CN101363860A - Syphilis helicoid antibody chemiluminescence immune assay determination kit and method for preparing same - Google Patents
Syphilis helicoid antibody chemiluminescence immune assay determination kit and method for preparing same Download PDFInfo
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Abstract
The invention discloses a kit of chemiluminescent immunological analysis measurement of a Treponema pallidum antibody, and preparation method thereof, which belongs to the technical field of immunological analysis medical diagnosis. The kit comprises (1) a carrier coated with a treponema pallidum specific recombination protein antigen; (2) treponema pallidum antibody negative and positive reference substances; (3) an enzyme labeled treponema pallidum specific recombination protein antigen; and (4) an enzyme acted chemiluminescent primer. Furthermore, the preparation method of the kit based on the invention comprises the following steps of (1) preparing TP antibody negative and positive reference substances; (2) labeling the TP specific recombination protein antigen with an enzyme; (3) coating with the carrier; (4) sub-packaging; and (5) assembling. The kit of the detection of the Treponema pallidum antibody has the advantages of easy and simple operation, easy popularization, high sensitivity, strong specificity, good repeatability, safety, non toxicity, and no pollution.
Description
Technical field
The invention belongs to immunoassay medical diagnostic techniqu field, particularly, the invention provides a kind of syphilis helicoid antibody chemiluminescence immune assay determination kit and preparation method thereof.
Background technology
Syphilis (syphilis) is the common sexually transmitted disease that is caused by microspironema pallidum (claiming spirochaeta pallida again), clinical manifestation is very complicated, almost can invade the tissue and the organ of whole body, and produce the clinical symptoms and the Signs of varied complexity, early stage main invade mucocutaneously, invade internal organs late period, severe patient can cause the serious cardiovascular system nervous system damage of unifying, cause maimed person even threat to life, very harmful.The main trafficability characteristic behavior of syphilis infects to other people, also can pass to fetus by placenta, and congenital syphilis takes place; Only a few patient can by kiss, lactation, blood transfusion, contact be infectious the infringement patient articles for daily use infect.Early syphilis is infectious, and does not then have infectiousness late period.The early syphilis result of treatment is good, and late period, then result of treatment was poor.The global in recent years syphilis incidence of disease is ascendant trend year by year, it is reported that the whole world has the new cases of infection of 12,000,000 examples every year approximately.Therefore, WHO has classified syphilis as the global public health problem of serious harm human health.In China, the syphilis report of infectious disease presented year after year quick rising tendency-2006 year national syphilis number of reports first above gonorrhoea in nearest 3 years after continuous 6 years of the nineties in 20th century continuing to rise, and the general report number reaches 174506 examples.In order effectively to contain the popular of syphilis, the hygiene department of China has assigned indication, each medical institutions must to marry, donate blood, blood transfusion or preoperative personnel carry out the examination of syphilis serology, join the army, participate in the personnel of recruitment for those, and be engaged in the personnel of tourism industry, food, beverage trade and gutter man etc. and also must carry out the examination of syphilis serology.
At present, common syphilis serology examination comprises that rapid plasma reagin ring-type card test (RPR), toluidine red do not heat serum test (TRUST), the test of microspironema pallidum hemagglutination (TPHA), microspironema pallidum GAT (TPPA), enzyme linked immunosorbent assay (ELISA) etc.Wherein, RPR and TRUST method are used to detect the non-specific antibody of microspironema pallidum, its sensitivity and specificity relatively poor (having false positive and false negative rate more than 30%).The specificity of TPHA is higher, but sensitivity is not so good as the ELISA method, and the reagent cost height, needs manual operation, can not realize robotization.The TPPA method is the upgrading products of TPHA, though its specificity and sensitivity are all higher, employed is natural leptospira antigen, costs an arm and a leg, and the reaction time is long, and test findings is subjected to interference from human factor bigger, is unsuitable for large batch of detection.Immunoassay is used in the detection of microspironema pallidum now mostly, and along with going deep into to understanding (antibody specificity and the Changing Pattern) of microspironema pallidum specific antibody, new detection target spot also can occur, people are making great efforts to study the antigenic specificity of microspironema pallidum specific proteins fragment, and the antibody that causes makes progress the characteristics in each period in syphilis.According to present research, the specific antigen fragment that can contain all kinds and disease progression each period in the human body is (distinguishing according to molecular weight): 15 (TpN15), 17 (TpN17), 33,37 (TpN37), 39,43,45 (TmpA), 47 (TpN47) and 97Kda.And in the syphilis serology detects most worthy be: 15 (TpN15), 17 (TpN17), 33,37 (TpN37), 39,43,45 (TmpA) and 47 (TpN47), they can be with different applied in any combination in the EIA enzyme immunoassay detection.The antigen that facts have proved these reorganization has fabulous sensitivity and specificity.In recent years, ELISA is acknowledged as the common method that detects syphilis helicoid antibody.
