CN106990057A - One kind 5 hydroxyindole acetic acid reagents of detection and preparation method - Google Patents
One kind 5 hydroxyindole acetic acid reagents of detection and preparation method Download PDFInfo
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- CN106990057A CN106990057A CN201710311846.0A CN201710311846A CN106990057A CN 106990057 A CN106990057 A CN 106990057A CN 201710311846 A CN201710311846 A CN 201710311846A CN 106990057 A CN106990057 A CN 106990057A
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Abstract
The present invention relates to a kind of preparation method for the reagent and reagent for detecting 5 hydroxyindole acetic acids, the reagent that purpose is to provide should have the characteristics of simple to operate, detection time is short, the degree of accuracy is high, repeated and stability is good, and the preparation method of reagent thereof provided, which should have, prepares the characteristics of convenient and cost is not high.Technical scheme is：A kind of reagent for detecting 5 hydroxyindole acetic acids, including following component and content：A, 5 hydroxyindole acetic acid reagents 1；B, 5 hydroxyindole acetic acid reagents 2；C, the hydroxyindole acetic acid reference calibrations product of liquid-type 5.A kind of preparation method for detecting 5 hydroxyindole acetic acid reagents, is followed the steps below：1) 5 hydroxyindole acetic acid reagents 1 are prepared；2) 5 hydroxyindole acetic acid reagents 2 are prepared；3) the hydroxyindole acetic acid reference calibrations product of liquid-type 5 are prepared.
Description
Technical field
The present invention relates to a kind of preparation method for the reagent and reagent for detecting 5-hydroxyindoleacetic acid, increase especially with latex
The reagent of 5-hydroxyindoleacetic acid in strong immunoturbidimetry detection body fluid (serum, blood plasma or urine etc.) and corresponding preparation side
Method, can be widely used in medical science and technological field of biochemistry.
Background technology
5-hydroxyindoleacetic acid (5-hydroxyindole acetic acid, 5-HIAA) is the derivative of heteroauxin, 5-
Hydroxyindole acetic acid be serotonin (5-hydroxytryptamine, 5-HT) monoamine oxidase (Monoamine oxidase,
MAO it is transformed under) acting on, is active material important in human body.5-HIAA is that normal in mammal urine is constituted into
Point, while existing in the body fluid tissue such as cerebrospinal fluid, blood platelet, finally discharged in urine.Clinically by the excessive of 5-HIAA
Secrete the biochemical indicia the most specific as carcinoid tumor.Class cancer (carcinoid) is also known as carcinoid tumor (carcinoid
), tumor the neuroendocrine tumor of to be one group betide intestines and stomach and other organ chromaffin cells, its clinical, histochemistry and
Biochemical character can be different because of its happening part and different, such a tumour can secrete the biological activities such as 5-HT, kassinin kinin class, histamine because
Son, causes vasomotor disturbance, gastrointestinal symptoms, heart and pulmonary lesion etc., referred to as carcinoid syndrome (carcinoid
syndrome).Because quasi-cancer cell discharges substantial amounts of 5-HT, and 5-HT is further metabolized generation 5-HIAA, so class cancer patient
5-HIAA excretions significantly increase in urine, typically in 156.9~3 138 μm of ol/24h (10.5~47.1 μm of ol/ of normal reference value
24h urinates), therefore 5-HIAA content detections are the strong evidences of diagnostics classes cancer in 24h urine, and 5-HIAA amounts are integrated with class cancer in urine
The frequency and the order of severity that symptom occurs have direct relation.Allen etc. also found that 5-HIAA contents can be as monitoring in blood plasma
The highly useful biomarker of class cancer patient.Early in 5-HIAA reductions in 1976, sberg report cerebrospinal fluid and certainly
There is correlation between killing.The nineteen eighty-threes such as Stanley prove 5-HIAA can as suicide patient biomarker, and 5-
There is correlation between the decline of HIAA levels and suicide probability.Other 5-HIAA contents increase can be as P-AH, liver
Hardening, a useful organisms mark of acute appendicitis examination.In summary, 5-HIAA is neurotransmitter important in human body
Metabolite, increasing or reduce all can some pathological change in antimer indirectly.Simultaneously with the day to its detection method research
Open up increasingly, 5-HIAA and the research of clinically relevant disease will be further in progress, 5-HIAA will be clinical diagnosis
More and more reliable foundation is provided with treatment
Laboratory method conventional detection 5-HIAA has colorimetric method, radioimmunology analytic approach (RIA), chemiluminescence at present
Immunoassay (CLIA), high performance liquid chromatography (HPLC), mass spectrography (MS), ELISA (ELISA).Wherein colorimetric method
Complex operation, and 5-HIAA loss is easily caused in extraction process, sensitiveness is relatively low.Radioimmunology analytic approach (RIA)
Time-consuming (19~22h) for detection, and has the pollution of radioactive element, and label stability is poor, and discarded object is difficult to handle and make
It is restricted.Chemiluminescence immunoassay (CLIA) is through catalyst and oxidant oxygen using chemiluminescent substance
Change, form the intermediate of an excitation state, when the intermediate of this excitation state returns to stable ground state, send photon, using luminous
Signal measurement instrument measures number of photons, so that indirect detection 5-HIAA concentration.There are tubular type and board-like two kinds of reagents in the market
Box, but this method exist the direct labelled antibody of acridinium ester, luminol, different luminol luminous efficiency it is low, label it is unstable lack
Point；And this direct labelling method belongs to moment light emitting-type, it is difficult to ensure that the stability and repeatability of test result, in addition it is also necessary to special
Different detecting instrument, is not easy to Routine Test Lab development.And utilize biotin-avidin system to detect that 5-HIAA electrochemistry is sent out
Light immunoassay, high performance liquid chromatography (HPLC) and mass spectrography (MS), are required to be equipped with expensive fully automatic electric chemistry hair
Optical detector, high performance liquid chromatography and mass spectrograph, Routine Test Lab and laboratories can not be carried out, and be brought not to patient
Necessary expense, increases patient burden, is not inconsistent national medical reform relevant policies.ELISA (ELISA) automaticity is not high,
And larger, poor repeatability is affected by human factors, the whole reaction detection time is also very long (at least needing 40 minutes).
