that step 2.2 is obtained
Sub- worm sporoderm protein 1G5 monoclonal antibodies are with 1xAphaLISA buffer solutions according to 1:200~1:400 volume ratio is diluted；
The AlphaLISA kits for detecting microsporidian are constituted together with streptavidin antibody microballoon；Wherein, donor microballoon is
Perkinelmer Products, article No.：6760002s.
The processing of 3.1 normative reference product and sample：
The processing of silkworm seed：" nontoxic " silkworm seed with PBS (0.01mol/L, pH7.4) grind, filtered through gauze, filtrate in
500r/min centrifuges 2min, collects supernatant, then centrifuges 10min with 3000r/min, collects precipitation, precipitation PBS
(0.01mol/L, pH7.4) is diluted to 5mL, is used as experiment silkworm seed lapping liquid.During detection, in the silkworm seed lapping liquid of standard item group
Purifying Nb spores (nosema bombycis spore) are added, make Nb spore contents from 107Spores/mL starts doubling dilution successively,
Concentration after dilution is followed successively by：106spores/mL、105spores/mL、104Spores/mL and 103spores/mL；It is negative right
According to using silkworm seed lapping liquid.
The foundation of 3.2AlphaLISA systems：
Standard items or detected sample, the acceptor microballoon dilution of the connection antibody of above-mentioned preparation are separately added into microwell plate
Each 25 μ L of liquid, biotinylated antibody dilution, are incubated 20min in 37 DEG C of vibrations, then add Streptavidin under the conditions of lucifuge
Donor microballoon (being diluted to final concentration of 40 μ g/mL using preceding 1 × AlphaLISA assay Buffer) l75 μ L of change, then at
37 DEG C of vibrations are incubated after 20min, at AlphaScreen/Lisa detectors 615nm, detected signal value.
Specific steps include：
(1) by the acceptor microballoon for having connected antibody and 1xAphaLISA buffer solutions according to 1:50~1:100 volume
Than being diluted, reagent 1 is used as；
(2) by the biotinylated antibody and 1xAphaLISA buffer solutions according to 1:200~1:400 volume ratio carries out dilute
Release, be used as reagent 2；
(3) 10 × AlphaLISA assay Buffer are diluted to 1 × AlphaLISA assay with ultra-pure water
Buffer, the donor microballoon of the streptavidin is diluted to 1 × AlphaLISA assay Buffer final concentration of
40 μ g/mL, are used as reagent 3；
(4) after Eggs of Silkworm is ground with PBS (0.01mol/L, pH7.4), with filtered through gauze, take filtrate in
500~600r/min centrifuge 2~3min, collect supernatant, and by supernatant then at 3000~3500r/min centrifugation 10~
11min, collects precipitation, and precipitation is diluted to 5mL with PBS (0.01mol/L, pH7.4), is used as silkworm seed lapping liquid；
(5) nosema bombycis is added in the silkworm seed lapping liquid prepared to step (4), nosema bombycis is ground in silkworm seed
Concentration in grinding fluid is followed successively by 107spores/mL、106spores/mL、105spores/mL、104Spores/mL and
103spores/mL；
(6) foundation of standard curve：Take silkworm seed lapping liquid, step (1) that step (5) each concentration contains nosema bombycis
Each 25 μ L of reagent 2 prepared by the reagent 1 and step (2) of preparation, are well mixed, and constitute standard items experimental group；Step (4) is taken to prepare
Silkworm seed lapping liquid, step (1) prepare reagent 1 and step (2) prepare each 25 μ L of reagent 2, be well mixed, as feminine gender it is right
According to；The sample of the standard items experimental group and negative control is incubated 20min in 37 DEG C of vibrations, then added under the conditions of lucifuge
Enter the reagent 3 of 175 μ L steps (3) preparation, be incubated then at 37 DEG C of vibrations after 20min, on AlphaScreen/Lisa detectors
At 615nm, each signal value of examination criteria product experimental group；Logarithm value using each concentration of standard item group is abscissa, each concentration mark
The ratio of the signal value of quasi- product and the signal value of negative control is ordinate, draws standard curve；
(7) detection of testing sample：Testing sample obtains treating test sample using being handled with step (4) identical method
Savor lapping liquid；Reagent 1 prepared by testing sample lapping liquid, step (1) and each 25 μ L of reagent 2 prepared by step (2) are taken, mixing is equal
It is even, in 37 DEG C vibration be incubated 20min, 175 μ L steps 3 are then added under the conditions of lucifuge) prepare reagent 3, shaken then at 37 DEG C
It is dynamic to be incubated after 20min, on AlphaScreen/Lisa detectors at 615nm, the signal value of testing sample is detected, and according to step
Suddenly the standard curve that (6) are set up, is calculated, and obtains the concentration of microsporidian in testing sample lapping liquid, and then detection is become a Buddhist monk or nun
Microsporidian content in silkworm silkworm seed.