(Chemiluminescence Immunoassay's chemiluminescence immune assay CLIA) came out in 1977, and first generation chemical luminescence immune analysis reagent box was succeeded in developing and put on market in 1985.Enter the nineties, the production of the development of chemical luminescence immune analysis reagent box and automatic measurement instrument has obtained breakthrough, thereby enters the high speed development stage.Chemiluminescence immune assay is a new immuno analytical method that grows up after fluorescence, radioactive isotope and EIA enzyme immunoassay, according to lot of experiment results and clinical practice data, from practicality, stability, accuracy and development prospect, chemiluminescence immune assay maintains the leading position in nonradioactive labeling's analytical technology, the direction and the trend of world today's development have been represented, it not only has immunoreactive specificity, and (detection limit can reach 10 to have the high sensitivity of chemiluminescence reaction
-15～10
-18Mol/L).Advantages such as that chemiluminescence immunoassay technology has is highly sensitive, quick, accurate, good reproducibility, the effect phase is long and safety non-toxic is pollution-free become the first-selection that replaces radiommunoassay and EIA enzyme immunoassay technology.
Chemical luminescence immune analysis reagent box of the prior art is the luminous measuring system of closed full-automatic chemical, needs the expensive luminous measuring instrument of full-automatic chemical, promotes the use of thereby limited, and can't be widely used in clinical diagnosis and research work.Kit of the present invention adopts the microwell plate chemiluminescence immunoassay technology, both can be applicable to the open luminous measuring instrument of semi-automatic chemistry, also can be used for full automatic measuring system, can realize fast detection the in enormous quantities, and use cost is low, easier applying.
Summary of the invention
The present invention has solved the problems referred to above simultaneously, soon chemiluminescence is learned effectively with the immunoassay of syphilis and is combined, used simultaneously that luminous signal is strong, longer duration, the self-control chemical luminous substrate liquid of high s/n ratio more, a kind of kit that can easy, quick, sensitive, stably detect syphilis helicoid antibody is provided.The method that kit of the present invention adopts is more highly sensitive than enzyme immunoassay (EIA), can shorten the detection window phase, makes this kit be suitable for applying effectively on industry.
The purpose of this invention is to provide a kind of syphilis helicoid antibody chemiluminescence immune assay determination kit.
A further object of the present invention provides a kind of method for preparing the mentioned reagent box.
What syphilis helicoid antibody (Anti-TP) diagnostic kit (chemoluminescence method) of the present invention's development adopted is double antigens sandwich single stage method reaction pattern, adopt the microspironema pallidum specificity recombinant protein antigen of multiple combination to prepare solid phase, specific recombinant protein antigen purity height, no infectiousness, non-specific binding reaction is little, does not need special experimental laboratory just can produce in enormous quantities.
The reaction principle of kit is the recombinant syphilis spirochete specific antigen that adds enzyme labeling in wrapping by micropore, add positive and negative contrast or test serum then, form the compound of solid phase antigen-antibody-enzyme-labelled antigen by the short time incubation, after washing irrelevant antibody and other compositions behind the incubation off, add chemical luminous substrate liquid, in 5～30 minutes, measure its luminous intensity (RLU), according to whether containing the microspironema pallidum specific antibody in the critical value judgement sample.
