CN102944679A - Kit for performing retinol binding protein detection by using latex turbidimetry - Google Patents
Kit for performing retinol binding protein detection by using latex turbidimetry Download PDFInfo
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Abstract
The invention relates to the technical field of biotechnology, and particularly discloses a kit for detecting retinol binding protein content by using latex immunoturbidimetry. The kit comprises a reagent R1, a reagent R2 and a standard product, wherein the reagent R1 is buffer solution with pH (Potential of Hydrogen) value of 6-9; the reagent R2 is latex reagent coated by anti-retinol binding protein double antibodies; and the standard product is retinol binding protein solution with pH value of 5-8. According to the kit, the retinol binding protein content in a sample can be detected by using the latex immunoturbidimetry; the sensitivity is high and can reach 0.042 mg/L; and the kit has the advantages of high stability, easiness and quickness in operation, high specificity, low probability of interference, accurate quantification and broad application prospect.
Description
Technical field
The present invention relates to biological technical field, particularly use double antibody and prepare the latex immunoreagent carries out Retinal-binding protein detection with the latex immunoturbidimetry kit.
Background technology
RBP ELISA (retinol binding protein, RBP) is the single peptide chain protein, and the about 21kD of molecular weight has a site-specific in conjunction with a part alltrans retinol, i.e. vitamin A.RBP ELISA falls into 5 types according to sequence homology: RBP in RBP, the nuclear receptor of retinoic acid, the specific extracellular RBP of visual organization and the specific cell of visual organization in secreting type RBP, the cell.Serum RBP4 is secreting type RBP, is the topmost transport protein in extracellular, is responsible for the retinol in combination, the transhipment blood.
RBP ELISA is the transport protein of vitamin in the blood, synthetic by liver, be distributed widely in human serum, cerebrospinal fluid, urine and other body fluid, but the RBP ELISA in the adipose tissue still accounts for 15%～30% of its synthetic quantity, the retinol of body about 20% is stored in adipose tissue.RBP and retinol, transthyretin (TTR) formed the macromolecule protein compound with 1: 1: 1 in blood circulation, and its biological action is that retinol is transported to target tissue from liver cell, and the intracellular transport metabolism that realizes VitA.The RBP ELISA normal value is the man in the serum: 36～56mg/L (36.0～56.0 μ g/ml), woman: 26.7～57.9mg/L (26.7～57.9 μ g/ml).
90%RBP is combined with retinol in the body, form retinol, RBP ELISA and thyroxinic polymer composite, can not be leached by glomerulus, the RBP of 10% free state then can leach through glomerulus, 99.97% is heavily absorbed by proximal tubular epithelial cells, and being broken down into amino acid, the synthetic utilization in the donor only has on a small quantity and drains from urine.Content is extremely low among the healthy human urine, less than 0.7mg/L.When the reabsorption obstacle, urine RBP discharges and to increase, and thinks at present to reflect one of the near-end renal tubular function preferably mark.Recent study shows: urine RBP content and renal tubular interstitium be impaired preferably correlativity, and think that interstitial disease of renal tubule early causes renal dysfunction than the glomerulopathy change, renal glomerular disease more than 30% is with interstitial disease of renal tubule, may make progress in this case to be renal failure.So urine RBP measures the attention that the importance of interstitial disease of renal tubule in carrying out property glomerulus is impaired is subject to more and more Chinese scholars.
The ephrosis albuminuria is often without any clinical manifestation, and routine inspection urine albumen mostly is negative, and serum urea nitrogen and Cr are as the index of tradition reflection renal function, and both sensitivitys are relatively poor.Because the compensatory capacity of glomerulus is very strong, only has the rising that when the glomerulus more than 50% is impaired, just can cause both.Abroad be reported in the early diagnosis of Kidney Damage by Hypertensive Disease, RBP is sensitiveer than β 2-MG, mAlb.Early stage at diabetic nephropathy, urine RBP4 drains to increase prior to microalbuminuria and occurs, and is considered to reflect the sign of Renal Injury.It is the good index of evaluate renal function earlier damage that urine RBP detects, and has preferably clinical value.
The method of present detection urine RBP, by the qualitative detection of initial immunoelectrophoresis, to the quantitative detection that unidirectional immunity diffusion RID, Enzyme-linked Immunosorbent Assay EIA, fluorescence immunoassay are arranged, and the robotization detection of arriving immunoturbidimetry, develop very rapid.At present clinical method of carrying out the normal employing of great amount of samples detection is immunoturbidimetry.
