CN102749446A - Chlamydia pneumoniae IgM (immunoglobulin M) colloidal golden method kit and preparation method thereof - Google Patents
Chlamydia pneumoniae IgM (immunoglobulin M) colloidal golden method kit and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a chlamydia pneumoniae IgM (immunoglobulin M) colloidal golden method kit which comprises recombinant chlamydia pneumoniae antigens enveloped on a nitrocellulose membrane detection line, goat anti-rat IgG (immunoglobulin G) antibodies enveloped on a quality control line and rat anti-human IgM monoclonal antibodies which have colloidal gold labels and are enveloped on a gold label pad, the concentration of the chlamydia pneumoniae antigens ranges from 1mg/ml to 2mg/ml and is measured by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), the concentration of the goat anti-rat IgG (immunoglobulin G) antibodies ranges from 1mg/ml to 3mg/ml, and the concentration of the rat anti-human IgM monoclonal antibodies ranges from 5g/mL to 30g/mL and is measured by the SDS-PAGE. The chlamydia pneumoniae IgM colloidal golden method kit has the advantages that the kid is speedy, simple, convenient and accurate and is high in sensitivity, and a judgment result can be read after integral operation time of dozens of minutes; and a colloidal gold quick detecting paper strip is made of the multi-epitope recombinant antigens and is simple and convenient to operate, low in cost, good in specificity and high in sensitivity, can be used for single-component detection and is popularized easily, and detection and control effects for chlamydia pneumoniae IgM are obvious.
Description
Technical field
The present invention relates to a kind of antibody assay kit and preparation method thereof, be specifically related to a kind of CPN IgM antibody colloidal gold method detection kit and preparation method thereof.
Background technology
CPN is the important pathogen of human breathing tract disease, can cause the acute and chronic breathing problem, and investigation shows that pneumonia, bronchitis and nasosinusitis 5-10% that community obtains are caused by CPN.Often cause adult and teen-age non-model's pneumonia, also can cause acute respiration road transmissions such as bronchitis, pharyngitis and tonsillitis.Its atypical clinical manifestations of Chlamydia Pneumonia Respiratory Tract Infections, usually with pharyngalgia and hoarsen onset, several days to week back appearance cough; Compare with other breathing problems, from onset to the time of seeking medical advice with infection involving chlamydia pneumoniae for the longest, therefore sick low-heat at the beginning; How to have reduced to during inspection normally, abnormal breathing sound and nasal sinus district tenderness are modal characteristic, and white blood cell count(WBC) is normal mostly; The erythrocyte sedimentation rate speedup, normal one-sided segmental pneumonia, the similar atypical pneumonia of showing of X line rabat; The severe patient pathology is more extensive, even involves two lungs, also can be with pleurisy or hydrothorax.
Country area, the torrid zone is higher than the north country that makes a good deal of money, and 5～14 years old age group incidence of disease in the area that has is higher than the adult.In causing the cause of disease of pneumonia, be the 3rd the catatonia substance that after pneumococcus, haemophilus influenzae, causes community acquired pneumonia according to statistics.
About 300,000 examples of the annual morbidity of U.S.'s report, and only about 150 examples of the pneumonia that chlamydia psittaci causes.
Also there is identical report in Hong Kong, and in the patient that respiratory system infects, detecting serum chlamydia pneumoniae (cp) positive rate is 54.8%; Nervous infector is 24.8%; Many 1: 256 of antibody titer, and the positive rate of Chlamydia psittaci antibody is merely 0.9%, and be mostly non-recent infection.
It is obviously relevant also to realize that CPN infects with the generation of angiocardiopathy such as hat unpleasantness, myocardial infarction and intumescent cardiomyopathy and cranial vascular disease etc.This disease is hidden 10～65 days phases.Poor specificity clinical manifestation, asymptomatic infection and patient with slight symptoms are common.
1. the infection of acute respiration system is its nervous performance, like pharyngitis, laryngitis, nasosinusitis, tympanitis, bronchitis and pneumonia, accounts for more than 50% so that pneumonia is the most common, and bronchitis is taken second place.The elderly is common with pneumonia, the teenager below 20 years old, and then mostly is that the bronchitis and the upper respiratory tract infect.Often with fever, uncomfortable, pharyngalgia and hoarseness onset all over the body, present cough after a few days, this moment, how usual body temperature is.Also bronchitis, bronchial astehma can be caused, after the patient of original bronchial astehma is infected CPN, the state of an illness can be increased the weight of.Also can cause pharyngitis, nasosinusitis and tympanitis, how this exists with pneumonia and bronchitis simultaneously.Pathology is usually all lighter, although use antibiotic therapy, the state of an illness is restored to norm slower, but cough and all over the body the symptom consecutive numbers week such as discomfort to the several months.
State of an illness anxiety person can increase the weight of or dead because of producing complication such as bacterial infection because of former basic disease.
2. typhoid fever type small number of patients shows as high heat, headache, relative infrequent pulse and liver spleen eve of the lunar New Year, is prone to concurrent myocarditis, endocarditis and meningitis, the patient with severe symptoms appear faint and acute kidney exhausted, the performance heavy typhoid fever that duplicates.
