CN101833009A - Double antibody complex retinol-binding protein assay kit - Google Patents
Double antibody complex retinol-binding protein assay kit Download PDFInfo
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Abstract
A retinol-binding protein assay kit in the market at present has good specificity and insufficient sensitivity or has high sensitivity and poor specificity because the purity of antibodies is insufficient or the potency cannot meet the requirement. The invention provides a double antibody complex retinol-binding protein assay kit, which consists of three parts, namely, a reagent R1, a reagent R2 and calibration materials. The reagent R1 is a phosphate buffer system which consists of phosphate buffer solution with the pH of 7.2 to 7.6, polyethylene glycol 6000-8000 and ethylene diamine tetraacetic acid; the reagent R2 is antibody solution which consists of mouse anti-human monoclonal antibody, rabbit anti-human polyclonal antibody, phosphate buffer solution with the pH of 7.2 to 7.6 and the ethylene diamine tetraacetic acid. By adopting a complex antibody of the polyclonal antibody and monoclonal antibody, the sensitivity and high linearity are guaranteed, and the accuracy of the measured result is greatly improved.
Description
Technical field
The present invention relates to medical immunology in-vitro diagnosis field, particularly a kind of RBP ELISA that detects in serum or the urine is measured kit.
Background technology
RBP ELISA (RBP) is the transport protein of retinol in the blood (vitamin A).Berggard found at α in immunoelectrophoresis in 1961
2-globulin zone can form the protein of a long precipitation line, once is called long α
2-globulin.Afterwards in decades, people have carried out comprehensive research to the molecular structure of this protein, biological characteristics etc., find that this protein is distributed in normal person's body fluid widely, belong to α
1-globulin has and transport the function of retinol to surrounding tissue from liver cell.Think that now RBP mainly exists with the composite form of retinol, prealbumin combination in the blood, when retinol in the compound with after target cell combines, RBP just separates with prealbumin, leach from glomerulus, by the near-end renal cells absorb, degraded.Further investigation in recent years shows that the change of RBP content can reflect near-end renal tubular function, hepatic disorder degree sensitively, is the sensitive indicator that reflection kidney, liver and nutritive disease develop, lapse to.
The assay method of RBP mainly contains euzymelinked immunosorbent assay (ELISA) (EIA) at present, radioimmunology (RIA) reaches (ITA), immune tem analysis method is because of its stable reagent in above-mentioned three kinds of methods, operation automation, testing result fast, accurately, reliable, therefore become the assay method of the tool prospect of clinical labororatory.The purity of antibody is not enough or tire and do not reach requirement but the existing market RBP ELISA is measured kit, simultaneously, adopt a kind of antibody merely in the reagent process for preparation, cause the special good and insufficient sensitivity of kit, or sensitive spirit is higher but specificity is relatively poor.
Summary of the invention
Technical matters to be solved by this invention is to overcome the defective that above-mentioned available reagent box exists, provide the compound mensuration kit of a kind of double antibody to detect the content of RBP ELISA in serum or the urine, with reach easy and simple to handle, highly sensitive, specificity is good, fast, measurement result purpose accurately and reliably.
For achieving the above object, the technical solution used in the present invention is: the Retinal-binding protein detection kit that a kind of double antibody is compound, form by reagent R1, reagent R2 and calibration object three parts, reagent R1 is a phosphate buffer, and it consists of phosphate buffer 35-60mmol/L, Macrogol 6000-8000 60-95mmol/L and the disodium ethylene diamine tetraacetate 6-13mmol/L of pH=7.2-7.6; Reagent R2 is an antibody liquid, and it consists of phosphate buffer 35-60mmol/L and the disodium ethylene diamine tetraacetate 6-13mmol/L of mouse-anti human monoclonal antibodies 2-4ml/L (tiring greater than 1: 128), the anti-people's polyclonal antibody of rabbit 3-5ml/L (tiring greater than 1: 16), pH=7.2-7.6; Calibration object is the dried frozen aquatic products of 110mg/L human serum matrix, and it also comprises Sodium azide 0.2-2.2%, Tween-20 1-10%, bovine serum albumin(BSA) 1-3%, the number percent of the long-pending consumption of above-mentioned number percent behaviour serotonin plastid.Above-mentioned Sodium azide also can adopt other antiseptic, and Tween-20 also can adopt other stabilizing agent, and bovine serum albumin(BSA) also can adopt other surfactant.