The method of carrying out clinically the great amount of samples detection at present is immune turbidimetry and immune scattering turbidimetry.Immunoturbidimetry is that antigen-antibody is in conjunction with the dynamic measurement method.Its ultimate principle is: when antigen and antibody reacts in special dilution system and during ratio suitable (general provision antibody excess), under the effect of the short poly-agent (polyglycol etc.) of the soluble immune complex that forms in dilution system, separate out from liquid phase, form particulate, make reactant liquor turbidity occur.When antibody concentration fixedly the time, the amount of the immune complex of formation increases along with the increase of antigen amount in the sample, and the turbidity of reactant liquor also increases thereupon.By turbidity and the contrast of series of standards product of assaying reaction liquid, can calculate the content of antigen in the sample.But when the antigen amount was very low, the antigen-antibody bond was less, and the turbidity of formation is low, was difficult for being detected, thereby caused sensitivity low.The latex enhancing immune turbidimetry then be with antibody labeling on latex particle, when antigen-antibody reaction, in the bond of formation is included in latex, thereby amplify the turbidity effect, be easy to be detected.
The kit that panimmunity transmittance purifying method is arranged in the market, can detect simultaneously the RBP concentration of serum and urine, but because Healthy Human Serum RBP concentration is 26.7～68.6mg/L, RBP concentration is less than 0.7mg/L in the urine, the range of linearity of kit is wide: detectability 10～140mg/L, cause when measuring urine RBP low concentration, all having the under-sensitive problem.Therefore select the suitable range of linearity, the kit that development can be accurately, sensitivity is carried out urine RBP concentration determination has a very big significance.
Summary of the invention
The object of the invention is to overcome the deficiency of at present existing RBP detection kit, provide the latex enhancing immune of a kind of highly sensitive detection RBP than turbid kit.
For realizing purpose of the present invention, the invention provides a kind of kit that adopts the latex immunoturbidimetry to detect RBP ELISA content, comprise reagent R1, reagent R2, calibration object; Described reagent R1 is the damping fluid of pH value 6-9, the emulsion reagent that the double antibody that described reagent R2 is anti-RBP ELISA is coated, and described calibration object is the RBP ELISA solution of pH value 5-8.
Double antibody is a strain monoclonal antibody and a kind of polyclonal antibody of pairing among the described reagent R2, and monoclonal antibody affinity is high, with the combination of RBP elder generation, plays the effect of catching, and polyclonal antibody contains many strains can in conjunction with the antibody of the different epi-positions of RBP, play measuring ability.Monoclonal antibody is selected from mouse-anti people, the anti-human antibody of rabbit, preferably, is selected from the mouse-anti human monoclonal antibodies; Polyclonal antibody is selected from mouse-anti people, anti-human, the goat anti-human antibody of rabbit, preferably, is selected from the anti-human polyclonal antibody of rabbit.
Preferably, the damping fluid of reagent 1, reagent 2, calibration object solution is selected from a kind of in Tris/HCl damping fluid, phosphate buffer, HEPES damping fluid, glycine buffer, barbitol buffer solution, MOPSO damping fluid, DIPSO damping fluid, the HEPPS damping fluid, and the pH scope of reagent R1 is 6-9; The pH scope of reagent R2 is 6-8; The pH scope of calibration object is 5-8, is preferably 5.9-6.1.
Preferably, reagent R1 contains the PEG 6000 of NaCl, the 1%-6% of 0.7%-0.9%, the Tween80 of 0.01%-0.1%.
Preferably, among the reagent R2, the RBP ELISA antibody concentration of selecting is 0.1-1mg/ml, is a strain RBP ELISA monoclonal antibody and a kind of RBP ELISA polyclonal antibody of pairing.Monoclonal antibody is selected from mouse-anti people or the anti-human monoclonal antibody of rabbit, preferred mouse monoclonal antibody; Polyclonal antibody is selected from the mouse-anti people, rabbit is anti-human or goat-anti people polyclonal antibody, the preferred anti-human polyclonal antibody of rabbit.
Described reagent R2 is prepared by following methods: after will resisting the monoclonal antibody of RBP ELISA and polyclonal antibody to mix by weight 1:1-4:1 and latex particle fully hatch, adding the sealing fluid-tight closes, the centrifugal supernatant that goes is dispersed to latex particle concentration to 2mg/ml with the Tris/HCl damping fluid.The preferred ratio of monoclonal antibody and polyclonal antibody is 1.5:1-3:1,
More preferably, the latex particle that preparation reagent R2 selects is hydrophobicity latex particle polyvinyl benzyl chloride latex, and on latex particle, the particle size range of latex particle is 50-250nm to the method for employing chemical crosslinking with antibody labeling, preferred 60-140nm.