3. the coherence of the morbidity of CPN infection and artery sclerosis, hat unpleasantness and acute myocardial infarction AMI is according to statistics in 50% the chronic hat unpleasantness and 68% patients of acute myocardial infarction serum; Can detect anti-chlamydia pneumoniae (cp) (IgG and IgA), comparative group only 17%.
With the group dyeing of CPN monoclonal antibody immunity or use the PCR method, at coronary artery or in the harden plaque of artery, can detect CPN antigen or its DNA, confirm in focus, to exist pathogen, and in usual artery structure, do not detecting.
The monoclonal antibody immunity fluorescence method is used in report such as Gloria, takes leave of and in the sample of artery and coronary sclerosis, is detecting CPN antigen, and it is 13% and 79% that positive rate is taken leave of, and is 4% from artery usually.
So it is relevant with arteriosclerotic generation to think that CPN infects, be the crisis status that produces the hat unpleasantness, CPN infects except should noting hat unpleasantness patient, and feel to prevent and treat CPN infect have the eve of the lunar New Year slightly reduction be preced with the generation of unpleasantness.
It was reported; Cardiovascular patients such as atherosclerotic, hat unpleasantness and peripheral artery embolization, normal merger CPN infects, with the cyclic lactone class antibiotic therapy eve of the lunar New Year such as ROX; And through agreed 2～7 years following up a case by regular visits to, progress that can obvious low cardiovascular disease.Realize the hat unpleasantness patient who has kidney exhausted simultaneously, the infectious rate of its CPN is higher, and more is prone to promote the progress of cardiovascular disease.
4. other can cause iritis, hepatitis, endocarditis, meningitis and erythema nodosum etc.
Diagnostic method generally has:
Because this sick poor specificity clinical manifestation, so,, can do aetology or immunology detection is made a definite diagnosis as doubting and this disease to the patient of pneumonia or above-mentioned clinical manifestation is arranged.
The implementation chamber is detected:
1. how usual blood picture blood leukocytes counting is, and the patient with severe symptoms can raise.Erythrocyte sedimentation rate speeds more.
2. to make an inventory be the reliable method of making a definite diagnosis this disease to aetology.Clinical diagnosis is of little use.
(1) direct smear: with Giemsa or the dyeing of immunofluorescence monoclonal antibody, detect CPN inclusion body and substance behind the smear, method is light and handy, but positive rate is low.
(2) structure cultivation: the chick embryo yolk sac inoculation is low seldom used because of detecting positive rate.Available cell culture method is got throat swab or is collected the lower respiratory tract sample, with HEP-2 cell (laryngeal cancer cell) or Hela229 cellular incubation 24 h, again with the dyeing of CPN monoclonal antibody specific, detection specificity inclusion body.
Method is more numerous and diverse, and low than other Chlamydia recall rates.
3. immunology detection: be the diagnostic method of using always.
(1) DIF: with the dyeing of CPN monoclonal antibody, DIF detects CPN antigen, and method is special flexible and sharply light and handy.
(2) microimmunofluorescence (MIF) method: detect chlamydia pneumoniae (cp), specific IgM titre >=1: 16 with (or) IgG >=l: 512 or paired sera titre rising person more than 4 times, all diagnosable acute infection.Like IgM≤1: 16 or IgG≤1: 512, then be previously to infect.
The dirigibility of this method specificity is all higher, and can be used for distinguishing primary infection and infect, is nowadays the most frequently used and serological method the most flexibly.But remove helping to change of rheumatoid factor in the blood samsara.
(3) complement associating antibody test: titre >=l: 64 with (or) paired sera titre rising person more than 4 times, which kind of Chlamydia all diagnosable acute infection, but can not be used for early diagnosis also can not be divided into and infect.
4.PCR method: detect CPN DNA, dirigibility is higher, and can distinguish with other kinds Chlamydia, and its specificity dirigibility is higher than additive method.According to statistics, PCR method recall rate is 50%～55%, and DIF and smear method to take leave of be 24%～27% and 6%～10%.Detect with connecting polymerase chain repercussion (LCR), can further improve cleverness property and recall rate, but as yet not in clinical practice.It was reported that the PCR-EIA method is a kind of rapid, light and handy enzyme immunoassay, the PCR that can improve is superior to the PCR method to the augmentation detection effect of CPN DNA, more is superior to cultivation.
Summary of the invention
The object of the invention is exactly to above-mentioned defective of the prior art, and a kind of CPN IgM antibody colloidal gold method detection kit that can fast qualitative detects the CPN IgM antibody in the serum sample is provided.
To achieve these goals; Technical scheme provided by the invention is: a kind of CPN IgM antibody colloidal gold method detection kit; The mouse-anti people IgM monoclonal antibody that comprises the colloid gold label that encapsulates on the sheep anti-mouse igg antibody that encapsulates on the reorganization CPN antigen that encapsulates on the nitrocellulose filter detection line, the nature controlling line and the gold mark pad; Said CPN antigen concentration is 1-2mg/ml, measures with SDS-PAGE; Said sheep anti-mouse igg antibody concentration is 1-3mg/ml; Said mouse-anti people IgM monoclonal antibody concentration is 5-30
/mL, measures with SDS-PAGE.