Chinese patent (open (bulletin) number CN104390975A), utilizes the mercurous ion and 5-hydroxyindoleacetic acid in reagent
Reaction produces red, type of red precipitation, so as to tentatively judge whether to carry cancer cell accordingly, the method can only be qualitative, it is impossible to fixed
Amount detection, less be can be automated operation, and easily be disturbed, and specificity, which also differs, to be set.For another example Chinese patent (is disclosed (bulletin)
Number CN105131106A) using homogeneous enzyme immunoassay method detection 5-HIAA, although it can realize in full-automatic biochemical point
High flux, rapid detection are carried out in analyzer.But the enzyme mark conjugate that this method is used is G-6-P dehydrogenation
Enzyme-haptens enzyme mark conjugate, this enzyme conjugates heat endurance is poor, and the term of validity of reagent is short.
The content of the invention
The technical problems to be solved by the invention are to overcome the shortcomings of above-mentioned background technology there is provided a kind of 5-hydroxyindoleacetic acid
(5-HIAA) detection reagent；The reagent provided should have that simple to operate, detection time is short, the degree of accuracy is high, repeated and stably
Property it is good the characteristics of, and be applicable to the 5-hydroxyindoleacetic acid detection of various types of automatic clinical chemistry analyzers or special protein instrument
Reagent；The preparation method of reagent thereof provided, which should have, prepares the characteristics of convenient and cost is not high.
The technical scheme that the present invention is provided is：
A kind of reagent for detecting 5-hydroxyindoleacetic acid, including following component and content：
A, 5-hydroxyindoleacetic acid reagent 1：
The pH of reagent 1 is 4.0-11.0；
B, 5-hydroxyindoleacetic acid reagent 2：
The latex particle with anti-human 5-hydroxyindoleacetic acid antibody, carboxylated latex microspherulite diameter therein is 20-
500nm；
C, liquid-type 5-hydroxyindoleacetic acid reference calibrations product：
The liquid-type 5-hydroxyindoleacetic acid reference calibrations product include the reference calibrations product of 5 parts of formation series concentrations；5 parts of ginsengs
It is 2 to examine the formula that the 5-hydroxyindoleacetic acid content in calibration object arranges from low to highn-1x mg/L；N=1,2 ... ... 5；X according to
It needs to be determined that (span is 0.5-2.5)；Or,
The 5-hydroxyindoleacetic acid that the liquid-type 5-hydroxyindoleacetic acid reference calibrations product may also be single-point high concentration refers to school
Quasi- product.
The buffer solution is phosphate buffer, trishydroxymethylaminomethane (Tris) buffer solution, 1,4- piperazine diethyl sulphurs
Sour (PIPES) buffer solution, 4- (2- ethoxys) piperazine -1-1 ethane sulfonic acids (HEPES) buffer solution, the sulphur of Pehanorm base third
Sour (TAPS) buffer solution, glycine buffer, borate buffer, 3- N-morpholinyls (MOPS) buffer solution, 3- (N- morpholinyls)-
One or both of 2- hydroxy-propanesulfonic acids (MOPSO) buffer solution, MES (MES) buffer solution any of the above ratio
Mixing.
The concentration of buffer solution is 20-200mmol/L in the reagent 1, and the pH value of buffer solution is 7.0-8.0.
The reaction accelerator is polyethylene glycol or dextran sulfate (DS-50).
The reaction accelerator concentration is 0.01-50g/L.
The preferred 0.05-30g/L of reaction accelerator concentration.
The electrolyte is anion electrolyte or cationic polyelectrolyte, and concentration is 50-200mmol/L.
Sodium chloride in the electrolyte preferred cationic.
Described 5-HIAA- conjugates are 5 hydroxyindole acetic acids-bovine serum albumin (5-HIAA-BSA, as follows) or 5- hydroxyls
Heteroauxin-hemocyanin (5-HIAA-KHA, as follows), preferably 5-HIAA- bovine serum albumins, the preferred 0.01-10g/ of concentration
L.Described conjugate is not particularly limited herein, as long as the purpose of the present invention can be reached.
The glass or plastic containers, cationic surfactant, anion surfactant or
Zwitterionic surfactant；The surfactant concentration is 0.5-1g/L.
The nonionic surfactant is Theist, Tween, polyoxyethylene laurel ether, polyoxyethylene phenyl ether, poly-
One or both of oxygen ethene octyl group phenylate, polyoxyethylene alkyl phenyl ether, ethylene nonyl phenyl ether any of the above ratio
The mixing of example.
