CN109781990A - A kind of β-trace protein detection kit and preparation method - Google Patents
A kind of β-trace protein detection kit and preparation method Download PDFInfo
- Publication number
- CN109781990A CN109781990A CN201811594811.3A CN201811594811A CN109781990A CN 109781990 A CN109781990 A CN 109781990A CN 201811594811 A CN201811594811 A CN 201811594811A CN 109781990 A CN109781990 A CN 109781990A
- Authority
- CN
- China
- Prior art keywords
- reagent
- buffer
- trace protein
- trace
- stabilizer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to a kind of β-trace protein assay reagents, including reagent R1 independent of each other and reagent R2, the reagent R1 to be mainly made of buffer 1, stabilizer 1, preservative 1, EDTA, the solidifying agent of increasing and protective agent 1；The reagent R2 is mainly made of crosslinking β-trace protein antibodies polystyrene latex microspheres, buffer 2, stabilizer 2, preservative 2, protective agent 2, is wherein connected in a manner of covalent cross-linking between polystyrene latex microspheres and β-trace protein antibodies.The invention further relates to this kind of β-trace protein assay reagent preparation methods.The detection reagent has many advantages, such as that detection is sensitive, stability is high, result is accurate, can be widely applied to kidney trouble using detection kit prepared by the reagent and carries out screening detection, state of illness monitoring, prognosis evaluation etc..
Description
Technical field
The present invention relates to technical field of medical detection, and in particular to it is a kind of be used to prepare detection kidney disease agent box reagent and
Preparation method.
Background technique
Clinical evaluation kidney trouble progress and severity are generally reference with renal function, and renal function is generally with glomerulus
Filtration rate (GFR) reflection.It is the reflection most important index of renal function.According to glomerular filtration rate (GFR) marker source, divide
Exogenous and endogenous.Exogenous marker includes inulin (inulin), Iohexol (iohexol), 51Cr-EDTA, 99mTc-
DTPA etc..Endogenous marker includes serum creatinine (Scr), urea (Urea), β2-microglobulin (β 2-M), β-trace albumen
(BTP) and serumβ trace albumen (CystatinC, CysC).
In endogenous marker, such as 1 microglobulin of α, β2-microglobulin small molecular protein, because immune, infection, diet,
Influence that the kidneys such as hepatopathy and tumour other factor generates it and the index that cannot function as evaluation GFR.
The index for being clinically most commonly used to reflection detection of glomeruli filtration function variation at present is Scr and creatinine clearance rate (Ccr).
Scr measuring method is easy, but the metabolins such as glucose, uric acid, and the drugs such as cephalosporin, dopamine are likely to interference Scr and survey
It is fixed.Different eating habit and muscle capacity also will affect Scr level, to influence Scr reflection GFR sensibility and specificity.
Ccr measures sensitivity compared with Scr, but the collection of patient's urine volume is difficult to accomplish accurately, especially for the patient of renal insufficiency, since kidney is small
Pipe compensatory excretion creatinine increases, and can lead to over-evaluating for Ccr.
CysC is proved to be the index of a valuable reflection GFR, and especially equation GFR based on by creatinine is not
When accurate estimation can be provided (cirrhosis, muscle loss etc.).It is also used for the early detection of renal insufficiency simultaneously.However, CysC
Concentration influenced by the non-kidney factor such as glucocorticoid and abnormal thyroid function.
β trace albumen (beta trace protein, BTP, Hoffmann A, Nimlz M, Conradt
S.Molecular characterization of beta-trace protein in human serum and urine:a
Protential diagnostic marker for renal diease.Glycobiology, 1997,7:499-506.),
Also known as prostaglandin D synthetase is a low molecular weight protein, belongs to Lipocalin protein superfamilies member, contain
168 amino acid, molecular weight find BTP most early in 1961 in 23~29KD (its molecular size range depends on saccharification degree)
It is the major protein in human cerebrospinal fluid, while can also be measured in human serum and urine.
Research finds that serumβ-TP is related with detection of glomeruli filtration function, it can reflect the slight damage of renal function earlier
Wound is sensitiveer than clinically common serum creatinine (Scr).β-TP is not by gender, age, inflammation, weight, nutrition, sense in serum
Dye, muscle etc. influence, and are a kind of markers of comparatively ideal reaction glomerular filtration filter.
The antibody that uses of N Latex BTP kit that Siemens Company develops prepares that (mouse is anti-, rabbit-anti, sheep to be conventional
It is anti-), there is the risk with cross reactions such as rheumatoid factor in serum or HAMA, interference is brought to testing result.
The shortcomings that in view of the prior art, the present invention provide a kind of β-trace protein detection kit and preparation method thereof, should
Kit has a better detection sensitivity, linear width good stability and the small feature of interference, and the kit is at low cost, is suitable for
Clinical expansion.
