CN107966567B - A kind of haptoglobin assay kit - Google Patents
A kind of haptoglobin assay kit Download PDFInfo
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- CN107966567B CN107966567B CN201711161909.5A CN201711161909A CN107966567B CN 107966567 B CN107966567 B CN 107966567B CN 201711161909 A CN201711161909 A CN 201711161909A CN 107966567 B CN107966567 B CN 107966567B
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention belongs to field of biotechnology, and in particular to Protein Assay Kit is more particularly to a kind of haptoglobin measurement reagent.The haptoglobin assay kit stated includes the reagent 1 and reagent 2 of independent packaging.The formula of mentioned reagent 1 includes: 50-150mM TRIS buffer, 3-12g/L polyoxyethylene laurel ether -35,3-8g/L bovine serum albumin(BSA), 4-8g/L sodium chloride and 0.1-0.3mL/L liquid bio preservative.It further include reinforcing agent in mentioned reagent 1 and reagent 2, after reinforcing agent is added, the stability of reagent is greatly improved.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to Protein Assay Kit is more particularly to a kind of touching pearl egg
White measurement reagent.
Background technique
Haptoglobin is also known as hoptoglobin, is a kind of acute phase protein, synthesizes in liver cell, is by two light chains (α
Chain) and two heavy chains (β chain) composition glycoprotein.Haptoglobin can be in conjunction with free hemoglobin caused by haemolysis, and is transported to
Liver cell is degraded.When haemolysis occurs, since than liver to synthesize new haptoglobin speed faster for its removing speed, so
Any the case where capable of making erythroclasis that hemoglobin be caused to increase, can all be such that haptoglobin concentration in serum reduces.Hemolytic
Concentration significantly reduces when anaemia, hepatocellular damage；Concentration is significantly raised when inflammation, wound and coronary heart disease.Therefore haptoglobin
Measurement has important biology and medical significance.Haptoglobin antibody can generate agglutinating reaction, shape with the haptoglobin in serum
At antigen antibody complex, turbidity height is directly proportional to haptoglobin in serum in the presence of a certain amount of antibody.Pass through measurement
The absorbance value of specific wavelength can calculate the concentration of haptoglobin in serum referring to calibration curve.
In one level, appear on the market at present or disclosed haptoglobin detection reagent or the type of kit and few.One side
The range that face select user can is narrow；On the other hand, the product that appears on the market means that market competition is small less, and price is high,
And then customer using cost is high；Another aspect, but also the product of this type be difficult to it is competing by active market
It strives to form effective technology promotion, so that the type product is technically for a long time without new development.
In another level, just at present from the point of view of published haptoglobin measurement product, although product category is insufficient, its
Serious technical problem has been exposed in practical applications.For example, State Food and Drug Administration is in November, 2012
Issue within 26th: Ortho-Clinical Diagnostics, Inc company is to haptoglobin assay kit (immunoturbidimetry)
Recalled, due to user using when frequently occur sample treatment and the problem of can not obtain measurement result, it is given birth to by the said firm
The haptoglobin assay kit (immunoturbidimetry) of some models batch of production (number of registration: eat medicine and supervise tool (into) word 2010 by state
No. 2402877) actively recall.Although specifically recalling without reference to Chinese market, but still current touching pearl is reflected
There are certain technological deficiency and unstability for protein assay reagent (box).
There are also haptoglobin measurement products to appear on the market for the country of China at present.Such as Zhejiang Kangte Biotech Co., Ltd.
