CN103940816B - A kind of kit and preparation method measuring human body content of glycocholic acid - Google Patents
A kind of kit and preparation method measuring human body content of glycocholic acid Download PDFInfo
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Abstract
Measure kit and the preparation method of human body content of glycocholic acid, kit comprises: glycocholic acid R1 reagent, glycocholic acid R2 reagent and glycocholic acid calibration object, glycocholic acid R1 reagent is fully mixed to get glycocholic acid-protein conjugate and damping fluid, glycocholic acid R2 reagent is mixed to get by the latex particle of glycocholic acid-protein antibodies bag quilt and buffer suspension liquid, glycocholic acid calibration object is made up of humanized's glycocholic acid and damping fluid, the method is by the human plasma containing glycocholic acid, serum sample and glycocholic acid-protein conjugate competition binding glycocholic acid-protein antibodies latex particle, the glycocholic acid be combined with glycocholic acid-protein antibodies latex particle does not produce turbidity, and turbidity can be produced with glycocholic acid-protein conjugate cholylglycine-protein antibodies latex particle and change, by the reduction degree of system turbidity after assaying reaction, quantitative test is carried out to glycocholic acid.Present invention greatly enhances immunoreactive signal intensity, make low content material immunity in conjunction with time also can produce the reaction of stronger turbidity, for detection.
Description
Technical field
The present invention relates to technological field of biochemistry, be specifically related to a kind of Immune competition method, latex-enhanced turbidimetry of adopting and measure reagent of glycocholic acid (CG) content in human body and preparation method thereof.
Background technology
Serum CG (Cholyglycine, CG) is cholic acid and the mating type cholic acid one of of glycocoll in conjunction with secondary, in liver cell, cholesterol through and the enzymatic reaction of complexity, be transformed into primary bile acid.Wherein there are cholic acid (CA) and chenodeoxycholic acid (CD-CA).The steroids core of cholic acid has three hydroxyls (C3, C7, C12), the hydroxyl of side chain terminal is combined with glycocoll with peptide bond, and CG is synthesized by liver cell, enters gall-bladder through bile capillaries, bile duct, enters duodenum in company with bile, helps food digestion.95% cholic acid heavily absorbs at terminal ileum, and trans-portal vein returns liver again, absorbs recycling by liver cell.Mainly exist with protein-bound form in serum.Under normal circumstances, in peripheral blood, cholic acid content is very micro-, and adult normal no matter on an empty stomach or after the meal, its change of serum C G concentration stabilize is in low-level.When liver cell is impaired, liver cell picked-up CG ability declines, and causes CG content in blood to increase; During cholestegnosis, hepatic excretion cholic acid generation obstacle, and the sanguimotor CG content that backflows increases, and also makes blood CG content increase.Measuring Serum CG (CG) is one of sensitive indicator evaluating hepatocyte function and liver and gall system material recycle function thereof.Pregnant woman is in the middle and later periods of pregnancy in addition, due to the increase of baby, liver can be oppressed, make hepatic excretion cholic acid generation obstacle and cause glycocholic acid higher, increase because gestational period placenta synthesizes and secrete a large amount of estrogen and progestational hormone and metabolic burden in addition, the change of liver and gall can be brought out, pregnant woman is made easily to suffer from intrahepatic cholestasis of pregnancy (ICP), fetus is produced serious influence, increase with ICP patients serum CG and make amniotic fluid-pollution rate, early productive rate, fetal distress in uterus rate and cesarean delivery rate increase, severe patient can cause the death of baby, therefore pregnancy serum glycocholic acid is measured, to finding that the order of severity of ICP and understanding ICP has very important meaning early, the mensuration of glycocholic acid can be used as examination and follows up a case by regular visits to the index of ICP.Thus reduction the perinatal fetal mortality.Glycocholic acid is the organic acid of maximum in hepatic secretion to bile, enters enteric cavity and helps fat digestion to absorb, and heavily absorbed at ileum and colon major part, trans-portal vein enters liver.Liver cell can absorb a large amount of glycocholic acid from portal vein high-effectly, so that the glycocholic acid amount in blood is less than 1.9g/ml.Re-absorbed glycocholic acid enters hepato-enteric circulation again, and by this mechanism, body can make full use of glycocholic acid.Once liver cell lesion, the glycocholic acid concentration in blood raises, and wherein oxyhepatitis, chronic hepatitis slightly raise, cirrhosis, and hepatocarcinoma patient significantly raises.When liver cell is impaired, liver cell picked-up CG ability declines, and causes CG content in blood to increase; During cholestegnosis, hepatic excretion cholic acid generation obstacle, and the sanguimotor CG content that backflows increases, and also makes blood CG content increase.Therefore, measuring Serum CG (CG) is one of sensitive indicator evaluating hepatocyte function and liver and gall system material recycle function thereof.
