CN102161716A - Method and reagent for latex sensitization - Google Patents
Method and reagent for latex sensitization Download PDFInfo
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- CN102161716A CN102161716A CN 201010615283 CN201010615283A CN102161716A CN 102161716 A CN102161716 A CN 102161716A CN 201010615283 CN201010615283 CN 201010615283 CN 201010615283 A CN201010615283 A CN 201010615283A CN 102161716 A CN102161716 A CN 102161716A
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- Prior art keywords
- latex
- reagent
- antigen
- damping fluid
- sensitization
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Links
- 239000004816 latex Substances 0.000 title claims abstract description 150
- 229920000126 latex Polymers 0.000 title claims abstract description 150
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 105
- 238000000034 method Methods 0.000 title claims abstract description 46
- 206010070834 Sensitisation Diseases 0.000 title claims abstract description 36
- 230000008313 sensitization Effects 0.000 title claims abstract description 36
- 239000000427 antigen Substances 0.000 claims abstract description 43
- 108091007433 antigens Proteins 0.000 claims abstract description 43
- 102000036639 antigens Human genes 0.000 claims abstract description 43
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 19
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000000126 substance Substances 0.000 claims abstract description 15
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 9
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Natural products NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 39
- 238000013016 damping Methods 0.000 claims description 38
- 239000012530 fluid Substances 0.000 claims description 38
- 238000006243 chemical reaction Methods 0.000 claims description 36
- 230000002708 enhancing effect Effects 0.000 claims description 32
- 239000000872 buffer Substances 0.000 claims description 27
- 239000004471 Glycine Substances 0.000 claims description 22
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 17
- 239000004793 Polystyrene Substances 0.000 claims description 16
- 229920002223 polystyrene Polymers 0.000 claims description 16
- 230000001235 sensitizing effect Effects 0.000 claims description 15
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 14
- 108010075210 streptolysin O Proteins 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- -1 salt ion Chemical class 0.000 claims description 12
- 125000003636 chemical group Chemical group 0.000 claims description 9
- 239000007983 Tris buffer Substances 0.000 claims description 8
- 230000006798 recombination Effects 0.000 claims description 8
- 238000005215 recombination Methods 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 8
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 8
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 7
- 238000012360 testing method Methods 0.000 claims description 7
- 239000002245 particle Substances 0.000 claims description 6
- 229920000642 polymer Polymers 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 229910001415 sodium ion Inorganic materials 0.000 claims description 6
- 239000008118 PEG 6000 Substances 0.000 claims description 5
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 claims description 5
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 5
- 239000013543 active substance Substances 0.000 claims description 5
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims description 5
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 claims description 5
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 5
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 5
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 4
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 3
- 239000007995 HEPES buffer Substances 0.000 claims description 3
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims description 3
- FKNQFGJONOIPTF-UHFFFAOYSA-N Sodium cation Chemical compound [Na+] FKNQFGJONOIPTF-UHFFFAOYSA-N 0.000 claims description 3
- 230000000890 antigenic effect Effects 0.000 claims description 3
- 108060003552 hemocyanin Proteins 0.000 claims description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 3
- 229910001425 magnesium ion Inorganic materials 0.000 claims description 3
- 229910021645 metal ion Inorganic materials 0.000 claims description 3
- 150000001455 metallic ions Chemical class 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 229920000136 polysorbate Polymers 0.000 claims description 3
- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 claims description 2
- 238000010521 absorption reaction Methods 0.000 claims description 2
- 229920001223 polyethylene glycol Polymers 0.000 claims description 2
- 229920001184 polypeptide Polymers 0.000 claims description 2
- 239000004302 potassium sorbate Substances 0.000 claims description 2
- 235000010241 potassium sorbate Nutrition 0.000 claims description 2
- 229940069338 potassium sorbate Drugs 0.000 claims description 2
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 2
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 2
- 230000035939 shock Effects 0.000 claims description 2
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 claims description 2
- 235000010234 sodium benzoate Nutrition 0.000 claims description 2