(commercially available 1) or Ningbo Ruiyuan Biotechnology Co., Ltd. (the haptoglobin assay kit (immunoturbidimetry) of commercially available 2) production,
Both kits supply the content of haptoglobin in external quantitative detection human serum.The product condition of storage of both kits
And validity period is equal are as follows: (drying is protected from light) storage under the conditions of 2-8 DEG C, validity period 12 months.Through actual verification, both kits
After unlatching, reagent therein is kept in dark place at 2-8 DEG C at most to be stablized 30 days, and calibration object therein is kept in dark place most at 2-8 DEG C
It is multistable 7 days fixed.Have commercially available haptoglobin measurement product sealing validity period even only 6 months less than.And a product from
Factory, to undergoing dealers at different levels, then to hospital or other use mechanism, circulation experienced or many situations of haulage time
It is up to the several months long down.That is mentioned reagent box its practical validity period when reaching in user hand has greatly shortened,
Along with reagent once opens only a couple of days its stationary phase, most dozens of days.The above reason will lead to a large amount of postmature deliverys before breaking a seal
Fail product after product or Kaifeng, and theoretically these expired or failure products should be discarded or recycled, but one actually therein
Divide and is still mingled in circulating pathway.This aspect causes the significant wastage of resource, on the other hand causes the confusion in market, more
Notably these expired failure products are once used for tester and are likely to cause the measurement result of mistake so as to cause serious medical thing
Therefore.
Although technical staff can not participate in the management of commodity circulation, can technological layer reduce as far as possible
State a possibility that happening.Therefore, improving the stability of haptoglobin measurement product, simplifying its preservation condition should be the neck
Research staff endeavours an important directions of research.
The newest technology innovation that can be retrieved at present is a kind of touching disclosed in Chinese patent application 201510407469
Globin assay kit, however the emphasis of the invention is the mechanical realization of kit, discloses only haptoglobin inspection
Part conventional reagent in survey, such as polyethylene glycol, phosphate buffer, sheep anti-human haptoglobin antibody, bovine serum albumin(BSA)
Deng.And any new approaches are not provided to the stability for how improving detection kit.
In conclusion how to further increase the stability of haptoglobin measurement reagent (box), sensitivity, accuracy
It can be the research direction that this field merits attention.
Summary of the invention
To make up the deficiencies in the prior art, the present invention provides a kind of haptoglobin assay kits.With prior art phase
Than the performances such as accuracy, sensitivity of the kit are improved to some extent, while the stability of the kit obtains substantially
It improves, preservation condition is simplified.
The technical solution adopted by the present invention are as follows:
A kind of haptoglobin assay kit, it is characterised in that: the haptoglobin assay kit includes independent packet
The reagent 1 and reagent 2 of dress.
The formula of mentioned reagent 1 includes: 50-150mM TRIS buffer, the 3-12g/L polyoxyethylene moon
Osmanthus ether -35,3-8g/L bovine serum albumin(BSA), 4-8g/L sodium chloride and 0.1-0.3mL/L liquid bio preservative.
Preferably, the formula of mentioned reagent 1 includes: 70-130mM TRIS buffer, 5-11g/L polyoxy
Ethylene laurel ether -35,4-7g/L bovine serum albumin(BSA), 5-7g/L sodium chloride and 0.1-0.3mL/L liquid bio preservative.
It is further preferred that the formula of mentioned reagent 1 includes: 80-110mM TRIS buffer, 7-
10g/L polyoxyethylene laurel ether -35,4-6g/L bovine serum albumin(BSA), 5-6g/L sodium chloride and 0.1-0.3mL/L liquid bio are anti-
Rotten agent.
In a preferred embodiment, the formula of mentioned reagent 1 includes: 100mM trishydroxymethylaminomethane buffering
Liquid, 10g/L polyoxyethylene laurel ether -35,5g/L bovine serum albumin(BSA), 5.85g/L sodium chloride and 0.2mL/L liquid bio anti-corrosion
Agent.
In another preferred embodiment, the formula of mentioned reagent 1 includes: 90mM trishydroxymethylaminomethane buffering
Liquid, 8.5g/L polyoxyethylene laurel ether -35,4.5g/L bovine serum albumin(BSA), 5.35g/L sodium chloride and 0.1mL/L liquid bio
Preservative.