Glycocholic acid assay method known at present has radioimmunology (RIA), chemoluminescence method, enzyme linked immunosorbent assay (ELISA) method, latex-enhanced turbidimetry etc., radio immunoassay complex steps, reagent is expensive, need use supporting instrument and there is radioactive contamination.There is length detection time, complicated operation, poor repeatability, be unsuitable for the needs that emergency treatment and clinical patient diagnose in time in enzyme linked immunosorbent assay.Although but the sensitivity simple to operate, quick of existing latex enhancing immune turbidimetry is low, low value poor repeatability.
Summary of the invention
The weak point that the present invention exists in order to avoid prior art, provides one can be applied to glycocholic acid (CG) kit and preparation method thereof on full-automatic biochemical analyzer.This reagent can detect the content of glycocholic acid in human plasma sample fast and accurately.
The present invention is by being suspended in specific damping fluid with the glycocholic acid bag be coupled on albumen making R2 reagent by latex particle.Because glycocholic acid molecular weight is very little, only has an antigen site, emulsion reagent can not be made, the reaction of antigen and antibody can not form glue and joins and produce turbidity, glycocholic acid can only be allowed first to be coupled on high molecular weight protein by chemical bond, form multiple site, and the larger albumen of molecular weight better can be combined with latex particle, coupling has the protein concentration of glycocholic acid to be 0.1-2.0mg/mL.Coupling has the albumen of glycocholic acid to be coated on latex particle surface further by Chemical Crosslinking Methods.
The present invention makes R1 reagent by adding glycocholic acid-protein conjugate in specific damping fluid, will add inorganic salts, set accelerator, protein and antiseptic in specific damping fluid.First selected glycocholic acid-protein complexes will be detected by indirect ELISA method, and confirmation can not produce nonspecific reaction.Wherein require that the surge capability of damping fluid is for regulating pH scope between 7-9, various damping fluid can be selected, preferred HEPES, glycocoll, Tris etc., compared with other damping fluids, above-mentioned damping fluid has surge capability Strong, high, the good biological fitness of solubleness and reaction.Wherein Tris-HCl can control constant pH scope the long period, and therefore more preferably Tirs-HCl, its concentration range is 10-100mM, pH scope is 7-8.PEG-6000 selected by set accelerator, can accelerate immune response speed, and shorten detection time, its final concentration is 2-6%.Protein selects bovine serum albumin(BSA), and final concentration is 0.1-1%.Sodium chloride concentration is 0.7-0.9%, and Sodium azide concentration is 0.05-0.1%.
The present invention is respectively 0 by adding glycocholic acid concentration in specific damping fluid, 2.5,10,20,40,80ug/mL makes multiple spot calibration object, wherein require that the surge capability of damping fluid is for regulating pH scope between 7-9, various damping fluid can be selected, preferred HEPES, glycocoll, Tris etc., compared with other damping fluids, there is surge capability Strong, high, the good biological fitness of solubleness and reaction.Wherein Tris-HCl can control constant pH scope the long period, and therefore more preferably Tirs-HCl, its concentration range is 10-200mM, pH scope is 7-8.Wherein containing specific protein stabiliser such as bovine serum albumin(BSA) concentration is 0.1-1%, containing specific antiseptic if PC-300 concentration is 0.05-0.5%, containing specific antioxidant if EDTA-2Na concentration is 5-50mM.