- 239000004299 sodium benzoate Substances 0.000 claims description 2
- 235000010288 sodium nitrite Nutrition 0.000 claims description 2
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical class [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 14
- 238000004879 turbidimetry Methods 0.000 abstract description 3
- 238000004132 cross linking Methods 0.000 abstract description 2
- 238000002360 preparation method Methods 0.000 description 12
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 5
- 238000004140 cleaning Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000035484 reaction time Effects 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 238000010219 correlation analysis Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 239000002872 contrast media Substances 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229930002839 ionone Natural products 0.000 description 1
- 150000002499 ionone derivatives Chemical class 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- BHTJEPVNHUUIPV-UHFFFAOYSA-N pentanedial;hydrate Chemical compound O.O=CCCCC=O BHTJEPVNHUUIPV-UHFFFAOYSA-N 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
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- Peptides Or Proteins (AREA)
Abstract
The invention relates to a method and a reagent for latex sensitization. Specifically, the method for latex sensitization comprises the following steps: combining latex with an unrelated protein by a chemical bond; and then, crosslinking an antigen or an antibody on the unrelated protein by glutaraldehyde so as to cause latex sensitization. The antistreptolysin O latex prepared with the method enhances an immune turbidimetry reagent. With the method disclosed by the invention, time for preparing the reagent can be shortened, and the sensitivity of the reagent is improved.
Description
Technical field
The present invention relates to the method and the reagent of latex sensitization.More particularly, the present invention relates to earlier by chemical bond latex nothing to do with protein binding is re-used glutaraldehyde with antigen or antibody linked on irrelevant albumen, thereby makes the method for latex sensitization.The invention still further relates to the latex enhancing immune of latex that utilizes this method to obtain the latex of sensitization and used this sensitization than turbid reagent.
Background technology
It is micron-sized particle that Singer in 1956 and plotz bring into use diameter, and slide agglutination detects by carrying out qualitatively attached to the antigen on the latex or antibody to the antibody in the serum or antigen.The latex enhancing immune turbidimetry that grows up has on this basis improved the sensitivity of common immunoturbidimetry, and simple to operate, can use common Biochemical Analyzer to measure, and is a kind of new methods for clinical diagnosis.
Latex enhancing immune than turbid reagent in, the sensitization of latex just combines antigen or antibody with latex, this step be the preparation reagent in most crucial steps.The sensitization of latex has physisorphtion and chemical crosslink technique usually.Physisorphtion is by the interaction between the hydrophobic grouping on hydrophobic grouping on the protein molecular structure and latex surface, and antigen or antibody are adsorbed onto the latex surface.Chemical crosslink technique is the chemical group reaction by antigen or antibody and latex surface, with antigen or antibody linked on latex.Chemical crosslink technique now is widely used in the external diagnosis reagent owing to characteristics such as good, the easy preservation of specificity, favorable dispersities.But chemical crosslink technique can influence the sensitivity of reagent because chemical reaction causes antigen or antibody loss of activity in cross-linking process.The step that chemical crosslink technique is taked usually is, earlier with antigen or antibody by direct crosslinked its sensitization that on latex, makes of chemical bond, add the unconjugated sites of protein or polymer sealing latex surface such as BSA, polyoxyethylene glycol again, entire reaction is generally more than two days and two days, and the time is longer.And because the condition of this reaction is stronger and the reaction times is longer, the loss of activity of antigen or antibody is bigger, and the sensitivity that causes reacting also is subjected to bigger influence.So, need a kind of milder, the method and reagent of latex sensitization faster of reacting.
Summary of the invention
Therefore, technical purpose of the present invention is to seek a kind of milder, the method and reagent of latex sensitization faster of reacting.
Therefore, a first aspect of the present invention relates to a kind of method of latex sensitization, it is characterized in that earlier by chemical bond latex nothing to do with protein binding, re-use glutaraldehyde with antigen or antibody linked on irrelevant albumen, thereby make latex sensitization, wherein said irrelevant albumen is meant water miscible and with described antigen or antibody immunoreactive albumen does not take place, preferably, described irrelevant albumen is selected from BSA or hemocyanin, and preferably, described latex is polystyrene latex.
