CN105891504A - Colloidal gold immunocolorimetry kit for detecting lipoprotein (Lpa) and preparation method of kit - Google Patents

Abstract

The invention provides a colloidal gold immunocolorimetry kit for detecting lipoprotein (Lpa) and a preparation method of the kit and belongs to the field of in-vitro diagnostic reagents. The kit is used for judging whether the situation of individual acute kidney injury occurs or not. According to the method, colloidal gold particles are adopted as a carrier, a lipoprotein (Lpa for short) antibody and the colloidal gold particles are coupled, Lpa protein in a sample makes contact with the coupled gold particles in a corresponding detection device, an antigen-antibody-gold compound is formed, the light absorbance of the gold particles under detection wavelength is changed, the change amount of the light absorbance is positively correlated with the concentration of Lpa protein, and then the purpose of detecting Lpa protein in the sample is achieved. The kit has high analysis sensitivity.

Description

For detecting colloid gold immune colorimetric kit of LP(a) (Lpa) and preparation method thereof

Invention field

The invention belongs to external diagnosis reagent field, be particularly used for detecting the colloid gold immune colorimetric method of LP(a) (Lpa) Kit

Technical background

LP(a) (lipoprottein a, Lpa) is one of lipoprotein big molecule important member, is also the heat of current lipoprotein research Point.

1963, the Norway geneticist Berg in Northern Europe human ldl (LDL) immunizing rabbit of experimenter, produce one Planting the antiserum for certain antigenic component in LDL component, Berg is by this named Lpa of newfound antigenic component.1972 Year, first Dalhen etc. finds that the plasma lipoprotein of a lot of patients with coronary heart disease has a prebeta-lipoprotein in composing, and confirms this zone It is Lpa.The research such as Dahlen in 1975 thinks that Lpa is the risk factor of AS.The research such as McLean in 1987 finds apo (a) With plasminogen (PLG), there is high homology, thus think that Lpa is not only the hazards of AS, and may be with fibrinolytic system Unite relevant.Lpa is set to coronary heart disease (CHD) independent hazard factor by international Lpa meeting in 1988.Therefore, people couple are caused Lpa science of heredity, metabolism, function and further investigate with contacting of disease.

The species distribution of Lpa is narrow, only in the mankind, Old World Monkeys and the internal discovery of Europe hedgehog, therefore limits basis and grinds The progress studied carefully.The physiological action of Lpa lacks abundant proof, thus it is speculated that the physiological action of Lpa mainly repairs the vascular system of damage With negative regulation revascularization.

Lpa really can enter and take advantage of the occasion on atherosclerotic tube wall.Lpa by rich in cholesterol LDL particulate and with fibre Former similar apo (a) composition of lyase, this design feature determinism Lpa is not only sent out in atherosclerotic progress Wave effect, it is also possible to formation and myocardial infarction with thrombus have important relationship.

In vitro study display Lpa causes the atherosclerotic main mechanism to be: 1) promote expression of adhesion molecule, in induction of vascular Skin produces MCF；2) picked-up Lpa, the Lpa increase macrophage that the Lpa inducing macrophage aoxidized is active The generation of interleukin 8 (IL-8), promotes patch confirmatory reaction；3) tumorgrowthfactor-β (TGF β) of suppression activation Generation, cause smooth muscle cell proliferation to migrate and more preferential than LDL carry the mechanism such as oxidized phospholipids (tool pro-inflammatory matter).