The invention provides for measuring at human plasma, glycocholic acid competition immunoturbidimetry assay method in blood serum sample, the method is by the human plasma containing glycocholic acid, serum sample and glycocholic acid-protein conjugate competition binding glycocholic acid-protein antibodies latex particle, the glycocholic acid be combined with glycocholic acid-protein antibodies latex particle does not produce turbidity, and turbidity can be produced with glycocholic acid-protein conjugate cholylglycine-protein antibodies latex particle and change, glycocholic acid is combined the efficiency being better than glycocholic acid-protein conjugate cholylglycine-protein antibodies latex particle and combining with glycocholic acid-protein antibodies latex particle, estimate content of glycocholic acid in sample higher, react the turbidity produced lower.The degree declined is directly proportional to the content of glycocholic acid in human plasma, serum, uses automatic clinical chemistry analyzer device can calculate the content of glycocholic acid in human plasma, serum sample.
The concrete technical scheme of the present invention is as follows:
Measure a kit for human body content of glycocholic acid, its feature is to comprise: glycocholic acid R1 reagent; Glycocholic acid R2 reagent and glycocholic acid calibration object;
Described glycocholic acid R1 reagent is made up of glycocholic acid-protein conjugate and damping fluid, and described damping fluid is the Tirs-HCl solution of concentration 10-100mM, and the pH value of described damping fluid is 7-8; The sodium chloride of PEG-6000,0.7-0.9wt% of concentration 2-6wt%, the Sodium azide of 0.05-0.1wt%, PC-300,0.1-1wt% bovine serum albumin(BSA) of 0.3wt% and 20mMEDTA is contained in described damping fluid; In described glycocholic acid R1 reagent, the concentration of glycocholic acid-protein conjugate is 0.05-2mg/mL; Described glycocholic acid-protein conjugate is obtained after glycocholic acid and albumen coupling by coupling agent; In described glycocholic acid-protein conjugate, protein concentration is 0.1-2.0mg/mL;
Described glycocholic acid R2 reagent is made up of the latex particle of buffer suspension liquid and glycocholic acid-protein antibodies bag quilt, the glycocoll-Na0H damping fluid of described buffer suspension liquid to be concentration containing stabilizing agent and antiseptic be 20-100mM; Described stabilizing agent is made up of the bovine serum albumin(BSA) of final concentration 0.1-1.0wt%, the sodium chloride of 0.7-0.9wt%, the disodium ethylene diamine tetraacetate of 5-50mM, volumetric concentration 0.1-1.0%TX-100; Described antiseptic is PC-300, and final volume concentration is 0.05-0.1%; In described glycocholic acid R2 reagent, the massfraction of the latex particle of glycocholic acid-protein antibodies bag quilt is 0.05-1.0%;
Described glycocholic acid calibration object is made up of humanized's glycocholic acid and damping fluid, described damping fluid is the Tirs-HCl solution of concentration 50-200mM, the disodium ethylene diamine tetraacetate containing the bovine serum albumin(BSA) of final concentration 0.1-1wt%, PC-300,5-50mM of 0.05-0.5wt% in described damping fluid; Described glycocholic acid calibration object comprise humanized's glycocholic acid concentration be respectively 0,2.5,5,10,20,40, one group of calibration object of 80ug/ml;
Kit of the present invention, its feature is also:
Mixing of a kind of hypotype in human serum albumins, bovine serum albumin(BSA), ox transferrins, hemocyanin or multiple hypotype is selected from the albumen of glycocholic acid coupling; Described coupling agent be selected from carbodiimides, glutaraldehyde, two isocyanic acid compounds or dihalide dinitro benzene one or more.
Described latex particle is nucleocapsid structure, and diameter range is 100-450nm, and concentration is 1-5mg/mL; The surperficial with of described latex particle has the chemical group modified by carboxyl, amino, hydroxyl, hydrazide group or chloromethyl; The chemical group that described glycocholic acid-protein antibodies and latex particle itself carry is combined in latex particle surface by chemical bonded refractory.