The content of the latex particle with anti-human 5-hydroxyindoleacetic acid antibody is 0.1- in the 5-hydroxyindoleacetic acid reagent 2
50g/L。
A kind of preparation method for detecting 5-hydroxyindoleacetic acid reagent, is followed the steps below：
1) 5-hydroxyindoleacetic acid reagent 1：
By formula ratio each composition is mixed, then adjust pH value；
2) 5-hydroxyindoleacetic acid reagent 2：
(1) carboxylated latex microballoon is taken to be diluted to 5-50g/L suspension with buffer solution；
(2) 5-30g EDAC (ethyldimethyl amine carbodiimide) are added by 1 liter of carboxylated latex microsphere suspension liquid
With 5-80g NHS (n-hydroxysuccinimide sulfonate sodium) ratio, this mixture is reacted in room temperature after mixing immediately
15-30 minutes, it is stirred continuously；
(3) with buffer solution or purifying water washing carboxylated latex microballoon, unreacted NHS and EDAC is removed, and latex is micro-
Ball suspends in purified water, and it is 0.1-50g/L to make latex microsphere content；
(4) anti-human 5-hydroxyindoleacetic acid antibody is dissolved in cushioning liquid, makes the albumen of anti-human 5-hydroxyindoleacetic acid antibody
Matter concentration is 0.1-0.5g/L；
(5) the carboxylated latex microsphere suspension liquid 500ml in step (3) is taken to add anti-human 5- hydroxyindoles in step (4) immediately
Mixture, is then stirred continuously 3-5 hours by acetic acid antibody 500ml at 37 DEG C；
(7) ratio for adding 50-100g trishydroxymethylaminomethanes in the above-mentioned reactant mixtures of 1L adds trihydroxy methyl amino
Methane, then reacts 30-60 minutes, and be stirred continuously at 37 DEG C；
(8) centrifugal treating removes uncombined protein and trishydroxymethylaminomethane, then is diluted with buffer solution, makes mixing
The concentration of the latex particle with anti-human 5-- hydroxyindole acetic acids antibody is 10-500ml/L in liquid, then adds stabilizer, anti-corrosion
After agent dissolving mixing；
3) liquid-type 5-hydroxyindoleacetic acid reference calibrations product：
First preservative, stabilizer, buffer solution and purified water are mixed by formula ratio and are divided into 5 portions of mixed liquors, then according to
The 5-hydroxyindoleacetic acid reference calibrations product concentration needed, the 1000mg/L of respective amount 5-hydroxyindoleacetic acid sterling is separately added into
In above-mentioned mixed liquor, the reference calibrations product of 5 parts of formation series concentrations are obtained；5-hydroxyindoleacetic acid in 5 parts of reference calibrations product contains
It is 2 to measure the formula arranged from low to highn-1x mg/L；N=1,2 ... ... 5；X is determined as needed；Or,
Mixed liquor is obtained after mixing preservative, stabilizer and buffer solution by formula ratio, is then added in mixed liquor certain
The 1000mg/L of amount 5-hydroxyindoleacetic acid sterling, obtains the 5-hydroxyindoleacetic acid reference calibrations product of single-point high concentration.
The anti-human 5-hydroxyindoleacetic acid antibody is goat-anti people's 5-hydroxyindoleacetic acid antibody or rabbit-anti people's 5-hydroxyindoleacetic acid
Antibody or the anti-human 5-hydroxyindoleacetic acid antibody of mouse or the anti-human 5-hydroxyindoleacetic acid antibody of chicken.
The centrifugal treating uses centrifuge, and centrifuge speed is 15000 revs/min.
The operation principle for the 5-HIAA detection reagents that the present invention is provided is：Carrying in the 5-HIAA and reagent in sample is anti-
Antigen-antibody reaction occurs for the latex particle of people's 5-HIAA antibody, makes the increase of reaction solution turbidity, reaction solution is turbid within the specific limits
The amount of degree and antigen in sample is linear, and Biochemical Analyzer or other optical detecting instruments can be used in 540-600nm ripples
Strong point detects reaction solution absorbance, and reaction solution absorbance is directly proportional to surveyed 5-HIAA concentration.
The beneficial effects of the invention are as follows：Using the 5-hydroxyindoleacetic acid detection reagent of latex immunoturbidimetry, by by 5-
HIAA antibody is combined with carboxylated latex microballoon, is exaggerated the surface area of 5-HIAA antigens and antibody response, not only accuracy it is high (with
The correlation of chemiluminescence immunoassay is 0.9965-0.9998), it is reproducible, and detection it is quick (since detection to
Obtain a result and at most only need to 10 minutes, or even shorter), high-volume pattern detection can be carried out on routine biochemistry instrument, significantly
Improve detection operating efficiency；The term of validity of reagent is also up to 12 months.The preparation method of the reagent is easy to operate, and raw material is easy to get
(all from outsourcing raw material), cost is not high, is adapted to all kinds of medical research units and Routine Test Lab application.
Brief description of the drawings
Fig. 1 show in the embodiment of the present invention 1 as the method for the invention obtained by the detected value of 5-HIAA reagents with it is chemical
The correlation of 5-HIAA measured values obtained by luminescent immunoassay.Through regression analysis：Y=1.0022x+0.0411, R2=
0.9998.Show that the embodiment of the present invention 1 and chemiluminescence immunoassay have good correlation.
Fig. 2 show in the embodiment of the present invention 2 as the method for the invention obtained by the detected value of 5-HIAA reagents with it is chemical
The correlation of 5-HIAA measured values obtained by luminescent immunoassay.Through regression analysis：Y=1.0007x+0.0179, R2=
0.9998。
Show that the embodiment of the present invention 2 and chemiluminescence immunoassay have good correlation.