Much studying confirmation, blood plasma Lpa level raises closely related with AS and thrombotic diseases.Dahlem (1986) etc. study Display, blood plasma Lpa concentration ＞ 300mg/L person, the danger suffering from coronary heart disease is high 1.75 times compared with blood plasma Lpa ＜ 300mg/L person, and The former degree of stenosis is the most heavier.The research such as Rhoads also shows, blood plasma Lpa concentration ＞ 200mg/L person suffers from hat The relative risk of worry is 2.5 times of ＜ 200mg/L person.Rosengren (1990) etc. measures blood to 766 general populations of Sweden Slurry Lpa level, and tracing study 6 years, wherein 26 people suffer from non-lethal miocardial infarction or die from coronary heart disease；Analyze and find Blood plasma Lpa level (278mg/L) of this 26 example patient is apparently higher than collator (173mg/L).This research also finds, blood plasma The incidence of Lpa ＞ 365mg/L person's coronary heart disease is 2 times of Lpa ＜ 365mg/L person.Multivariat analysis shows, blood plasma Lpa Level and the independence advance notice factor that known coronary risk factor is coronary heart disease as LDL-C level.Document is had to report, hat After shape arterial bypass, patients blood plasma's Lpa concentration of Saphenous ISR relatively doubles without narrow person.Along with blood plasma Lpa level raises, and transplanted hemadostewnosis degree increases the weight of the most accordingly；When blood plasma Lpa concentration reaches 316mg/L, 92% Patient have the narrow of grafting vessel.Also it was observed, it is that coronary artery is again after percutaneous coronary plasty that blood plasma Lpa level raises Narrow independent hazard factor.As blood plasma Lpa ＞ 190mg/L, the Relative hazard (OR) of ISR is 5.9；And work as blood plasma During Lpa ＞ 400mg/L, the OR value of ISR reaches 11.4.The dangerous critical level of Lpa is typically at 200-300mg/L, as super Crossing the dangerous of 300mg/L then AS and rise 2 times, as risen with LDL-C simultaneously, the relative risk of coronary heart disease rises 5-6 Times.And Lpa level is the highest, occur coronary heart disease then the most early.A perspective study is separately had to point out, the Lpa people higher than 450mg/L In Qun, many 2.6 times of cardiovascular death, many 4 times of outbreak.When LDL-C is higher, the coronary heart disease differentiation that Lpa is best refers to Mark.

Summary of the invention

Problem to be solved by this invention is to improve the sensitivity for analysis of Lpa protein content in detection human body fluid sample further, carries Good for a species specificity, the collaurum even phase immunity colorimetric reagent box of highly sensitive analysis Lpa albumen, may be used for accurately surveying Determine the Lpa protein content of 0-1400mg/L in body fluid sample.Additionally the present invention also resides in offer one about the examination of Lpa Protein Detection The preparation method of agent box.

In order to solve the problems referred to above, the present invention uses collaurum even phase immune response method, the Lpa albumen in detection body fluid sample to contain Amount.The colloid gold particle of label L pa antibody has maximum absorption band at 540nm, when environment exists Lpa albumen, and can shape Become Ag-Ab-Au composite, and now the rising along with concentration of specimens is weakened by maximum absorption band at 540nm, and Maximum absorption band at 660nm will increase therewith.The minimizing by detection and calculating 540nm absorbance and 660nm absorbance Increase, be associated with the sample of concentration known, and then form calibration curve, thus i.e. can reach Lpa in quantitative measurment body fluid sample The content of albumen.

The present invention provides techniques below scheme:

A kind of about Lp (a) detection collaurum even phase immunity detection reagent, its main purpose is for detecting individual human body fluid sample The content of Lp (a) albumen in Ben, mainly by the calibration object of known Lp (a) albumen for forming calibration curve in kit of the present invention (Lp (a) calibration object), reaction buffer reagent (reagent 1), colloid gold label thing reagent (reagent 2) three part forms.Its Middle Lp (a) calibration object is to be configured to variable concentrations gradient according to a certain percentage by calibration object dilution and Lp (a) albumen, its concentration model Enclose for 0mg/L-1400mg/L, reaction buffer reagent be by phosphate and bovine serum albumin(BSA) formulated this can be provided Bright required immunological response and the solution with pH buffer capacity, colloid gold label thing reagent be by gold mark anti-human The antibody of source Lp (a) albumen and the buffer solution composition of suspension label.

First, need to prepare the colloid gold particle that main absworption peak is 450nm ± 10nm according to kit detection.

Secondly reagent preparation 1, it mainly comprises and should be 50mM PBS pH8.0 solution, be wherein dissolved with PEG6000-20000, A kind of material in the polymers such as ficoll 400 and bovine serum albumin(BSA) or large biological molecule or two kinds and more than, and control PH value should be between 7.0-9.0.

3rd, reagent preparation 2 is, it mainly comprises and should be 50mM PBS pH8.0 solution, is wherein dissolved with PEG6000-20000, ficoll 400, polysorbas20, polymer or the large biological molecule such as bovine serum albumin(BSA) and Triton X-100 In a kind of material or two kinds and more than, and control ph should be between 7.0-9.0.By the gold after label L p (a) antibody Grain concentrates, and the solution prepared by this step hangs again.Multiple outstanding rear colloid gold label thing reagent (examination is diluted with deionized water or ultra-pure water Agent 2) after 50 times, and do blank with deionized water or ultra-pure water, the detection absorbance at 540nm ± 10nm, if absorbance In the range of 0.2000Abs-0.3000Abs, then it is assumed that the gold grain concentration marking Lp (a) antibody in reagent 2 is qualified, if not Meeting above-mentioned requirements, adjusting gold grain concentration in reagent 2 the most further, until meeting the requirements.