Summary of the invention
In order to achieve the above objects and other related objects, the present invention provides a kind of β-trace protein assay reagent, including that
This independent reagent R1 and reagent R2, which is characterized in that the reagent R1 mainly by buffer 1, stabilizer 1, preservative 1,
EDTA, increase solidifying agent and the composition of protective agent 1；The reagent R2 is mainly micro- by crosslinking β-trace protein antibodies polystyrene latex
Ball, buffer 2, stabilizer 2, preservative 2, protective agent 2 form, wherein polystyrene latex microspheres and β-trace protein antibodies it
Between connected in a manner of covalent cross-linking.The reagent can be used for preparing the kit of detection kidney trouble.
More preferably, the β-trace protein antibodies are the anti-human β trace protein antibodies of mouse, goat-anti people β-trace protein I gG
The combination of antibody, rabbit-anti people β-trace protein I gG antibody, the anti-human β of chicken-trace protein I gY antibody one or more.
More preferably, the β-trace protein antibodies are the anti-human β of chicken-trace protein I gY antibody, have higher specificity
And affinity.Because in immunoassays, IgY cannot identify complement and people's Fc receptor in the mammalian body, will not be with class wind
The wet factor and HAMA (human anti-mouse antibody) reaction can avoid these factors and made in addition, IgY will not be combined with albumin A and G
At false positive or false negative result.
More preferably, the buffer 1 in the reagent R1 is selected from Hepes buffer, Tris-HCl buffer, MOPS buffering
Liquid, PBS buffer solution, glycine buffer, any one in borate buffer solution, the pH of buffer is 7.0~9.0, and concentration is
25~500mmol/L；Buffer 2 in the reagent R2 be selected from PBS buffer solution, borate buffer solution, glycine buffer,
Hepes buffer, GOODS buffer, any one in MOPS buffer, the pH of buffer are 7.0~9.0, concentration is 25~
500mmol/L。
More preferably, it includes following component that agent is coagulated in the increasing in the reagent R1: stachyose, alum, Fructose Diphosphate, six are partially
Sodium phosphate and glycine, and the total concentration of stachyose, alum, Fructose Diphosphate, calgon, glycine be 3.5~
7.5g/L, the pH value of buffer 1 are 7.2-7.6.
More preferably, any one or a few in KCl, NaCl, CaCl2 of the stabilizer 1 in the reagent R1, matter
Measuring concentration is 0.5%~10%；Stabilizer 2 in the reagent R2 is used cooperatively using ion stabilizer and suspension stabilizer；
Wherein ion stabilizer is any one or a few in NaCl, KCl, Na2CO3, Na2SO4 or K2SO4, suspension stabilizer
For PEG8000, sucrose, glycerol or glucose.
More preferably, the preservative 1 in the reagent R1 is Sodium azide, thimerosal or ProClin300, the reagent R2
In preservative 2 be Sodium azide, thimerosal or ProClin300；It is that PEG8000 or Portugal are poly- that agent is coagulated in increasing in the reagent R1
Sugar；Protective agent 1 in the reagent R1 is bovine serum albumin(BSA)；Protective agent 2 in the reagent R2 is bovine serum albumin(BSA).It is anti-
Rotten agent 1 and 2 refers to a kind of reagent that can inhibit bacterium and microbial contamination in reagent, has the work of antisepsis and sterilization to reagent
With.Protective agent 2 in reagent R2 is the reagent of a kind of antibody that can protect latex particle surface.Stabilizer 1 and 2 can be kept
Charge balance in reagent.Such preservative 1 and 2 has excellent antisepsis and sterilization performance.Increase the solidifying more preferable PEG8000 of agent,
It is to belong to non-ionic water-soluble polymer due to PEG8000, dissolubility in water is bigger, and adjustable reagent R1's is viscous
Degree promotes antigen and antibody molecule to be combined into compound.
More preferably, the surface functional group of the polystyrene latex microballoon in the reagent R2 is amino, carboxyl, hydrazides, aldehyde
Base or epoxy group, the partial size of polystyrene latex microballoon is in 100~600nm.It is preferred that surface functional group is the polystyrene of carboxyl
Latex microsphere, this is because polystyrene latex microspheres surface be carboxyl functional group be easy to be activated by EDC, thus quickly with
β-trace protein antibodies combine, and increase coupling effect, guarantee the stability and accuracy of test result.
More preferably, the concentration of the EDTA in the reagent R1 is 10~100mmol/L.