Fig. 3 show in the embodiment of the present invention 3 as the method for the invention obtained by the detected value of 5-HIAA reagents with it is chemical
The correlation of 5-HIAA measured values obtained by luminescent immunoassay.Through regression analysis：Y=0.9947x+0.0598, R2=
0.9995, show that the embodiment of the present invention 3 and chemiluminescence immunoassay have good correlation.
Fig. 4 show in the embodiment of the present invention 4 as the method for the invention obtained by the detected value of 5-HIAA reagents with it is chemical
The correlation of 5-HIAA measured values obtained by luminescent immunoassay.Through regression analysis：Y=1.0081x-0.0398, R2=
0.999.Show that the embodiment of the present invention 4 and chemiluminescence immunoassay have good correlation.
Embodiment
The present invention principle be：5-hydroxyindoleacetic acid-the coupling contained in 5-hydroxyindoleacetic acid reagent 1 in the present invention
Thing can form turbidity with the latex particle of the anti-human 5-hydroxyindoleacetic acid antibody in 5-hydroxyindoleacetic acid reagent 2；When in sample
During containing 5-hydroxyindoleacetic acid, 5-hydroxyindoleacetic acid small molecule and 5-hydroxyindoleacetic acid in sample-conjugate competition and 5- hydroxyls
The latex particle reaction of anti-human 5-hydroxyindoleacetic acid antibody in heteroauxin reagent 2.5-hydroxyindoleacetic acid concentration in sample
Higher, the antibody on the latex particle of anti-human 5-hydroxyindoleacetic acid antibody is fewer with 5-hydroxyindoleacetic acid-conjugate reaction, that is, inhales
Light varience is lower, therefore can be compared the 5- hydroxyls Yin calculated in sample with known calibration condition according to the change of reaction absorbance
The content of indolylbutyric acid.
Primary raw material needed for detection reagent of the present invention and calibration object has：
1st, anti-human 5-hydroxyindoleacetic acid antibody, has the anti-human 5-hydroxyindoleacetic acid antibody of many commercializations available, such as
DaKo companies of Finland, UNF companies of Japan；The antibody only reacts with people's 5-hydroxyindoleacetic acid, anti-without immunological cross with other antigens
Should, it can be polyclonal antibody or monoclonal antibody, be not particularly limited here, as long as can be coupled with latex microsphere.Its
Anti-human 5-hydroxyindoleacetic acid antibody can be goat-anti (i.e. goat-anti people 5-hydroxyindoleacetic acid antibody；It is as follows), can also be rabbit-anti,
Chicken is anti-or mouse is anti-.As long as using known immunoelectrophoresis detection, the antibody only presents single heavy fixed between people 5-HIAA
Line, the titre of anti-human 5-hydroxyindoleacetic acid antibody is more than or equal to 1.0mg/ml.
2nd, latex microsphere, has the nanoparticle of many commercializations available, and such as Merck companies of Germany, Japan UNF is public
Department, JSR companies of Japan and Thermo companies；Its basic material is polystyrene or co polystyrene, surface carboxylic group
(COOH) modify, can also be not used as any modification or other groups.Here will be according to the combination side for selecting antibody and microballoon
Method is different and selects different balls.Chemical crosslink technique is such as selected, then (is handed over microballoon of the surface through modifications such as carboxyl, amino, aldehyde radicals
Join agent preferred：Ethyldimethyl amine carbodiimide).
The latex microsphere of the preferred carboxylic group modification of this law, the preferred 20nm-500nm of particle diameter of carboxylated latex microballoon more enters
One step, preferably 70nm-300nm.
Anti-human 5-HIAA antibody latexs particle can be prepared using physisorphtion or chemical crosslink technique in reagent 2, this hair
Bright preferred chemical crosslink technique (conventional method).
3rd, 5-hydroxyindoleacetic acid (5-HIAA) sterling：Purchased from Sigms companies of the U.S., school is referred to for preparing this reagent place
Quasi- product.
4th, 5-HIAA- bovine serum albumins, purchased from Sigms companies.
5-HIAA reference calibrations product concentration, can set the 5 reference calibrations product to form series concentration, 5 reference calibrations
5-HIAA contents are respectively 2.5mg/L, 5.0mg/L, 10.0mg/L, 20.0mg/L, 40.0mg/L from low to high in product；
The 5-HIAA reference calibrations product of single-point high concentration (such as 100.0mg/L, or 500.0mg/L) can also be prepared into, are made
Used time uses normal saline dilution into the reference calibrations product of multiple various concentrations again；Here have no particular limits, as long as obtained
5-HIAA reference calibrations product can be compared with sample, can detect the content of 5-HIAA in sample.
The present invention is further illustrated below in conjunction with embodiment, but these embodiments are not considered as determining for the limitation present invention
Justice.
Embodiment one
One) 5-hydroxyindoleacetic acid reagent 1
After above-mentioned raw materials are dissolved with purified water 800ml, then add purified water and be settled to 1000ml, then with hydrochloric acid or hydrogen
Sodium oxide molybdena adjusts PH to 7.4.
Two) 5-hydroxyindoleacetic acid reagent 2
1. take 80nm carboxylated latex microballoon to be diluted to 20g/L suspension with 50mmol/L, PH6.0 MES buffer solutions；
2. by 1 liter carboxylated latex microsphere suspension liquid add 10g EDAC (ethyldimethyl amine carbodiimide) and
50g NHS (n-hydroxysuccinimide sulfonate sodium) mix immediately after room temperature by this mixture react 15-30 minutes, no
Disconnected stirring；
3. with 50mmol/L, PH6.0 MES buffer solutions or purifying water washing carboxylated latex microballoon, remove unreacted NHS
And EDAC, and latex microsphere suspended in purified water carboxylated latex microsphere suspension liquid, wherein carboxylated latex microballoon content
For 5g/L.
