CN102721818A - Competitive inhibitory enzyme-linked immunoassay kit of visfatin and method - Google Patents
Competitive inhibitory enzyme-linked immunoassay kit of visfatin and method Download PDFInfo
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- CN102721818A CN102721818A CN2012102158078A CN201210215807A CN102721818A CN 102721818 A CN102721818 A CN 102721818A CN 2012102158078 A CN2012102158078 A CN 2012102158078A CN 201210215807 A CN201210215807 A CN 201210215807A CN 102721818 A CN102721818 A CN 102721818A
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Abstract
The invention discloses a competitive inhibitory enzyme-linked immunoassay kit of visfatin. The kit comprises (1) anti-visfatin antibodies and visfatin polypeptides containing local or whole peptide segment of holoprotein of visfatin, wherein the visfatin polypeptides are in specific antigen-antibody reaction with the anti-visfatin antibodies; (2) a reactant and a detector for detecting whether a sample to be tested has visfatin or not by competitive inhibitory enzyme-linked immunoassay and determining the contained substances, wherein the visfatin polypeptides are polypeptides with amino acid sequence shown as SEQ ID NO:1. The invention further discloses a quantitative and qualitative method for visfatin in the sample to be tested by using the kit. The scheme provided by the invention can be used for directly measuring visfatin in human serum, blood plasma and other biological liquids quantitatively and qualitatively, and has the advantages of rapidness and accuracy, high sensitivity and throughput, low cost and the like, and is of huge guiding significance for prevention and treatment of diseases related with visfatin.
Description
Technical field
The present invention relates to field of biomedicine technology, relate in particular to the plain competition inhibitory enzyme linked immune assay kit of a kind of lactones and adopt this kit that lactones element in the testing sample is carried out qualitative and quantitative methods.
Technical background
Obesity is that fat is a kind of symptom of characteristic too much accumulation of tissue, has become one of important diseases that threatens public health.The not only concurrent lung that causes of obesity is pressed increase, joint and bone overload, and finally cause life-threatening important diseases to take place, and like angiocardiopathy, type ii diabetes and cancer etc.Many-sided research confirms, and the lactones element is the associated biomolecule bioactive molecule that disease takes place of causeing fat.
Lactones plain (Visfatin is also referred to as PBEF and Nampt) is the interior albumen of the cytoplasm of 52 kDa size, is made up of 473 amino acid, is enriched in interior fat; Be to separate the earliest, further discover that it is a kind of novel adipocyte factor that influences glycolipid metabolism, have the lipogenesis of promotion and similar insulin active effect through the cDNA library that makes up active periphery lymphocyte.In the evolution of obesity, the plain level of blood plasma lactones obviously increases.Research finds that also its mRNA highly expresses, and at liver, skeletal muscle, marrow, lymphoid tissue and fetal membrane expression is arranged all in the interior fat of animal used as test such as mouse, same, metabolism has the effect of adjusting to tallow.
Moreover, the lactones element also has other biological function.It can make, and the niacinamide phosphoribosyltransferase is active to raise, and is just regulating the biosynthesizing of icotinamide-adenine dinucleotide.The research report is also arranged, and the lactones element promotes the B cell maturation and suppresses neutrophil apoptosis.Current research is found, in the artery sclerosis macrophage, measures the plain existence of lactones, and the lactones element can activated mononuclear cell metalloproteinases 9, infers that thus the lactones element also is the active factors that very big correlativity is arranged with inflammation; In addition, the lactones element can stimulate TNF-α, and inflammatory factors such as interleukin 1 β (IL-1 β) and IL-6 are expressed.
Discoveries such as Berndt, concentration that the blood plasma interior fat is plain and body mass index (BMl) and body fat percentage (Percent body fat) are closely related.Research shows that the lactones element can promote the differentiation and maturation of adipocyte, promotes the generation and the gathering of fat, and the plain concentration of lactones also obviously raises in the blood plasma, thereby causes fat.There are some researches show that also the overweight people is after losing weight, its blood plasma lactones element presents decline.The clinical research result of Chen etc. confirms that also the lactones element has obvious rising in the type ii diabetes patient, and with waist-to-hipratio (Waist-to-Hip Ratio, WHR) directly related.