The formula of mentioned reagent 2 includes: 50-150mM TRIS buffer, 10-30mL/L goat-anti people touching
Globin antibodies, 3-8g/L bovine serum albumin(BSA) and 0.1-0.3mL/L liquid bio preservative.
Preferably, the formula of mentioned reagent 2 includes: 70-130mM TRIS buffer, 15-25mL/L sheep
Anti-human haptoglobin antibody, 4-7g/L bovine serum albumin(BSA) and 0.1-0.3mL/L liquid bio preservative.
It is further preferred that the formula of mentioned reagent 2 includes: 80-110mM TRIS buffer, 18-
20mL/L sheep anti-human haptoglobin antibody, 4-6g/L bovine serum albumin(BSA) and 0.1-0.3mL/L liquid bio preservative.
In a preferred embodiment, the formula of mentioned reagent 2 includes: 100mM trishydroxymethylaminomethane buffering
Liquid, 20mL/L sheep anti-human haptoglobin antibody, 5g/L bovine serum albumin(BSA) and 0.2mL/L liquid bio preservative.
In another preferred embodiment, the formula of mentioned reagent 2 includes: 80mM trishydroxymethylaminomethane buffering
Liquid, 20mL/L sheep anti-human haptoglobin antibody, 4g/L bovine serum albumin(BSA) and 0.2mL/L liquid bio preservative.
The liquid bio preservative being related in the present invention can selected from ProClin150, ProClin200 or
Any one in ProClin300；It preferably is selected from any one in ProClin200 or ProClin300.
Except it is expected that, after reinforcing agent is added in mentioned reagent 1 or reagent 2, not only make touching provided by the invention
The detection accuracy of globin assay kit and sensitivity slightly improve, it is often more important that substantially increase its stability, make
Kit preservation condition is obtained to simplify.
Above-mentioned reinforcing agent can be carbohydrate；The carbohydrate is trisaccharide；The trisaccharide is preferably gossypose or its derivative
Object.
Gossypose (Raffinose) be widely present in nature, it is fragrant such as in the vegetables such as beet, potato, cabbage
In the fruit such as any of several broadleaf plants, grape, the cereal such as rice, wheat are medium.Gossypose is combined by galactolipin, fructose and glucose, is
A kind of functional oligose.
Gossypose has effects that adjust or strengthen immunity, moisturizing beauty, adjust the functions such as blood pressure or, therefore answered extensively
Field for food, medicine, cosmetics etc..
Present invention discovery also unexpected in implementation process, when gossypose be present in certain content range it is above-mentioned
When in reagent 1 or reagent 2, relatively better reinforcing effect can be played, this content range is 0.5-3g/L, preferably 0.7-
1.9g/L, further preferably 0.9-1.3g/L；When its content is less than 0.5g/L, do not observe to reagent 1 or reagent 2
There is apparent reinforcing effect, thus it is speculated that a kind of possible reason is point of the gossypose in reagent when its content is less than 0.5g/L
Subnumber amount is not enough to be formed effective intermolecular contacts, so that effective reinforcing effect can not be played；When its content is greater than 3g/L
When, there is apparent reinforcing effect to reagent 1 or reagent 2 also without observing, thus it is speculated that a kind of possible reason is when its content
When greater than 3g/L, the molecular amounts in reagent are excessive, influence other formula (such as liquid bio preservative) molecules and exist
Interaction in reagent, to can not also play effective reinforcing effect.
It may occur to persons skilled in the art that other with gossypose there is the trisaccharide of same or similar molecular structure to answer
For can also play above-mentioned technical effect in the present invention, therefore should be contained within the scope of protection of present invention.