4. goat-anti people's 5-hydroxyindoleacetic acid antibody is dissolved in 50mmol/L, PH8.5 HEPES cushioning liquid, make goat-anti
The protein concentration of people's 5-hydroxyindoleacetic acid antibody is 0.25mg/ml；
5. the carboxylated latex microsphere suspension liquid 500ml in step (3) is taken, the goat-anti people's 5- hydroxyindoles added in step (4)
Acetic acid antibody 500ml；Then mixture is reacted 3 hours at 37 DEG C, be stirred continuously；
7. adding 50g trishydroxymethylaminomethane by 1 liter of reactant mixture, react 20 minutes, be stirred continuously；
8. be centrifuged off uncombined protein and trishydroxymethylaminomethane with 15000 revs/min, with 50mmol/L,
PH7.2 MOPSO buffer solutions dilution, the concentration for making the latex particle with goat-anti people's 5-HIAA antibody in mixed liquor is 20ml/
L；Then add after bovine serum albumin(BSA) (stabilizer) 3mmol/L, 3mmol/L Sodium azide (preservative) dissolving is mixed and produce 5- hydroxyls
Heteroauxin reagent 2.
Three) liquid-type 5-HIAA reference calibrations product
Corresponding 5-HIAA sterlings 1000mg/L is added above-mentioned buffering by 5-HIAA reference calibrations product concentration as required
In liquid, be prepared into the reference calibrations product of 5 series concentrations, the reference calibrations product of 5 series concentrations 5-HIAA contents by it is low to
Height is respectively that (obviously, 5 reference calibrations product are according to formula by 2.5mg/L, 5.0mg/L, 10.0mg/L, 20.0mg/L, 40.0mg/L
2n-1Xmg/L determines that 2.5) wherein x is.
Pattern detection (with 30 samples of Chemiluminescence immunoassay difference contrasting detection)：
According to laboratory operating procedures, 200 μ L 5-hydroxyindoleacetic acid reagent 1 is added into 10 μ L samples, 3 are incubated at 37 DEG C
Minute, add after 2,37 DEG C of 50 μ L5- hydroxyindole acetic acids reagent is incubated 10 seconds and read absorbance A in wavelength 600nm1, reaction to 5
Absorbance A is read during minute2, calculate absorbance change value △ A=A twice2-A1.Compared by the calibration solution with equally handling,
Calculate the 5-HIAA concentration in sample.It can be seen that the present invention is provided from table 1 and Fig. 1 (Fig. 1 is drawn according to the data of table 1) result
Method although simple to operate, required time only within 10 minutes, but with Chemiluminescence immunoassay detect contrast, display very
Good correlation (correlation is 0.9998).
Embodiment two
One) 5-hydroxyindoleacetic acid reagent 1
PC300 (preservative) 5mmol/L
5-HIAA-BSA (conjugate) 10g/L
Disodium ethylene diamine tetraacetate (stabilizer) 8mmol/L
After above-mentioned raw materials are dissolved with purified water 800ml, then purified water is added to 1000ml (1L), then use sodium hydroxide
Adjust PH to 8.0.
Two) 5-hydroxyindoleacetic acid reagent 2
1. take 150nm carboxylated latex microballoon to be diluted to the outstanding of 0.01mg/ml with 50mmol/L, PH6.0 MES buffer solutions
Supernatant liquid；
2. 30g EDAC and 80g NHS are added by 1 liter of carboxylated latex microsphere suspension liquid, will in room temperature after mixing immediately
Mixture reacts 30 minutes, is stirred continuously；
3. with 50mmol/L, PH6.0 MES buffer solutions or purifying water washing carboxylated latex microballoon, remove unreacted NHS
And EDAC, and latex microsphere suspended in purified water carboxylated latex microsphere suspension liquid, wherein carboxylated latex microballoon content
For 50g/L；
4. rabbit-anti people's 5-hydroxyindoleacetic acid antibody is dissolved in 50mmol/L, PH8.5 borate buffer solution, make rabbit-anti
The protein concentration of people's 5-hydroxyindoleacetic acid antibody is 0.25mg/ml；
5. the rabbit-anti people's 5- hydroxyls Yin for taking the carboxylated latex microsphere suspension liquid 500ml in step (3) to add immediately in step (4)
Indolylbutyric acid antibody 500ml；Then mixture is stirred continuously 5 hours at 37 DEG C；
6. adding 100g trishydroxymethylaminomethanes by 1 liter of reactant mixture, react 60 minutes, be stirred continuously；
7. be centrifuged off uncombined protein and trishydroxymethylaminomethane with 15000 revs/min, with 50mmol/L,
PH7.4, phosphate buffer dilution, make the concentration of latex particle with rabbit-anti people's 5-HIAA antibody in mixed liquor be
400ml/L；Then add after bovine serum albumin(BSA) (stabilizer) 4mmol/L, 2mmol/L Sodium azide (preservative) dissolving is mixed i.e.
Obtain 5-hydroxyindoleacetic acid reagent 2.