The acceptor microballoon and donor microballoon are nano-scale particle；The acceptor microsphere surface is coated with chemiluminescence agent
With lanthanide series europium；The donor microsphere surface is coated with streptavidin and sensitising agent.
The optimization of 3.3AlphaLISA systems：
3.3.1 dose-response curve
The logarithm of normative reference product concentration in AlphaLISA kits with above-mentioned self-control for detecting microsporidian
(lg10nSpores/mL it is) abscissa, the ratio (Bx/B0) of measuring samples signal value and negative sample signal value is vertical seat
Mark, draws normative reference product dose-response curve (Fig. 4) in kit, in measurement range, makes standard items dose-response by oneself
Curvilinear equation is Y=10.95X+0.9785, and correlation coefficient r=0.9757 shows homemade sporozoite photo-induced chemiluminescence immunoassay
Detection reagent has good dose response.
3.3.2 Precision Experiment
Standard items are prepared into low middle high 3 concentration, 10 parallel holes are respectively set, it is multiple in interior and different experiments with once testing
Determine.As a result show：The present embodiment is homemade to be used to detect that the coefficient of variation is in the AlphaLISA kit assays of microsporidian
4.56%~6.74%, the coefficient of variation is 6.45%~8.52% between analysis, and precision is good.
3.3.3 specificity experiments
Present invention selection nosema bombycis (Nb), deformation microsporidian (Shandong strain), bombyx mori nuclear polyhydrosis virus
(NPV), bacillus thuringiensis, Escherichia coli (E coli), 7 kinds of pathogen of black chest sepsis bacillus and Serratieae,
Feminine gender is normal silkworm seed lapping liquid, is used to detect that the AlphaLISA kits of microsporidian are carried out with the present embodiment is homemade
AlphaLISA is detected, the specificity of this method is determined with this, and testing result as shown in table 4, as a result shows only Nosema bombycis
Worm detected value is substantially higher, and result judgement is the positive, and other pathogens detected value is relatively low, is feminine gender by criterion, shows
This method specificity is higher.
The specificity experiments result of table 4
Remarks：Criterion is more than 2.0 for the signal value of measuring samples with the ratio of negative (NC), you can be judged to the positive.
3.3.4 stability experiment
The AlphaLISA kits room temperature made by oneself for detecting microsporidian is placed after 7d, detected again, therewith
Preceding testing result is compared, and finds each batch of reagent signal value changes less, linear relationship is good, and normative reference product signal value is not
See significantly raised, explanation has good stability.
4th, the evaluation of AlphaLISA detection methods
The present invention learns from else's experience 156 parts of the sample of female moth Microscopical Method For Detection detection, it has been determined that wherein 95 parts of positive, negative sample 61
Part；Above-mentioned 156 parts of sample the present embodiment are used for the AlphaLISA kits for detecting microsporidian, entered through AlphaLISA methods
Row detection, as a result as shown in table 5, finds to detect 93 parts of positive in 95 parts of positives, finds to examine in 61 parts of negative samples
58 parts of negative sample is surveyed, the sensitivity of detection is 97.9%, specificity 95.1%, and positive predictive value is 96.9%, negative prediction
It is worth for 96.7%.As a result show that set up AlphaLISA detection methods have higher sensitivity and specificity, can meet height
Flux, the requirement of quick detection, with potential application value.
The AlphaLISA detection methods of table 5 are evaluated
Finally illustrate, the above embodiments are merely illustrative of the technical solutions of the present invention and it is unrestricted, although with reference to compared with
The present invention is described in detail good embodiment, it will be understood by those within the art that, can be to the technology of invention
Scheme is modified or equivalent substitution, and without departing from the objective and scope of technical solution of the present invention, it all should cover in this hair
Among bright right.