Goat-anti people polyclonal antibody is tired greater than 1: 16 in the compound antibody that the present invention adopts, and the mouse-anti human monoclonal antibodies is tired greater than 1: 128, many anti-linear and high values that guarantee, and monoclonal antibody guarantees sensitivity and low value; The antibody that high-purity, height are tired can adopt single-point calibration line sexual norm can guarantee the accuracy of measurement result fully in 1-110mg/L, operation is easy, quick than the multiple spot calibration pattern that other producer's kit adopts, save cost, measurement result accurately and reliably.The present invention adopts the compound antibody of polyclonal antibody and monoclonal antibody to guarantee sensitivity with high linear, and the RBP ELISA kit used with present clinical labororatory compares, and the measurement result accuracy improves greatly.
Among the present invention, described antibody has been carried out purification process, its method is as follows: be linked to Ago-Gel 4B with crude antigen or purifying antigen and make affinity column, thick antibody is passed through described affinity column, the unconjugated nonspecific proteins of flush away, use the potassium rhodanide wash-out again, the wash-out effluent is pure polyclonal antibody.The affinity chromatography The Application of Technology, the purity that has improved antibody greatly with tire, avoided nonspecific reaction to greatest extent, make as a result that accuracy is greatly improved, precision is better.
The preservation of antibody: lyophilized antibodies has characteristics such as good stability, long shelf-life, is suitable for long preservation, can add an amount of glycerine and bovine serum albumin(BSA) (BSA) used as stabilizers after being made into reagent.
The buffer system of kit of the present invention is that pH value is the 7.2-7.6 phosphate buffer, because the time limit that is formed with of immune complex changes, after antigen-antibody meets, be combined into immediately little compound (＜19S), a few minutes just formed by several hours visible compound (＞19S).As quick turbidimetric assay, this speed is too slow, therefore, adds polymerizer (or short poly-agent) and can quicken big immune complex formation.The present invention adds short poly-agent and is the polyglycol (MW6000～8000) of using more, and concentration is about 4-6%.
The stability factor of reagent mainly contains the purity of antibody, the quality of damping fluid, the suspension granule in the reagent.Granule is invisible to the naked eye, and floats in the reagent, influences the absorption of light, and can't remove with conventional filtration, and its Main Ingredients and Appearance is impurity, staple fibre and bacterium, and this is to cause between bottle and one of big reason of difference between batch.Common filtration, can't remove the impurity that is invisible to the naked eye, the present invention is in order to improve the kit content of effective on to greatest extent, adopted the mocromembrane filtration in process of production, by discovering to the filtering membrane aperture, the precision that can significantly improve reagent is filtered in the small-bore, prolongs to calibrate stationary phase, dwindles difference between batch.
The relation (n=20) of the filter membrane aperture and the coefficient of variation, the calibration term of validity sees the following form
| Aperture (μ m) | ??0.25 | ??0.45 | ??0.6 | ??0.8 | ??1.0 | ??1.2 |
| ??CV(％) | ??3.2 | ??4.5 | ??5.1 | ??5.6 | ??7.5 | ??8.2 |
| The calibration term of validity (my god) | ??30 | ??14 | ??10 | ??8 | ??7 | ??5 |
When aperture during, can remove general contaminant particles substantially at 1 μ m; When aperture during, can remove short and small fiber substantially at 0.45 μ m; When aperture during, can remove microorganism such as degerm substantially at 0.25 μ m.The present invention selects in used filter membrane aperture 0.25 μ m or 0.45 μ m for use.
The calibration object that kit of the present invention adopted is the freeze-drying human serum, this human serum matrix is obtained after concentrating by the healthy human serum of no HBV, HIV, HAV, HCV, the TP infection sources, by one-level reference material and the reference method definite value of tracing to the source, further reduced the influence of matrix effect to measurement result.
Owing to adopted the antibody purification technology of immunochromatography, adopted single-point calibration linear equation when using in clinical labororatory.Can not only guarantee measurement result accurately and reliably, and cumbersome calibration object doubling dilution process when having avoided adopting the multiple spot calibration, thereby also reduced cost of determination, therefore, be particularly suitable for the application of carrying out of domestic clinical labororatories at different levels.