Three) liquid-type 5-HIAA reference calibrations product
Table 2
Corresponding 5-HIAA sterlings 1000mg/L is added above-mentioned buffering by 5-HIAA reference calibrations product concentration as required
In liquid, 5 reference calibrations product for forming series concentration are obtained；5-HIAA contents are respectively from low to high in 5 reference calibrations product
1.0mg/L、2.0mg/L、4.0mg/L、8.0mg/L、16.0mg/L.Obviously, 5 reference calibrations product are according to formula 2n-1x mg/L
It is determined that, 1.0) wherein x is
Pattern detection (with 30 samples of Chemiluminescence immunoassay difference contrasting detection)：
According to laboratory operating procedures, 240 μ L 5-hydroxyindoleacetic acid reagent 1 is added into 10 μ L samples, 3 are incubated at 37 DEG C
Minute, add after 2,37 DEG C of 60 μ L5- hydroxyindole acetic acids reagent is incubated 10 seconds and read absorbance A in wavelength 600nm1, reaction to 5
Absorbance A is read during minute2, calculate absorbance change value △ A=A twice2-A1.Compared by the calibration solution with equally handling,
Calculate the 5-HIAA concentration in sample.It can be seen that from table 2 and Fig. 2 (Fig. 2 is drawn according to the data of table 2) result, the inventive method
Although simple to operate, required time is only within 10 minutes, also with Chemiluminescence immunoassay very good correlation (correlation
For 0.9998).
Embodiment three
One) 5-hydroxyindoleacetic acid reagent 1
After above-mentioned raw materials are dissolved with purified water 800ml, then purified water is added to 1000ml (1L), then use sodium hydroxide
Adjust PH to 8.0.
Two) 5-hydroxyindoleacetic acid reagent 2
1. take 300nm carboxylated latex microballoon to be diluted to 5g/L suspension with 50mmol/L, PH6.0 MES buffer solutions.
2. 20g EDAC and 40g NHS are added by 1 liter of carboxylated latex microsphere suspension liquid, will in room temperature after mixing immediately
This mixture reacts 30 minutes, is stirred continuously；
3. with 50mmol/L, PH6.0 MES buffer solutions or purifying water washing carboxylated latex microballoon, remove unreacted NHS
And EDAC, and latex microsphere suspended in purified water carboxylated latex microsphere suspension liquid, wherein carboxylated latex microballoon content
For 20g/L；
4. the anti-human 5-hydroxyindoleacetic acid antibody of mouse is dissolved in 50mmol/L, PH8.5 TAPS salt buffer solutions, resist mouse
The protein concentration of people's 5-hydroxyindoleacetic acid antibody is 0.1g/L；
5. the anti-human 5- hydroxyindoles of mouse for taking the carboxylated latex microsphere suspension liquid 1ml in step (3) to add immediately in step (4)
Acetic acid antibody 1ml；Then mixture is stirred continuously 3 hours at 37 DEG C；
7. adding 10g trishydroxymethylaminomethane by 1 liter of reactant mixture, react 15 minutes, be stirred continuously；
8. be centrifuged off uncombined protein and trishydroxymethylaminomethane with 15000 revs/min, with 100mmol/L,
PH8.0 PIPES buffer solutions dilution, the concentration for making the latex particle with the anti-human 5-HIAA antibody of mouse in mixed liquor is 300ml/
L；Then add after bovine serum albumin(BSA) (stabilizer) 2mmol/L, 4mmol/L Sodium azide (preservative) dissolving is mixed and produce 5- hydroxyls
Heteroauxin reagent 2.
Table 3
Three) liquid-type 5-HIAA reference calibrations product
Corresponding 5-HIAA sterlings 1000mg/ml is added above-mentioned buffering by 5-HIAA reference calibrations product concentration as required
In liquid, be prepared into the reference calibrations product of 5 series concentrations, the reference calibrations product of 5 series concentrations 5-HIAA contents by it is low to
Height is respectively 2.0mg/L, 4.0mg/L, 8.0mg/L, 16.0mg/L, 32.0mg/L.Obviously, 5 reference calibrations product are according to formula
2n-1Xmg/L determines that 2.0) wherein x is
Pattern detection (with 30 samples of Chemiluminescence immunoassay difference contrasting detection)：
According to laboratory operating procedures, 240 μ L 5-hydroxyindoleacetic acid reagent 1 is added into 10 μ L samples, 3 are incubated at 37 DEG C
Minute, add after 2,37 DEG C of 60 μ L5- hydroxyindole acetic acids reagent is incubated 10 seconds and read absorbance A in wavelength 600nm1, reaction to 5
Absorbance A is read during minute2, calculate absorbance change value △ A=A twice2-A1.Compared by the calibration solution with equally handling,
Calculate the 5-HIAA concentration in sample.It can be seen that from table 3 and Fig. 3 (Fig. 3 is drawn according to the data of table 3) result, the present invention
Method is although simple to operate, and required time is also very good with Chemiluminescence immunoassay only within 10 minutes
Correlation (correlation is 0.9998).
Example IV
One) 5-hydroxyindoleacetic acid reagent 1
After above-mentioned raw materials are dissolved with purified water 800ml, then purified water is added to 1000ml, then with hydrochloric acid or hydroxide
Sodium adjusts PH to 7.6.
