CN107102153A - LP(a) priority double reagent assay method in serum - Google Patents
LP(a) priority double reagent assay method in serum Download PDFInfo
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- CN107102153A CN107102153A CN201710353228.2A CN201710353228A CN107102153A CN 107102153 A CN107102153 A CN 107102153A CN 201710353228 A CN201710353228 A CN 201710353228A CN 107102153 A CN107102153 A CN 107102153A
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6806—Determination of free amino acids
- G01N33/6812—Assays for specific amino acids
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Abstract
The invention discloses a kind of priority double reagent assay method of LP(a) in serum, belong to and utilize visible ray, color change is produced come the method for test material by the result of test reaction.The technical scheme is that：LP(a) forms antigen antibody complex in 3~5 minutes with determining antibody in reagent in 37 DEG C of warm bath in serum, reaction solution turbidity is occurred, then add LP(a) and determine reagent, when LP(a) is superfluous in reaction solution, to occur reaction peak again, and show LP(a) antigen excess；When there is power, then show that reaction has reached terminal, instrument is detected at 334~340nm wavelength, the content that the turbidity produced calculates LP(a) is reacted with the first step.Antigen excess phenomenon is can interpolate that during present invention detection, it is economical convenient and easy, it is a kind of higher LP(a) detection method of accuracy.
Description
Technical field
The invention belongs to a kind of LP(a) immunoturbidimetry assay method；Or visible ray is utilized, pass through the knot of test reaction
Fruit produces the method that color change carrys out test material, more particularly to a kind of to detect LP(a) in serum with Biochemical Analyzer
Priority double reagent assay method.
Background technology
LP(a) (LPa) is the independent hazard factor of cardiovascular and cerebrovascular diseases, and it is in atherosclerosis (AS) and thrombosis
In play important the role as a bridge and a link, LPa has obvious polymorphism, due to the particularity of its structure so that at present survey
The result determined between LPa distinct methods, different reagents, different system lacks comparativity, it need to be determined and is standardized.Survey
Determine LPa immunochemistry detection method, such as radial immunodiffusion (RID), electroimmunodiffusion (EID), radiommunoassay
Method (RIA), EUSA (ELISA), immune turbidimetry (including Immune scatter turbidimetry (INA) and immune transmission
Turbidimetry (ITA), dissociation enhancing ligand fluorescence immunoassay (DELFIA) etc..RID methods, because easy to operate, are not required to EID methods
Special installation, still has some grass-roots unit laboratories to use, but have the disadvantage that sensitivity is low.The shortcoming of RIA methods is complex operation, is had
Radionuclide contamination.DELFIA methods are because needing specific apparatus, domestic also less application.Various LPa methods determine gained reference value
Close, current domestic and international used criterion is essentially identical.It is generally acknowledged that 300mg/L is critical level, more than 300mg/
More than L is Pathological levels, and the clinical value of LPa measure has caused the attention of more and more researchers.
International LPa Quality controls show for several times for the standardization that LPa is determined, LPa result differences between different experiments room
It is very big, illustrate that detection quality problems are serious, urgent need is standardized work, and LPa compositions, size, the property of density unevenness one are LPa's
Polymorphism is one of greatest difficulty of LPa bioassay standards.The internationally recognized two grades of ginsengs of analytical performance difference, shortage of kit
It is also to influence the key factor of LPa standardization to examine material, LPa report manner etc..
Various haemocyanins, complement, immunoglobulin, Acute radiation reaction, hemagluttinin proteins and urine micro protein, LPa etc.
Antigen (Ag)-antibody (Ab) compound can be all formed in the presence of their specific antibody.These immune complexs can be not
In the case of marking, light property (light-scattering) is scattered based on it turbid (Turbidimetry) by transmittance
Or scattering turbidimetry (Nephelometry) method direct reference standard (correction) thing is determined.
Ag and its special Ab combination are non-covalent, are carried out by following reaction laws：
Ag+Ab→AgAbcomplex
Binding constant is K.The power of this adhesion is decided by affinities of the Ag to Ab, and the active force for participating in combining includes
Hydrogen bond, Van der Waals force (Van Der Waals Bond) and hydrophobic bond etc..In [Ab] one timing, increase [Ag], react to Ag-Ab
Compound formation direction is carried out.With the increase of [Ag], in [Ab] excessive area, as [Ag] increase immune complex formation increases
Plus, reaction turbidity rise, until highest reacts equilibrium area；Hereafter, further increase [Ag] will cause precipitation to increase, and react turbid
Degree declines on the contrary, and this is the superfluous areas of Ag.The reason for these three stages occur be：Most of Ab are bivalent (Multivalent).