The present invention also provides a kind of β-trace protein assay reagent preparation methods, which is characterized in that this method includes such as
Lower step:
(1) preparation of reagent R1: stabilizer 1 being added in buffer 1, increases solidifying agent, preservative 1, protective agent 1 and EDTA,
It is uniformly mixed to get reagent R1；
(2) preparation of reagent R2:
Step 1: β-trace protein I gY antibody is subjected to 2-8 DEG C of dialysis, then is resisted β-trace protein I gY with buffer 2
Body is diluted to 0.5-2mg/ml, obtains β-trace protein I gY antibody diluent；By polystyrene latex microspheres distilled water from
The heart, washing 3 times；
Step 2: the polystyrene latex microspheres after step 1 is washed, which are diluted to mass concentration, with buffer 2 is
1%, the EDC that mass concentration is 0.01%-0.1% is added, reaction 15-45min is stirred at room temperature, after reaction
Then β-trace protein I gY antibody that step 1 obtains is added to remove unreacted EDC in 10000-15000rpm centrifuge washing
Dilution is stirred to react 15-45min at room temperature, adds terminate liquid and terminates reaction, the reaction solution 10000- that will be obtained
15000rpm centrifugation, is washed with buffer 2 and is precipitated, and repeated centrifugation is washed 3 times, is eventually adding buffer 2, preservative 2, stabilizer
2, protective agent 2 is uniformly mixed up to reagent R2.
Compared with existing detection technique or detection reagent, the invention has the following beneficial effects:
1. using latex enhancing immune turbidimetry, when the β trace albumen in blood is reacted with β trace protein antibodies, band
It has moved polystyrene latex aggregation and has generated certain turbidity, and the β trace protein content in turbidity and blood is in a certain range
It is directly proportional, it can be detected under the wavelength of 400~800nm, detection is more convenient, is easy to apply in clinic.
2. the surface functional group of polystyrene latex microspheres is amino, carboxyl, hydrazides or epoxy group etc., the official on surface
Can roll into a ball can form covalent coupling structure with combinations such as the amino of antibody surface, combine β trace protein antibodies firmly in glue
Newborn microsphere surface ensure that the stability of R2, extend reagent validity period.
3. using partial size for the bulky grain polystyrene latex particles of 100~600nm, there is biggish partial size, increase
Turbidity when β trace albumen and β trace protein antibodies react in blood, to increase the sensitivity of test reaction, shorten instead
Between seasonable.
4. being used in combination in reagent R2 using ion stabilizer and suspension stabilizer, makes the charge balance state of reagent, mention
The high stability of β trace albumen latex intensified Immunoturbidimetric kit, so that β trace albumen latex intensified immunoturbidimetry reagent
The stability of box was up to 18 months.
Specific embodiment
In order to be more clearly understood that the technology contents of the invention patent, spy lifts following embodiment and is described in detail.
The preparation process of the primary membranous nephropathy detection reagent of the invention patent is summarized as follows, it should be appreciated that these implementations
Example is only illustrative of the invention and is not intended to limit the scope of the invention.The experiment side of actual conditions is not specified in the following example
Method, usually according to normal condition, for example (,) Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring
Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.
The composition of embodiment 1: β-trace protein assay reagent
Kit citing of the invention is double reagent, in which:
Reagent R1:
Stachyose 0.8-3g/L, alum 0.1-1g/L, Fructose Diphosphate 0.8-3g/L calgon 0.05-0.5g/
L, glycine 1.5-2.25g/L, solvent are 50mmol/L PBS, PH7.4.
PEG80002.7g/L, Sodium azide 1.6g/L, bovine serum albumin(BSA) 2.2g/L, ethylenediamine tetraacetic are added in buffer 1
Acetic acid disodium 4.5g/L, sodium chloride 2.5g/L, are uniformly mixed to get reagent R1
Reagent R2:
It is coated with the anti-β of chicken-trace protein antibodies sensitization polystyrene latex particles, partial size 200nm, concentration 1%.
Calibration object: β as required-trace albumen reference calibrations product concentration distinguishes corresponding β trace protein standard substance
Above-mentioned buffer is added, one group of calibration object of various concentration is prepared.
Embodiment 2: β-trace protein assay reagent uses and effect assessment
1. reagent prepares, reagent is liquid double reagent, and corkage is used, in which:
(1) preparation of reagent R1:
Configure buffer 1: stachyose 0.8-3g/L, alum 0.1-1g/L, Fructose Diphosphate 0.8-3g/L hexa metaphosphoric acid
Sodium 0.05-0.5g/L, glycine 1.5-2.25g/L, solvent 50mmol/L, PH7.4.