The present invention adopts the compound mensuration kit of a kind of double antibody to detect the content of RBP ELISA in serum or the urine, has improved the accuracy of measurement result greatly by the Application of composite of two groups of antibody; Simultaneously, in reagent, add suitable surfactant disodium ethylene diamine tetraacetate, can make the binding site of antigen-antibody fully exposed, thereby further improved the specificity of reaction.In addition, because the antibody titer height that this kit adopts, and purity is good, can adopt the equation model of single-point calibration fully, thereby the use of convenient clinical labororatory has also reduced cost, has bigger using value clinically.
Below in conjunction with specification drawings and specific embodiments the present invention is further described.
Description of drawings
Fig. 1 is that RBP ELISA is measured kit single-point calibration curve figure among the embodiment 1.
Fig. 2 is that RBP ELISA is measured kit multiple spot calibration curve figure among the embodiment 1.
Fig. 3 is that RBP ELISA is measured calibration of kit single-point and multiple spot calibration measurement result dependent linearity figure among the embodiment 1.
Fig. 4 is that RBP ELISA is measured kit single-point calibration curve figure among the embodiment 2.
Fig. 5 is that RBP ELISA is measured kit multiple spot calibration curve figure among the embodiment 2.
Fig. 6 is that RBP ELISA is measured calibration of kit single-point and multiple spot calibration measurement result dependent linearity figure among the embodiment 2.
Fig. 7 is that RBP ELISA is measured kit single-point calibration curve figure among the embodiment 3.
Fig. 8 is that RBP ELISA is measured kit multiple spot calibration curve figure among the embodiment 3.
Fig. 9 is that RBP ELISA is measured calibration of kit single-point and multiple spot calibration measurement result dependent linearity figure among the embodiment 3.
Embodiment
Measure the composed as follows of kit:
R1：
PH7.4 phosphate buffer 45mmol/L
Macrogol 6000 78mmol/L
Disodium ethylene diamine tetraacetate 10.2mmol/L
R2：
Mouse-anti human monoclonal antibodies 2ml/L
The anti-people's polyclonal antibody of rabbit 3ml/L
PH7.4 phosphate buffer 45mmlo/L
Disodium ethylene diamine tetraacetate 10.2mmlo/L
Human serum matrix calibration object:
Concentration: 110mg/L
Sodium azide 0.2%
Tween-20 1%
Bovine serum albumin(BSA) 1%
The RBP ELISA that present embodiment is described is measured kit, is suitable for various types of automatic clinical chemistry analyzers, is example with Hitachi's 7060 automatic clinical chemistry analyzers, and its operation is as follows:
Result to gained carries out single-point calibration and multiple spot calibration.The Spline equation in the nonlinear calibration is adopted in multiple spot calibration, and instrument generates calibration curve automatically, can calculate RBP concentration in the sample automatically according to calibration curve.
From the calibration curve figure of Fig. 2 as can be seen, the RPB calibration object of 110mg/L carried out the doubling dilution of 8 concentration after, the absorbance that records basic with dilute after concentration be directly proportional, multiple spot calibration curve figure is a straight line.And the absorbance of the calibration object concentration of 110mg/L was basic identical when the calibration object concentration of 110mg/L and multiple spot were calibrated when single-point was calibrated among Fig. 1.Can analyze thus, kit of the present invention can adopt single-point linear scaled equation to substitute cumbersome multiple spot calibration equation.