In the incipient stage of reaction, there is more Ab to be incorporated on antigen.Therefore, all binding sites on Ag are all tied by Ab
Close, only simple solubility Ag-Ab compounds, [AgAb] can be quantified by the property of light scattering, through with standard (correction)
Thing is relatively obtained [Ag], but in clinical examination measure, how to confirm that measurement result appearance is inclined caused by antigen concentration surplus
Difference.
The content of the invention：
There is measurement result caused by LPa excessive concentrations to solve the assay method of LPa in serum in the prior art
There is deviation, the present invention provides a kind of economical convenient and easy, and accuracy is higher, can judge serum lipid by real time reaction curve
Method superfluous in determining albumen a.
Solving the technical scheme of technical problem use is：Using priority double reagent assay method, reagent I and reagent II are equal
For anti-human LP(a) antibody reagent, LP(a) and LP(a) determine in reagent antibody in 37 DEG C of warm bath 3~5 minutes in serum
Antigen antibody complex is formed, reaction solution turbidity is occurred, LP(a) is then added and determines reagent, when fat egg in reaction solution
During white a surpluses, will occur reaction peak again, and show LP(a) antigen excess；When there is power, then reaction is shown
Terminal is reached, instrument is detected at 334~340nm wavelength, reacting the turbidity produced with the first step calculates LP(a)
Content.Antigen excess phenomenon is can interpolate that during present invention detection, it is economical convenient and easy, it is a kind of higher LP(a) of accuracy
Detection method.
Reagent I each component concentration is：0.10~0.24mmol/L of glycine, the mmol/L of sodium chloride 0.05~0.15, coating
The Latex Particles suspension 2~8% of anti-LPa antibody, the μ l of μ l of Proclin-300 preservatives 100~300；Reagent II each component is dense
Spend and be：0.10~0.24mmol/L of glycine, 0.05~0.15mmol/L of sodium chloride, the latex particle for being coated with anti-LPa antibody hang
Supernatant liquid 2~8%, the μ l of μ l of Proclin-300 preservatives 100~300.The pH of phosphate buffer in mentioned reagent I and reagent II
It is worth for 8.0 ± 0.2.
The volume ratio of reactant is in said determination：Sample: reagent I: reagent II=1: 40~60: 5~10.
Though the reagent of this method is identical with original LPa reagent components, priority double reagent assay method, examination will be simply used
Agent I and reagent II are anti-human LP(a) antibody reagent, and LPa excess phenomenons can be monitored by real time reaction curve, are determined
Effect is significantly different.
The present invention has the following advantages compared with prior art：Original LPa assay methods can not monitor that LPa surpluses are existing
As, and the inventive method can monitor LPa excess phenomenons, be a kind of higher LPa detection methods of accuracy.
Brief description of the drawings
Accompanying drawing 1. is single agents method real time reaction curve；
Accompanying drawing 2 is real time reaction curve when the inventive method has reached reaction end；
Real time reaction curve when accompanying drawing 3 is the inventive method antigen excess.
Embodiment：
The present invention is described in further details below by embodiment and accompanying drawing.
Embodiment 1
The composition of reagent：
A. reagent I：
Glycine 0.17mmol/L, sodium chloride 0.10mmol/L, are coated with the Latex Particles suspension 5% of anti-LPa antibody,
The μ l of Proclin-300 preservatives 200.
B. reagent II：
Glycine 0.17mmol/L, sodium chloride 0.10mmol/L, are coated with the Latex Particles suspension 5% of anti-LPa antibody,
The μ l of Proclin-300 preservatives 200.
C. titer：Multiple spot calibration is carried out using 1000mg/L definite values serum.
The volume ratio of reactant is in said determination：Sample: reagent I: reagent II=1: 50: 5.
Embodiment 3
Mensuration program
Research object is in hospital and out-patient 45 carries out blood sampling 2.0mL on an empty stomach, and serum is separated after centrifugation and carries out LPa surveys
It is fixed, LPa measure is carried out using single agents method and the inventive method, and real time reaction curve is observed, below by single agents
Method and Patent Law of the present invention illustrate the Detection results of the present invention
1. double reagent method：On the full-automatic Biochemical Analyzers of Japanese OLYMPUS AU2700, instrument is automatically by 5 μ l samples
Product be added in 250 μ l reagent Is mix, 37 DEG C be incubated 3 minutes, add 50 μ l reagent IIs mix, 37 DEG C be incubated 5.1 minutes, entirely
Automatic analyzer is detected at 340nm wavelength.Instrument calculates LPa results automatically, is specifically shown in Table 1：
The present invention automation Biochemical Analyzer test condition of table 1.