PEG80002.7g/L, Sodium azide 1.6g/L, bovine serum albumin(BSA) 2.2g/L, ethylenediamine tetraacetic are added in buffer 1
Acetic acid disodium 4.5g/L, sodium chloride 2.5g/L, are uniformly mixed to get reagent R1；
(2) preparation of reagent R2:
Step 1: being diluted to 2mg/ml for β-trace protein antibodies with buffer 2, obtains β-trace protein antibodies dilution
Liquid；Polystyrene latex microspheres distilled water is centrifuged, washing 3 times；
Step 2: will be after step 1 is washed with buffer 2 (tromethamine buffer 75mmol/L, PH7.2)
It is 1% that polystyrene latex microspheres, which are diluted to mass concentration, the EDC that mass concentration is 0.01%-0.1% is added, in room temperature
Lower reaction 30min, after reaction then the β that step 1 obtains is added to remove unreacted EDC in 15000rpm centrifuge washing
Trace protein antibodies dilution, concussion reaction 30min, adds terminate liquid and terminates reaction, the reaction solution that will be obtained at room temperature
15000rpm centrifugation, is washed with buffer 2 (tromethamine buffer 75mmol/L, PH7.2) and is precipitated, repeated centrifugation washing 3
It is secondary, it is eventually adding buffer 2 (tromethamine buffer 75mmol/L, PH7.2), PEG80002.7g/L, Sodium azide is added
1.6g/L, bovine serum albumin(BSA) 2.2g/L, disodium ethylene diamine tetraacetate 4.5g/L, sodium chloride 2.5g/L, are uniformly mixed i.e.
Obtain reagent R2.
(3) preparation of calibration object: β as required-trace albumen reference calibrations product concentration is by corresponding β trace albumen mark
Quasi- product are separately added into above-mentioned buffer, and one group of calibration object of various concentration is prepared.
2. full automatic biochemical apparatus parameter setting (by taking HITACHI 7180 as an example)
(a) Detection wavelength: dominant wavelength 600nm, a length of none of complementary wave；
(b) detection temperature: 37 DEG C；
(c) reaction time: 10min, wherein the absorbance A 1 of measurement reading immediately is added after reagent R2 in incubation time 5min,
Absorbance A 2 is read after five minutes, calculates absorbance change Δ A=A2-A1；
(d) the Direction of Reaction: negative direction
3. detecting step
(a) 200ul reagent R1 and 5ul serum sample (avoiding haemolysis) is taken to mix；
(b) by the solution after mixing in 37 DEG C of incubation time 5min；
(c) 50ul reagent R2 is added, absorbance A 1 is read in measurement immediately, reads absorbance A 2 after five minutes, calculates extinction
Spend changes delta A=A2-A1.
4. calculating β in sample-trace albumen content by calibration curve.
Following tests are the performance test to 1 kit of embodiment
β-trace protein detection kit prepared by embodiment 1 is tested for the property, it is mainly tested and analyzes minimum inspection
Rising limit, accuracy, repeatability and anti-interference etc..
1) minimum detection limit: using 5%BSA normal saline solution as blank sample, blank sample should be free of measured object.
Detection 20 times is continuously repeated on Biochemical Analyzer, records testing result.Its lowest detection is limited to 0.01ug/L as the result is shown.
2) accuracy: the conventional detection sample of suitable concentration is selected, different amounts of numeraire is added into conventional sample
Product are fabricated to recycling sample, and definite value sample uses deionized water as solvent；The deionized water of same amount is added in conventional sample
It is fabricated to basic sample, the amount of the numeraire product of addition is no more than the 1/10 of total volume, is each repeated 3 times detection and takes its mean value
To recycle concentration.Average recovery rate is 98.34% as the result is shown, and accuracy is higher.
3) stability: taking β-trace protein detection kit to carry out normal storage stability test, and 2-8 DEG C of placement is pressed respectively
It was detected within time 2,4,6,8,9,10,11,12,13,14 months；Stability test uncap respectively by 2-8 DEG C of placement 0 day, 7
It, be measured within 14 days, 16 days, 18 days, 20 days, 22 days, 24 days, 26 days, 28 days, 30 days, 32 days.β trace egg as the result is shown
White detection kit is stored in 2-8 DEG C, in light protected environment, and validity period is 18 months.It uncaps and is stored in 2-8 DEG C, in light protected environment,
Validity period is 60 days.
4) anti-interference: the chaff interferent of various concentration is added in serum specimen, and distilled water is added in control group, to be added
The serum of interfering substance detects average value three times, as measured value, be added the control group of serum of distilled water detect three times it is flat
It is worth on the basis of mean value.Measured value subtracts a reference value and then obtains deviation divided by a reference value.Sample mesobilirubin≤300 μ as the result is shown
The interference of β trace protein detection kit testing result is less than when mol/L, hemoglobin≤1mg/mL, chyle≤0.30%
10%.
3 β of embodiment-trace protein assay reagent uses and effect assessment
The preparation and use of contrast agent box:
Buffer in reagent R1 uses Tris-HCl 100mmol/L, PH8.5.2, other reagents and experimental method are same
Examples 1 and 2.