Preferably, described chemical bond forms by the proteic corresponding chemical group of the chemical group nothing to do with on polystyrene latex surface, and preferably, chemical group is selected from carboxyl, sulfonic group or hydroxyl.
Preferably, described antigen is selected from one or more of natural antigen, gene recombinant antigens, chemosynthesis antigen or polypeptide.
Preferably, described antibody is selected from one or more in polyclonal antibody or the monoclonal antibody.
Preferably, the particle diameter of described polystyrene latex is 50-200nm.
Preferably, the concentration of latex in reaction system does not have special restriction, preferred 0.1～2% weightmeasurement ratio, more preferably 0.5～1% weightmeasurement ratio; And/or the irrelevant concentration of albumen in reaction system does not have special restriction, preferred 0.05～2mg/ml, more preferably 0.1～0.3mg/ml; And/or reaction conditions is preferably at 4～40 ℃, more preferably at 25～37 ℃ of reaction 0.5～1hr; And/or, being reflected at and carrying out in the damping fluid of shock absorption, described damping fluid is selected from MES damping fluid, Tris damping fluid, phosphoric acid buffer, preferred MES damping fluid, pH5.5-7; And/or being combined with irrelevant proteic latex concentration does not have special restriction, preferred 0.1～2% weightmeasurement ratio, more preferably 0.2～1% weightmeasurement ratio; And/or antigen or the antibody concentration in reaction system does not have special restriction, preferred 0.05～2mg/ml, more preferably 0.1～0.2mg/ml; And/or, preferred 0.001%～0.02% weightmeasurement ratio of glutaraldehyde concentration in reaction system, more preferably 0.001～0.008% weightmeasurement ratio; And/or reaction conditions is preferably at 4～40 ℃, more preferably at 25～37 ℃ of reaction 0.5～3hr.
A second aspect of the present invention relates to a kind of latex, wherein, described latex uses the method sensitization of aforesaid latex sensitization, preferably, described latex is that the polystyrene latex of carboxyl modified utilizes carboxyl in conjunction with BSA earlier, utilizes glutaraldehyde that gene recombination streptolysin O antigen is combined in BSA then and goes up the latex that obtains.
A third aspect of the present invention relates to a kind of latex enhancing immune than turbid reagent, and wherein, described latex uses the method sensitization according to above-mentioned latex sensitization.
Preferably, described latex enhancing immune is that the antistreptolysin O (ASO) latex enhancing immune is than turbid reagent than turbid reagent, it comprises reagent R1, reagent R2, reagent R1 is the damping fluid that contains salt ion, reagent R2 the has been crosslinked antigenic sensitizing latex solution of streptolysin O, preferably, described streptolysin O antigen is gene recombination streptolysin O antigen, preferably, described latex is the polystyrene latex of carboxyl modified.
Preferably, described salt ion is one or more in monovalent metallic ion and the divalent-metal ion, and preferably, described salt ion is selected from sodium ion and/or magnesium ion, and more preferably, described salt ion is the Na ion; Preferably, the preferred 10～300mM of the concentration of described salt ion in reaction system, more preferably 100～200mM; And/or the concentration of latex in reaction system is at 0.08～0.2% weightmeasurement ratio.
Preferably, the damping fluid of described reagent R1, R2 is selected from glycine buffer, Tris damping fluid, HEPES damping fluid or phosphoric acid buffer, preferably, the damping fluid of described reagent R1, R2 is a glycine buffer, preferably, the pH of the damping fluid of described reagent R1, R2 is between 6.5～9, and more preferably, the pH of the damping fluid of described reagent R1, R2 is between 8～8.6; And/or, described reagent R1, R2 contain tensio-active agent, sanitas, polymer, preferably, described tensio-active agent is a nonionogenic tenside, and more preferably, described nonionogenic tenside is selected from TWEEN series, SPAN series, TRITON series, preferably, described sanitas is selected from one or more among potassium sorbate, Sodium Benzoate, sodium azide, Sodium Nitrite, the PC300, and preferably, described polymer is selected from PEG series, PVP series.