During final utilization, Lp (a) calibration object (or sample), reagent 1 and reagent 2 are mixed according to certain ratio and order, The change of detection absorbance, can reflect Lp (a) content to be detected in sample.

Use the inventive method, there is high sensitivity characteristic, Lp (a) content sample at below 1400mg/L can be analyzed.This Invention can be simultaneously used for detecting the body fluid sample of different substrates, and such as serum, blood plasma, in urine, the content of Lp (a) albumen, is not required to Sample or reagent done other technical finesse, including test sample amount is increased or decreased, to increase or reduce reagent 1 and reagent 2 Ratio, possesses the advantages such as high sensitivity, high specific, high accuracy and good repeatability, is suitable for full automatic biochemical apparatus, Semi-automatic biochemical analyzer and ultraviolet-visible spectrophotometer use.

Present invention is primarily aimed at and improve for the specific and sensitivity for analysis of Lpa protein content detection in human body fluid sample, In the present invention by controlling reaction condition and the size of gold grain of label, and suitably measuring condition, thus reach State purpose.

Specifically, the present invention can provide the Lpa detection kit of a kind of high analyte sensitivity, and its key property is Possess high analyte sensitivity in the range of 0mg/L-30mg/L, possess the most linear in the range of 0mg/L-1400mg/L, There is for clinical diagnosis preferable directive significance.Reagent 1, reagent 2 and the calibration object wherein comprised in kit, with Volume calculates, and calibration object consumption (or sample to be tested consumption) is 2uL-10uL, and reagent 1 consumption is 200uL-250uL, reagent 2 consumptions are 50uL-100uL.

Heretofore described gold grain size should control with maximum absorption band and absworption peak peak width, by adjusting gold chloride and reduction The ratio of agent, makes the maximum absorption band of colloid gold particle of system in the range of 540nm ± 20nm, and optimal conditions makes granular size Controlled further, system the maximum absorption band of colloid gold particle in the range of 540nm ± 10nm, further to experiment bar Piece optimization, the maximum absorption band of the colloid gold particle of system is in the range of 540nm ± 5nm, and now colloid gold particle size is the most Properly.

In the even phase reagent of collaurum 1 illustrated in the present invention and reagent 2, its buffer solution selects PBS, pH value control System is between 7.0-9.0, and further by optimum experimental buffer condition, pH value controls between 7.5-8.5, Lpa albumen and mark Antibody response is more suitable, and further Optimal Experimental condition finds, pH controls when 8.0 ± 0.2, Lpa albumen and mark Antibody response is the most suitable.And polymer and large biological molecule described in reagent 1 mainly include PEG6000-20000, Ficoll 400 and bovine serum albumin(BSA), and addition content is about the Sodium azide of 0.1% (mass volume ratio) wherein.Reagent 2 Described in polymer and large biological molecule PEG6000-20000, ficoll 400, polysorbas20, bovine serum albumin(BSA) and Triton X-100, and addition content is about the Sodium azide of 0.1% (mass volume ratio) wherein.Antibody in reagent 2, after mark Content in reagent 2 is controlled in the way of absorbance, will be containing reagent 2 deionized water or the ultra-pure water of label After diluting 50 times, doing blank (reference) with deionized water or ultra-pure water, the absorbance under detection 540nm should be In the range of 0.20Abs-0.25Abs.In reagent 2, Lpa antibody and the mark of collaurum rely on gold grain surface charged to Lpa antibody Absorption and complete.

Being used for described in the present invention detects or monitors the kit of Lpa protein content in body fluid sample, including Lpa egg White calibration object and calibration object dilution two parts composition.Calibration object dilution is by PBS, bovine serum albumin(BSA) and Sodium azide Composition, wherein PBS control ph is between 7.0-9.0, and the pH after optimization controls 8.0 ± 0.5, adds ox blood The amount of pure albumen and Sodium azide is 0.1%-0.5% (mass volume ratio).Calibration object is pure by calibration object diluted Lpa albumen Product are made, and content is 0mg/L, 112.5mg/L, 225mg/L, 350mg/L, 700mg/L, 1400mg/L.