Polyvinyl benzyl chloride latex particle is a kind of latex particle of nucleocapsid form, and its latex nuclear is the vinyltoluene polymkeric substance, and the latex shell is the polymkeric substance of vinyl chloride, is a kind of hydrophobicity latex.Have the chloromethyl reactive group on the surface of shell, these reactive groups can react by amino groups direct and antibody, antigen or other parts in the aqueous solution of gentleness, produce a kind of stable covalent compound by single step reaction.
In the specific embodiment of the present invention, standard items RBP ELISA solution concentration is respectively 8mg/L, 4mg/L, 2mg/L, 1mg/L, 0.5mg/L, 0mg/L.The pH scope of calibration object is 5-8, and the pH value of Healthy People urine is about 6, so be preferably 5.9-6.1
Preferably, contain stabilizing agent and antiseptic in the kit of the present invention.Described stabilizing agent is selected from one or more in protein, amino acid, inorganic salts, surfactant, suspending agent or the antioxidant; Described antiseptic is selected from 0.1% Sodium azide, Proclin 300 or gentamicin.
The principle that the present invention measures sample is the latex enhancing immune turbidimetry, selected sample is human urine, sample and reagent R1 (the pH value is the damping fluid of 6-9) preincubate (fully exposed the antibody combining site in the sample) after 35 minutes, add reagent R2 (RBP ELISA antibody latex reagent), continued to hatch 35 minutes, RBP ELISA in the urine is combined with the antibody of latex particle, form insoluble antigen-antibody-latex compound, produce certain turbidity, its turbidity height is directly proportional with specific antibody concentration in detecting sample.Under provision wavelengths, measure the absorbance of this insoluble antigen antibody complex, compare with the RBP ELISA calibration object of concentration known, then can calculate the concentration of RBP ELISA in the sample.
Adopt monoclonal antibody and the polyclonal antibody of pairing coated to latex particle among the present invention, coated antibody to latex particle is combined with RBP to be measured, forms complex compound, can detect turbidity and change.By detecting 6 calibration objects, drawing standard curve.When detecting sample, calculate the concentration of the RBP in the sample to be tested by the reaction absorbance.
This kit adopts the latex enhancing immune turbidimetry, and the complex compound that forms with RBP is than the immunoturbidimetry that does not adopt latex intensified, and turbidity changes obviously, and the signal reaction value is high, and the detection of denier albumen is also had signal response well.
This kit adopts a strain monoclonal antibody and a kind of Anti-TNF-α mark latex of high-affinity.When alone labeling of monoclonal antibody latex, the antigen in the sample is caught fully by antibody, forms latex-Antibody-antigen complex, but the epi-position that antigen can binding antibody is occupied, can not continue with antibody not form the variation of turbidity in conjunction with forming complex compound.When only with the how anti-mark latex of rabbit, can form the complex compound of latex-antibody-Ag-Ab-latex, good detection sensitivity is arranged, but linear bad, variation and the concentration of reaction absorbance are not proportional relationships.The present invention uses monoclonal antibody and polyclonal antibody together, has both guaranteed the sensitivity when how anti-independent use is, has obtained again good linearity.
Description of drawings
Fig. 1 shows the correlativity of kit 0.005～8mg/L range of linearity of the present invention.
Embodiment
The invention discloses a kind of latex turbidimetry and carry out the kit of Retinal-binding protein detection, those skilled in the art can use for reference this paper content, realize by suitable improvement technological parameter.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are deemed to be included within the present invention's technical scheme required for protection.Product of the present invention and method are described by preferred embodiment, the related personnel obviously can change or suitably change and combination methods and applications as herein described within not breaking away from content of the present invention, spirit and scope, realizes and use the technology of the present invention.
For making the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention, these embodiment only are not used in for explanation the present invention and limit the scope of the invention.Embodiment 1: the preparation of RBP ELISA latex immune reagent kit
1, the preparation of reagent R1
First with distilled water dissolving NaCl (7.0～9.0g), add again the Tris-HCl damping fluid, add at last distilled water to 1000ml, making the Tris final concentration is 0.05mol/L, fully shakes up, and adds a small amount of PEG 6000 and Tween80 again, mixes to get final product.
Those skilled in the art also can select other conventional damping fluid, such as in phosphate buffer, HEPES damping fluid, glycine buffer, the barbitol buffer solution one or more.