Preferably, described antistreptolysin O (ASO) latex enhancing immune is as follows than the composition of turbid reagent:
Reagent R1: glycine buffer concentration is 0.01-0.5M pH8.4, preferred 0.05-0.2MpH8.4, NaCl concentration is 10-600mM, preferred 100-500mM, more preferably 200-400mM, Tween 20 concentration are the 0.01%-1% weightmeasurement ratio, preferred 0.02-0.5% weightmeasurement ratio, more preferably 0.05%-0.2% weightmeasurement ratio, PEG6000 concentration is the 0.01-5% weightmeasurement ratio, preferred 0.1-3.5% weightmeasurement ratio, more preferably 0.5-3% weightmeasurement ratio, NaN
3Concentration is the 0.01%-1% weightmeasurement ratio, preferred 0.02%-0.5% weightmeasurement ratio, more preferably 0.05%-0.2% weightmeasurement ratio;
Reagent R2: glycine buffer concentration is 0.01-0.5M pH8.4, preferred 0.05-0.2MpH8.4, sensitizing latex concentration is the 0.01%-1% weightmeasurement ratio, preferred 0.2%-0.5% weightmeasurement ratio, more preferably 0.1%-0.3% weightmeasurement ratio, BSA concentration is the 0.01%-2% weightmeasurement ratio, preferred 0.1%-1% weightmeasurement ratio, more preferably 0.2%-0.6% weightmeasurement ratio, NaN
3Concentration is the 0.01%-1% weightmeasurement ratio, preferred 0.02%-0.5% weightmeasurement ratio, more preferably 0.05%-0.2% weightmeasurement ratio.
Wherein said sensitizing latex is that the polystyrene latex of carboxyl modified utilizes carboxyl in conjunction with BSA earlier, utilizes glutaraldehyde that gene recombination streptolysin O antigen is combined in BSA then and goes up acquisition.
Most preferably, described antistreptolysin O (ASO) latex enhancing immune is as follows than the composition of turbid reagent:
Reagent R1: glycine buffer 0.1M pH8.4,
NaCl 300mM，
Tween 200.1% weightmeasurement ratio,
NaN
30.1% weightmeasurement ratio,
Reagent R2: glycine buffer 0.1M pH8.4,
Sensitizing latex 0.17% weightmeasurement ratio,
BSA 0.5% weightmeasurement ratio,
NaN
30.1% weightmeasurement ratio,
Wherein said sensitizing latex is that the polystyrene latex of carboxyl modified utilizes carboxyl in conjunction with BSA earlier, utilizes glutaraldehyde that gene recombination streptolysin O antigen is combined in BSA then and goes up acquisition.
A fourth aspect of the present invention relates to a kind of latex enhancing immune than turbid test kit, wherein, described test kit used as the described latex of second aspect present invention or as the described latex enhancing immune of third aspect present invention than turbid reagent.
In other words, the invention provides a kind of method of latex sensitization, it is characterized in that earlier by chemical bond latex nothing to do with protein binding is re-used glutaraldehyde with antigen or antibody linked on irrelevant albumen, thereby makes latex sensitization.Like this, owing to taked the chemical site of elder generation in conjunction with the latex surface, crosslinked again antigen or antibody, make latex sensitization, therefore, general several hrs just can be finished reaction, saved long sealing step consuming time in the traditional method, reduced preparation time, saved time than traditional method.By adopting glutaraldehyde method crosslinked,, therefore in reaction, can keep the immunocompetence of antigen-antibody because glutaraldehyde is a kind of protein-crosslinking agent of gentleness.Simultaneously, many irrelevant proteic connections between latex and antigen or the antibody, the connection of an arm that how has been equivalent to the latex surface has increased the space divergence degree of antigen or antibody, has reduced sterically hinderedly, makes antigen antibody reaction rapider, has improved sensitivity.
Latex is meant the polystyrene latex that the surface has chemical group to modify, and chemical group can be carboxyl, hydroxyl, sulfonic group etc., and preferred latex is the polystyrene latex of carboxyl modified, and preferred particle diameter is 50-200nm.