The content of the Lpa albumen in employing collaurum even phase immunity colorimetric method for determining sample in the present invention.It is marked with particle homogeneous, The Lpa antibody of size to fit gold grain, with the Lpa albumen in sample, forming Lpa Protein-antibody-gold utensil in buffer solution has The compound of three-dimensional structure, so that gold grain weakens in the absorbance of 540nm originally, and the absorbance at 660nm adds By force, by by corresponding for known with calibration object for this change Lpa protein concentration, and then form the calibration curve of detecting system, When the body fluid sample of unknown Lpa protein concentration reacts with label with same reaction pattern, by calculating above-mentioned absorbance Change, corresponding calibration curve thus reach the purpose of quantitative measurment.

Accompanying drawing explanation

Fig. 1 reagent of the present invention and contrast agents linear verification result, with dilution factor as abscissa, map with testing result for ordinate, Reagent of the present invention has and is better than the feature that similar detection project kit is linear.

Fig. 2 reagent of the present invention and contrast agents calibration curve experimental result, in sample, Lpa protein content often changes a concentration unit, The absorbance signal of reagent of the present invention is changed to about 11 times of contrast agents, and sensitivity of the present invention is substantially better than contrast agents.

Fig. 3 label sedimentation test experience result, reagent label of the present invention did not occurred substantially to settle in 40 days.

Detailed description of the invention

Embodiment

If not doing specified otherwise in the present invention, then the percent concentration stating content all represents mass volume ratio, i.e. g/100mL

Embodiment 1

The preparation of Lpa calibration object

It is dissolved in calibration object dilution (bovine serum albumin(BSA) 0.5%, Sodium azide 0.1%, PBS with recombined human Lpa albumen (commercially available) PH of buffer 8.0) prepare the calibration object of variable concentrations gradient, use the detection of the last nine Lpa latex immunity colorimetric reagent box to prepare Calibration object, each concentration gradient detect 3 times, take the mean value of testing result, definite value be 0mg/L (calibration object dilution, not Add Lpa albumen), 112.5mg/L, 225mg/L, 350mg/L, 700mg/L, 1400mg/L.

The preparation of reaction buffer reagent (reagent 1)

Taking addition PEG6000-200005g in the buffer solution of 80mL 50mM PBS pH8.0, ficoll-400 1g, ox blood is pure Albumen 0.5g, Sodium azide 0.1g, be finally settled to 100mL with corresponding buffer solution, and use 0.22um filtering with microporous membrane, I.e. make and react buffer reagent (reagent 1), wherein PEG6000-200005%, ficoll-400 1%, bovine serum albumin(BSA) 0.5%, Sodium azide 0.1%.

The preparation of colloid gold label thing reagent (reagent 2)

The preparation process of colloid gold label thing reagent (reagent 2) is broadly divided into three parts, i.e. label and prepares, plain buffer Preparation, label hang three steps again.

The preparation of label

Commercially available gold chloride and trisodium citrate are dissolved, and be configured to respectively 1% the aqueous solution, by the deionized water or super of 1L Boiling pure water, the chlorauric acid solution being subsequently added into 10mL keeps seething with excitement after about 1min, adds the citric acid three sodium solution of 10mL, Change solution over time seethes with excitement again, and solution colour is changed into atropurpureus by bright yellow therewith, and then to transfer wine to red the short time Look, after keeping boiling 10min afterwards, cools down standby.

By prepare colloidal gold solution concentration be 20%K₂CO₃Solution adjusts pH value to after 8.0, adds in proportion as necessarily The Lpa antibody (commercially available) of volume, is sufficiently stirred for 1h, i.e. completes the labeling process of antibody.

After antibody labeling completes, available 10%NaCl solution check mark amount is the most suitable, when according to 1: 10 volume ratio After adding NaCl solution, the antibody minimum amount when occurring without black precipitate, for optimum mark amount.

The preparation of label plain buffer

Take addition PEG6000-2000020g, ficoll-400 5g, cow's serum in the buffer solution of 80mL 50mM PBS pH8.0 Albumin 0.5g, Sodium azide 0.1g, polysorbas20 1g, Triton X-100 1g, finally it is settled to 100mL with corresponding buffer solution, And use 0.22um filtering with microporous membrane, i.e. make reaction buffer reagent (reagent 1), wherein PEG6000-2000020%, poly-sugarcane Sugar-400 5%, bovine serum albumin(BSA) 0.5%, Sodium azide 0.1%, polysorbas20 1%, Triton X-100 1%.