As stated, people have accumulated great deal of research results to the research of obesity, and have obtained great successes, but still do not have a kind of fast, accurately, carry out the method for predictive diagnosis and lactones element relevant disease.Traditional method is to measure multiple indexs such as blood pressure, blood sugar, blood fat, insulin, leptin and urine protein, with the generation and the development of comprehensive assessment and the plain relevant disease of lactones.Also have through non-directly related indexs such as WBV, plasma viscosity, packed cell volume, erythrocyte aggregation index, red blood cell rigidity indexs and accomplish assessment obesity; But the shortcoming of these methods is to be difficult to decision making through a certain detection, and must be the synthetic determination of several different methods: so test item is various, recall rate be low, and workload is big, and expense is high.In recent years, also utilize biochip technology, and analyzed and assess the polymorphism of genes of individuals, promptly genotype is predicted the hereditary probability that obesity takes place, but, and its accuracy, practicality, economy subject to confirmation and checking.
In view of this; Develop the plain immunodiagnosis kit of lactones element/interior fat in a kind of ability directly qualitative and quantitatively determining human serum, blood plasma and other biofluid; It has quick and precisely, highly sensitive, high flux, advantage such as cheap, and prevention and treatment with the plain relevant disease of lactones are had great directive significance.
Summary of the invention
Deficiency to prior art; Technical matters to be solved by this invention is to provide the plain competition inhibitory enzyme linked immune assay kit of lactones in a kind of ability directly qualitative and quantitatively determining human serum, blood plasma and other biofluid; It has quick and precisely, highly sensitive, high flux, advantage such as cheap, and prevention and treatment with the plain relevant disease of lactones are had great directive significance.
In order to solve the problems of the technologies described above; The competition inhibitory enzyme linked immune assay kit that a kind of lactones provided by the invention is plain comprises: the plain antibody of (1) anti-lactones with comprise the local or whole peptide section of the plain holoprotein of lactones and can with the plain polypeptide of lactones of the plain antibody generation of said anti-lactones specific anti antigen-antibody reaction; (2) reactant and detection agent are used for existing with not and confirm the material of its content through competition inhibition enzyme linked immunosorbent detection testing sample lactones is plain.
Preferably, the plain polypeptide of said lactones is the polypeptide of amino acid sequence shown in SEQ ID NO:1:
Plain polypeptide: the NH2-CVTKSYSFDEIRKNAQLNIELEAAHH (SEQ ID NO:1) of lactones.
Preferably, this kit also comprises the plain polypeptide of standard lactones, and the plain polypeptide of said standard lactones is to be formed by plain polypeptide of lactones and KLH key azurin chelating.More preferably; The plain polypeptide of said standard lactones prepares in the following manner: with the plain polypeptide of biotin labeling lactones, then biotin labeled lactones element polypeptide is placed bag filter, go out micromolecular compound wherein through dialysis; Hatch in the room temperature lucifuge in the KLH of equimolar amounts key azurin again; And place the 1 * PBS dialysed overnight that contains 0.1% Sodium azide, then quantitative its concentration, freeze-drying to preserve or prepare through doubling dilution in use the plain polypeptide of standard lactones of step concentration at once.
Preferably, the plain polypeptide marker of said lactones has biotin, and said reactant also comprises the biotin labeled Streptavidin of identification, and said marked by streptavidin has horseradish peroxidase.
Preferably, this kit also comprises and is coated with special two anti-ELISA Plates, said special two anti-can with the antigen-antibody reaction of the plain antibody generation of said anti-lactones specific anti.More preferably, said ELISA Plate is 96 hole ELISA Plates, and said special two anti-being generally to the kind of immune animal special two resist.
In addition, the present invention also provides a kind of lactones plain competition inhibitory enzyme linked immune detection method, comprises the steps:
Step 1, the plain competition inhibitory enzyme linked immune assay kit of preparation lactones, this kit comprises: the plain antibody of (1) anti-lactones with comprise the local or whole peptide section of the plain holoprotein of lactones and can with the plain polypeptide of lactones of the plain antibody generation of said anti-lactones specific anti antigen-antibody reaction; (2) reactant and detection agent are used for existing with not and confirm the material of its content through competition inhibition enzyme linked immunosorbent detection testing sample lactones is plain.
Step 2, exist with not and confirm its content through lactones in the competition inhibition enzyme linked immunosorbent detection testing sample is plain.
Preferably, the plain polypeptide of said lactones is the polypeptide of amino acid sequence shown in SEQ ID NO:1:
Plain polypeptide: the NH2-CVTKSYSFDEIRKNAQLNIELEAAHH (SEQ ID NO:1) of lactones.