Second purpose of the present invention provided a kind of preparation method of CPN IgM antibody colloidal gold method detection kit, may further comprise the steps:
(1) preparation of reaction film: use phosphate buffer that recombinant C pn-Ag is diluted to and encapsulate concentration and be 1.0mg/ml; It is 2.0mg/ml that sheep anti-mouse igg antibody is diluted to concentration; Two kinds of coating buffers are drawn respectively on the nitrocellulose filter, with preserving after the nitrocellulose filter drying that has encapsulated;
(2) preparation of mouse-anti people IgM monoclonal antibody gold bond pad: with label concentration is that mouse-anti people IgM monoclonal antibody and the collaurum of 5-30 μ g/ml carries out coupling and make colloid gold label mouse-anti people IgM monoclonal antibody solution; With the rotating speed is the centrifugal 40min of 12000r/min; Abandon supernatant, add collaurum bond dilution, then with mixed liquid dipping gold mark pad to 1/2 of original volume; Be prepared into Cpn-IgM gold bond pad, with Cpn-IgM gold bond pad kept dry;
(3) cutting assembling: the wide self-adhesive paper of 1.5cm is thrown off on the top of offset plate at the reaction film place, and robust fibre filter paper in the stickup makes the bottom of paper push down reaction film; Throw off the self-adhesive paper of the 0.9cm of reaction film bottom, stick Cpn-IgM gold bond pad, and reaction film 1mm is pushed down on the top of golden bond pad; Throw off again golden bond pad bottom self-adhesive paper, stick sample pad, the bottom 1mm of golden bond pad is pushed down on the top of sample pad; Process reaction plate; Be cut into the 4mm test strips with cutting cutter again, after being installed, process product in the aluminium foil bag of packing into.
Further, among the preparation method of said CPN IgM antibody colloidal gold method detection kit, the drying condition of said step (1) and (2) is 37 ℃ of dryings 3 hours.
Further, among the preparation method of said CPN IgM antibody colloidal gold method detection kit, it is characterized in that: the label concentration of the mouse-anti people IgM monoclonal antibody of said step (2) is 15 μ g/ml.
Beneficial effect of the present invention is: CPN IgM antibody colloidal gold method detection kit has quick, easy, accurate and highly sensitive characteristics, and the whole operation time only needs the dozens of minutes just can sentence read result.Colloidal gold fast detecting test paper strip is a raw material with the recombinant antigen of multi-epitope, and it is easier, with low cost to have an operation, and specificity is good, and highly sensitive characteristics can single part of detection, is easy to popularize, and are obvious for detection and the control effect of CPN IgM.
Description of drawings
Fig. 1 is preparation technology's schematic flow sheet of a kind of CPN IgM antibody colloidal gold method detection kit provided by the invention.
Embodiment
One, manufacturing condition confirms
1. the selection of colloid gold particle:
1.1 principle:Prepare collaurum according to gold chloride under the condition of boiling and trisodium citrate generation redox reaction.Can change and control the grain size of collaurum through the additional proportion of regulating gold chloride and trisodium citrate.
Operation:In the 100mL purified water, add 1% citric acid three sodium solution that 0.1mL 10% gold chloride boils, under stirring condition, adds different volumes, boiled 5 minutes, the colloid gold particle of preparation different condition treats that its natural cooling returns back to original volume.With the collaurum of mouse-anti people IgM monoclonal antibody mark variable grain, and then detect with reference to article with enterprise.Its result such as table 1:
The selection experiment (particle diameter selection) of table 1:1% trisodium citrate consumption
Reaction film proportioning with the four kinds of colloid gold label mouse-anti people IgM monoclonal antibodies and the recombinant C pn-Ag of preparation encapsulates detects with reference to article serum with enterprise again.Like table 2:
The different colloid gold label mouse-anti of table 2 people's IgM monoclonal antibody and recombinant C pn-Ag film pairing testing result
Interpretation of result: can know by table 2 result, the collaurum of No. 3 system gold bar spare systems, color is scarlet, limpid, no muddiness and floating thing, its specificity of the collaurum behind the mark, susceptibility are better.So select system gold bar spare to be: 1/0,000 gold chloride 100ml+1.8ml, 1% trisodium citrate
2. the optimization of colloid gold label mouse-anti people IgM monoclonal antibody condition:
2.1 principle:According to the colloidal gold-labeled method characteristic,, form red collaurum mouse-anti people IgM monoclonal antibody bond with the mouse-anti people IgM monoclonal antibody stable bond of gold grain trickle in the collaurum and purifying.
The selection of colloid gold label optimum pH:Adopt ocular estimate.Get several 1.5mL test tubes, add 1 mL collaurum respectively; Use 0.1M K
2CO
3Or 5M hydrochloric acid is adjusted to 3,4,5,6,7,8,9,10 respectively with pH; Get 96 hole ELISA Plates, respectively above-mentioned collaurum is got 100 respectively from low to high by pH
L adds in the hand-hole, triplicate; Every hole adds 3 respectively
L concentration is the mouse-anti people IgM monoclonal antibody of 1 mg/mL, mixes room temperature held 40 min; Every hole adds 20 respectively
L concentration is 10% NaCl solution, mixing, room temperature held 2 hours; Observing colloid gold change color, record keeps red minimum pH (X).Adjustment pH is X-1.0, X-0.5, X, X+0.5, X+1.0, repeats above step, keeps red minimum pH value to be the optimal pH of mark.Result such as table 3:
The selection experimental result of table 3 colloid gold label mouse-anti people IgM monoclonal antibody optimum PH
Conclusion: can know by table 3 result, when pH value is 8.5, promptly add 0.1MK
2CO
3During 20 μ l/ml, the effect of colloid gold label mouse-anti people IgM monoclonal antibody is better, good stability, and therefore selected pH value 8.5 (promptly adds 0.1MK
2CO
320 μ l/ml) be the optimum PH of colloid gold label mouse-anti people IgM monoclonal antibody.