Therefore, as a preferred embodiment, haptoglobin assay kit of the present invention includes independent packet
The reagent 1 and reagent 2 of dress；The formula of the reagent 1 includes: 50-150mM TRIS buffer, 3-12g/L
Polyoxyethylene laurel ether -35,3-8g/L bovine serum albumin(BSA), 4-8g/L sodium chloride, 0.1-0.3mL/L liquid bio preservative and
0.5-3g/L gossypose；The formula of the reagent 2 includes: 50-150mM TRIS buffer, 10-30mL/L
Sheep anti-human haptoglobin antibody, 3-8g/L bovine serum albumin(BSA), 0.1-0.3mL/L liquid bio preservative and 0.5-3g/L cotton seed
Sugar.
Further, haptoglobin assay kit provided by the invention further includes calibration object, and the calibration object includes
Independent packaging loads in mixture haptoglobin, sodium chloride, bovine serum albumin(BSA), sucrose and liquid biological preservative together.
Therefore, as another preferred embodiment, haptoglobin assay kit of the present invention includes independent
Reagent 1, reagent 2 and the calibration object of packaging；The formula of the reagent 1 includes: 50-150mM trishydroxymethylaminomethane buffering
Liquid, 3-12g/L polyoxyethylene laurel ether -35,3-8g/L bovine serum albumin(BSA), 4-8g/L sodium chloride, 0.1-0.3mL/L liquid are raw
Object preservative and 0.5-3g/L gossypose；The formula of the reagent 2 includes: 50-150mM trishydroxymethylaminomethane buffering
Liquid, 10-30mL/L sheep anti-human haptoglobin antibody, 3-8g/L bovine serum albumin(BSA), 0.1-0.3mL/L liquid bio preservative and
0.5-3g/L gossypose；The calibration object includes that independent packaging or the haptoglobin, sodium chloride, the ox blood that load in mixture together are pure
Albumen, sucrose and liquid biological preservative.
The application method of haptoglobin assay kit provided by the invention is preferred are as follows:
(1) basic parameter:
1) reaction type: end-point method；2) reaction temperature: 37 DEG C；3) dominant wavelength: 340nm；4) secondary wavelength: 700nm；5) sample
This amount: 3 μ l；6) reagent 1 (R1) is measured: 240 μ l；7) reagent 2 (R2) is measured: 60 μ l；8) the Direction of Reaction: reaction is risen；
(2) step: reagent 1 being added into sample, and reaction measures absorbance after five minutes and is calculated as A1；Reagent 2 is added,
Reaction measures absorbance after five minutes and is calculated as A2；Absorbance change value: △ A=A2-A1 is calculated as follows；With the suction of calibration object
Light varience value △ A is ordinate, and corresponding concentration C is that abscissa draws calibration curve, bent in calibration according to the △ A of sample
Haptoglobin concentration in corresponding sample is found out on line.
In above-mentioned application method, the type of sample is serum, preferably Diagnostic Value of Fasting Serum.
All formula ranges being related in the present invention include all integers and decimal in the range, such as 3-12g/
L polyoxyethylene laurel ether -35 includes all decimal g/L polyoxyethylene moons between 3,4,5,6,7,8,9,10,11,12 or 3-12
Osmanthus ether -35.
Specific embodiment
The explanation of following embodiment is merely used to help understand method and its core concept of the invention.It should be pointed out that pair
For those skilled in the art, without departing from the principle of the present invention, the present invention can also be carried out
Some improvements and modifications, these improvements and modifications also fall within the scope of protection of the claims of the present invention.To disclosed implementation
The following the description of example, enables those skilled in the art to implement or use the present invention.Various modifications to these embodiments
It will be readily apparent to those skilled in the art, the general principles defined herein can not depart from this
In the case where the spirit or scope of invention, realize in other embodiments.Therefore, the present invention is not intended to be limited to illustrated herein
These embodiments in, but can be applied to meet broader model consistent with the principles and novel features disclosed in this article
It encloses.
Unless otherwise defined, all technical and scientific terms used herein have and the technical field of the invention
The normally understood identical meaning of those of ordinary skill.
It is such as not particularly illustrated, substance (including organic substance and inorganic substances) involved in the present invention is to pass through business
Approach obtains.