Reagent R1:
Stachyose 0.8-3g/L, alum 0.1-1g/L, Fructose Diphosphate 0.8-3g/L calgon 0.05-0.5g/
L, glycine 1.5-2.25g/L, solvent are 100mmol/L Tris-HCl buffer, pH 8.5.
PEG80002.7g/L, Sodium azide 1.6g/L, bovine serum albumin(BSA) 2.2g/L, ethylenediamine tetraacetic are added in buffer 1
Acetic acid disodium 4.5g/L, sodium chloride 2.5g/L, are uniformly mixed to get reagent R1
Reagent R2:
It is coated with the anti-β of chicken-trace protein antibodies sensitization polystyrene latex particles, partial size 100nm, concentration 1%.
Calibration object:
β as required-trace albumen reference calibrations product concentration adds corresponding β-trace protein antibodies standard items respectively
Enter above-mentioned buffer, one group of calibration object of various concentration is prepared.
β-trace protein antibodies detection kit prepared by embodiment 3 is tested for the property, method is the same as embodiment 2.
1) minimum detection limit: using 5%BSA normal saline solution as blank sample, blank sample should be free of measured object.
Detection 20 times is continuously repeated on Biochemical Analyzer, records testing result.Its lowest detection is limited to 0.1ug/L as the result is shown.
2) accuracy: the conventional detection sample of suitable concentration is selected, different amounts of numeraire is added into conventional sample
Product are fabricated to recycling sample, and definite value sample uses deionized water as solvent；The deionized water of same amount is added in conventional sample
It is fabricated to basic sample, the amount of the numeraire product of addition is no more than the 1/10 of total volume, is each repeated 3 times detection and takes its mean value
To recycle concentration.Average recovery rate is 100.05% as the result is shown, and accuracy is higher.
3) stability: taking β-trace protein detection kit to carry out normal storage stability test, and 2-8 DEG C of placement is pressed respectively
It was detected within time 2,4,6,8,9,10,11,12,13,14 months；Stability test uncap respectively by 2-8 DEG C of placement 0 day, 7
It, be measured within 14 days, 16 days, 18 days, 20 days, 22 days, 24 days, 26 days, 28 days, 30 days, 32 days.β trace egg as the result is shown
White detection kit is stored in 2-8 DEG C, in light protected environment, and validity period is 15 months.It uncaps and is stored in 2-8 DEG C, in light protected environment,
Validity period is 32 days.
4) anti-interference: the chaff interferent of various concentration is added in serum specimen, and distilled water is added in control group, to be added
The serum of interfering substance detects average value three times, as measured value, be added the control group of serum of distilled water detect three times it is flat
It is worth on the basis of mean value.Measured value subtracts a reference value and then obtains deviation divided by a reference value.Sample mesobilirubin≤300 μ as the result is shown
The interference of β trace protein detection kit testing result is less than when mol/L, hemoglobin≤1mg/mL, chyle≤0.30%
15%.
4 β of embodiment-trace protein assay reagent uses and effect assessment
The preparation and use of contrast agent box:
Polystyrene latex particles in reagent R2, partial size use 200nm, other reagents and the same embodiment of experimental method
1 and 2.
Reagent R1:
Stachyose 0.8-3g/L, alum 0.1-1g/L, Fructose Diphosphate 0.8-3g/L calgon 0.05-0.5g/
L, glycine 1.5-2.25g/L, solvent are 50mmol/L PBS, pH7.4.
PEG80002.7g/L, Sodium azide 1.6g/L, bovine serum albumin(BSA) 2.2g/L, ethylenediamine tetraacetic are added in buffer 1
Acetic acid disodium 4.5g/L, sodium chloride 2.5g/L, are uniformly mixed to get reagent R1
Reagent R2:
It is coated with the anti-β of chicken-trace protein antibodies sensitization polystyrene latex particles, partial size 200nm, concentration 1%.
Calibration object:
β as required-trace albumen reference calibrations product concentration adds corresponding β-trace protein antibodies standard items respectively
Enter above-mentioned buffer, one group of calibration object of various concentration is prepared.
β-trace protein antibodies detection kit prepared by embodiment 4 is tested for the property, method is the same as embodiment 2.
1) minimum detection limit: using 5%BSA normal saline solution as blank sample, blank sample should be free of measured object.
Detection 20 times is continuously repeated on Biochemical Analyzer, records testing result.Its lowest detection is limited to 1ug/L as the result is shown.
2) accuracy: the conventional detection sample of suitable concentration is selected, different amounts of numeraire is added into conventional sample
Product are fabricated to recycling sample, and definite value sample uses deionized water as solvent；The deionized water of same amount is added in conventional sample
It is fabricated to basic sample, the amount of the numeraire product of addition is no more than the 1/10 of total volume, is each repeated 3 times detection and takes its mean value
To recycle concentration.Average recovery rate is 96.37% as the result is shown, and accuracy is higher.