The selection of mouse-anti people IgM monoclonal antibody optimum mark amount:
2.3.1 minimum mark amount: salt precipitation method and gradient method.At first adopt salt precipitation method to select the minimum mark amount of mouse-anti people IgM monoclonal antibody, select the optimum mark amount of mouse-anti people IgM monoclonal antibody then with the intersection matching method.Get 96 hole ELISA Plates; Add collaurum 100
L of best pH respectively, repeat 3 times; Each hole adds the albumen of different amounts, mixing, room temperature held 40min successively; Adding 20
L 10%NaCl, room temperature held 2h; Color still keeps the red minimum albumen consumption of mark that is.Result such as table 4:
The experimental result that table 4 mouse-anti people IgM monoclonal antibody minimum mark amount is selected
Can know by table 4 experimental result: the minimum mark amount of mouse-anti people IgM monoclonal antibody be 5
g/mL.
2.3.2 the optimum mark amount is selected: use 0.1M K
2C0
3Solution is transferred PH to 8.5, adds 5 respectively then
G-30
G/mL mark stirred 40 minutes with mouse-anti people IgM monoclonal antibody, and adding 10% bovine serum albumin(BSA) to final concentration again is 1%, stirs 15 minutes.Centrifugal 40 minutes of 4 ℃ of 12000rpm, careful abandoning supernatant adds collaurum bond dilution.The shop gold, after the drying, the Cpn-IgM reaction film pairing with encapsulating with the righttest condition of encapsulating detects its specificity and sensitivity.Result such as table 5:
The experimental result that table 5 mouse-anti people IgM monoclonal antibody minimum mark amount is selected
Can be known by table 5 result: under pH value 8.5 conditions, when mouse-anti people IgM monoclonal antibody label concentration was 10 μ g/mL, the specificity and the susceptibility of test strips were better, and sensitivity is higher.So the flag condition that we confirm is: every milliliter of collaurum adds 0.1M solution of potassium carbonate 20 μ L, and mouse-anti people IgM monoclonal antibody label concentration is that 15 μ g/mL are the final production process conditions.
Detection line encapsulates the selection of concentration and colloid gold label mouse-anti people IgM dilution ratio:
Carry out mark according to above-mentioned definite good flag condition; Its volume is concentrated into 1/10 of original volume (original volume is with the collaurum volume calculation); Again with different dilution ratio dilution concentrates; Soak gold mark pad with dilution, the 1.5ml/ bar, dry back with encapsulate concentration with different detection lines; 0.1 the Cpn-IgM reaction film that
package amount of l/mm encapsulates matches, and detects internal control serum.Experimental program such as table 6, experimental result such as table 7,8:
The design of table 6 experimental program
The positive enterprise of table 7 is with reference to article and minimum detectability testing result
The negative enterprise of table 8 is with reference to article check result
Can know that through table 7,8 experiments two groups of experimental results of A3B3 and A3B4 are more satisfactory.
The concentrate dilution ratio is 1:5, and it is 1.0mg/ml during to 2.0mg/ml that detection line encapsulates concentration, and Cpn-IgM reaction film nature controlling line and detection line color developing effect are better, and sensitivity, and specificity is better.Select the A3B2 combinations of pairs at this.That is:
Collaurum bond dilution is added to 1/2 of original volume (original volume is with the collaurum volume calculation), soaks gold mark pad 1.5mL/ bar, is golden bond pad preparation condition behind the colloid gold label.
It is 1.0mg/ml that detection line encapsulates concentration, 0.1
l/mm specking amount.
Nature controlling line encapsulates the selection of concentration:
Sheep anti-mouse igg is made into variable concentrations; With recombinant C pn-Ag 1.0mg/ml; With 0.1
l/mm specking amount; Be coated on respectively on the nature controlling line and detection line position of nitrocellulose filter, carry out drying after, with the golden bond pad combo for preparing; Detect result such as table 9 with reference to article serum with enterprise:
Table 9 nature controlling line testing result
Can know by table 9 result: when nature controlling line concentration is 1.0mg/ml, 1.5mg/ml, nature controlling line show line than its concentration a little less than, and when detecting the strong positive sample nature controlling line show line more a little less than, this competition mainly due to detection line and nature controlling line causes.When nature controlling line concentration is 3.0mg/ml when above, it is thicker and irregular that nature controlling line shows line.Comprehensive above experimental result, we choose the encapsulate concentration of 2.0mg/ml as nature controlling line.