Label hangs again

The colloid gold particle centrifugal 40min under 6000rpm that will have marked, collects precipitation, delays by above-mentioned label blank afterwards Rush the liquid outstanding precipitation collected again, and label is adjusted to suitable concentration according to the claim (4) of the present invention by concentration.

Linear verification

1, taking an example and have the clinical patient serum sample about 2mL of acute injury of kidney presentation, the Lpa albumen being added thereto to people source is pure Product, prepare a linear high level sample.

2, the Lpa albumen latex microsphere turbidimetry kit that on market, technology is more ripe is selected to do contrast agents, at Toshiba 120FR Typing latex turbidimetry contrast agents box parameter on full automatic biochemical apparatus, and calibrate its reagent with the supporting calibration object of its kit, Detecting above-mentioned high level sample three times, average 1380mg/L of three testing results is final definite value result.

3, on Toshiba's 120FR full automatic biochemical apparatus, the experiment parameter of the typing present invention, and with the calibration object having corrected that through row examination Agent is calibrated.

4, above-mentioned linear high level sample physiological saline being done doubling dilution, dilution ratio is 1/2,1/4,1/8,1/16,1/32, 1/64, form a gradient sample group with former linear high level sample.

5, detecting above-mentioned sample respectively with the reagent of above-mentioned contrast agents and the present invention, each pattern detection of each reagent three times, with three The testing result that secondary result average is final, then compares the linear, shown in result table 1 and Fig. 1 of the present invention and contrast agents again.

Table 1 linear verification result

Table 1 is added up for Linear Experiment result data, in the detection range of the present invention and contrast agents box, by high level sample multiple proportions 6 Secondary dilution, detects respectively with contrast agents and reagent of the present invention

Sensitivity for analysis is verified

1, the Lpa albumen latex microsphere turbidimetry kit that on market, technology is more ripe is selected to do contrast agents, at Toshiba 120FR Typing latex turbidimetry contrast agents box parameter on full automatic biochemical apparatus, and calibrate its reagent with the supporting calibration object of its kit.

2, on Toshiba's 120FR full automatic biochemical apparatus, the experiment parameter of the typing present invention, and with the calibration object having corrected that through row examination Agent is calibrated.

3, comparing the change in the calibration point absorbance of below 5mg/L concentration of two reagent, result is as shown in table 2 and Fig. 2.Note: Owing to the Cleaning Principle of contrast agents with reagent of the present invention differs, so contrast agents absorbance being changed when drawing Fig. 2 Do opposite number to process.

Table 2 sensitivity for analysis experimental result

Table 2 is added up for sensitivity for analysis experimental result data, and experiment sample analyzes reagent of the present invention and contrast agents sensitivity for analysis,

Label settlement monitoring

Owing to label solution is colloidal solution, so should verify after long-time static placement whether it occurs particle sedimentation existing As.This example purpose is to verify whether the present invention can exist label sedimentation phenomenon.

1, take two 50mL beakers, after soaking 24 hours with acid potassium bichromate, clean up by deionized water, 100 DEG C of bakings Dry-cure 4h.

2, the label reagent in contrast agents box is poured into wherein in a beaker, pour reagent label of the present invention into another In beaker, two beakers stir 1h simultaneously.

3, at solution surface 2-5mm, draw contrast agents and each 60uL of reagent of the present invention respectively with micropipettor, and spend Ionized water dilution is only to 3mL, and concussion mixes.

4, using deionized water as reference, detecting the absorbance after two reagent label dilutions at 540nm, each reagent is examined Survey three times, with the average of three results, as final detection result.

5, by above-mentioned two beakers sealing, stand every detection in 10 days once at 2-8 DEG C, repeat the above steps 3 and step 4, Before and after contrast stands, absorbance changes, and experimental result is as shown in table 3 and Fig. 3.

Table 3 label settlement monitoring experimental result

Reaction cup is polluted experiment by label

Owing to label solution is gold grain colloidal solution, so adsorption phenomena can be there is on reaction cup surface.This example is to test Demonstrate,prove whether reagent of the present invention there will be absorption contamination phenomenon.