The present invention discloses a kind of preparation method measuring the kit of human body content of glycocholic acid, its feature is levied and is to comprise the steps:
One, the preparation of glycocholic acid-protein conjugate:
1) accurately take 2mg glycocholic acid, be dissolved in 100 μ lDMF reagent, gained solution is A liquid; Prepare the NHS solution of 89.4mg/mL with DMF, solution be B liquid; Prepare the dicyclohexylcarbodiimide solution of 60.2mg/mL with DMF, gained solution is C liquid; Take 16mg hemocyanin, be dissolved in 1mLpH7.2, concentration 0.02mol/L phosphate buffer; Gained solution is D liquid;
2) B liquid is got respectively and each 30.7 μ L of C liquid are added in 100 μ lA liquid, stirred at ambient temperature 90min;
3) the complete mixed liquor of stirring is added in D liquid, under 4 DEG C of conditions, stirs 12h;
4) by step 3) process reactant liquor puts into pH7.4, concentration 0.01mol/L phosphate buffer dialyses 12h, namely obtains glycocholic acid-protein conjugate;
Prepared by the latex particle of two, glycocholic acid-protein antibodies bag quilt:
1) with the glycocholic acid of step one gained-protein conjugate subcutaneous inoculation mouse; The Mouse spleen cells selecting Post-immunisation serum to tire to be greater than 10000 merges with SP2/0 myeloma cell, is screened obtain glycocholic acid monoclonal cell antibody by BSA-CG plate;
2) 1.5g latex particle is added in the 2-morpholino ethanesulfonic acid buffer of 150mlpH5.5, concentration 80mmol/L, stir 30 minutes; Then the N N-Hydroxysuccinimide 20ml of carbodiimides 20ml and 0.033mg/ml of 0.067mg/ml is added, normal-temperature reaction 45 minutes;
3) through step 2) reacted potpourri, with 15000 turns/mim centrifugal 15 minutes, removes supernatant, then uses the phosphate buffer of 150mlpH8.2, concentration 0.1mol/L resuspended; Obtain latex particle suspension;
4) the latex particle suspension of gained is repeated step 3), the latex particle suspension obtained after repeating cleans; After cleaning, with the phosphate buffer of concentration 0.1mol/L, volume is adjusted to 700ml;
5) 180mg glycocholic acid monoclonal cell antibody is joined in 300mlpH8.2,0.1mol/L phosphate buffer, stir; Join step 4) latex solution in; Then the shaking table of 37 degree is placed in, reaction 2-4 hour; Add 0.5g/ml glycocoll 10ml again, again react 30 minutes;
6) step 5) reaction terminate after, 5g bovine serum albumin(BSA) is added in above-mentioned mixed liquor, after stirring, room temperature place 12h; Then centrifugal 15 minutes of 9000 turns/mim, removes supernatant, resuspended with the phosphate buffer of 500mlpH8.2, concentration 0.1mol/L; Obtain latex particle suspension;
7) by obtained latex particle suspension through 9000 turns/mim centrifugal 15 minutes again, supernatant is removed, resuspended with the phosphate buffer of 500mlpH8.2, concentration 0.1mol/L; By the latex particle suspension pH8.2 of resuspended rear gained, the phosphate buffer of concentration 0.1mol/L are adjusted to 1000ml volume again; Gained solution is the latex particle of glycocholic acid-protein antibodies bag quilt;
Three, the preparation of glycocholic acid R1 reagent
Glycocholic acid-protein conjugate step one prepared fully mixes with damping fluid and obtains glycocholic acid R1 reagent, and described damping fluid is the Tirs-HCl solution of concentration 10-100mM, and the pH value of described damping fluid is 7-8; The sodium chloride of PEG-6000,0.7-0.9wt% of concentration 2-6wt%, the Sodium azide of 0.05-0.1wt%, PC-300,0.1-1wt% bovine serum albumin(BSA) of 0.3wt% and 20mMEDTA is contained in described damping fluid; In described glycocholic acid R1 reagent, the concentration of glycocholic acid-protein conjugate is 0.05-2mg/mL;
Four, the preparation of glycocholic acid R2 reagent
The latex particle of glycocholic acid step 2 prepared-protein antibodies bag quilt mixes with buffer suspension liquid and obtains glycocholic acid R2 reagent, the glycocoll-Na0H damping fluid of described buffer suspension liquid to be concentration containing stabilizing agent and antiseptic be 20-100mM; Described stabilizing agent is made up of the bovine serum albumin(BSA) of final concentration 0.1-1.0wt%, the sodium chloride of 0.7-0.9wt%, the disodium ethylene diamine tetraacetate of 5-50mM, volumetric concentration 0.1-1.0%TX-100; Described antiseptic is PC-300, and final volume concentration is 0.05-0.1%; In described glycocholic acid R2 reagent, the mass concentration of the latex particle of glycocholic acid-protein antibodies bag quilt is 0.05-1.0%.