A kind of haptoglobin assay kit of embodiment 1
Reagent 1, reagent 2 and calibration object comprising independent packaging.
Wherein, calibration object include the haptoglobin of independent packaging, sodium chloride, bovine serum albumin(BSA), sucrose and
Proclin300。
Wherein, the formula of reagent 1 and reagent 2 is as shown in the table:
A kind of haptoglobin assay kit of embodiment 2
Reagent 1, reagent 2 and calibration object comprising independent packaging.
Wherein, calibration object include the haptoglobin of independent packaging, sodium chloride, bovine serum albumin(BSA), sucrose and
Proclin300。
Wherein, the formula of reagent 1 and reagent 2 is as shown in the table:
A kind of haptoglobin assay kit of embodiment 3
Reagent 1, reagent 2 and calibration object comprising independent packaging.
Wherein, calibration object include the haptoglobin of independent packaging, sodium chloride, bovine serum albumin(BSA), sucrose and
Proclin300。
Wherein, the formula of reagent 1 and reagent 2 is as shown in the table:
Such as it is not particularly illustrated, in the formula of the embodiment or comparative example that are related in embodiment of the present invention, addition
The mother liquid concentration of sheep anti-human haptoglobin antibody is 20mg/mL.
A kind of haptoglobin assay kit of embodiment 4
The present embodiment and embodiment 3 the difference is that: further include 0.5g/ in the reagent 1 of the present embodiment and the formula of reagent 2
The gossypose of L, remaining is the same as embodiment 3.
A kind of haptoglobin assay kit of embodiment 5
The present embodiment and embodiment 3 the difference is that: further include 3g/L in the reagent 1 of the present embodiment and the formula of reagent 2
Gossypose, remaining is the same as embodiment 3.
A kind of haptoglobin assay kit of embodiment 6
The present embodiment and embodiment 3 the difference is that: further include 1g/L in the reagent 1 of the present embodiment and the formula of reagent 2
Gossypose, remaining is the same as embodiment 3.
A kind of haptoglobin assay kit of embodiment 7
The present embodiment and embodiment 6 the difference is that: will be in the reagent 1, reagent 2 and calibration object of embodiment 6
Proclin300 replaces with Proclin200, remaining is the same as embodiment 6.
A kind of haptoglobin assay kit of embodiment 8
The present embodiment and embodiment 6 the difference is that: will be in the reagent 1, reagent 2 and calibration object of embodiment 6
Proclin300 replaces with Proclin150, remaining is the same as embodiment 6.
A kind of haptoglobin assay kit of embodiment 9
The present embodiment and embodiment 6 the difference is that: by the trihydroxy methyl amino first in the reagent 1 and reagent 2 of embodiment 6
The concentration of alkane buffer is changed to 70mM, remaining is the same as embodiment 6.
A kind of haptoglobin assay kit of comparative example 1
The present embodiment and embodiment 6 the difference is that: gossypose is dense in the reagent 1 of the present embodiment and the formula of reagent 2
Degree is 0.4g/L's, remaining is the same as embodiment 6.
A kind of haptoglobin assay kit of comparative example 2
The present embodiment and embodiment 6 the difference is that: gossypose is dense in the reagent 1 of the present embodiment and the formula of reagent 2
Degree is 3.5g/L's, remaining is the same as embodiment 6.