For this reason reaction film finally the condition of encapsulating be:
The specking amount is 0.1 μ L/mm,
Nature controlling line encapsulates concentration: sheep anti-mouse igg antibody is 2.0mg/ml
Detection line encapsulates concentration: recombinant C pn-Ag is 1.0 mg/ml
The selection of 5 Cpn-IgM reaction films and golden bond pad drying condition:
By above-mentioned definite reaction film and golden bond pad working condition,, put into drying box with the reaction film alloy mark pad that encapsulates; After dry different time, take out golden bond pad and reaction film, be assembled into test strips; Detect with reference to article serum with enterprise, and carry out stability test.The result is as showing 10-14:
Table 10: dry back testing result
Table 11: testing result after dry 3 days
Table 12: testing result after dry 6 days
Table 13: testing result after dry 9 days
Table 14: testing result after dry 14 days
Suitable drying condition; Not only influencing the sensitivity of kit, and be related to the stability of kit. the result shows, is under 37 ℃ of conditions at dry environment; The test strips of preparation in dry 3 hours; Its susceptibility, stability are all better, so drying 3 hours was the drying condition of reaction film and the production of gold mark pad under 37 ℃ of conditions of selection.
Two, reaction system research
1. the selection of reaction time and application of sample amount:
According to above-mentioned definite condition, produce the Cpn-IgM test strips, its reaction time and application of sample amount are confirmed.Result such as table 15:
The experimental result that table 15 reaction time and application of sample amount are selected
Can know by table 15 experimental result: when the application of sample amount be 10
during L serum and 100
L sample dilution; The 15-20min sentence read result, it is better that the Cpn-IgM test strips detects effect.Therefore confirm the application of sample amount be 10
L serum and 100
L sample dilution, the interpretation time is 15-20min.
The test of the different anti-coagulants sample of serum and blood plasma contrast verification:
Gather blood with different anti-coagulants heparin, sodium citrate, EDTA processing blood sample and preparation serum, the test strips with above-mentioned definite working condition prepares, detect the sample after handling, testing result such as table 16:
The blood testing result that table 16 different disposal method is handled
Can be known by table 16 experimental result: the blood sample that anti-coagulants heparin, sodium citrate, EDTA handle does not have influence to testing result, detects consistent with serum.Therefore this kit can be used for the detection of serum and plasma sample.
Embodiment 1:
1. raw material sources
Each raw material of CPN IgM antibody colloidal gold method detection kit preparation and dilution source and consumption see the following form 17:
Table 17
2, ingredient requirement:
2.1 recombinant C pn antigen
(1) outward appearance: colourless transparent liquid;
(2) concentration and purity requirement: concentration is measured with SDS-PAGE greater than 2mg/ml, and applied sample amount is only deposited a band under 10 μ l conditions;
2.2 sheep anti-mouse igg antibody
(1) outward appearance: colourless transparent liquid;
(2) concentration requirement: concentration is greater than 4mg/ml;
2.3 mouse-anti people IgM monoclonal antibody
(1) outward appearance: colourless transparent liquid;
(2) concentration and purity requirement: concentration detects with the SDS-PAGE protein electrophoresis greater than 2mg/ml, and heavy chain/light chain is single district band;
3. liquid dosage: as shown in Figure 1, the concrete preparation flow of kit is following:
3.1 the preparation of 0.05M PBS damping fluid:
(1) standard recipe: according to 100mL amount meter.
(2) compound method:
Accurately take by weighing Na according to the standard recipe content
2HPO
412H
2O, NaH
2PO
42H
2O, NaCl add purified water 80.00mL, and after stirring made abundant dissolving, using pH meter to measure liquid pH value should be in 7.30～7.50 scope, and the conductivity of diluting solution that detection is joined with the purified water twice should be 9000
S/cm～13000
S/cm is settled to 100.00mL with purified water, mixes.2～8 ℃ of preservations are subsequent use, the term of validity 14 days.
The preparation of chlorauric acid solution:
(1) standard recipe: according to 100mL amount meter.
(2) compound method:
Get the gold chloride that a 1g/ props up, open bottle; Measure the 2ml purified water and pour in the container, get purified water and clean up the gold chloride dissolving and the reagent bottle that will hold gold chloride, cleaning fluid is poured in the container, add purified water again to 100ml; Cover tight bottle cap, fully rocked 15 minutes, mix, wrap with aluminium foil, 2～8 ℃ of preservations are subsequent use, the term of validity 12 months.
The solution of potassium carbonate preparation:
(1) standard recipe: according to 20mL amount meter.
(2) compound method:
Measure treat dosing volume 80% purified water in reagent bottle, accurately take by weighing K2CO3 according to the standard recipe content and add in the reagent bottle, add purified water to 20.0mL, make abundant dissolving.2～8 ℃ of preservations are subsequent use, the term of validity 14 days.
The preparation of bovine serum albumin solution:
(1) standard recipe: according to 10mL amount meter.
(2) compound method:
Measure treat dosing volume 80% purified water in reagent bottle, accurately take by weighing bovine serum albumin(BSA) according to the standard recipe content and add in the reagent bottle, treat to dissolve fully, add purified water to 10.00mL, make abundant dissolving, instant joining.
The preparation of collaurum bond dilution:
(1) standard recipe: according to 100mL amount meter.