Five, the preparation of glycocholic acid calibration object
The humanized's glycocholic acid adding different amount in damping fluid obtain concentration be respectively 0,2.5,5,10,20,40, one group of calibration object of 80ug/ml; Described damping fluid is the Tirs-HCl solution of concentration 0-200mM, the disodium ethylene diamine tetraacetate containing the bovine serum albumin(BSA) of final concentration 0.1-1wt%, PC-300,5-50mM of 0.05-0.5wt% in described damping fluid.
Compared with the prior art, beneficial effect of the present invention is embodied in:
1, latex of the present invention drastically increases immunoreactive signal intensity than turbid reagent, make low content material immunity in conjunction with time also can produce the reaction of stronger turbidity, for detection.
2, the benefit that the present invention competes immunoturbidimetry assay method is that anti-interference is stronger, with traditional immunization turbidimetry Reagent evaluation ratio, larger in low magnitude portion signal intensity, because in human body, the normal range of glycocholic acid is 0-2.5ug/ml, the accuracy of low magnitude portion, be most important part for clinical practice, the repeatability that competition law reagent just allows low value test and accuracy are greatly improved.
3, the present invention is based on a specificity for the monoclonal antibody of glycocholic acid and the crosslinked latex particle suspending liquid having glycocholic acid-human serum albumins, in specific reaction system, there is antigen-antibody reaction form certain turbidity, can be detected by based on spectrophotometric Biochemical Analyzer.And if have the glycocholic acid of free state to exist in human plasma, can and the glycocholic acid competition binding monoclonal antibody being fixed on latex particle surface, turbidity is declined, by the reduction degree of system turbidity after assaying reaction, quantitative test is carried out to glycocholic acid.
5, the present invention solves the specificity and the low problem of automaticity that glycocholic acid detects simultaneously, can wide popularization and application.
Accompanying drawing explanation
Fig. 1 is kit of the present invention and commercial reagent box testing result correlativity equation of linear regression.
Fig. 2 is the equation of linear regression that kit of the present invention detects glycocholic acid sterling.
Embodiment
Below by way of specific embodiment, explanation is further explained to technical solution of the present invention.
In following examples, the reagent that uses is commercially available prod, and authentication method, screening technique etc. involved in embodiment are this area routine techniques means.
Embodiment 1 liver and gall acid-protein conjugate preparation
One, reagent prepares:
A liquid: accurately take 2mg glycocholic acid, is dissolved in 100 μ lDMF reagent; B liquid: the NHS solution preparing 89.4mg/mL with DMF; C liquid: DCC (dicyclohexylcarbodiimide) solution preparing 60.2mg/mL with DMF; D liquid: take 16mgBSA (bovine serum albumin(BSA)), being dissolved in 1mLpH is in the 0.02mol/L phosphate buffer of 7.2.
Phosphate buffer in the embodiment of the present invention is all selected from commercially available sodium hydrogen phosphate-sodium dihydrogen phosphate or dipotassium hydrogen phosphate-potassium phosphate buffer.
Two, reagent configuration:
1, B liquid is got respectively and each 30.7 μ L of C liquid are added in 100 μ lA liquid, stirred at ambient temperature 90min.
2, stir complete mixed liquor and be added to also violent mixing in D liquid, under 4 DEG C of conditions, stir 12h.
3, reactant liquor being put into pH is that 7.4 concentration 0.01mol/LPBS solution are dialysed 12h, namely obtains glycocholic acid-protein conjugate.
Three, the qualification of liver and gall acid-protein complexes:
1, the ultraviolet qualification ultraviolet spectrophotometry of artificial antigen carries out UV scanning to BSA standard items, coupled product and glycocholic acid, judges whether coupling success according to the displacement of coupled product maximum absorption band.If maximum absorption band is consistent with BSA standard items or glycocholic acid, represents non-coupling success, if coupled product maximum absorption band and BSA standard items maximum absorption band produce offset, then represent coupling success.The inventive method gained coupled product yield is adopted to be 80%.
Prepared by embodiment 2 glycocholic acid monoclonal antibody
1, prepare
With subcutaneous inoculation BalB/c mouse after KLH albumen coupling CG (BalB/c is commercially available mouse strain), through four immunity (once just exempting from three booster immunizations), the BalB/c Mouse spleen cells getting immune effect ideal (namely adopt indirect elisa method qualification Post-immunisation serum to tire and be greater than 10000) merges with SP2/0 myeloma cell's (being provided by ATCC), is selected obtain glycocholic acid monoclonal cell antibody by BSA-CG plate sieve (existing instrument).