Preferably, this kit also comprises the plain polypeptide of standard lactones, and the plain polypeptide of said standard lactones is to be formed by plain polypeptide of lactones and KLH key azurin chelating.More preferably; The plain polypeptide of said standard lactones prepares in the following manner: with the plain polypeptide of biotin labeling lactones, then biotin labeled lactones element polypeptide is placed bag filter, go out micromolecular compound wherein through dialysis; Hatch in the room temperature lucifuge in the KLH of equimolar amounts key azurin again; And place the 1 * PBS dialysed overnight that contains 0.1% Sodium azide, then quantitative its concentration, freeze-drying to preserve or prepare through doubling dilution in use the plain polypeptide of standard lactones of step concentration at once.
Preferably, the plain polypeptide marker of said lactones has biotin, and said reactant also comprises the biotin labeled Streptavidin of identification, and said marked by streptavidin has horseradish peroxidase.
This kit also comprises and is coated with special two anti-ELISA Plates, said special two anti-can with the antigen-antibody reaction of the plain antibody generation of said anti-lactones specific anti.More preferably, said ELISA Plate is 96 hole ELISA Plates, and said special two anti-being generally to the kind of immune animal special two resist.
In principle; In a concrete scheme of the present invention, with the kind special two anti-enzyme mark microwell plates that encapsulate, after the washing sealing; Add the plain antibody of anti-lactones; Carry out the room temperature incubation reaction, after the washing, add the plain polypeptide of biotin labeled lactones and be standard association reaction group again with the normal plain polypeptide of standard lactones (not using biotin labeling).Simultaneously, sample determination reaction group is also mixed and is added plain peptide of biotin labeled lactones and detected sample.Like this; In each reaction group potpourri all with the anti-lactones element antibody generation molecule that is combined in two high degree of specificity that resist the association reaction of vying each other; After competing the association reaction completion, fully washing has only the plain polypeptide of the biotin labeling lactones that is combined in the plain antibody of anti-lactones through the Ag-Ab specific binding reaction fully to form the strong compound that combines with the Streptavidin of horseradish peroxidase-labeled; Also and then add the enzyme chromogenic substrate through last washing, finally through enzymic catalytic reaction.Like this, the intensity of reaction solution is anti-with two-the plain antibody of lactones-biotin labeled lactones compound that plain polypeptide-the horseradish peroxidase Streptavidin forms is directly proportional, and with plain polypeptide of standard lactones and sample in the plain amount of lactones be inversely proportional to.According to the concentration known of confirming the plain polypeptide of adding standard lactones, draw out the typical curve of each measuring, thus, calculate the plain actual amount of corresponding lactones in the sample.
Than prior art; The present invention is the specificity of abundant conjugated antigen antibody response and the quick and susceptibility of enzyme reaction; A kind of enzyme immunity detection method that can accurately measure the lactones element of setting up and having developed; Compete the plain polypeptide of the lactones that combines the plain antibody ligand of anti-lactones through adopting, to combine the advantage of competition inhibition enzyme linked immunosorbent detection, the competition inhibitory enzyme linked immune assay kit of lactones element in ability direct qualitative and quantitatively determining human serum, blood plasma and other biofluid; It has quick and precisely, highly sensitive, high flux, advantage such as cheap, and prevention and treatment with the plain relevant disease of lactones are had great directive significance.
Embodiment
For making the present invention be more prone to understand,, further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.
Embodiment 1: the plain detection test of lactones in the testing sample
That adopts in the present embodiment is provided with 1 96 ELISA Plate by the production of U.S. Rui Boao bio tech ltd, has encapsulated kind special two in each orifice plate and has resisted, and then carries out the operation of following each step successively:
1, application of sample
Tear the diaphragm on the ELISA Plate, 100 μ l are added in the orifice plate through the sample that sample diluting liquid A diluted, be placed on then and shook under the room temperature on the shaking table 1 to 2 hour, perhaps also can under 4 ° of C, react 12-18 hour.
2, wash film
Cleansing solution cleans: sucking-off sample in the orifice plate, and with the cleansing solution cleaning of 1 times of 300 μ l, be placed on the shaking table room temperature afterwards and shook 5 minutes, repeat twice of this cleaning step again.Also can directly use and wash plate machine washing and wash.