Syphilis helicoid antibody chemiluminescence immune assay determination kit according to the present invention comprises: the 1) solid phase carrier of microspironema pallidum specificity recombinant protein antigen bag quilt; 2) syphilis helicoid antibody feminine gender and positive reference substance; 3) the microspironema pallidum specificity recombinant protein antigen of enzyme labeling; And 4) chemical luminous substrate that above-mentioned enzyme acted on.
Preferably, described microspironema pallidum specificity recombinant protein antigen is selected from and comprises in TpN15, TpN17, TpN47, TpN37 and the TmpA group one or more, more preferably one or more among TpN15, TpN17 and the TpN47.
According to kit of the present invention, wherein, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle; Described enzyme is alkaline phosphatase or horseradish peroxidase; Described chemical luminous substrate is 1,2-two oxidative ethane analog derivatives, luminol or different luminol, preferably, described 1,2-two oxidative ethane analog derivatives are (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 "-the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star.
The present invention also provides a kind of method for preparing the mentioned reagent box, may further comprise the steps:
1) with the human serum preparation syphilis helicoid antibody positive reference substance of the syphilis helicoid antibody test positive of high titre; Detect negative human serum preparation syphilis helicoid antibody negative control product with syphilis helicoid antibody;
2) with enzyme labeling microspironema pallidum specificity recombinant protein antigen;
3) use microspironema pallidum specificity recombinant protein antigen bag by solid phase carrier;
4) chemical luminous substrate that acted on of microspironema pallidum specificity recombinant protein antigen and this enzyme of the negative and positive control of the above-mentioned microspironema pallidum of packing, enzyme labeling; And
5) be assembled into finished product.
Preferably, the bag described in the said method is adopted following method by the step 3) of solid phase carrier:
1) bag quilt
With 0.050M pH value is the Avidin coating buffer that 9.6 carbonate buffer solution is mixed with desired concn, and coating buffer is carried on the solid phase carrier;
2) with physiological saline washing above-mentioned solid phase carrier; And
3) sealing
With containing 1%BSA, the pH value is 7.0～7.5, and concentration is that the phosphate buffer of 0.010M seals solid phase carrier after the above-mentioned washing as confining liquid.
Particularly, described method for coating comprises and can comprise
1) bag quilt
The preparation coating buffer, the sodium carbonate and the 2.94g sodium bicarbonate that comprise 1.59g based on the described coating buffer of 1000mL, the pH value 9.6 of described coating buffer, the coating buffer with preparation mixes with microspironema pallidum specificity recombinant protein antigen then, and the gained mixed liquor is carried on the solid phase carrier;
2) with physiological saline washing above-mentioned solid phase carrier; And
3) sealing
The preparation confining liquid comprises 0.2g NaH based on the described confining liquid of 1000mL
2PO
42H
2O, 2.9gNaH
2PO
412H
2O, 10g BSA and 1mL biological preservative, the pH value of described confining liquid is 7.0～7.5, then the gained confining liquid is carried on the solid phase carrier after the above-mentioned washing.
Preferably, described microspironema pallidum specificity recombinant protein antigen is selected from and comprises in TpN15, TpN17, TpN47, TpN37 and the TmpA group one or more; More preferably, described microspironema pallidum specificity recombinant protein antigen is one or more among TpN15, TpN17 and the TpN47.
Preferably, in said method, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle; Described enzyme is alkaline phosphatase or horseradish peroxidase.Described chemical luminous substrate is 1,2-two oxidative ethane analog derivatives, luminol or different luminol, preferably described 1,2-two oxidative ethane analog derivatives are (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star.