2, about screening and the qualification of liver and gall acid monoclonal antibody
Qualification Small molecular monoclonal antibody, is selected " BSA-CG bag is by elisa plate " to be tested by competitive ELISA, has the antibody of competitive inhibition reaction to be exactly the monoclonal antibody of specificity for CG with free CG.Competitive relation is more obvious, illustrates CG affinity higher, otherwise illustrates that affinity is more weak.
Prepared by embodiment 3 liver and gall acid-protein antibodies and reactant emulsion thing
1,1.5g latex particle, (this particle is nucleocapsid structure, its newborn core is poly styrene polymer, breast shell is made up of styrene, n-butyl acrylate and methacrylic acid copolymer, and the chemical group difference of its surperficial institute with can be selected from the one of carboxyl, amino, hydroxyl, hydrazide group, chloromethyl modification; Latex particle diameter range is 100-450nm, and market is bought and obtained, and the model provided as JSR company is P0220 type carboxyl microballoon) to add pH be 5.5, concentration is in the 2-morpholino ethanesulfonic acid buffer 150ml of 80 mMs/L, stirs 30 minutes;
2, toward in above-mentioned solution, add carbodiimides 20 milliliters (concentration is 0.067mg/ml) and N N-Hydroxysuccinimide 20 milliliters (concentration is 0.033mg/ml), react 45 minutes.
3, said mixture, being placed on rotating speed is centrifugal 15 minutes of 15000 turns/mim, and removing supernatant, is 8.2 with pH, and concentration is that the phosphate buffer 1 50ml of 0.1mol/L is resuspended.As solution cannot be resuspended immediately, ultrasonic instrument can be used to carry out ultrasonic to solution.
4, repeat the 3rd step, resuspended rear gained latex particle suspension is 8.2 with pH, and concentration is that the phosphate buffer of 0.1mol/L is adjusted to 700ml volume.
5,180mg glycocholic acid monoclonal cell antibody is joined in 300mlpH8.2,0.1mol/L phosphate buffer, stir.Then join slowly in the latex solution of the 4th step.
6, after antibody adds, above-mentioned latex mixture is placed in the shaking table of 37 degree, reaction 2-4 hour.
7, after reacting completely, 0.5g/ml glycocoll 10ml is added in above-mentioned mixing, reacts 30 minutes
8, after reacting completely, 5g bovine serum albumin(BSA) is added in above-mentioned mixing, after stirring, room temperature places 12h.
9, said mixture, being placed on rotating speed is centrifugal 15 minutes of 9000 turns/mim, and removing supernatant, is 8.2 with pH, and concentration is that the phosphate buffer 500ml of 0.1mol/L is resuspended, obtains latex particle suspension.As solution cannot be resuspended immediately, ultrasonic instrument can be used to carry out ultrasonic to solution.
10, by obtained latex particle suspension through 9000 turns/mim centrifugal 15 minutes again, supernatant is removed, resuspended with the phosphate buffer of 500mlpH8.2, concentration 0.1mol/L; By the latex particle suspension pH8.2 of resuspended rear gained, the phosphate buffer of concentration 0.1mol/L are adjusted to 1000ml volume again; Gained solution is the latex particle of glycocholic acid-protein antibodies bag quilt.
The preparation of embodiment 4, glycocholic acid R1 reagent
Glycocholic acid-protein conjugate step one prepared fully mixes with damping fluid and obtains glycocholic acid R1 reagent, and described damping fluid is the Tirs-HCl solution of concentration 10-100mM, and the pH value of described damping fluid is 7-8; The sodium chloride of PEG-6000,0.7-0.9wt% containing concentration 2-6wt% in described damping fluid, the Sodium azide of 0.05-0.1wt%, the PC-300 (commercially available prod of 0.3wt%, supelco company, trade name proclin300), 0.1-1wt% bovine serum albumin(BSA) and 20mMEDTA; In described glycocholic acid R1 reagent, the concentration of glycocholic acid-protein conjugate is 0.05-2mg/mL;
The preparation of embodiment 5 glycocholic acid R2 reagent
The latex particle of glycocholic acid step 2 prepared-protein antibodies bag quilt mixes with buffer suspension liquid and obtains glycocholic acid R2 reagent, the glycocoll-Na0H damping fluid of described buffer suspension liquid to be concentration containing stabilizing agent and antiseptic be 20-100mM; Described stabilizing agent is made up of the bovine serum albumin(BSA) of final concentration 0.1-1.0wt%, the sodium chloride of 0.7-0.9wt%, the disodium ethylene diamine tetraacetate of 5-50mM, volumetric concentration 0.1-1.0%TX-100; Described antiseptic is PC-300, and final volume concentration is 0.05-0.1%; In described glycocholic acid R2 reagent, the mass concentration of the latex particle of glycocholic acid-protein antibodies bag quilt is 0.05-1.0%.