Calculation formula is：
ODLPa=OD2-OD1
LP(a) concentration=F × ODLPa
Wherein ODLPaIt is the absorbance that LP(a) is produced, OD1It is the absorbance measured before sample is added, OD2It is to add examination
The absorbance that agent I is measured after reacting, F is correction factor.
2. single agents method：On the full-automatic Biochemical Analyzers of Japanese OLYMPUS AU2700, instrument is automatically by 5 μ l
Sample is added in 225 μ l reagents and mixed, and 37 DEG C are incubated 3 minutes, and fully-automatic analyzer is detected at 340nm wavelength, and instrument is certainly
It is dynamic to calculate LPa results.
Research shows that there was no significant difference for two methods measurement result, n=45, t in 0.8~926.5mg/L of LPa
=3.23, P=0.3338, real time reaction curve such as Fig. 1, shown in 2, measurement result is reliable, and the inventive method adds reagent II
Flat curve such as Fig. 2 is may occur in which afterwards；When LPa is more than 926.5mg/L, single agents method measurement result shows 926.5mg/L,
The inventive method adds real time reaction curve after reagent II and may occur in which Double-peak Phenomenon, can point out in addition to measurement result is calculated
There is superfluous, reaction not up to terminal in LPa, and accurate result can be quoted by being resurveyed after should diluting, as shown in Figure 3.The inventive method
In the excessive real time reaction curves different from showing in right amount of LPa, such as Fig. 2,3 compare.
Compare more than and can be seen that,, can be with because of use priority double reagent method though the present invention is identical with original formulation composition
Antigen phenomenon superfluous in reaction solution is monitored, the purpose of accurate detection has been reached.
Claims (4)
1. LP(a) priority double reagent assay method in a kind of serum, it is characterised in that：LP(a) is with determining reagent in serum
Middle antibody forms antigen antibody complex in 3~5 minutes in 37 DEG C of warm bath, reaction solution turbidity is occurred, then adds LP(a)
Reagent is determined, when LP(a) is superfluous in reaction solution, will occur reaction peak again, and show antigen excess；When there is power
When, then show that reaction has reached terminal, instrument is detected at 334~340nm wavelength, the nephelometer produced is reacted with the first step
The content of LP(a) is calculated, antigen excess phenomenon is can interpolate that during present invention detection, is a kind of higher LP(a) of accuracy
Detection method.
Calculation formula is：
ODLPa=OD2-OD1
LP(a) concentration=F × ODLPa
Wherein ODLPaIt is the absorbance that LP(a) is produced, OD1It is the absorbance measured before sample is added, OD2It is that addition reagent I is anti-
Should after the absorbance that measures, F is correction factor.
2. LP(a) priority double reagent assay method in serum according to claim 1, it is characterised in that reagent I each group
Point concentration is：0.10~0.24mmol/L of glycine, 0.05~0.15mmol/L of sodium chloride, are coated with the latex of anti-LPa antibody
Grain suspension 2~8%, the μ l of μ l of Proclin-300 preservatives 100~300；Reagent II each component concentration is：Glycine 0.10~
0.24mmol/L, 0.05~0.15mmol/L of sodium chloride, are coated with the Latex Particles suspension 2~8% of anti-LPa antibody,
The μ l of μ l of Proclin-300 preservatives 100~300.
3. LP(a) priority double reagent assay method in serum according to claim 1, it is characterised in that using successively double
Reagent assay method, reagent I and reagent II are anti-human LP(a) antibody reagent.
4. LP(a) priority double reagent assay method in serum according to claim 1, it is characterised in that the volume of measure
Than for：Sample: reagent I: reagent II=1: 40~60: 5~10.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710353228.2A CN107102153A (en) | 2017-05-18 | 2017-05-18 | LP(a) priority double reagent assay method in serum |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710353228.2A CN107102153A (en) | 2017-05-18 | 2017-05-18 | LP(a) priority double reagent assay method in serum |
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| CN107102153A true CN107102153A (en) | 2017-08-29 |
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| CN201710353228.2A Pending CN107102153A (en) | 2017-05-18 | 2017-05-18 | LP(a) priority double reagent assay method in serum |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112255421A (en) * | 2020-10-13 | 2021-01-22 | 黎法飓 | Lipoprotein a detection kit and detection method |
| CN112352161A (en) * | 2018-07-13 | 2021-02-09 | 美国西门子医学诊断股份有限公司 | Method for detecting abnormal results caused by incomplete dispersion of immunoassay reagents |
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Application publication date: 20170829 |
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