Concrete mentioned reagent box can comprise syphilis helicoid antibody negative and positive reference substance, antigen coated microplate, enzyme labeling thing and chemical luminous substrate liquid, 20 times of concentrated cleaning solutions etc.Wherein, described antigen coated microplate is that micropore lath, the enzyme labeling microspironema pallidum specificity recombinant protein antigen in 48 or 96 holes are that coupling horseradish peroxidase, chemical luminous substrate liquid are that luminol, concentrated cleaning solution are Tris-HCl or PBST.
What the present invention's " syphilis helicoid antibody (Anti-TP) chemical luminescence immune assay determination reagent kit " adopted is double antigens sandwich single stage method reaction pattern, adopt the microspironema pallidum specificity recombinant protein antigen of multiple combination to prepare solid phase and enzyme labeling thing, both effectively utilize the chemiluminescence principle, guaranteed the sensitivity that detects again.It has advantages such as easy, quick, sensitive, stable.Every index that this syphilis helicoid antibody is measured kit (chemoluminescence method) all reaches or is better than enzyme immunoassay (EIA).And detection system of the present invention is an open-sky technique, does not easyly fast need the expensive luminous measuring instrument of full-automatic chemical, is particularly suitable for vast middle and small hospital and promotes the use of, for clinical diagnosis and research work provide a kind of very valuable detection means.
What kit of the present invention was used is the enzymatic luminous substrate, by the chromogenic substrate in the light signal replacement EIA enzyme immunoassay that detects the luminous substrate generation, thereby have a specificity equal with EIA enzyme immunoassay, and sensitivity improves greatly, the sensitivity of ratio EIA enzyme immunoassay now improves about 10 times, and the diagnosis that can be syphilis provides more special, quick, reliable foundation.
Description of drawings
Fig. 1 has illustrated determining of syphilis helicoid antibody chemiluminescence immune assay determination kit critical value of the present invention.
Embodiment
Embodiment 1 preparation syphilis helicoid antibody chemiluminescence immune assay determination kit of the present invention
One, enzyme-labelled antigen preparation
Adopt sodium periodate oxidation labelling method three kinds of microspironema pallidum differential proteins 15 of mark (TpN15), 17 (TpN17), 47 (TpN47) and horseradish peroxidase.Add packing behind 50% glycerine ,-20 ℃ of preservations.
Two, the preparation of TP feminine gender and positive reference substance
Positive control serum: the high titre positive human serum of determining through definite method is mixed into positive blood plasma (PositivePool, the blood plasma umber is greater than 5), after 56 ℃ of deactivations of heating in 1 hour, use NBCS suitably to dilute, the Food Red that adds biological preservative and ten thousand/(W/V) makes it to become redness, aseptic filtration, 2～8 ℃ of preservations.
Negative control sera: after selecting for use syphilis antibody to detect negative human serum mixing (the blood plasma umber is greater than 10) 56 ℃ of deactivations of heating in 1 hour, aseptic filtration, 2～8 ℃ of preservations.
Three, enzyme-labelled antigen concentration is selected
Adopting the square formation method to select the dilutability of three kinds of enzyme labeling thing the bests all is 1:6000
Four, the preparation of solid-phase coating plate
(1) bag quilt
Sodium carbonate 1.59g
Sodium bicarbonate 2.94g
Distilled water 1000mL
Behind the dissolving mixing, adjust PH to 9.6, add an amount of microspironema pallidum specificity recombinant protein antigen 15 (TpN15), 17 (TpN17), 47 (TpN47) mixing, add in each hole of microwell plate every hole 100 μ L, 4 ℃ of 24h then.
(2) washing: it is inferior to give a baby a bath on the third day after its birth with physiological saline.
(3) sealing
NaH
2PO
4·2H
2O 0.2g
Na
2HPO
4·12H
2O 2.9g
BSA 10g
Proclin300 1mL
Distilled water is settled to 1000mL
The mentioned reagent weighing is put into clean container well, add the distilled water constant volume, the dissolving mixing, measuring the pH value is 7.0.
Every hole adds confining liquid 180 μ L respectively, and room temperature was placed 2 hours.Get rid of confining liquid, on thieving paper, pat dry.Room temperature removal moisture drying 24 hours.Carry out vacuum sealing bag immediately.Place behind the envelope and checked not have gas leakage in 15 minutes, if there is gas leakage to need envelope again.2～8 ℃ of preservations of labeling postposition.