The preparation of embodiment 6 glycocholic acid calibration object
The humanized's glycocholic acid adding different amount in damping fluid obtain concentration be respectively 0,2.5,5,10,20,40, one group of calibration object of 80ug/ml; Described damping fluid is the Tirs-HCl solution of concentration 0-200mM, the disodium ethylene diamine tetraacetate containing the bovine serum albumin(BSA) of final concentration 0.1-1wt%, PC-300,5-50mM of 0.05-0.5wt% in described damping fluid.
In above embodiment, antiseptic used is selected from one or more in Sodium azide, thimerosal, PC-300 and ethyl mercury sodium thiosulfate.Set accelerator is selected from the one in PEG-4000, PEG-6000, PEG-8000, sodium dextran sulfate, tygon pyrrole network alkane ketone.
Three, the assay method of kit
Analytical approach: Two point end assay
The Direction of Reaction: reaction of rising
Calibrating mode: logit-log (4p) or SPLINE
Measure wavelength: 570nm
Measure temperature: 37 DEG C
Sample: glycocholic acid R1 reagent: glycocholic acid R2 reagent=5:200:50 (ul)
Operation steps: 200ul reagent R1 is added 5ul sample, 37 DEG C hatch 5 minutes after add 50ul reagent R2, read absorbance A 1 after 1 minute, after 3 minutes, read absorbance A 2.
Adopt 6 scaling methods, detect with Hitachi 7180 automatic clinical chemistry analyzer, calibration object concentration is respectively: 0,2.5,10,20,40,80ug/mL
Four, the henchnmrk test of kit
1, linear dependence
The kit of the present invention prepared by embodiment 4-6 and commercially available glycocholic acid chemoluminescence method detection kit A contrast detection 102 parts of serum samples, compare the correlativity of kit of the present invention and commercially available glycocholic acid chemoluminescence method detection kit testing result.(the results are shown in Figure 1, X, Y-axis is measured value, unit mg/L.) correlation coefficient r=0.9940, equation of linear regression is: y=1.019x-0.014, and result shows reagent of the present invention and enzyme linked immunological kit testing result good relationship.Experimental data is as shown in table 1, and regression equation is shown in accompanying drawing 1.
Table 1
2, range of linearity experiment
The high level sample of concentration 160mg/L is mixed with, with physiological saline rare 160mg/L, 80mg/L, 40mg/L, 20mg/L, 10mg/L, 5mg/L, 2.5mg/L, 1.25mg/L, 0.5mg/L, 0mg/L (water) respectively by glycocholic acid sterling.According to kit detection method separately, each sample replication three times, obtains the average (yi) of measurement result.With dilute concentration (xi) for independent variable, with measurement result average (yi) for dependent variable obtains equation of linear regression, as shown in Figure 2, the related coefficient (r) of linear regression is calculated.Substitute into dilute concentration (xi) and obtain equation of linear regression, calculate the estimated value of yi and the relative deviation of yi and estimated value.Result shows that the kit range of linearity of the present invention can reach 100mg/L, and the relative deviation of the estimated value of yi and yi and estimated value is all less than 10%, in table 2.
Table 2
3, accuracy
Get the Quality Control of serum high level and each portion of low value Quality Control with traceability, carry out detection six times with invention kit, average, contrast with Quality Control target value.Result shows, testing result average is close to target value, and relative deviation is less, and accuracy is better.The results are shown in Table 3.