<110>Southwestern University；Chongqing University of Technology；
<120>A kind of bombyx mori egg microsporidiosis light activating chemical luminescence luminous immune detecting method；
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 316
<212> PRT
<213>Artificial sequence
<220>
<223>SEQ ID NO.1 amino acid sequence
<400> 1
MKTSVVILAM SYITSIFANN SYDFCDGEFA ISEKDDKCGF SFNPCTLYKQ IQKEYSNCVI 60
LRFYLKAISN MLESICDNNK RCLPFSIDSL YRPLYNREQF KHKHHHTLYA LFRNCLYLVN 120
PIYFKEYQDA LACNQLDVCY FLIRRTVCPK YGVTKYIECL KKRCEDRFDE KELAIFICDS 180
ETFVNIKLEI DACRKICPAD CTLDICKIYS LDFWERRDLL KAIFDYHYCH VLSSDNTRDE 240
IIKKIEEIYL NGTERDIKFT SFIIYQLFTD LVACKSSDKN IKRILDLICL YEKKDKCPSG 300
VMVMIEFFVK VCSYQC 316
Claims (2)
1. a kind of bombyx mori egg microsporidiosis light activating chemical luminescence luminous immune detecting method, comprises the following steps：
1）The preparation of cultivated silkworm microsporidian sporoderm protein antigen：It is first that the spore of microsporidian is broken 12 ~ 13 hours with bead
Afterwards, DAPI dyeing observations are carried out, and are screened, the spore core for sending blue light are not seen in the visual field, by observing multiple regard
Open country statistics, the purity of prepared spore shell is more than 99%, obtains microsporidian spore shell；Then, the microsporidian spore shell is used again
Bead is broken 12 ~ 13 hours, obtains spore shell fragmin, i.e. cultivated silkworm microsporidian sporoderm protein antigen；
2）The foundation of mouse resisting silkworm microsporidian sporoderm protein monoclonal antibody hybridoma cell strain：With step 1）Obtained spore shell
Fragmin is antigen, and immune balb/c mice using PEG1500 cell-fusion techniques, and by subclone screening, is divided
Secrete the hybridoma cell strain of monoclonal antibody；
3）The ascites of cultivated silkworm microsporidian sporoderm protein monoclonal antibody is prepared and purified：Recovery step 2）Obtained silkworm is micro-
Sporozoite sporoderm protein monoclonal antibody hybridoma cell, when cultivating to exponential phase, is injected intraperitoneally BALB/C mice, collects
Ascites, and using the purifying of caprylic acid-ammonium progress antibody；By the cultivated silkworm microsporidian sporoderm protein monoclonal antibody of purifying
It is divided into two parts, portion biotin labeling, another and acceptor microballoon are coupled；
4）The foundation of nosema bombycis AlphaLISA detection methods：Microsporidian is detected using double antibody sandwich method；
1. cultivated silkworm microsporidian sporoderm protein monoclonal antibody is connected with biotin：Take step 3）The antibody purification l mg of preparation
Add in the centrifuge tube with filter membrane, 8 min are centrifuged with 9000 r/min；With buffer solution repeated washing is marked 6 times, mark is buffered
Liquid is 0.1mol/L Na2CO3/NaHCO3, pH9.5；Antibody is collected, is configured in 50 μ L antibody-solutions, the antibody-solutions
Include the biotin of cultivated silkworm microsporidian sporoderm protein monoclonal antibody, mark buffer solution and the mg/mL of 5 μ L 22, matched somebody with somebody with DMSO
System；The antibody-solutions are incubated 4 h in shaking at room temperature, unnecessary biotin is removed using the centrifuge tube with filter membrane, is given birth to
Thing elementization antibody, and be 0.5 mg/mL with 1xAphaLISA cushioning liquid adjustment concentration by biotinylated antibody；
2. cultivated silkworm microsporidian sporoderm protein monoclonal antibody is coupled with acceptor microballoon：Take step 3）The antibody purification of preparation
0.2 mg is added in the centrifuge tube with filter membrane, 8 min is centrifuged with 9 000 r/min, after buffer solution repeated washing 6 times
Antibody is collected, buffer solution is PBS 0.13 mol/L, pH 8.0；1 mg acceptors microballoon, 10 μ L 25 are added in antibody-solutions
mg/mL NaBH3CN and Tween-20, wherein NaBH that 1.25 μ L mass concentrations are 10%3CN is prepared with PBS, is now matched somebody with somebody
Now use；Cumulative volume is then added into 200 μ L with buffer solution, in 37 DEG C of h of lucifuge oscillating reactions 48；It is eventually adding 10 μ L
65 mg/mL carboxymethyl hydroxylamine hydrochloride solution, the carboxymethyl hydroxylamine hydrochloride solution is prepared with 0.8 mol/L NaOH, existing
With existing use；The uncombined site of 1 h closings is incubated in 37 DEG C of lucifuges, centrifuge washing has been connected the acceptor microballoon of antibody, used
1xAphaLISA buffer solutions adjustment acceptor microballoon concentration is 5 mg/mL；
3. the foundation of method：By step 2. the obtained acceptor microballoon for having connected antibody and 1xAphaLISA buffer solutions according to 1:50
~1:100 volume ratio is diluted, step 1. obtained biotinylated antibody and 1xAphaLISA buffer solutions according to 1:200~