RBP ELISA is measured the contrast of calibration of kit single-point and multiple spot calibration measurement result among the embodiment 1
Determination data sees the following form:
| Mark this shop | Single-point calibration measurement result (mg/L) | Multiple spot calibration measurement result (mg/L) |
| ??1 | ??55.8 | ??53.9 |
| ??2 | ??45.6 | ??46.7 |
| ??3 | ??23.9 | ??23.7 |
| ??4 | ??44.6 | ??44.9 |
| ??5 | ??88.5 | ??89.6 |
| ??6 | ??19.8 | ??21.8 |
| ??7 | ??66.3 | ??65.5 |
| ??8 | ??34.2 | ??33.8 |
| ??9 | ??38.9 | ??39.4 |
| ??10 | ??44.7 | ??45.1 |
| Mark this shop | Single-point calibration measurement result (mg/L) | Multiple spot calibration measurement result (mg/L) |
| ??11 | ??54.6 | ??55.6 |
| ??12 | ??40.9 | ??40.8 |
| ??13 | ??29.7 | ??28.9 |
| ??14 | ??33.9 | ??34.1 |
| ??15 | ??67.8 | ??68.2 |
| ??16 | ??90.7 | ??92.8 |
| ??17 | ??34.5 | ??33.9 |
| ??18 | ??56.4 | ??57.4 |
| ??19 | ??63.3 | ??64.5 |
| ??20 | ??29.7 | ??30.7 |
| ??21 | ??35.6 | ??34.5 |
| ??22 | ??44.2 | ??45.3 |
| ??23 | ??65.3 | ??64.7 |
| ??24 | ??54.2 | ??55.6 |
| ??25 | ??48.6 | ??47.8 |
| ??26 | ??41.2 | ??40.6 |
| ??27 | ??53.0 | ??53.9 |
| ??28 | ??37.9 | ??38.6 |
| ??29 | ??42.4 | ??43.2 |
| ??30 | ??48.7 | ??49.5 |
Calculate the related coefficient of two groups of experimental datas: r=0.998488, the dependent linearity figure (see figure 3) of result and two groups of data shows by experiment, kit single-point calibration measurement result of the present invention and multiple spot calibration measurement result height correlation, therefore, measurement result is in the 0-110mg/L scope, and kit of the present invention can adopt the single-point linear scaled to substitute cumbersome multiple spot nonlinear calibration equation fully.
This measuring sample is: the calibration object to 110mg/L carries out a series of doubling dilutions, and dilution ratio sees the following form:
| Dilution ratio | The present invention measures kit measurement result (mg/L) | Domestic certain commercially available reagent box measurement result (mg/L) | Theoretical value (mg/L) |
| ??1 | ??113.5 | ??106.2 | ??110 |
| ??0.9 | ??101.3 | ??90.1 | ??99 |
| ??0.8 | ??89.7 | ??85.4 | ??88 |
| ??0.7 | ??78.9 | ??76.9 | ??77 |
| ??0.6 | ??66.8 | ??68.6 | ??66 |
| ??0.5 | ??54.8 | ??54.8 | ??55 |
| ??0.4 | ??44.7 | ??46.8 | ??44 |
| ??0.3 | ??34.2 | ??39.6 | ??33 |
| ??0.2 | ??22.5 | ??29.7 | ??22 |
| ??0.1 | ??12.5 | ??23.2 | ??11 |
| ??0.05 | ??5.74 | ??12.89 | ??5.5 |
| ??0.025 | ??2.689 | ??10.96 | ??2.75 |
| ??0.0125 | ??1.124 | ??6.87 | ??1.375 |
Above-mentioned experimental data shows, the measurement result of domestic goods kit is below 33mg/L, measurement result is higher more than at least 20%, measurement result is on the low side more than 9% more than the 99mg/L. because the sensitivity of the mensuration that compound antibody technology such as kit of the present invention employing polyclonal antibody, monoclonal antibody not only guarantee, simultaneously, also guaranteed the setting-out line scope. in 0-110mg/L, the deviation of measurement result is all less than 4%.
Embodiment 2
It is composed as follows that RBP ELISA is measured kit:
R1：
PH7.6 phosphate buffer 60mmol/L
Macrogol 6000 95mmol/L
Disodium ethylene diamine tetraacetate 13mmol/L
R2：
Mouse-anti human monoclonal antibodies 3ml/L
The anti-people's polyclonal antibody of rabbit 4ml/L
PH7.6 phosphate buffer 60mmol/L
Disodium ethylene diamine tetraacetate 13mmol/L
Human serum matrix calibration object:
Concentration: 110mg/L
Sodium azide 1.2%
Tween-20 5.5%
Bovine serum albumin(BSA) 2%
Experimentation is with embodiment 1.