Table 3
4, precision
Choose low value serum sample and each portion of high level serum sample, use kit to a serum sample METHOD FOR CONTINUOUS DETERMINATION 10 times, calculate the coefficient of variation that kit detects sample.Precision Experiment data are as shown in table 4: testing result shows, invention kit detects high level, low value sample coefficient of variation is respectively: 4.80%, 2.08%, and precision is better.The outstanding advantages of this kit has better precision at low magnitude portion, thus meet the repeatability of sample in low value region, the better clinical demand of accuracy.Data are in table 4
Table 4
| Testing time | Result (high level) | Result (low value) |
| 1 | 31.54 | 0.85 |
| 2 | 32.23 | 0.87 |
| 3 | 28.75 | 0.86 |
| 4 | 31.83 | 0.88 |
| 5 | 28.94 | 0.86 |
| 6 | 29.10 | 0.85 |
| 7 | 31.13 | 0.88 |
| 8 | 29.23 | 0.87 |
| 9 | 28.71 | 0.82 |
| 10 | 31.47 | 0.86 |
| Mean value | 30.29 | 0.86 |
| SD | 1.453 | 0.018 |
| CV | 4.80％ | 2.08％ |
Claims (3)
1. measure a kit for human body content of glycocholic acid, it is characterized in that comprising: glycocholic acid R1 reagent; Glycocholic acid R2 reagent and glycocholic acid calibration object;
Described glycocholic acid R1 reagent is made up of glycocholic acid-protein conjugate and damping fluid, and described damping fluid is the Tirs-HCl solution of concentration 10-100mM, and the pH value of described damping fluid is 7-8; The sodium chloride of PEG-6000,0.7-0.9wt% of concentration 2-6wt%, the Sodium azide of 0.05-0.1wt%, PC-300,0.1-1wt% bovine serum albumin(BSA) of 0.3wt% and 20mMEDTA is contained in described damping fluid; In described glycocholic acid R1 reagent, the concentration of glycocholic acid-protein conjugate is 0.05-2mg/mL; Described glycocholic acid-protein conjugate is obtained after glycocholic acid and albumen coupling by coupling agent; In described glycocholic acid-protein conjugate, protein concentration is 0.1-2.0mg/mL;
Described glycocholic acid R2 reagent is made up of the latex particle of buffer suspension liquid and glycocholic acid-protein antibodies bag quilt, the glycocoll-Na0H damping fluid of described buffer suspension liquid to be concentration containing stabilizing agent and antiseptic be 20-100mM; Described stabilizing agent is made up of the bovine serum albumin(BSA) of final concentration 0.1-1.0wt%, the sodium chloride of 0.7-0.9wt%, the disodium ethylene diamine tetraacetate of 5-50mM, volumetric concentration 0.1-1.0%TX-100; Described antiseptic is PC-300, and final volume concentration is 0.05-0.1%; In described glycocholic acid R2 reagent, the massfraction of the latex particle of glycocholic acid-protein antibodies bag quilt is 0.05-1.0%;
Described glycocholic acid calibration object is made up of humanized's glycocholic acid and damping fluid, described damping fluid is the Tirs-HCl solution of concentration 50-200mM, the disodium ethylene diamine tetraacetate containing the bovine serum albumin(BSA) of final concentration 0.1-1wt%, PC-300,5-50mM of 0.05-0.5wt% in described damping fluid; Described glycocholic acid calibration object comprise humanized's glycocholic acid concentration be respectively 0,2.5,5,10,20,40, one group of calibration object of 80ug/ml.
2. a kind of kit measuring human body content of glycocholic acid according to claim 1, it is characterized in that, be selected from mixing of a kind of hypotype in human serum albumins, bovine serum albumin(BSA), ox transferrins, hemocyanin or multiple hypotype with the albumen of glycocholic acid coupling; Described coupling agent be selected from carbodiimides, glutaraldehyde, two isocyanic acid compounds or dihalide dinitro benzene one or more.
3. a kind of kit measuring human body content of glycocholic acid according to claim 1, is characterized in that, described latex particle is nucleocapsid structure, and diameter range is 100-450nm, and concentration is 1-5mg/mL; The surperficial with of described latex particle has the chemical group modified by carboxyl, amino, hydroxyl, hydrazide group or chloromethyl; The chemical group that described glycocholic acid-protein antibodies and latex particle itself carry is combined in latex particle surface by chemical bonded refractory.
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