Irrelevant albumen used in the present invention does not have special restriction, and satisfy two conditions and get final product, the one, water-soluble protein, the 2nd, not with crosslinked antigen-antibody generation immune response.Preferred irrelevant albumen is BSA, hemocyanin etc.
With latex nothing to do with albumen use chemical bond in conjunction with after, to latex carry out centrifugal, cleaning, resuspended, disperse series of steps, obtain being combined with irrelevant proteic latex.Cleaning can be used MES damping fluid, Tris damping fluid, phosphoric acid buffer etc., but is not limited thereto.
After will being combined with irrelevant proteic latex and antigen or antibody using glutaraldehyde to combine, latex is carried out centrifugal, cleaning, resuspended, dispersion series of steps, obtain sensitizing latex.Cleaning can be used MES damping fluid, Tris damping fluid, phosphoric acid buffer etc., but is not limited thereto.Resuspended liquid is glycine buffer, Tris damping fluid etc. preferably, but is not limited thereto.
The present invention also provides the latex enhancing immune that uses present method preparation than turbid reagent, and the antistreptolysin O (ASO) latex enhancing immune that particularly uses present method preparation is than turbid reagent.
Among the reagent R1 of antistreptolysin O (ASO) latex enhancing immune of the present invention than turbid reagent, described salt ion can be in monovalent metallic ion and the divalent-metal ion one or more, preferred sodium ion and magnesium ion, more preferably Na ion, preferred 10～the 300mM of its concentration in reaction system, more preferably 100～200mM.In reagent R2, the crosslinked concentration of the antigenic sensitizing latex of streptolysin O in reaction system is at 0.08～0.2% weightmeasurement ratio.
In the present invention, the damping fluid of reagent R1, R2 is glycine buffer preferably, Tris damping fluid, HEPES damping fluid, phosphoric acid buffer etc., and more preferably glycine buffer, pH is between 6.5～9, more preferably between 8～8.6.In reagent R1, R2, can also contain tensio-active agent, sanitas, polymer etc. as required.
Latex of the present invention and latex enhancing immune can be used to prepare latex enhancing immune than turbid test kit than turbid reagent.
The method of latex sensitization of the present invention is saved time with respect to prior art latex sleeves sensitization method, and detection sensitivity improves, and has strengthened the clinical value of latex enhancing immune turbidimetry.
Description of drawings
Fig. 1: adopt reagent of the present invention and commercial reagent A respectively, adopt Olympus AU400 automatic clinical chemistry analyzer, measure, measured value is carried out correlation analysis by each autoregressive parameter to 50 parts of Freshman serum (comprising normal and exceptional sample).What wherein X-axis was represented is the measured value of reagent of the present invention; What Y-axis was represented is the measured value of A reagent, coefficient R
2=0.9988, regression equation is y=0.981x+5.864.
Embodiment
To further specify the present invention by following non-limiting example below, as well known to those skilled in the art, without departing from the spirit of the invention, can make many modifications to the present invention, such modification also falls into scope of the present invention.
Following experimental technique is ordinary method if no special instructions, and employed experiment material all can easily be obtained from commercial company if no special instructions.
Embodiment
Embodiment 1: the proteic combination of latex nothing to do with
Raw material:
The polystyrene latex of carboxyl modified, particle diameter 124nm, concentration 10% (weightmeasurement ratio), surperficial carboxyl-content is 100 μ mol/g.
(analytical pure sigma) is dissolved in the distilled water EDAC, is mixed with 5mg/ml solution, preparation half an hour before using.
Use 10mM MES, the pH6.1 damping fluid is 0.5% (weightmeasurement ratio) with the latex solution dilution, adds BSA solution again, make the concentration of BSA in system reach 200ug/ml, the EDAC solution that adds 5mg/ml at last makes EDAC concentration in system reach 75ug/ml, makes the reaction beginning.Use thermostat water bath, keep 37 ℃ of temperature of reaction, reaction times 3hr.After reaction finished, centrifugal (12000rpm 30min), added initial 0.1M PBS with amount again, the pH7.4 damping fluid, ice-bath ultrasonic makes latex disperse recentrifuge, latex is scattered in 10mMTris, and in pH 8.4 (25 ℃) damping fluid, latex concentration is 0.5% (weightmeasurement ratio).