(2) compound method:
Accurately take by weighing trishydroxymethylaminomethane in container according to the standard recipe content; Add purified water 80mL; After stirring makes abundant dissolving; Dripping hydrochloric acid makes its pH value be modulated to 9.0 then; Again load weighted other recipe ingredients are added in the above-mentioned solution successively; Fully after the stirring and dissolving; Measuring Tween-20 according to prescription with sample injector adds in the reagent bottle; Use purified water to be settled to 100.00mL, batch pH value of using pH meter to measure liquid is 8.60～8.80, the conductivity of solution that detection is joined should 2300
s/cm～3000
s/cm.2～8 ℃ of preservations are subsequent use, the term of validity 14 days.
The preparation of sample dilution:
(1) standard recipe: according to 100mL amount meter.
(2) preparation method: accurately take by weighing NaH according to the standard recipe content
2PO
42H
2O and Na
2HPO
412H
2O adds purified water 80.00mL, stirs to make abundant dissolving back add 0.85g sodium chloride, and fully mixing is settled to 100.00mL, and batch pH value of using pH meter to measure liquid is 7.30～7.50,4～30 ℃ of preservations, the term of validity 14 months.
The preparation of collaurum:
(1) standard recipe: according to 100mL amount meter.
(2) preparation method:
Accurately measure 1% chlorauric acid solution 1.0mL according to the standard recipe content, join in the 99mL purified water, heat while stirring to boiling; After 5 minutes, accurately measure 1% citric acid three sodium solution of 1.8ml, rapid, disposable joining in the container continued heated and boiled 5min.The adding purified water returns to original volume after being cooled to room temperature.2～8 ℃ of preservations are subsequent use, the term of validity 14 days.
The preparation of kit detection line coating buffer:
(1) standard recipe: according to 10mL amount meter.
(2) preparation method:
Accurately measure recombinant C pn-Ag 10mg according to the standard recipe content, join in the 0.05M PBS damping fluid of respective volume.Abundant mixing 15min.Instant joining.
The preparation of kit nature controlling line coating buffer:
(1) standard recipe: according to 10mL amount meter.
(2) preparation method:
Accurately measure sheep anti-mouse igg antibody 20mg according to the standard recipe content; Join in the 0.05M PBS damping fluid of respective volume, fully mixing 15min is settled to final volume 10.0mL with the 0.05PBS damping fluid; The final concentration that makes sheep anti-mouse igg antibody is 2.0mg/ml, instant joining.
The preparation of colloid gold label mouse-anti people IgM monoclonal antibody:
(1) related reagent standard consumption: according to 100mL amount meter.
(2) preparation method:
The collaurum of measuring the main formula ormal weight according to reagent standard consumption accurately adds the 0.1M solution of potassium carbonate of main formula ormal weight in triangular flask, mixing left standstill 10 minutes.Accurately measure the mouse-anti people IgM monoclonal antibody of main formula ormal weight, stir fast down, mouse-anti people IgM monoclonal antibody is dropwise joined in the triangular flask, room temperature left standstill 40 minutes.Accurately measure 10% bovine serum albumin solution of main formula ormal weight, dropwise join in the triangular flask under stirring fast, room temperature left standstill 15 minutes.12000rpm, 4 ℃ centrifugal 40 minutes, careful abandoning supernatant, it is subsequent use to 1/2,2～8 ℃ of preservations of original volume to add collaurum bond dilution, the term of validity 14 days.
Embodiment 2
The preparation one of kit
(1) preparation of reaction film: use phosphate buffer that recombinant C pn-Ag is diluted to and encapsulate concentration and be 1.0mg/ml; It is 2.0mg/ml that sheep anti-mouse igg antibody is diluted to concentration; Two kinds of coating buffers are drawn respectively on the nitrocellulose filter, with preserving after the nitrocellulose filter drying that has encapsulated;
(2) preparation of mouse-anti people IgM monoclonal antibody gold bond pad: with label concentration is that mouse-anti people IgM monoclonal antibody and the collaurum of 5 μ g/ml carries out coupling and make colloid gold label mouse-anti people IgM monoclonal antibody solution; With the rotating speed is the centrifugal 40min of 12000r/min; Abandon supernatant, add collaurum bond dilution, then with mixed liquid dipping gold mark pad to 1/2 of original volume; Be prepared into Cpn-IgM gold bond pad, with Cpn-IgM gold bond pad kept dry;
(3) cutting assembling: the wide self-adhesive paper of 1.5cm is thrown off on the top of offset plate at the reaction film place, and robust fibre filter paper in the stickup makes the bottom of paper push down reaction film; Throw off the self-adhesive paper of the 0.9cm of reaction film bottom, stick Cpn-IgM gold bond pad, and reaction film 1mm is pushed down on the top of golden bond pad; Throw off again golden bond pad bottom self-adhesive paper, stick sample pad, the bottom 1mm of golden bond pad is pushed down on the top of sample pad; Process reaction plate; Be cut into the 4mm test strips with cutting cutter again, after being installed, process product in the aluminium foil bag of packing into.