From the calibration curve figure of Fig. 5 as can be seen, the RPB calibration object of 110mg/L carried out the doubling dilution of 8 concentration after, the absorbance that records basic with dilute after concentration be directly proportional, multiple spot calibration curve figure is a straight line.And the absorbance of the calibration object concentration of 110mg/L was basic identical when the calibration object concentration of 110mg/L and multiple spot were calibrated when single-point was calibrated among Fig. 4.Can analyze thus, kit of the present invention can adopt single-point linear scaled equation to substitute cumbersome multiple spot calibration equation.
RBP ELISA is measured the contrast of calibration of kit single-point and multiple spot calibration measurement result among the embodiment 2
Determination data sees the following form:
| Mark this shop | Single-point calibration measurement result (mg/L) | Multiple spot calibration measurement result (mg/L) |
| ??1 | ??38.9 | ??39.8 |
| ??2 | ??27.8 | ??26.9 |
| ??3 | ??42.5 | ??43.5 |
| ??4 | ??53.6 | ??52.8 |
| ??5 | ??35.4 | ??36.5 |
| ??6 | ??46.3 | ??45.7 |
| ??7 | ??68.9 | ??68.7 |
| ??8 | ??72.5 | ??73.5 |
| ??9 | ??95.6 | ??95.1 |
| ??10 | ??120.5 | ??121.5 |
| ??11 | ??68.7 | ??67.9 |
| Mark this shop | Single-point calibration measurement result (mg/L) | Multiple spot calibration measurement result (mg/L) |
| ??12 | ??23.8 | ??24.7 |
| ??13 | ??33.7 | ??34.5 |
| ??14 | ??47.8 | ??46.8 |
| ??15 | ??35.7 | ??36.4 |
| ??16 | ??44.7 | ??43.5 |
| ??17 | ??78.9 | ??79.8 |
| ??18 | ??83.4 | ??84.5 |
| ??19 | ??85.1 | ??86.1 |
| ??20 | ??62.1 | ??63.1 |
| ??21 | ??50.4 | ??49.8 |
| ??22 | ??65.4 | ??66.5 |
| ??23 | ??42.6 | ??43.5 |
| ??24 | ??35.6 | ??36.7 |
| ??25 | ??29.8 | ??30.2 |
| ??26 | ??36.8 | ??35.8 |
| ??27 | ??42.1 | ??43.5 |
| ??28 | ??25.7 | ??24.8 |
| ??29 | ??71.5 | ??72.5 |
| ??30 | ??62.5 | ??63.7 |
Calculate the related coefficient of two groups of experimental datas: r=0.999, the dependent linearity figure (see figure 6) of result and two groups of data shows by experiment, kit single-point calibration measurement result of the present invention and multiple spot calibration measurement result height correlation, therefore, measurement result is in the 0-110mg/L scope, and kit of the present invention can adopt the single-point linear scaled to substitute cumbersome multiple spot nonlinear calibration equation fully.
Embodiment 2 RBP ELISAs are measured kit and the contrast of contrast agents experimental data
This measuring sample is: the calibration object to 110mg/L carries out a series of doubling dilutions, and dilution ratio sees the following form:
| Dilution ratio | The present invention measures kit measurement result (mg/L) | Domestic certain commercially available reagent box measurement result (mg/L) | Theoretical value (mg/L) |
| ??1 | ??112.4 | ??103.5 | ??110 |
| ??0.9 | ??102.3 | ??89.6 | ??99 |
| ??0.8 | ??90.2 | ??80.9 | ??88 |
| ??0.7 | ??78.1 | ??77.5 | ??77 |
| ??0.6 | ??65.9 | ??66.5 | ??66 |
| ??0.5 | ??56.9 | ??56.2 | ??55 |
| ??0.4 | ??43.9 | ??47.5 | ??44 |
| ??0.3 | ??33.1 | ??36.8 | ??33 |
| ??0.2 | ??21.8 | ??30.2 | ??22 |
| ??0.1 | ??10.9 | ??20.8 | ??11 |
| ??0.05 | ??5.49 | ??11.6 | ??5.5 |
| ??0.025 | ??2.73 | ??9.87 | ??2.75 |
| ??0.0125 | ??1.47 | ??6.78 | ??1.375 |
Above-mentioned experimental data shows that the measurement result of domestic goods kit is below 33mg/L, and measurement result is higher more than 11.5%, and measurement result is at 99mg/L, more than on the low side more than 10.5%.Because kit of the present invention adopts compound antibodies such as polyclonal antibody, monoclonal antibody, the not only sensitivity of the mensuration of Bao Zhenging has also guaranteed the setting-out line scope in 0-110mg/L simultaneously, and the deviation of measurement result is all less than 4%.