Two) 5-hydroxyindoleacetic acid reagent 2
1. take 220nm carboxylated latex microballoon to be diluted to 40g/L suspension with 50mmol/L, PH6.0 MES buffer solutions
Liquid；2. add 30g EDAC and 60g NHS by 1 liter of carboxylated latex microsphere suspension liquid, in room temperature by this mixture after mixing immediately
Reaction 25 minutes, is stirred continuously；
3. with 50mmol/L, PH6.0 MES buffer solutions or purifying water washing carboxylated latex microballoon, remove unreacted NHS
And EDAC, and latex microsphere suspended in purified water carboxylated latex microsphere suspension liquid, wherein carboxylated latex microballoon content
For 40g/L；
4. the anti-human 5-hydroxyindoleacetic acid antibody of chicken is dissolved in 50mmol/L, PH8.5 borate buffer solution, resist chicken
The protein concentration of people's 5-hydroxyindoleacetic acid antibody is 0.5g/L；
5. the anti-human 5- hydroxyls Yin of chicken for taking the carboxylated latex microsphere suspension liquid 500ml in step (3) to add immediately in step (4)
Indolylbutyric acid antibody 500ml；Then mixture is stirred continuously 3 hours at 37 DEG C；
7. adding 60g trishydroxymethylaminomethane by 1 liter of reactant mixture, react 40 minutes, be stirred continuously；
8. be centrifuged off uncombined protein and trishydroxymethylaminomethane with 15000 revs/min, with 50mmol/L,
PH7.6, HEPES buffer solution dilution, make in mixed liquor with the anti-human 5-HIAA antibody of chicken latex particle concentration be 40ml/
L；Then i.e. 5- hydroxyls Yin after bovine serum albumin(BSA) (stabilizer) 3mmol/L, 3mmol/L Sodium azide (preservative) dissolving is mixed is added
Indolylbutyric acid reagent 2.
3) liquid-type 5-HIAA reference calibrations product
Corresponding 5-HIAA sterlings 1000mg/L is added above-mentioned buffering by 5-HIAA reference calibrations product concentration as required
In liquid,
Obtain 5 reference calibrations product for forming series concentration；5-HIAA contents are distinguished from low to high in 5 reference calibrations product
For 0.5mg/L, 1.0mg/L, 2.0mg/L, 4.0mg/L, 8.0mg/L.Obviously, 5 reference calibrations product are according to formula 2n-1x mg/L
It is determined that, 0.5) wherein x is
Pattern detection (with 30 samples of Chemiluminescence immunoassay difference contrasting detection)：
According to laboratory operating procedures, 240 μ L 5-hydroxyindoleacetic acid reagent 1 is added into 10 μ L samples, 3 are incubated at 37 DEG C
Minute, add after 2,37 DEG C of 60 μ L5- hydroxyindole acetic acids reagent is incubated 10 seconds and read absorbance A in wavelength 600nm1, reaction to 5
Absorbance A is read during minute2, calculate absorbance change value △ A=A twice2-A1.Compared by the calibration solution with equally handling,
Calculate the 5-HIAA concentration in sample.It can be seen that from table 4 and Fig. 4 (Fig. 4 is drawn according to the data of table 4) result, the inventive method
Although simple to operate, required time shows very good correlation (phase only within 10 minutes, also with Chemiluminescence immunoassay
0.9993) closing property is.
Table 4
Operating procedure is detected the following is 5-hydroxyindoleacetic acid reagent of the present invention and is specifically divided on automatic clinical chemistry analyzer
Analyse parameter：
Detecting step
Analytical parameters (by taking the parameter of the biochemical instruments of Hitachi 7080 as an example)
Method：Two point end assay, reaction temperature：37℃
Dominant wavelength：570-600nm commplementary wave lengths：800nm (can not be selected)
Sample size：10 μ L, the amount of 1 amount of reagent/reagent 2：240μL/60μL
The Direction of Reaction：Positive (rising), calibration mode：Multiple spot is non-linear
The read point time：Respectively 5 points and 21 points (total reaction time is suitable 420 seconds)
By taking the parameter of the biochemical instruments of Hitachi 7080 as an example
Instrument can carry out 5-HIAA detection after being automatically performed calibration, and it is dense that instrument calculates corresponding 5-HIAA automatically
Degree.
It is pointed out that above-described is only the preferred embodiments of the invention, the common of city is led for this technology
For technical staff, under the premise without departing from the principles of the invention, some modifications and adaptations are also used as, these are improved
Adjustment also should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of reagent for detecting 5-hydroxyindoleacetic acid, including following component and content：
A, 5-hydroxyindoleacetic acid reagent 1：
The pH of reagent 1 is 4.0-11.0；
B, 5-hydroxyindoleacetic acid reagent 2：
The latex particle with anti-human 5-hydroxyindoleacetic acid antibody, carboxylated latex microspherulite diameter therein is 20-500nm；
C, liquid-type 5-hydroxyindoleacetic acid reference calibrations product：
The liquid-type 5-hydroxyindoleacetic acid reference calibrations product include the reference calibrations product of 5 parts of formation series concentrations；5 parts refer to school
The formula that 5-hydroxyindoleacetic acid content in quasi- product is arranged from low to high is 2n-1x mg/L；N=1,2 ... ... 5；X is as needed
It is determined that；Or,
The liquid-type 5-hydroxyindoleacetic acid reference calibrations product are the 5-hydroxyindoleacetic acid reference calibrations product of single-point high concentration.
2. the reagent of described detection 5-hydroxyindoleacetic acid is required according to claim 1, it is characterised in that：The buffer solution is phosphorus
Phthalate buffer, trishydroxymethylaminomethane (Tris) buffer solution, the ethyl sulfonic acid of 1,4- piperazines two (PIPES) buffer solution, 4- (2- hydroxyls
Ethyl) piperazine -1-1 ethane sulfonic acids (HEPES) buffer solution, Pehanorm base propane sulfonic acid (TAPS) buffer solution, glycine delay
Fliud flushing, borate buffer, 3- N-morpholinyls (MOPS) buffer solution, 3- (N- morpholinyls) -2- hydroxy-propanesulfonic acids (MOPSO) buffering
The mixing of one or both of liquid, MES (MES) buffer solution any of the above ratio；Buffer solution in the reagent 1
Concentration be 20-200mmol/L, the pH value of buffer solution is 7.0-8.0.