3) stability: taking β-trace protein detection kit to carry out normal storage stability test, and 2-8 DEG C of placement is pressed respectively
It was detected within time 2,4,6,8,9,10,11,12,13,14 months；Stability test uncap respectively by 2-8 DEG C of placement 0 day, 7
It, be measured within 14 days, 16 days, 18 days, 20 days, 22 days, 24 days, 26 days, 28 days, 30 days, 32 days.β trace egg as the result is shown
White detection kit is stored in 2-8 DEG C, in light protected environment, and validity period is 15 months.It uncaps and is stored in 2-8 DEG C, in light protected environment,
Validity period is 26 days.
4) anti-interference: the chaff interferent of various concentration is added in serum specimen, and distilled water is added in control group, to be added
The serum of interfering substance detects average value three times, as measured value, be added the control group of serum of distilled water detect three times it is flat
It is worth on the basis of mean value.Measured value subtracts a reference value and then obtains deviation divided by a reference value.Sample mesobilirubin≤300 μ as the result is shown
The interference of β trace protein detection kit testing result is less than when mol/L, hemoglobin≤1mg/mL, chyle≤0.30%
18%.
In conclusion β of the invention-trace protein assay reagent reliable performance easy to operate, can satisfy clinical application
Requirement.
It should be noted that the foregoing is merely illustrative of the preferred embodiments of the present invention, it is not intended to restrict the invention
Range, made any modification, equivalent replacement and improvement etc., should be included in all within the spirits and principles of the present invention
Within protection scope of the present invention.
Claims (10)
1. a kind of β-trace protein assay reagent, including reagent R1 independent of each other and reagent R2, which is characterized in that the reagent
R1 is mainly made of buffer 1, stabilizer 1, preservative 1, EDTA, the solidifying agent of increasing and protective agent 1；The reagent R2 is mainly by being crosslinked
β-trace protein antibodies polystyrene latex microspheres, buffer 2, stabilizer 2, preservative 2, protective agent 2 form, wherein polyphenyl
It is connected in a manner of covalent cross-linking between ethylene latex microballoon and β-trace protein antibodies.
2. β according to claim 1-trace protein assay reagent, which is characterized in that the β-trace protein antibodies are
The anti-human β of mouse-trace protein antibodies, goat-anti people β-trace protein I gG antibody, rabbit-anti people β-trace protein I gG antibody, the anti-human β-of chicken
One or more kinds of combinations of trace protein I gY antibody.
3. β according to claim 2-trace protein assay reagent, which is characterized in that the β-trace protein antibodies are
The anti-human β of chicken-trace protein I gY antibody.
4. β according to claim 1-trace protein assay reagent, which is characterized in that the buffer 1 in the reagent R1
In Hepes buffer, Tris-HCl buffer, MOPS buffer, PBS buffer solution, glycine buffer, borate buffer solution
Any one, the pH of buffer is 7.0~9.0, and concentration is 25~500mmol/L；Buffer 2 in the reagent R2 is selected from
It is PBS buffer solution, borate buffer solution, glycine buffer, Hepes buffer, GOODS buffer, any one in MOPS buffer
Kind, the pH of buffer is 7.0~9.0, and concentration is 25~500mmol/L.
5. β according to claim 1-trace protein assay reagent, which is characterized in that agent packet is coagulated in the increasing in the reagent R1
Include following component: stachyose, alum, Fructose Diphosphate, calgon and glycine, and stachyose, alum, two phosphorus of fructose
Sour sodium, calgon, glycine total concentration be 3.5~7.5g/L, the pH value of buffer 1 is 7.2-7.6.
6. β according to claim 1-trace protein assay reagent, which is characterized in that the stabilizer 1 in the reagent R1
Any one or a few in KCl, NaCl, CaCl2, mass concentration are 0.5%~10%；It is steady in the reagent R2
Determine agent 2 to be used cooperatively using ion stabilizer and suspension stabilizer；Wherein ion stabilizer be NaCl, KCl, Na2CO3,
Perhaps any one or a few suspension stabilizer in K2SO4 is PEG8000, sucrose, glycerol or glucose to Na2SO4.
7. β according to claim 1-trace protein assay reagent, which is characterized in that the preservative 1 in the reagent R1
For Sodium azide, thimerosal or ProClin300, the preservative 2 in the reagent R2 be Sodium azide, thimerosal or
ProClin300；It is PEG8000 or glucan that agent is coagulated in increasing in the reagent R1；Protective agent 1 in the reagent R1 is ox
Seralbumin；Protective agent 2 in the reagent R2 is bovine serum albumin(BSA).