Embodiment 2: the preparation of sensitizing latex
Being combined with in the irrelevant proteic latex solution in embodiment 1 adds streptolysin O antigen.The streptolysin O antigen that uses is the gene recombination product.The concentration of antigen in system that adds is 100ug/ml.Add 2% glutaraldehyde water solution again and make the reaction beginning, the ratio of interpolation is 0.002%.25 ℃ of temperature of reaction, reaction times 3hr, after reaction finishes, centrifugal (12000rpm, 30min), add initial 0.1M glycine again, pH8.4 damping fluid, ice-bath ultrasonic with amount, latex is disperseed, recentrifuge is scattered in the 0.1M glycine that contains 0.5% (weightmeasurement ratio) BSA with latex, preserves in the pH8.4 damping fluid.
Embodiment 3: the antistreptolysin O (ASO) latex enhancing immune is than the preparation of turbid reagent
The antistreptolysin O (ASO) latex enhancing immune is specifically more composed as follows than turbid reagent:
Reagent R1: glycine buffer 0.1M pH8.4
NaCl 300mM
Tween 20 0.1% (weightmeasurement ratio)
PEG6000 1% (weightmeasurement ratio)
NaN
30.1% (weightmeasurement ratio)
Reagent R2: glycine buffer 0.1M pH8.4
Sensitizing latex (preparation among the embodiment 2) 0.17% (weightmeasurement ratio)
BSA 0.5% (weightmeasurement ratio)
NaN
30.1% (weightmeasurement ratio).
Contrast is commercially available import reagent A, and its information is as follows:
Name of product: streptococcus O detection kit (immunoturbidimetry)
Principle: the latex particle reagent react of ASO in the sample and hypersensization, form immunocomplex, detect the variation of its turbidity at wavelength 570nm place, the ASO content in its intensity of variation and the sample is proportional.
Reagent R1 ': glycine buffer
Reagent R2 ': the damping fluid that contains 0.12% (weightmeasurement ratio) sensitizing latex.
Embodiment 4: the reagent measuring method
Analytical procedure: 2 end-point methods, i.e. reagent R1: reagent R2 consumption is respectively 100ul and 170ul, sample consumption 3ul.100ul reagent R1 adds the 3ul sample, adds 170ul reagent R2 behind 37 ℃ of 5min, promptly begins reading, reads another point behind the reaction 5min, and the detection wavelength is 540nm.
Use Olympus AU400 automatic biochemistry analyzer.
Adopt the single-point calibrating method, reference liquid adopts commercial reagent A planning standard liquid, and concentration is 500IU/ml.
Commercial reagent A by specification operation.
1 correlation test
Detect 50 parts of Freshman serum (comprise normal and exceptional sample), measure, measured value is carried out correlation analysis (the results are shown in Figure 1, X, Y-axis is measured value, the IU/ml of unit) by each autoregressive parameter.Coefficient R
2=0.9988, regression equation is y=0.981x+5.864, and the result shows that this reagent and commercial reagent dependency are good, has excellent specificity and accuracy.
2 sensitivity tests
Table 1 shows the sensitivity result of reagent of the present invention and commercial reagent A, and concrete operation method is the same.
The result shows that reagent sensitivity of the present invention is better than commercial reagent A.
Table 1: reagent of the present invention and commercial reagent A sensitivity test result
This reagent lowest detectable limit (LLD)=12.5+3*1.90567=18.21701
Sensitivity: 50*18.21701/275.85=3.3IU/ml
Contrast agents lowest detectable limit (LLD)=105.15+3*24.16887=177.65661
Sensitivity: 50*177.65661/346.1=25.7IU/ml.