Five, enzyme-labelled antigen dilution
Tris 12.120g
BSA 5g
Proclin 1mL
Distilled water 1000mL
Six, chemical luminous substrate liquid
A liquid: add 3.29gTris, 3.26g borax, 1.0g BSA, 1.772g luminol, 0.5g benzofluoranthrene, 1mL Proclin 300 in distilled water, be settled to 1000mL with distilled water, mix, its pH value is 8.2～8.8;
B liquid: in distilled water, add 36.72g Na
2HPO
412H
2The H of O, 10.21g citric acid, 1.0mL30%
2O
2, 1mL Proclin 300, water is settled to 100mL, its pH value is 5.0～5.8.
Using method: before using A liquid is mixed the back with B liquid in the 1:1 ratio and use.
Seven, 20 times of cleansing solutions
Tris 24g
NaCl 160g
KCl 4g
HCl 15mL
Deionized water 1000mL
Adjust pH to 7.4
Or
Na
2HPO
4·12H
2O 58g
NaH
2PO
4·2H
2O 2g
NaCl 160g
Tween-20 1mlL
Proclin 30 1mlL
Deionized water 1000mlL
Adjust pH to 7.2～7.4
Eight, semi-manufacture and finished product are formed
The packing of above-mentioned steps products obtained therefrom is semi-manufacture.Extract three parts of process specificitys, accuracy, sensitivity and stable assay approvals out and just can be assembled into syphilis helicoid antibody chemiluminescence immune assay determination kit.Be assembled into also need inspect by random samples behind the kit and just can dispatch from the factory after qualified.
To sum up, in research process of the present invention, the present inventor has at first carried out shaker test and Quality Identification to used starting material, comprises the luminous intensity of activity, chemical luminous substrate of the absorption property of activity, solid phase carrier (as lighttight white microwell plate) of labelled antigen and envelope antigen and variation size, HRP and luminous duration etc.Then method for coating is studied, be cushioned liquid and protect liquid to test, select optimal bag and be cushioned liquid and protection liquid, tested by concentration by the different bags of antibody and find best concentration conditions with different bags.Mark for HRP can have diverse ways, by explore repeatedly and contrast test finally found easy, productive rate is high, cost is low, the reliable quality labeling method, and different enzyme dilutions has been carried out long-term investigation tested, made and can make the activity stabilized dilution of the long-term maintenance of enzyme labeling thing.
The present inventor has also carried out experimental study to the reaction pattern and the reaction conditions of kit, finally determined double antigens sandwich single stage method reaction pattern, and the influence of test findings is tested with regard to the different reaction time, determine the optimal reaction time.By the influence test to measured value shows to the mensuration of luminous duration of chemical luminous substrate liquid and different fluorescent lifetime: be measured as the best between 5～30 minutes after adding chemical luminous substrate liquid, its result is also comparatively accurate.
Embodiment 2～3 preparations TP of the present invention measures kit
Divided by alkali phosphatase enzyme mark TP specificity recombinant antigen, with AMPPD as chemical luminous substrate, with plastic bead as the pH value of carrier, coating buffer be 8.4, the pH value of confining liquid is outside 7.5, all the other all prepare TP with the method identical with embodiment 1 and measure kit.
Embodiment 4 preparations TP of the present invention measures kit
As outside the carrier, all the other all prepare TP with the method identical with embodiment 1 and measure kit divided by magnetic-particle.
The using method of embodiment 5 kits of the present invention
The concrete operations of the TP mensuration kit of above embodiment 1 preparation are as follows:
1. in 4 ℃ of refrigerators, take out kit, equilibrium at room temperature 15 minutes.
2. every hole adds sample 50 μ L to be measured, establishes positive and negative and contrasts each two hole, and every hole adds each 50 μ L of reference substance, establishes blank one hole.