2, the preparation of reagent R2
Antibody mixed liquor preparation: get the anti-RBP ELISA mouse monoclonal antibody of 1mg/ml solution 2ml, get the anti-RBP ELISA rabbit polyclonal antibody solution 1ml of 1mg/ml, mix two solution for subsequent use.
Get 500ul 4%(g/ml) polyvinyl benzyl chloride particle (latex particle particle diameter 110nm) suspension, add the dilution of 2.5ml deionized-distilled water, under the 300rpm magnetic agitation, add the antibody-solutions that mixes in the latex solution, at room temperature (20-25 ℃) lower continuation stirred 45 minutes, hatched 3 hours at 37 ℃ again, form stable latex particle-antibody complex.Then the 1XPBS damping fluid that adds 1ml confining liquid (containing 0.2(g/ml) B SA, pH7.4) 37 ℃ of sealings 1 hour, the centrifugal supernatant that inclines, 1XPBS with pH=7.4 washs latex twice again, use at last 10ml 20mol/L Tris/HCl damping fluid (containing 0.1%BSA, 0.8%NaCl, 0.1% Sodium azide, 0.1% Tween 80) to disperse latex, making latex particle concentration is 2mg/ml.
3, the preparation of calibration object (also can select commercially available RBP ELISA)
The phosphate buffer of preparation pH=6, with the dissolving RBP ELISA, making it concentration is 8mg/ml, then with this solution doubling dilution, get a series of gradient solutions: 8mg/ml, 4mg/ml, 2mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0mg/ml place 4 ℃ for subsequent use.
Embodiment 2: the assay method of Retinal-binding protein detection reagent
This kit is applicable to Beckman, Hitachi, Olympus, Toshiba, Luo Shi, Abbott Laboratories, Siemens, step the full-automatic or semi-automatic biochemical analyzer of the brand such as auspicious, and assay method is as follows:
1, condition determination
The condition determination of kit of the present invention
Temperature: 37 ℃
Predominant wavelength: 560nm
Commplementary wave length: 800nm
Sample size: 5 μ L
R1 consumption: 200 μ L
R2 consumption: 40 μ L
Analysis type: Two point end assay
2, assay method is as follows:
Measure blank absorbency, add 5 μ L samples in 200ul R1 reagent, preincubate 5 minutes behind the mensuration blank absorbency, adds 40 μ LR2 reagent, hatches the assaying reaction absorbance 5 minutes.
Sample size, R1 consumption and R2 consumption can by the requirement of different model Biochemical Analyzer, be adjusted in the sample of regulation and the ratio of reagent dosage in " condition determination ".As in the instrument without specified wavelength, can select and the input of the immediate numerical value of specified wavelength.
(3) calibration and sample are measured
Use the calibration object in the kit to calibrate by the calibration procedure requirement in the use analytical instrument instructions, pattern is the multiple spot calibration, sets up working curve.After the calibration, measure serum sample, can calculate antibody concentration in the sample according to working curve.
Embodiment 3: the analytical performance assessment of RBP ELISA immunoreagent
1, sensitivity for analysis or minimum detectability
Contrast agents: external certain famous brand name
Detect principle: adopt Latex-enhanced immunoturbidimetric assay, detect its turbidity at 570nm and change, the RBP content in its intensity of variation and the sample is linear.
Sensitivity evaluation method: calibration object is diluted to a series of concentration near sensitivity, determine that through the ELISA method its concentration is 0.05mg/L, 0.10mg/L, 0.15mg/L, 0.21mg/L, increasing the zero standard product is sample, use two kinds of kits, according to measuring method separately, carry out replication 10 times, record reaction absorbance:
Table 1: embodiment 1 sensitivity measured value
The mean value of the zero standard product that replication is 10 times is-0.1, standard deviation S D is 2.81, (0.1+3X2.81) 8.43 as the minimum value of detecting take mean value and 3 times of standard deviation sums, because the reacting value of kit and concentration are proportional relations, so the sensitivity for analysis of kit is 8.43/10*0.05=0.042mg/L.
Table 2: contrast agents box sensitivity measured value
Evaluation criterion: because reacting value and the concentration of this kit is not proportional relation, so take (A-3SD) greater than the least concentration of (mean value 0+3SD) sensitivity as this detection kit.
Conclusion: the sensitivity of the kit of detection urine RBP of the present invention is 0.042mg/L, and the sensitivity of contrast agents box is 0.1mg/L, kit of the present invention highly sensitive in the sensitivity of contrast agents box.