1:400 volume ratio is diluted；Standard items or detected sample, the acceptor microballoon for connecting antibody are separately added into microwell plate
With each 25 μ L of biotinylated antibody, 20 min are incubated in 37 DEG C of vibrations, the confession of streptavidin is then added under the conditions of lucifuge
Body microballoon l75 μ L, are incubated after 20 min, the detected signal value on AlphaScreen/Lisa detectors in 37 DEG C of vibrations；It is described
The specific steps of setting up of step 3. method include：
（1）By the acceptor microballoon for having connected antibody and 1xAphaLISA buffer solutions according to 1:50~1:100 volume ratio is carried out
Dilution, is used as reagent 1；
（2）By the biotinylated antibody and 1xAphaLISA buffer solutions according to 1:200~1:400 volume ratio is diluted, and is made
For reagent 2；
（3）10 × AlphaLISA assay Buffer are diluted to 1 × AlphaLISA assay Buffer with ultra-pure water, will
The donor microballoon of the streptavidin is diluted to final concentration of 40 μ g/mL with 1 × AlphaLISA assay Buffer,
It is used as reagent 3；
（4）After Eggs of Silkworm is ground with PBS, with filtered through gauze, filtrate is taken to centrifuge 2 ~ 3 in 500 ~ 600 r/min
Min, collects supernatant, and supernatant is centrifuged into 10 ~ 11 min then at 3000 ~ 3500 r/min, collects precipitation, precipitation uses PBS
Buffer solution is diluted to 5mL, is used as silkworm seed lapping liquid；Wherein, PBS is 0.01mol/L, pH7.4；
（5）To step（4）Nosema bombycis is added in the silkworm seed lapping liquid of preparation, makes nosema bombycis in silkworm seed lapping liquid
In concentration be followed successively by 107 spores/mL、106 spores/mL、105 spores/mL、104Spores/mL and
103spores/mL；
（6）The foundation of standard curve：Take step（5）Each concentration contains the silkworm seed lapping liquid of nosema bombycis, step（1）Prepare
Reagent 1 and step（2）Each 25 μ L of reagent 2 of preparation, are well mixed, and constitute standard items experimental group；Take step（4）Prepare
Silkworm seed lapping liquid, step（1）The reagent 1 and step of preparation（2）Each 25 μ L of reagent 2 of preparation, are well mixed, right as feminine gender
According to；The sample of the standard items experimental group and negative control is incubated 20 min in 37 DEG C of vibrations, then under the conditions of lucifuge
Add 175 μ L steps（3）The reagent 3 of preparation, is incubated after 20 min then at 37 DEG C of vibrations, in AlphaScreen/Lisa detections
On instrument at 615 nm, each signal value of examination criteria product experimental group；Logarithm value using each concentration of standard item group is each dense as abscissa
The ratio for spending the signal value of standard items and the signal value of negative control is ordinate, draws standard curve；
（7）The detection of testing sample：Testing sample is used and step（4）Identical method is handled, and is obtained testing sample and is ground
Grinding fluid；Take testing sample lapping liquid, step（1）The reagent 1 and step of preparation（2）Each 25 μ L of reagent 2 of preparation, are well mixed,
20 min are incubated in 37 DEG C of vibrations, 175 μ L steps 3 are then added under the conditions of lucifuge）The reagent 3 of preparation, then at 37 DEG C of vibrations
It is incubated after 20 min, on AlphaScreen/Lisa detectors at 615 nm, detects the signal value of testing sample, and according to step
Suddenly（6）The standard curve of foundation, is calculated, and obtains the concentration of microsporidian in testing sample lapping liquid, and then detection is become a Buddhist monk or nun
Microsporidian content in silkworm silkworm seed.
2. bombyx mori egg microsporidiosis light activating chemical luminescence luminous immune detecting method as claimed in claim 1, it is characterised in that
The acceptor microballoon and donor microballoon are nano-scale particle；The acceptor microsphere surface is coated with chemiluminescence agent and group of the lanthanides member
Plain europium；The donor microsphere surface is coated with streptavidin and sensitising agent.
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| CN105759046A (en) * | 2016-04-08 | 2016-07-13 | 重庆理工大学 | Newcastle disease virus double-antibody sandwich AlphaLISA detection kit and detection method thereof |
| CN109706080A (en) * | 2018-12-28 | 2019-05-03 | 西南大学 | A kind of method for separating and concentrating of nosema bombycis sporoplasm |
| CN111239269B (en) * | 2020-01-16 | 2021-11-09 | 中国农业科学院兰州兽医研究所 | Method for analyzing coccidian sporozoite surface protein fingerprint spectrum and application of coccidian sporozoite surface protein fingerprint spectrum |
| CN112724223B (en) * | 2020-12-28 | 2022-10-21 | 华南农业大学 | Preparation and application of monoclonal antibody of spore wall protein of nosema enteromorpha |
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