8. β according to claim 1-trace protein assay reagent, which is characterized in that the polystyrene in the reagent R2
The surface functional group of latex beads is amino, carboxyl, hydrazides, aldehyde radical or epoxy group, and the partial size of polystyrene latex microballoon is 100
~600nm.
9. β according to claim 1-trace protein assay reagent, which is characterized in that EDTA's in the reagent R1 is dense
Degree is 10~100mmol/L.
10. preparing β according to any one of claims 1 to 9-trace protein assay reagent method, which is characterized in that the party
Method includes the following steps:
(1) preparation of reagent R1: stabilizer 1 is added in buffer 1, increases solidifying agent, preservative 1, protective agent 1 and EDTA, stirring
It is uniformly mixed to get reagent R1；
(2) preparation of reagent R2:
Step 1: β-trace protein I gY antibody is subjected to 2-8 DEG C of dialysis, then with buffer 2 that β-trace protein I gY antibody is dilute
It releases to 0.5-2mg/ml, obtains β-trace protein I gY antibody diluent；By the centrifugation of polystyrene latex microspheres distilled water, wash
It washs 3 times；
Step 2: it is 1% that the polystyrene latex microspheres after step 1 is washed, which are diluted to mass concentration, with buffer 2,
The EDC that mass concentration is 0.01%-0.1% is added, reaction 15-45min is stirred at room temperature, after reaction 10000-
Then β-trace protein I gY antibody dilution that step 1 obtains is added to remove unreacted EDC in 15000rpm centrifuge washing
Liquid is stirred to react 15-45min at room temperature, add terminate liquid terminate reaction, by obtained reaction solution 10000-15000rpm from
The heart is washed with buffer 2 and is precipitated, and repeated centrifugation is washed 3 times, is eventually adding buffer 2, preservative 2, stabilizer 2, protective agent 2
It is uniformly mixed up to reagent R2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811594811.3A CN109781990A (en) | 2018-12-25 | 2018-12-25 | A kind of β-trace protein detection kit and preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811594811.3A CN109781990A (en) | 2018-12-25 | 2018-12-25 | A kind of β-trace protein detection kit and preparation method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109781990A true CN109781990A (en) | 2019-05-21 |
Family
ID=66497704
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811594811.3A Pending CN109781990A (en) | 2018-12-25 | 2018-12-25 | A kind of β-trace protein detection kit and preparation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109781990A (en) |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0913405A1 (en) * | 1995-10-31 | 1999-05-06 | Maruha Corporation | Monoclonal antibody specific for prostaglandin d synthetase |
| CN1675543A (en) * | 2002-08-16 | 2005-09-28 | 安万特药物公司 | Method for assaying compounds or agents for ability to decrease the activity of microsomal prostaglandin E synthase or hematopoietic prostaglandin D synthase |
| CN101163971A (en) * | 2004-12-20 | 2008-04-16 | 免疫体公司 | Determination of neutrophil gelatinase-associated lipocalin (ngal) as a diagnostic marker for renal disorders |
| CN101937000A (en) * | 2010-08-05 | 2011-01-05 | 北京倍爱康生物技术有限公司 | Magnetic particle separation chemiluminescence immunoassay method of human cystatin C |
| CN102662061A (en) * | 2012-04-17 | 2012-09-12 | 北京九强生物技术股份有限公司 | Kit for determination of human alpha-fetoprotein content by latex-enhanced immunoturbidimetry |
| WO2012154889A1 (en) * | 2011-05-09 | 2012-11-15 | The Cleveland Clinic Foundation | Serum s100b and uses thereof |
| CN104360081A (en) * | 2014-12-05 | 2015-02-18 | 重庆中元生物技术有限公司 | Improved cystatin C detection kit |
| CN104407156A (en) * | 2014-12-05 | 2015-03-11 | 重庆中元生物技术有限公司 | Latex enhanced immunoturbidimetry kit for detecting lipopolysaccharide binding protein (LBP) |
| CN106093433A (en) * | 2016-06-17 | 2016-11-09 | 上海执诚生物科技有限公司 | Microalbumin test kit based on latex immunoturbidimetry and preparation method thereof |
| WO2017009869A1 (en) * | 2015-07-14 | 2017-01-19 | Nanosniff Technologies Pvt. Ltd. | Process for immobilizing one or more receptor biomolecules on one or more solid surfaces |
| CN107422131A (en) * | 2017-07-28 | 2017-12-01 | 江苏龙维生物科技有限公司 | Soluble ST2 detection method and detection kit |
-
2018
- 2018-12-25 CN CN201811594811.3A patent/CN109781990A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0913405A1 (en) * | 1995-10-31 | 1999-05-06 | Maruha Corporation | Monoclonal antibody specific for prostaglandin d synthetase |