2, accuracy and repeatability
Human urine take the calibration object of 4mg/L and sign value as 0.42mg/L is as sample, presses respectively replications 20 times of embodiment two described methods, calculates respectively and measures mean value X, standard deviation S D and coefficient of variation CV.The result shows that the coefficient of variation is respectively 2.11% and 2.45%.Investigate accuracy of measurement with relative deviation, relative deviation all in ± 15% scope, is investigated withinrun precision with the coefficient of variation, and the coefficient of variation of calibration object and urine specimen is respectively 3.9% and 4.6%.
3, betweenrun precision
With the reagent of the present invention of 3 lot numbers, measure respectively same urine specimen by embodiment two described methods, 5 times, calculate the coefficient of variation (CV) of 5 measurement results to investigate betweenrun precision, the result shows that the coefficient of variation is 6.7%.
4, the range of linearity
With the high value urine specimen (concentration is 7.86mg/L) near the range of linearity upper limit. press 1: 1,1: 3,1: 9,1: 27,1: 16,1:32 dilution with deionized water, altogether the solution of 6 variable concentrations, press each concentration determination of embodiment two described methods 3 times, actual measurement mean value and corresponding theory value are done regretional analysis, the calculating regression equation is y=0.9985x-0.0255, correlation coefficient r=0.9985, show kit of the present invention good relationship in (0.005～8) mg/L range of linearity, see accompanying drawing 1.
5, stability
With kit uncork of the present invention be placed on 2-8 ℃ preserved for 2 weeks after, taking out and measuring the sign values by example two described methods is the calibration object of 8.12mg/L, each replication 3 times calculates the relative deviation that detects mean value and sign value.Experimental result shows that the relative deviation that kit of the present invention detects mean value and sign value after 2 weeks of uncork is all less than 3%, and uncork stability better.
Place 2-8 ℃ of preservation after 16 months kit of the present invention, taking out and measuring the sign values by example two described methods is the calibration object of 8.12mg/L, and each replication 3 times calculates the relative deviation that detects average and sign value.The result shows the relative deviation of detected value and sign value all less than 4%, shows that it is more stable that kit of the present invention is preserved 16 months at 2-8 ℃.
The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a kit that adopts the latex turbidimetry to detect RBP ELISA content comprises reagent R1, reagent R2, calibration object; Described reagent R1 is the damping fluid of pH value 6-9, the emulsion reagent that the double antibody that described reagent R2 is anti-RBP ELISA is coated, and described calibration object is the RBP ELISA solution of pH value 5-8.
2. kit according to claim 1 is characterized in that: described damping fluid is selected from a kind of in Tris/HCl damping fluid, phosphate buffer, HEPES damping fluid, glycine buffer, barbitol buffer solution, MOPSO damping fluid, DIPSO damping fluid, the HEPPS damping fluid.
3. kit according to claim 1, reagent R1 also contains the PEG 6000 of NaCl, the 1%-6% of 0.7%-0.9%, the Tween80 of 0.01%-0.1%.
4. kit according to claim 1, it is characterized in that: described R2 reagent pH scope is 6-8, and the double antibody of described anti-RBP ELISA is the anti-RBP ELISA monoclonal antibody of a strain and a kind of anti-RBP ELISA polyclonal antibody of pairing.
5. kit according to claim 4, it is characterized in that: described monoclonal antibody is selected from mouse-anti people or the anti-human monoclonal antibody of rabbit; Described polyclonal antibody is selected from the mouse-anti people, rabbit is anti-human or goat-anti people polyclonal antibody.
6. each described kit according to claim 1-5, it is characterized in that: described reagent R2 is prepared by following methods: after will resisting the monoclonal antibody of RBP ELISA and polyclonal antibody to mix by weight 1:1-4:1 and latex particle fully hatch, adding the sealing fluid-tight closes, the centrifugal supernatant that goes is dispersed to latex particle concentration to 2mg/ml with the Tris/HCl damping fluid.
7. kit according to claim 6, it is characterized in that: described latex particle is hydrophobicity latex particle polyvinyl benzyl chloride latex, and its particle size range is 50-250nm, preferred 60-140nm.
8. kit according to claim 6 is characterized in that, the weight ratio of described monoclonal antibody and polyclonal antibody is 1.5:1-3:1; Described confining liquid is the 1XPBS damping fluid that contains 0.2g/ml BSA, pH7.4.
9. each described kit is characterized in that: also comprise stabilizing agent and antiseptic according to claim 1-8.
10. kit according to claim 9, it is characterized in that: described stabilizing agent is selected from one or more in protein, amino acid, inorganic salts, surfactant, suspending agent or the antioxidant; Described antiseptic is selected from 0.1% Sodium azide, Proclin 300 or gentamicin.
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