Embodiment 5: the antistreptolysin O (ASO) latex enhancing immune is than the preparation (2) of turbid reagent
The antistreptolysin O (ASO) latex enhancing immune is specifically more composed as follows than turbid reagent:
Reagent R1: glycine buffer 0.1M pH8.4
NaCl 200mM
Tween 20 0.05% (weightmeasurement ratio)
PEG6000 0.5% (weightmeasurement ratio)
NaN
30.1% (weightmeasurement ratio),
Reagent R2: glycine buffer 0.1M pH8.4
Sensitizing latex (preparation among the embodiment 2) 0.1% (weightmeasurement ratio)
BSA 0.5% (weightmeasurement ratio)
NaN
30.1% (weightmeasurement ratio).
The using method of this reagent is identical with the method described in the embodiment 4, after utilizing this reagent and commercial reagent A to measure 50 parts of Freshman serum, the result shows the difference (data summary) within normal limit of error between the data of described reagent of its correlation data and sensitivity data and embodiment 3.
Embodiment 6: the antistreptolysin O (ASO) latex enhancing immune is than the preparation (3) of turbid reagent
The antistreptolysin O (ASO) latex enhancing immune is specifically more composed as follows than turbid reagent:
Reagent R1: glycine buffer 0.1M pH8.4
NaCl 400mM
Tween 20 0.2% (weightmeasurement ratio)
PEG6000 3% (weightmeasurement ratio)
NaN
30.1% (weightmeasurement ratio)
Reagent R2: glycine buffer 0.1M pH8.4
Sensitizing latex (preparation among the embodiment 2) 0.3% (weightmeasurement ratio)
BSA 0.5% (weightmeasurement ratio)
NaN
30.1% (weightmeasurement ratio).
The using method of this reagent is identical with the method described in the embodiment 4, after utilizing this reagent and commercial reagent A to measure 50 parts of Freshman serum, the result shows the difference (data summary) within normal limit of error between the data of described reagent of its correlation data and sensitivity data and embodiment 3.
Claims (13)
1. the method for a latex sensitization, it is characterized in that earlier by chemical bond latex nothing to do with protein binding, re-use glutaraldehyde with antigen or antibody linked on irrelevant albumen, thereby make latex sensitization, wherein said irrelevant albumen is meant and with described antigen or antibody immunoreactive albumen does not take place that preferably, described irrelevant albumen is selected from BSA or hemocyanin water miscible, preferably, described latex is polystyrene latex.
2. the method for latex sensitization according to claim 1 is characterized in that described chemical bond forms by the proteic corresponding chemical group of the chemical group nothing to do with on polystyrene latex surface, and preferably, chemical group is selected from carboxyl, sulfonic group or hydroxyl.
3. the method for latex sensitization according to claim 1 and 2 is characterized in that described antigen is selected from one or more of natural antigen, gene recombinant antigens, chemosynthesis antigen or polypeptide.
4. according to the method for each described latex sensitization of claim 1 to 3, it is characterized in that described antibody is selected from one or more in polyclonal antibody or the monoclonal antibody.
5. according to the method for each described latex sensitization of claim 1 to 4, the particle diameter that it is characterized in that described polystyrene latex is 50-200nm.
6. according to the method for each described latex sensitization of claim 1 to 5, it is characterized in that preferred 0.1～2% weightmeasurement ratio of the concentration of latex in reaction system, more preferably 0.5～1% weightmeasurement ratio; And/or, the irrelevant preferred 0.05～2mg/ml of the concentration of albumen in reaction system, more preferably 0.1～0.3mg/ml; And/or reaction conditions is preferably at 4～40 ℃, more preferably at 25～37 ℃ of reaction 0.5～1hr; And/or, being reflected at and carrying out in the damping fluid of shock absorption, described damping fluid is selected from MES damping fluid, Tris damping fluid, phosphoric acid buffer, preferred MES damping fluid, pH5.5-7; And/or, be combined with irrelevant preferred 0.1～2% weightmeasurement ratio of proteic latex concentration, more preferably 0.2～1% weightmeasurement ratio; And/or, the preferred 0.05～2mg/ml of antigen or the antibody concentration in reaction system, more preferably 0.1～0.2mg/ml; And/or, preferred 0.001%～0.02% weightmeasurement ratio of glutaraldehyde concentration in reaction system, more preferably 0.001～0.008% weightmeasurement ratio; And/or reaction conditions is preferably at 4～40 ℃, more preferably at 25～37 ℃ of reaction 0.5～3hr.