3. every hole enzyme-added mark antigen bond 50 μ L (except the blank hole), abundant mixing, shrouding was put 37 ℃ of incubations 60 minutes.
4. abandon liquid in the hole, wash plate 5 times, on clean filter paper, buckle at last and do with the cleansing solution after the dilution.
5. every hole adds chemical luminous substrate liquid 50 μ L, measures the luminous intensity (RLU) in each hole in must the 5th～30 minute after adding chemical luminous substrate liquid, 1 second/hole of Measuring Time.
6. the RLU value in each hole of average and CUT OFF compare, and sample determination value 〉=2.1 * negative control then is judged as the positive, otherwise negative.
The methodology of embodiment 6 kits of the present invention is identified
According to manufacturing conventional in this area and vertification regulation the kit of preparation among the embodiment 1 is identified, is seen the following form 1:
Table 1
| Interventions Requested | Test stone | Assay |
| Accuracy | The feminine gender and the positive that detect in the syphilis helicoid antibody country reference material all detect | Conformance with standard |
| Accuracy CV (%) | ≤15％(n＝10) | Conformance with standard |
| Stability | Each reagent set split 37 ℃ at least 10 days | Conformance with standard |
Accuracy, accuracy, the stability that " syphilis helicoid antibody chemiluminescence immune assay determination kit " is described is fully qualified.
Determining of embodiment 7 normal persons' syphilis helicoid antibody content and critical value
Use " syphilis helicoid antibody chemiluminescence immune assay determination kit " of preparation among the embodiment 1～4 to detect whole negative sample and 400 routine healthy human serums in the syphilis helicoid antibody country reference material, try to achieve out the mean value and the standard deviation of healthy people's measured value, the mean value that calculates above-mentioned sample luminous value adds that 6 times standard deviation is as critical luminous counting, consider the change of each test, we advise adopting according to aforementioned definite critical luminous counting divided by the ratio of negative control average luminescence value as multiple, critical value is decided to be the mean value that this multiple multiply by the negative control luminous value, according to large sample test we determine that this radix is 2.1 as a result, i.e. the mean value of CUT OFF=2.1 * negative control luminous value.
Clinical practice and TPHA kit that embodiment 8 TP of the present invention measure kit compare
The microspironema pallidum serum hemagglutination test TPHA reagent that Chinese People's Liberation Army General Hospital Institute of Basic Medical Sciences (301 Hospital) uses the TP of embodiment 1 to measure kit and Britain OMEGA diagnostic companies detects 477 parts of clinical serum samples and compares, and examines the sensitivity and the specificity of " syphilis helicoid antibody (Anti-TP) diagnostic kit (chemoluminescence method) " of the present invention.
One, the source of clinical serum specimen
Through the patients with syphilis serum of clinical definite totally 139 examples (wherein primary syphilis patients serum 43 examples, mesosyphilis patients serum 96 examples); Antinuclear antibodies titre through making a definite diagnosis is greater than lupus erythematosus patients serum's 84 examples of 1:160, chronic persistant hepatitis patients serum 23 examples, rheumatoid arthritis patients serum 31 examples.Normal human serum 200 examples.
Annotate: 43 routine primary syphilis patients all had unclean contact history before 3～4 weeks, clinical all have typical chancre skin to decrease, wherein 35 examples are all positive through TPHA and chemiluminescence detection of the present invention, and 8 examples all show negative in addition, and serum T PHA occurs positive in the process but follow up a case by regular visits to after treatment.
Two, test findings
It is more as shown in table 2 that kit of the present invention and TPHA detect syphilis and other diseases and normal population serum result:
Table 2
Test findings shows: " syphilis helicoid antibody chemiluminescence immune assay determination kit " of the present invention is 90.65% to detecting the total sensitivity of patients with syphilis serum antibody, the overall detection sensitivity that is higher than TPHA 88.49%, this mainly is because " syphilis helicoid antibody chemiluminescence immune assay determination kit " is higher than TPHA reagent (62.79%) for primary syphilis positive rate 69.77%, that is: positive rate is better than the TPHA detection that patients with syphilis serum detects, and can be used for the Serological testing of clinical patients with syphilis.