Take three clean quartz cuvette, with air as reference, record its cup blank absorbency at 540nm respectively.

1, with reagent label of the present invention filling cuvette to its 3/5 height place, immersion 1h.

2, after taking out label, every cuvette deionized water cleans 10 times repeatedly

3, in every cuvette, load deionized water at cuvette 4/5 height, change water once every 1h, change water altogether three times.

4, naturally dry the moisture in cuvette, with air as reference, detect every cuvette absorbance at 540nm.

5, whether operate for contrast agents label repeat the above steps 1 to step 5, comparing two reagent labels can contrastive colours Cup causes absorption to pollute, and experimental result is as shown in table 4.

Table 4 label contrastive colours cup pollutes experimental data

According to interpretation, contrast agents label is positioned over for a long time in cuvette and can be adsorbed to cuvette surface, and the present invention tries Agent label non-contrastive colours cup within the identical time has obvious adsorption phenomena.

Claims (8)

1. Lp (a) detects a collaurum even phase immunity detection reagent, including Lp (a) calibration object, reaction buffer reagent, glue Body gold label reagent, it is characterised in that a kind of even phase quantitatively detect Lp (a) content in a corpse or other object for laboratory examination and chemical testing based on collaurum colorimetric method External diagnosis reagent.

Kit the most according to claim 1, it is characterised in that Lp (a) calibration object is by calibration object dilution and Lp (a) egg Proportionally being configured to variable concentrations gradient in vain, its concentration range is 0mg/L-1400mg/L, and reaction buffer reagent is By phosphate and bovine serum albumin(BSA) preparation and the solution with pH buffer capacity, colloid gold label thing reagent is by gold The antibody of anti-human source Lp (a) albumen of mark and the buffer solution composition of suspension label.

Kit the most according to claim 1, it is characterised in that the main absworption peak of colloid gold particle is 450nm ± 10nm.

4., according to the kit described in claim 1-3, it is characterized in that reaction buffer reagent is the controlling solution of reaction condition, Including 50mM PBS pH8.0 solution, PEG6000-20000, the polymer such as ficoll 400 and bovine serum albumin(BSA) or A kind of material in large biological molecule or two kinds and more than, and the pH value controlling reaction buffer reagent is 7.0-9.0。

5., according to the kit described in claim 1-3, it is characterized in that colloid gold label thing reagent is that the controlling of reaction condition is molten Liquid, including 50mM PBS pH8.0 solution, PEG6000-20000, ficoll 400, polysorbas20, bovine serum albumin A kind of material in the polymers such as white and Triton X-100 or large biological molecule or two kinds and more than, by label L p (a) Gold grain after antibody is suspended in this solution, and control ph should form colloid gold label between 7.0-9.0 Thing reagent.

Kit the most according to claim 5, it is characterised in that the foundation that the gold grain concentration of middle mark Lp (a) antibody is qualified After diluting colloid gold label thing reagent 50 times for ionized water or ultra-pure water, do blank with deionized water or ultra-pure water, its Absorbance at 540nm ± 10nm is in the range of 0.2000Abs-0.3000Abs.

Kit the most according to claim 6, it is characterised in that reaction system solution composition is that calibration solution or sample, reaction are delayed Rushing liquid reagent and colloid gold label thing reagent, wherein sample size scope is 2uL-20uL, the volume of reaction buffer reagent The volume at 2-10 times of colloid gold label thing reagent should be controlled.

8. preparation kit described in claim 1-7 any one, is characterized in that Lp (a) the protein-colloid even phase detection kit of gold Preparation method, including following step:

1) polymer or large biological molecule deionized water or ultra-pure water are dissolved, be configured to reaction buffer reagent.

2) gold chloride is mixed according to a certain percentage with reducing agent, heat with deionized water or ultra-pure water therewith and boil, system Main absworption peak is the colloid gold particle of 450nm ± 10nm.

3) Lp (a) antibody to be marked is mixed and made into mutually label L p (a) antibody-gold label with gold grain.

4) label made is mixed according to a certain percentage with the colloid gold label thing reagent not containing gold grain, make inspection Colloid gold label thing reagent required for test agent box

5) by commercially available people source Lp (a) albumen according to desired concn gradient, concentration is become to exist by calibration object diluent preparing Calibration object in the range of 0mg/L-1400mg/L, for complex reaction buffering agents and collaurum on instrument Label reagent forms calibration curve, to reach the purpose quantitatively detected.