| CN1675543A (en) * | 2002-08-16 | 2005-09-28 | 安万特药物公司 | Method for assaying compounds or agents for ability to decrease the activity of microsomal prostaglandin E synthase or hematopoietic prostaglandin D synthase |
| CN101163971A (en) * | 2004-12-20 | 2008-04-16 | 免疫体公司 | Determination of neutrophil gelatinase-associated lipocalin (ngal) as a diagnostic marker for renal disorders |
| CN101937000A (en) * | 2010-08-05 | 2011-01-05 | 北京倍爱康生物技术有限公司 | Magnetic particle separation chemiluminescence immunoassay method of human cystatin C |
| WO2012154889A1 (en) * | 2011-05-09 | 2012-11-15 | The Cleveland Clinic Foundation | Serum s100b and uses thereof |
| CN102662061A (en) * | 2012-04-17 | 2012-09-12 | 北京九强生物技术股份有限公司 | Kit for determination of human alpha-fetoprotein content by latex-enhanced immunoturbidimetry |
| CN104360081A (en) * | 2014-12-05 | 2015-02-18 | 重庆中元生物技术有限公司 | Improved cystatin C detection kit |
| CN104407156A (en) * | 2014-12-05 | 2015-03-11 | 重庆中元生物技术有限公司 | Latex enhanced immunoturbidimetry kit for detecting lipopolysaccharide binding protein (LBP) |
| WO2017009869A1 (en) * | 2015-07-14 | 2017-01-19 | Nanosniff Technologies Pvt. Ltd. | Process for immobilizing one or more receptor biomolecules on one or more solid surfaces |
| CN106093433A (en) * | 2016-06-17 | 2016-11-09 | 上海执诚生物科技有限公司 | Microalbumin test kit based on latex immunoturbidimetry and preparation method thereof |
| CN107422131A (en) * | 2017-07-28 | 2017-12-01 | 江苏龙维生物科技有限公司 | Soluble ST2 detection method and detection kit |
Non-Patent Citations (2)
| Title |
|---|
| GUIDO FILLER等: ""β-Trace Protein, Cystatin C,β2-Microglobulin, and Creatinine Compared for Detecting Impaired Glomerular Filtration Rates in Children"", 《CLINICAL CHEMISTRY》 * |
| 舒桂华 等: ""血清γ-痕迹蛋白对新生儿ABO溶血症肾功能损害的诊断价值"", 《临床儿科杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104360081B (en) | Cystatin C detection kit of a kind of improvement and preparation method thereof | |
| CN102759631B (en) | The latex enhancing immune of a kind of quantitative detection Procalcitonin PCT is than turbid kit | |
| CN101377492B (en) | Bladder chalone C determining reagent kit | |
| CN105510604A (en) | Method for improving sensitivity and linearity of latex reagent | |
| CN106932594A (en) | A kind of preferred cystatin C detection kit and preparation method thereof | |
| CN103823070A (en) | Cystatin C determination kit with high sensitivity | |
| CN102636653A (en) | Compounded latex particle-enveloped cystatin C detection kit | |
| CN105738617A (en) | Cystatin C latex-particle-enhanced turbidimetry detection reagent kit and application thereof | |
| CN103852584A (en) | Latex immune enhancement turbidimetric kit for detecting peptide C quantitatively | |
| CN111239418A (en) | Kit for determining ANA based on latex enhanced immunoturbidimetry and preparation and use methods thereof | |
| CN104535770A (en) | Myoglobin determination kit of compound antibody | |
| CN101699287A (en) | Homogeneous phase sol particle type cystatin C measuring kit and preparation method thereof | |
| CN105911298A (en) | Kit for determining myoglobin | |
| CN110716057A (en) | Kit for determining HBP (hepatitis B protein) based on latex enhanced immunoturbidimetry and preparation and use methods thereof | |
| CN106970226A (en) | The kit of the ECD concentration levels of HER 2 in a kind of measure human serum | |
| CN108627652B (en) | It is a kind of to detect based on simple grain diameter and simultaneously the kit of RBP ELISA in serum and urine specimen | |
| CN107228940A (en) | The detection reagent and method of a kind of bladder chalone C | |
| Van Lente et al. | Evaluation of a nephelometric assay for haptoglobin and its clinical usefulness. | |
| CN104459105A (en) | Latex-enhanced immunoturbidimetric assay kit for detecting leptin (LP) | |
| CN106053840A (en) | Kit for measuring alpha1-microglobulin and preparation method of kit | |
| CN107462729B (en) | A kind of Apolipoprotein A1 detection kit and detection method | |
| CN107942068A (en) | β2Microglobulin assay kit | |
| CN111190003A (en) | Retinol binding protein detection kit and preparation method thereof | |
| CN109781990A (en) | A kind of β-trace protein detection kit and preparation method | |
| CN107918020A (en) | Neutrophil gelatinase-associated lipocalin assay kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190521 |