Clinical practice and ELISA kit that embodiment 9 TP of the present invention measure kit compare
The syphilis spirochete antibody ELISA diagnostic kit that Beijing Friendship Hospital Attached to Capital Medical Univ. produces with mensuration kit of the present invention and Beijing Tso Biological Pharmaceutical Co detects 335 parts of clinical serum samples and compares, and kit of the present invention is carried out the clinical detection assessment.
One, the source of clinical serum specimen
155 parts of positive sample have been made a definite diagnosis in Beijing Friendship Hospital's venereal disease outpatient service, and wherein early latent syphilis serum is 10 parts, 42 parts of primary syphilis serum, 103 parts of mesosyphilis serum.180 parts of health check-up normal human serums.
Two, test findings
It is more as shown in table 3 that kit of the present invention and ELISA detect syphilis and other diseases and normal population serum result:
Table 3
| Clinical diagnosis | n | Anti-T.P.-ELISA (positive) | Anti-T.P.-CLIA (positive) |
| Primary syphilis mesosyphilis early latent syphilis adds up to | 42 103 10 155 | 42 103 8 153 | 42 103 9 154 |
| The normal person | 180 | 0 | 0 |
155 parts of syphilitic's serum after testing, it is good slightly that the sensitivity of syphilis helicoid antibody chemiluminescence immune assay determination kit of the present invention (99.35%) is exempted from (Anti-TP-ELISA) detection kit (98.71%) than syphilis spirochete antibody.With two kinds of detection systems 180 portions of normal human serums are detected none routine positive findings, specificity reaches 100%; This shows that syphilis helicoid antibody chemiluminescence immune assay determination kit can be applied to clinical.
Claims (16)
1. a syphilis helicoid antibody chemiluminescence immune assay determination kit is characterized in that, described kit comprises 1) solid phase carrier of microspironema pallidum specificity recombinant protein antigen bag quilt; 2) syphilis helicoid antibody feminine gender and positive reference substance; 3) the microspironema pallidum specificity recombinant protein antigen of enzyme labeling; And 4) chemical luminous substrate that above-mentioned enzyme acted on.
2. kit as claimed in claim 1 is characterized in that, described microspironema pallidum specificity recombinant protein antigen is selected from and comprises in TpN15, TpN17, TpN47, TpN37 and the TmpA group one or more.
3. kit as claimed in claim 2 is characterized in that, described microspironema pallidum specificity recombinant protein antigen is one or more among TpN15, TpN17 and the TpN47.
4. kit as claimed in claim 1 is characterized in that, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.
5. kit as claimed in claim 1 is characterized in that, described enzyme is alkaline phosphatase or horseradish peroxidase.
6. kit as claimed in claim 1 is characterized in that, described chemical luminous substrate is 1,2-two oxidative ethane analog derivatives, luminol or different luminol.
7. kit as claimed in claim 6, it is characterized in that described 1,2-two oxidative ethane analog derivatives are (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star.
8. kit as claimed in claim 1 is characterized in that, described chemical luminous substrate comprises A liquid and B liquid, wherein,
Based on the described chemical luminous substrate A of 1000mL liquid, comprise 3.29g Tris, 3.26g borax, 1.0gBSA, 1.772g luminol, 0.5g benzofluoranthrene, 1mL Proclin 300, its pH value is 8.2～8.8;
Based on the described chemical luminous substrate B of 100mL liquid, comprise 36.72g Na
2HPO
412H
2The H of O, 10.21g citric acid, 1.0mL30%
2O
2, 1mL Proclin 300, its pH value is 5.0～5.8.