Embodiment 3
It is composed as follows that RBP ELISA is measured kit:
R1：
PH7.2 phosphate buffer 35mmol/L
Macrogol 6000 60mmol/L
Disodium ethylene diamine tetraacetate 6mmol/L
R2：
Mouse-anti human monoclonal antibodies 4ml/L
The anti-people's polyclonal antibody of rabbit 5ml/L
PH7.2 phosphate buffer 35mmol/L
Disodium ethylene diamine tetraacetate 6mmol/L
Human serum matrix calibration object:
Concentration: 110mg/L
Sodium azide 2.2%
Tween-20 10%
Bovine serum albumin(BSA) 3%
Experimentation is with embodiment 1.
From the calibration curve figure of Fig. 8 as can be seen, the RPB calibration object of 110mg/L carried out the doubling dilution of 8 concentration after, the absorbance that records basic with dilute after concentration be directly proportional, multiple spot calibration curve figure is a straight line.And the absorbance of the calibration object concentration of 110mg/L was basic identical when the calibration object concentration of 110mg/L and multiple spot were calibrated during the calibration of the single-point among Fig. 7.Can analyze thus, kit of the present invention can adopt single-point linear scaled equation to substitute cumbersome multiple spot calibration equation.
RBP ELISA is measured the contrast of calibration of kit single-point and multiple spot calibration measurement result among the embodiment 3
Determination data sees the following form:
| Mark this shop | Single-point calibration measurement result (mg/L) | Multiple spot calibration measurement result (mg/L) |
| ??1 | ??87.5 | ??88.9 |
| ??2 | ??50.2 | ??49.8 |
| ??3 | ??29.7 | ??30.5 |
| ??4 | ??33.5 | ??34.5 |
| ??5 | ??24.7 | ??25.4 |
| ??6 | ??56.7 | ??57.8 |
| ??7 | ??66.1 | ??67.5 |
| ??8 | ??52.7 | ??51.2 |
| ??9 | ??48.9 | ??48.7 |
| ??10 | ??66.7 | ??66.1 |
| ??11 | ??90.5 | ??92.1 |
| ??12 | ??129.8 | ??128.7 |
| Mark this shop | Single-point calibration measurement result (mg/L) | Multiple spot calibration measurement result (mg/L) |
| ??13 | ??75.4 | ??76.8 |
| ??14 | ??42.5 | ??43.5 |
| ??15 | ??33.6 | ??34.5 |
| ??16 | ??51.4 | ??52.4 |
| ??17 | ??66.8 | ??67.8 |
| ??18 | ??89.7 | ??88.8 |
| ??19 | ??29.7 | ??30.2 |
| ??20 | ??33.7 | ??34.1 |
| ??21 | ??45.8 | ??46.5 |
| ??22 | ??96.7 | ??97.8 |
| ??23 | ??89.7 | ??90.2 |
| ??24 | ??23.5 | ??24.5 |
| ??25 | ??39.7 | ??40.1 |
| ??26 | ??42.7 | ??43.1 |
| ??27 | ??50.2 | ??51.2 |
| ??28 | ??62.3 | ??63.4 |
| ??29 | ??29.4 | ??30.2 |
| ??30 | ??33.8 | ??34.5 |
Calculate the related coefficient of two groups of experimental datas: r=0.999, the dependent linearity figure (see figure 9) of result and two groups of data shows by experiment, kit single-point calibration measurement result of the present invention and multiple spot calibration measurement result height correlation, therefore, measurement result is in the 0-110mg/L scope, and kit of the present invention can adopt the single-point linear scaled to substitute cumbersome multiple spot nonlinear calibration equation fully.