Performance test: in above-described embodiment and comparative example reagent 1 and reagent 2 make a service test
1, haptoglobin pattern detection above-described embodiment of known concentration and the linear model of comparative example the range of linearity: are utilized
It encloses, as a result as follows:
| Example | The range of linearity | Linearly dependent coefficient (r) |
| Embodiment 1 | 0.5-2.7g/L | ≥0.9900 |
| Embodiment 2 | 0.3-2.8g/L | ≥0.9900 |
| Embodiment 3 | 0.2-3.0g/L | ≥0.9900 |
| Embodiment 4 | 0.1-3.0g/L | ≥0.9900 |
| Embodiment 5 | 0.2-3.3g/L | ≥0.9900 |
| Embodiment 6 | 0.05-4.0g/L | ≥0.9999 |
| Embodiment 7 | 0.2-3.5g/L | ≥0.9900 |
| Embodiment 8 | 0.1-3.4g/L | ≥0.9900 |
| Embodiment 9 | 0.2-3.5g/L | ≥0.9900 |
| Comparative example 1 | 0.1-3.5g/L | ≥0.9900 |
| Comparative example 2 | 0.1-3.0g/L | ≥0.9900 |
2, stability
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (6)
1. a kind of haptoglobin assay kit, it is characterised in that: the haptoglobin assay kit includes independent packaging
Reagent 1 and reagent 2；The formula of the reagent 1 includes: 50-150mM TRIS buffer, 3-12g/L poly-
Ethylene oxide laurel ether -35,3-8g/L bovine serum albumin(BSA), 4-8g/L sodium chloride, 0.1-0.3mL/L liquid bio preservative and
0.5-3g/L gossypose；The formula of the reagent 2 includes: 50-150mM TRIS buffer, 10-30mL/L
Sheep anti-human haptoglobin antibody, 3-8g/L bovine serum albumin(BSA), 0.1-0.3mL/L liquid bio preservative and 0.5-3g/L cotton seed
Sugar.
2. haptoglobin assay kit as described in claim 1, it is characterised in that: the liquid bio preservative is selected from
Any one in ProClin150, ProClin200 or ProClin300.
3. haptoglobin assay kit as claimed in claim 2, it is characterised in that: the liquid bio preservative is selected from
Any one in ProClin200 or ProClin300.
4. haptoglobin assay kit as claimed in claim 3, it is characterised in that: the liquid bio preservative is
ProClin300。
5. haptoglobin assay kit as described in claim 1, it is characterised in that: the formula of the reagent 1 includes:
100mM TRIS buffer, 10g/L polyoxyethylene laurel ether -35,5g/L bovine serum albumin(BSA), 5.85g/L chlorine
Change sodium, 0.2mL/L liquid bio preservative and 1g/L gossypose；The formula of the reagent 2 includes: 100mM trihydroxy methyl ammonia
Methylmethane buffer, 20mL/L sheep anti-human haptoglobin antibody, 5g/L bovine serum albumin(BSA), 0.2mL/L liquid bio preservative
With 1g/L gossypose.
6. the haptoglobin assay kit as described in claim 1-4 any one, it is characterised in that: the haptoglobin
Assay kit includes reagent 1, reagent 2 and the calibration object of independent packaging；The formula of the reagent 1 includes: 50-150mM tri-
Hydroxymethyl aminomethane buffer, 3-12g/L polyoxyethylene laurel ether -35,3-8g/L bovine serum albumin(BSA), 4-8g/L chlorination
Sodium, 0.1-0.3mL/L liquid bio preservative and 0.5-3g/L gossypose；The formula of the reagent 2 includes: 50-150mM tri-
Hydroxymethyl aminomethane buffer, 10-30mL/L sheep anti-human haptoglobin antibody, 3-8g/L bovine serum albumin(BSA), 0.1-
0.3mL/L liquid bio preservative and 0.5-3g/L gossypose；The calibration object includes independent packaging or loads in mixture together
Haptoglobin, sodium chloride, bovine serum albumin(BSA), sucrose and liquid biological preservative.
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| CN116298317A (en) * | 2023-03-14 | 2023-06-23 | 浙江夸克生物科技有限公司 | Alpha fetoprotein determination kit based on latex immunoturbidimetry |
| CN117368466B (en) * | 2023-12-08 | 2024-03-12 | 安徽惠邦生物工程有限公司 | Kit for measuring urine binding globin by using biochemical analyzer and preparation method thereof |
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