9. method for preparing the described kit of claim 1 is characterized in that may further comprise the steps:
1) with the human serum preparation syphilis helicoid antibody positive reference substance of the syphilis helicoid antibody test positive of high titre, detects negative human serum preparation syphilis helicoid antibody negative control product with syphilis helicoid antibody;
2) with enzyme labeling microspironema pallidum specificity recombinant protein antigen;
3) use microspironema pallidum specificity recombinant protein antigen bag by solid phase carrier;
4) chemical luminous substrate that acted on of microspironema pallidum specificity recombinant protein antigen and this enzyme of the negative and positive reference substance of the above-mentioned microspironema pallidum of packing, enzyme labeling; And
5) be assembled into finished product.
10. method as claimed in claim 9 is characterized in that, described bag is adopted following method by the step 3) of solid phase carrier:
1) bag quilt
With 0.050M pH value is the Avidin coating buffer that 9.6 carbonate buffer solution is mixed with desired concn, and coating buffer is carried on the solid phase carrier;
2) with physiological saline washing above-mentioned solid phase carrier; And
3) sealing
With containing 1%BSA, the pH value is 7.0～7.5, and concentration is that the phosphate buffer of 0.010M seals solid phase carrier after the above-mentioned washing as confining liquid.
11. method as claimed in claim 9 is characterized in that, described microspironema pallidum specificity recombinant protein antigen is selected from and comprises in TpN15, TpN17, TpN47, TpN37 and the TmpA group one or more.
12. method as claimed in claim 11 is characterized in that, described microspironema pallidum specificity recombinant protein antigen is one or more among TpN15, TpN17 and the TpN47.
13. method as claimed in claim 9 is characterized in that, described solid phase carrier is microwell plate, plastic bead, plastic tube or magnetic-particle.
14. method as claimed in claim 9 is characterized in that, described enzyme is alkaline phosphatase or horseradish peroxidase.
15. method as claimed in claim 9 is characterized in that, described chemical luminous substrate is 1,2-two oxidative ethane analog derivatives, luminol or different luminol.
16. method as claimed in claim 15, it is characterized in that described 1,2-two oxidative ethane analog derivatives are (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star.
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| CN101858915A (en) * | 2010-05-19 | 2010-10-13 | 厦门大学附属中山医院 | Reagent strip for testing syphilis specific IgG antibodies through gold immunochromatographic assay and preparation method thereof |
| CN101858914A (en) * | 2010-05-19 | 2010-10-13 | 厦门大学附属中山医院 | Reagent strip for testing syphilis specific total antibodies through gold immunochromatographic assay and preparation method thereof |
| CN102081096A (en) * | 2011-01-25 | 2011-06-01 | 厦门大学附属中山医院 | Syphilis cardiolipin and specific antibody IgG (Immunoglobulin G) immunoblotting kit and preparation method thereof |
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| CN102081096A (en) * | 2011-01-25 | 2011-06-01 | 厦门大学附属中山医院 | Syphilis cardiolipin and specific antibody IgG (Immunoglobulin G) immunoblotting kit and preparation method thereof |
| CN102095859A (en) * | 2011-01-25 | 2011-06-15 | 厦门大学附属中山医院 | Immunoblotting reagent kit of syphilis specific total antibodies and preparing method thereof |
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| CN104330562A (en) * | 2014-11-10 | 2015-02-04 | 厦门大学附属中山医院 | High-throughput treponema pallidum specific antibody detection kit and preparation method thereof |
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| CN104698185A (en) * | 2015-02-10 | 2015-06-10 | 深圳市新产业生物医学工程股份有限公司 | Kit for detecting treponema pallidum antibody as well as detection method and application thereof |
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| CN111308073A (en) * | 2019-12-17 | 2020-06-19 | 郑州安图生物工程股份有限公司 | Treponema pallidum antibody detection kit |
| CN115975023A (en) * | 2022-12-16 | 2023-04-18 | 北京科跃中楷生物技术有限公司 | Preparation method of recombinant TP antigen and antibody detection reagent prepared by preparation method |
| CN115975023B (en) * | 2022-12-16 | 2023-06-13 | 北京科跃中楷生物技术有限公司 | Preparation method of recombinant TP antigen and antibody detection reagent prepared by preparation method |
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