Embodiment 3 RBP ELISAs are measured kit and the contrast of contrast agents experimental data
This measuring sample is: the calibration object to 110mg/L carries out a series of doubling dilutions, and dilution ratio sees the following form:
| Dilution ratio | The present invention measures kit measurement result (mg/L) | Domestic certain commercially available reagent box measurement result (mg/L) | Theoretical value (mg/L) |
| ??1 | ??111.5 | ??102.6 | ??110 |
| ??0.9 | ??101.9 | ??88.1 | ??99 |
| ??0.8 | ??89.9 | ??81.5 | ??88 |
| ??0.7 | ??79.1 | ??76.1 | ??77 |
| ??0.6 | ??66.5 | ??64.7 | ??66 |
| ??0.5 | ??54.9 | ??55.8 | ??55 |
| ??0.4 | ??44.8 | ??46.2 | ??44 |
| ??0.3 | ??33.3 | ??38.2 | ??33 |
| ??0.2 | ??22.5 | ??31.3 | ??22 |
| ??0.1 | ??10.8 | ??21.4 | ??11 |
| ??0.05 | ??5.48 | ??10.9 | ??5.5 |
| ??0.025 | ??2.71 | ??9.74 | ??2.75 |
| ??0.0125 | ??1.45 | ??6.58 | ??1.375 |
Above-mentioned experimental data shows that the measurement result of domestic goods kit is below 33mg/L, and measurement result is higher more than 15.8%, and measurement result is more than 99mg/L, and is on the low side more than 12.5%.Because kit of the present invention adopts compound antibodies such as polyclonal antibody, monoclonal antibody, the not only sensitivity of the mensuration of Bao Zhenging has also guaranteed the setting-out line scope in 0-110mg/L simultaneously, and the deviation of measurement result is all less than 4%.
Claims (3)
1. Retinal-binding protein detection kit that double antibody is compound, form by reagent R1, reagent R2 and calibration object three parts, reagent R1 is a phosphate buffer, and it consists of phosphate buffer 35-60mmol/L, Macrogol 6000-800060-95mmol/L and the disodium ethylene diamine tetraacetate 6-13mmol/L of pH=7.2-7.6; Reagent R2 is an antibody liquid, and it consists of phosphate buffer 35-60mmol/L and the disodium ethylene diamine tetraacetate 6-13mmol/L of mouse-anti human monoclonal antibodies 2-4ml/L, the anti-people's polyclonal antibody of rabbit 3-5ml/L, pH=7.2-7.6; Calibration object is the dried frozen aquatic products of 130mg/L human serum matrix, and it also comprises antiseptic 0.2-2.2%, stabilizing agent 1-10% and surfactant 1-3%, the number percent of the long-pending consumption of above-mentioned number percent behaviour serotonin plastid.
2. mensuration kit according to claim 1, it is characterized in that described antibody has been carried out purification process, its method is as follows: be linked to Ago-Gel 4B with crude antigen or purifying antigen and make affinity column, thick antibody is passed through described affinity column, the unconjugated nonspecific proteins of flush away, use the potassium rhodanide wash-out again, the wash-out effluent is pure polyclonal antibody.
3. mensuration kit according to claim 1 and 2 is characterized in that reagent has adopted the mocromembrane filtration in process of production, and the filter membrane aperture is selected 0.25 μ m or 0.45 μ m for use.
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| CN201010159837A CN101833009A (en) | 2010-04-29 | 2010-04-29 | Double antibody complex retinol-binding protein assay kit |
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|---|---|---|---|
| CN201010159837A CN101833009A (en) | 2010-04-29 | 2010-04-29 | Double antibody complex retinol-binding protein assay kit |
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Cited By (12)
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| CN102517379A (en) * | 2011-12-05 | 2012-06-27 | 西北工业大学 | Formula of 96-condition kit for protein crystallization screening |
| CN102841210A (en) * | 2012-08-30 | 2012-12-26 | 谢兵 | Retinol detection kit and preparation method thereof |
| CN102944679A (en) * | 2012-11-15 | 2013-02-27 | 北京康美天鸿生物科技有限公司 | Kit for performing retinol binding protein detection by using latex turbidimetry |
| CN103134934A (en) * | 2013-02-27 | 2013-06-05 | 宁波美康生物科技股份有限公司 | Kit for simultaneously detecting retinol-binding protein (RBP) in urine sample and serum sample |
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