Embodiment 3
The preparation two of kit
(1) preparation of reaction film: use phosphate buffer that recombinant C pn-Ag is diluted to and encapsulate concentration and be 1.0mg/ml; It is 2.0mg/ml that sheep anti-mouse igg antibody is diluted to concentration; Two kinds of coating buffers are drawn respectively on the nitrocellulose filter, with preserving after the nitrocellulose filter drying that has encapsulated;
(2) preparation of mouse-anti people IgM monoclonal antibody gold bond pad: with label concentration is that mouse-anti people IgM monoclonal antibody and the collaurum of 30 μ g/ml carries out coupling and make colloid gold label mouse-anti people IgM monoclonal antibody solution; With the rotating speed is the centrifugal 40min of 12000r/min; Abandon supernatant, add collaurum bond dilution, then with mixed liquid dipping gold mark pad to 1/2 of original volume; Be prepared into Cpn-IgM gold bond pad, with Cpn-IgM gold bond pad kept dry;
(3) cutting assembling: the wide self-adhesive paper of 1.5cm is thrown off on the top of offset plate at the reaction film place, and robust fibre filter paper in the stickup makes the bottom of paper push down reaction film; Throw off the self-adhesive paper of the 0.9cm of reaction film bottom, stick Cpn-IgM gold bond pad, and reaction film 1mm is pushed down on the top of golden bond pad; Throw off again golden bond pad bottom self-adhesive paper, stick sample pad, the bottom 1mm of golden bond pad is pushed down on the top of sample pad; Process reaction plate; Be cut into the 4mm test strips with cutting cutter again, after being installed, process product in the aluminium foil bag of packing into.
Embodiment 4
The preparation three of kit
(1) preparation of reaction film: use phosphate buffer that recombinant C pn-Ag is diluted to and encapsulate concentration and be 1.0mg/ml; It is 2.0mg/ml that sheep anti-mouse igg antibody is diluted to concentration; Two kinds of coating buffers are drawn respectively on the nitrocellulose filter, with preserving after the nitrocellulose filter drying that has encapsulated;
(2) preparation of mouse-anti people IgM monoclonal antibody gold bond pad: with label concentration is that mouse-anti people IgM monoclonal antibody and the collaurum of 15 μ g/ml carries out coupling and make colloid gold label mouse-anti people IgM monoclonal antibody solution; With the rotating speed is the centrifugal 40min of 12000r/min; Abandon supernatant, add collaurum bond dilution, then with mixed liquid dipping gold mark pad to 1/2 of original volume; Be prepared into Cpn-IgM gold bond pad, with Cpn-IgM gold bond pad kept dry;
(3) cutting assembling: the wide self-adhesive paper of 1.5cm is thrown off on the top of offset plate at the reaction film place, and robust fibre filter paper in the stickup makes the bottom of paper push down reaction film; Throw off the self-adhesive paper of the 0.9cm of reaction film bottom, stick Cpn-IgM gold bond pad, and reaction film 1mm is pushed down on the top of golden bond pad; Throw off again golden bond pad bottom self-adhesive paper, stick sample pad, the bottom 1mm of golden bond pad is pushed down on the top of sample pad; Process reaction plate; Be cut into the 4mm test strips with cutting cutter again, after being installed, process product in the aluminium foil bag of packing into.
Embodiment 5
Sample detection:
1, detects the requirement of sample
Serum sample is pressed conventional method by the vein collection.Plasma sample can adopt heparin, sodium citrate, EDTA to handle.The sample of measuring in 5 days can be placed 4 ℃ of preservations.Sample is placed on-20 ℃ and can preserves 3 months at least.Sample is avoided haemolysis or multigelation.Muddy or have the sample of deposition should be centrifugal or filter clarification after detect again.
, the method for inspection:
1) from aluminium foil bag, takes out test card;
2) be placed on the horizontal operation table top horizontal and carry out sample labeling;
3) with micropipettor get 10
L serum or plasma sample; Directly join in the well; Drip sample dilution 100
L (about 2-3 drips) again;
4) sentence read result in 15-20 minute, assay is invalid after 20 minutes.
The detector bar detection method:
1) from the original packing aluminium foil bag, takes out detector bar, be flat on the table top;
2) with micropipettor get 10
L serum or plasma sample;
3) be added to the application of sample place of detector bar arrow lower end; Drip the sample dilution again, 100
L (about 2-3 drips);
4) sentence read result in 15-20 minute, assay is invalid after 20 minutes.
, assay
1) feminine gender a: red stripes only occurs in nature controlling line (C) position.
2) positive: two red stripes appear in nature controlling line (C) and detection line (T) position.
3) invalid: red stripes does not appear in nature controlling line (C) position.
This product yin and yang attribute coincidence rate, accuracy, sensitivity etc. all meet the quality standard requirement, constant product quality in the effect phase.Rheumatoid factor, hepatitis B, syphilis can not cause interference to these article.
Embodiment 6
The selection of reaction time and application of sample amount
Produce the Cpn-IgM kit, its reaction time and application of sample amount are confirmed.The result sees table 18:
The selection experimental result of table 18 application of sample amount and interpretation time
Can be known by table 18 experimental result: what of application of sample amount can influence test strips and produce correct reaction result, and the application of sample amount is low omission or sample can be occurred and launch not enoughly, and the application of sample amount is easy to generate false positive when high.Through above experiment, consider error and the convenience of use of different users on using, selecting the application of sample amount is that 10 μ L serum add 100 μ L sample dilutions again, the 15-20min interpretation is an application of sample interpretation condition.