7. latex, it is characterized in that described latex uses the method sensitization according to each described latex sensitization of claim 1 to 6, preferably, described latex is that the polystyrene latex of carboxyl modified utilizes carboxyl in conjunction with BSA earlier, utilizes glutaraldehyde that gene recombination streptolysin O antigen is combined in BSA then and goes up the latex that obtains.
8. a latex enhancing immune is characterized in that than turbid reagent described latex uses the method sensitization according to each described latex sensitization of claim 1 to 6.
9. latex enhancing immune according to claim 8 is than turbid reagent, it is characterized in that described latex enhancing immune is that the antistreptolysin O (ASO) latex enhancing immune is than turbid reagent than turbid reagent, it comprises reagent R1, reagent R2, reagent R1 is the damping fluid that contains salt ion, reagent R2 the has been crosslinked antigenic sensitizing latex solution of streptolysin O, preferably, described streptolysin O antigen is gene recombination streptolysin O antigen, preferably, described latex is the polystyrene latex of carboxyl modified.
10. latex enhancing immune according to claim 9 is than turbid reagent, it is characterized in that described salt ion is one or more in monovalent metallic ion and the divalent-metal ion, preferably, described salt ion is selected from sodium ion and/or magnesium ion, more preferably, described salt ion is the Na ion; Preferably, the preferred 10～300mM of the concentration of described salt ion in reaction system, more preferably 100～200mM; And/or the concentration of latex in reaction system is at 0.08～0.2% weightmeasurement ratio.
11. according to claim 9 or 10 described latex enhancing immunes than turbid reagent, the damping fluid that it is characterized in that described reagent R1, R2 is selected from glycine buffer, Tris damping fluid, HEPES damping fluid or phosphoric acid buffer, preferably, the damping fluid of described reagent R1, R2 is a glycine buffer, preferably, the pH of the damping fluid of described reagent R1, R2 is between 6.5～9, and more preferably, the pH of the damping fluid of described reagent R1, R2 is between 8～8.6; And/or, described reagent R1, R2 contain tensio-active agent, sanitas, polymer, preferably, described tensio-active agent is a nonionogenic tenside, and more preferably, described nonionogenic tenside is selected from TWEEN series, SPAN series, TRITON series, preferably, described sanitas is selected from one or more among potassium sorbate, Sodium Benzoate, sodium azide, Sodium Nitrite, the PC300, and preferably, described polymer is selected from PEG series, PVP series.
12. than turbid reagent, it is characterized in that described antistreptolysin O (ASO) latex enhancing immune is as follows than the composition of turbid reagent according to each described latex enhancing immune of claim 9 to 11:
Reagent R1: glycine buffer 0.01-0.5M pH8.4,
NaCl 10-600mM，
Tween 20 0.01%-1% weightmeasurement ratios,
PEG6000 0.01-5% weightmeasurement ratio,
NaN
3The 0.01%-1% weightmeasurement ratio,
Reagent R2: glycine buffer 0.01-0.5M pH8.4,
Sensitizing latex 0.01%-1% weightmeasurement ratio,
BSA 0.01%-2% weightmeasurement ratio,
NaN
3The 0.01%-1% weightmeasurement ratio,
Wherein said sensitizing latex is that the polystyrene latex of carboxyl modified utilizes carboxyl in conjunction with BSA earlier, utilizes glutaraldehyde that gene recombination streptolysin O antigen is combined in BSA then and goes up acquisition.
13. a latex enhancing immune than turbid test kit, it is characterized in that its used latex as claimed in claim 7 or as each described latex enhancing immune of claim 8 to 12 than turbid reagent.
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| CN102161716B (en) | 2014-03-05 |
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