3. the reagent of described detection 5-hydroxyindoleacetic acid is required according to claim 1, it is characterised in that：The reaction accelerator
It is polyethylene glycol or dextran sulfate (DS-50)；The reaction accelerator concentration is 0.01-50g/L.
4. the reagent of described detection 5-hydroxyindoleacetic acid is required according to claim 1, it is characterised in that：The electrolyte is cloudy
Ionic electrolytes or cationic polyelectrolyte, concentration are 50-200mmol/L.
5. the reagent of described detection 5-hydroxyindoleacetic acid is required according to claim 1, it is characterised in that：Described 5-HIAA-
Conjugate is 5 hydroxyindole acetic acids-bovine serum albumin or 5-hydroxyindoleacetic acid-hemocyanin, the preferred 0.01-10g/L of concentration.
6. the reagent of described detection 5-hydroxyindoleacetic acid is required according to claim 1, it is characterised in that：The surfactant
It is nonionic surfactant, cationic surfactant, anion surfactant or zwitterionic surfactant；It is described
Surfactant concentration is 0.5-1g/L.
7. the reagent of described detection 5-hydroxyindoleacetic acid is required according to claim 1, it is characterised in that：The 5- hydroxyindoles second
The content of the latex particle with anti-human 5-HIAA antibody is 0.1-50g/L in acid reagent 2.
8. claim 1 requires the preparation method of described detection 5-hydroxyindoleacetic acid reagent, follow the steps below：
1) 5-hydroxyindoleacetic acid reagent 1：
By formula ratio each composition is mixed, then adjust pH value；
2) 5-hydroxyindoleacetic acid reagent 2：
(1) carboxylated latex microballoon is taken to be diluted to 5-50g/L suspension with buffer solution；
(2) 5-30g ethyldimethyl amines carbodiimide and 5-80g N- are added by 1 liter of carboxylated latex microsphere suspension liquid
The ratio of HOSu NHS sulfonate sodium, this mixture is reacted 15-30 minutes, constantly stir after mixing immediately in room temperature
Mix；
(3) with buffer solution or purifying water washing carboxylated latex microballoon, unreacted n-hydroxysuccinimide sulfonate sodium is removed
With ethyldimethyl amine carbodiimide, and latex microsphere is suspended in purified water, it is 0.1- to make latex microsphere content
50g/L；
(4) anti-human 5-hydroxyindoleacetic acid antibody is dissolved in cushioning liquid, makes the protein compression of anti-human 5-hydroxyindoleacetic acid antibody
Spend for 0.1-0.5g/L；
(5) the carboxylated latex microsphere suspension liquid 500ml in step (3) is taken to add anti-human 5-hydroxyindoleacetic acid in step (4) immediately
Mixture, is then stirred continuously 3-5 hours by antibody 500ml at 37 DEG C；
(7) ratio for adding 50-100g trishydroxymethylaminomethanes in the above-mentioned reactant mixtures of 1L adds trishydroxymethylaminomethane,
Then react 30-60 minutes, and be stirred continuously at 37 DEG C；
(8) centrifugal treating removes uncombined protein and trishydroxymethylaminomethane, then is diluted with buffer solution, makes in mixed liquor
The concentration of latex particle with anti-human 5-hydroxyindoleacetic acid antibody is 10-500ml/L, then adds stabilizer, preservative molten
After solution mixing；
3) liquid-type 5-hydroxyindoleacetic acid reference calibrations product：
First preservative, stabilizer, buffer solution and purified water are mixed by formula ratio and are divided into 5 portions of mixed liquors, then as required
5-hydroxyindoleacetic acid reference calibrations product concentration, the 1000mg/L of respective amount 5-hydroxyindoleacetic acid sterling is separately added into above-mentioned
In mixed liquor, the reference calibrations product of 5 parts of formation series concentrations are obtained；5-hydroxyindoleacetic acid content in 5 parts of reference calibrations product by
The formula of low to high arrangement is 2n-1x mg/L；N=1,2 ... ... 5；X is determined as needed；Or,
Mixed liquor is obtained after mixing preservative, stabilizer and buffer solution by formula ratio, is then added in mixed liquor a certain amount of
1000mg/L 5-hydroxyindoleacetic acid sterling, obtains the 5-hydroxyindoleacetic acid reference calibrations product of single-point high concentration.
9. the preparation method of described detection 5-hydroxyindoleacetic acid reagent is required according to claim 8, it is characterised in that：It is described anti-
People's 5-hydroxyindoleacetic acid antibody is goat-anti people's 5-hydroxyindoleacetic acid antibody or rabbit-anti people's 5-hydroxyindoleacetic acid antibody or the anti-human 5- of mouse
Hydroxyindole acetic acid antibody or the anti-human 5-hydroxyindoleacetic acid antibody of chicken.
10. the preparation method of described detection 5-hydroxyindoleacetic acid reagent is required according to claim 9, it is characterised in that：It is described
Centrifugal treating uses centrifuge, and centrifuge speed is 15000 revs/min.
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| CN110940663A (en) * | 2019-12-26 | 2020-03-31 | 欧迪玛(湖北)生物科技有限公司 | Urine 5-oxindole acetic acid detection reagent and preparation method thereof |
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