Embodiment 7
The test of the different anti-coagulants sample of serum and blood plasma contrast verification
Gather blood with different anti-coagulants heparin, sodium citrate, EDTA processing blood sample and preparation serum; Test strips with above-mentioned definite working condition preparation; Detect the sample after handling; The result shows: the blood sample that anti-coagulants heparin, sodium citrate, EDTA handle does not have influence to testing result, detects consistent with serum.Therefore this kit can be used for the detection of serum and plasma sample.
What should explain at last is: the above is merely the preferred embodiments of the present invention; Be not limited to the present invention; Although the present invention has been carried out detailed explanation with reference to previous embodiment; For a person skilled in the art, it still can be made amendment to the technical scheme that aforementioned each embodiment put down in writing, and perhaps part technical characterictic wherein is equal to replacement.All within spirit of the present invention and principle, any modification of being done, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (4)
1. CPN IgM antibody colloidal gold method detection kit; Comprise the mouse-anti people IgM monoclonal antibody of the colloid gold label that encapsulates on the sheep anti-mouse igg antibody that encapsulates on the reorganization CPN antigen that encapsulates on the nitrocellulose filter detection line, the nature controlling line and the gold mark pad, it is characterized in that:
Said CPN antigen concentration is 1-2mg/ml, measures with SDS-PAGE;
Said sheep anti-mouse igg antibody concentration is 1-3mg/ml;
Said mouse-anti people IgM monoclonal antibody concentration is 5-30
/mL, measures with SDS-PAGE.
2. the preparation method of CPN IgM antibody colloidal gold method detection kit according to claim 1 is characterized in that: may further comprise the steps:
(1) preparation of reaction film: use phosphate buffer that recombinant C pn-Ag is diluted to and encapsulate concentration and be 1.0mg/ml; It is 2.0mg/ml that sheep anti-mouse igg antibody is diluted to concentration; Two kinds of coating buffers are drawn respectively on the nitrocellulose filter, with preserving after the nitrocellulose filter drying that has encapsulated;
(2) preparation of mouse-anti people IgM monoclonal antibody gold bond pad: with label concentration is that mouse-anti people IgM monoclonal antibody and the collaurum of 5-30 μ g/ml carries out coupling and make colloid gold label mouse-anti people IgM monoclonal antibody solution; With the rotating speed is the centrifugal 40min of 12000r/min; Abandon supernatant, add collaurum bond dilution, then with mixed liquid dipping gold mark pad to 1/2 of original volume; Be prepared into Cpn-IgM gold bond pad, with Cpn-IgM gold bond pad kept dry;
(3) cutting assembling: the wide self-adhesive paper of 1.5cm is thrown off on the top of offset plate at the reaction film place, and robust fibre filter paper in the stickup makes the bottom of paper push down reaction film; Throw off the self-adhesive paper of the 0.9cm of reaction film bottom, stick Cpn-IgM gold bond pad, and reaction film 1mm is pushed down on the top of golden bond pad; Throw off again golden bond pad bottom self-adhesive paper, stick sample pad, the bottom 1mm of golden bond pad is pushed down on the top of sample pad; Process reaction plate; Be cut into the 4mm test strips with cutting cutter again, after being installed, process product in the aluminium foil bag of packing into.
3. the preparation method of CPN IgM antibody colloidal gold method detection kit according to claim 2 is characterized in that: the drying condition of said step (1) and (2) is 37 ℃ of dryings 3 hours.
4. the preparation method of CPN IgM antibody colloidal gold method detection kit according to claim 2 is characterized in that: the label concentration of the mouse-anti people IgM monoclonal antibody of said step (2) is 15 μ g/ml.
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| CN104280543A (en) * | 2013-07-11 | 2015-01-14 | 国家纳米科学中心 | Test paper and method for detecting IgG (immunoglobulin G) and IgM (immunoglobulin m) in liquid sample simultaneously |
| CN105974111A (en) * | 2016-07-20 | 2016-09-28 | 国家纳米科学中心 | Joint detection method for rheumatoid factors (IgM type) and other antigen-specific IgM antibodies |
| CN106404754A (en) * | 2016-06-30 | 2017-02-15 | 深圳市亚辉龙生物科技股份有限公司 | A chlamydiae pneumoniae IgM chemiluminescent immunoassay kit and a preparing method thereof |
| CN111521786A (en) * | 2020-04-28 | 2020-08-11 | 天津健博生物科技有限公司 | Respiratory tract pathogen IgM antibody joint detection kit and preparation method thereof |
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| CN106404754A (en) * | 2016-06-30 | 2017-02-15 | 深圳市亚辉龙生物科技股份有限公司 | A chlamydiae pneumoniae IgM chemiluminescent immunoassay kit and a preparing method thereof |
| CN105974111A (en) * | 2016-07-20 | 2016-09-28 | 国家纳米科学中心 | Joint detection method for rheumatoid factors (IgM type) and other antigen-specific IgM antibodies |
| CN111521786A (en) * | 2020-04-28 | 2020-08-11 | 天津健博生物科技有限公司 | Respiratory tract pathogen IgM antibody joint detection kit and preparation method thereof |
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Application publication date: 20121024 |