3, the plain polypeptide working fluid of the biotin labeled lactones of preparation
The plain polypeptide centrifuge tube of of short duration centrifugal biotin labeling lactones joins the plain polypeptide of the biotin labeling lactones of 5 μ l among the sample diluting liquid A of 5 ml, and piping and druming evenly up and down.The concentration of the plain polypeptide of final biotin labeling lactones should be 10ng/ml.
4, the standard items working fluid of preparation variable concentrations
The plain polypeptide standard items of of short duration centrifugal lactones centrifuge tube adds the plain polypeptide working fluid of lactones of 990 μ l with the plain polypeptide standard items of the lactones of 10 μ l, obtains the standard items of 1000ng/ml and presses 10 times of dilutions with the plain polypeptide working fluid of lactones; Obtain 100ng/ml respectively; 10ng/ml, 1ng/ml, 100pg/ml; 10pg/ml, the standard items working fluid of 0pg/ml.
5, preparation sample working fluid
Sample is introduced: specimen in use is taken from the preceding liver cancer patient blood serum of treatment in 36 to 76 years old; Sample serum is diluted 2 times (can dilute by different multiples according to different sample concentrations) with sample diluting liquid A; Equally the sample of 10 μ l is pressed 4 times of dilutions with the plain polypeptide working fluid of lactones, guarantee that the concentration of the plain polypeptide working fluid of lactones is 10ng/ml.
6, preparation positive control working fluid
Of short duration centrifugal over against according to centrifuge tube, add the plain polypeptide of 103 μ l sample diluting liquid B and 2 μ l, 10 * biotin labeling lactones.
7, add standard items, sample and positive control
Add the standard items of the different gradients of 100 μ l, sample and positive control are established two repetitions on microwell plate.Cover microwell plate with diaphragm and be placed on the shaking table and shook under the room temperature condition 2.5 hours, also can under 4 ° of C, react 12-18 hour.Sucking-off reactant liquor in the microwell plate then, repeating step 2 wash the film step.
8, the Streptavidin that adds horseradish peroxidase-labeled
Add the streptavidin of the horseradish peroxidase-labeled that 100 μ l diluted to each grid, be placed on then and shook under the room temperature condition on the shaking table 1 to 2 hour, also can under 4 ° of C, react 12-18 hour.The Streptavidin of sucking-off horseradish peroxidase-labeled in the grid then, repeating step 2 wash the film step.
Wherein, the Streptavidin of horseradish peroxidase-labeled is purchased from company of U.S. Powerleader company (production number 554066), before the use, need carry out 20,000 times of dilutions with confining liquid.
What need say in addition, is in step 1 to the step 4 of this enforcement, use following solution, but its composition compound method to be following:
Sample diluting liquid (20 * PBS): potassium chloride 16g, sodium chloride 640g, potassium dihydrogen phosphate 16g, sodium hydrogen phosphate 92g, be dissolved in the 2.6L purified water after, add purified water to 4 liter again.
Sample diluting liquid A (20 * PBS): potassium chloride 16g, sodium chloride 640g, potassium dihydrogen phosphate 16g, sodium hydrogen phosphate 92g, be dissolved in the 2.6L purified water after, add 1.8% Sodium azide, be settled to 4 liters with purified water again.
Sample diluting liquid B (5 * PBS): potassium chloride 4g, sodium chloride 160g, potassium dihydrogen phosphate 4g, sodium hydrogen phosphate 23g, be dissolved in the 2.6L purified water after, be settled to 4 liters with purified water again.
The compound method of confining liquid is following: earlier 20 * PBS is diluted to 1 * PBS (20 * PBS 200ml; Purified water 3800ml); Prepare 10% bovine serum albumin(BSA) (bovine serum albumin(BSA) 400g, 1 * PBS add to 4 liters) then, prepare confining liquid (4 liters of 10% bovine serum albumin(BSA)s at last; 4 liters of caseins, mixing).
(20 * TBS) partitions are following: 2M TRIS buffer (pH7.5) 800ml, 5M sodium chloride 4800ml (sodium chloride 1461g, 3.3 liters of purified water for 20 * cleansing solution; Dissolving back purified water adds to 5 liters); Behind the mixing, purified water adds to 8 liters, tween 160ml.During use, 20 * cleansing solution doubling dilution is got final product.
The preparation of the plain polypeptide of standard lactones is through peptide section NH2-CVTKSYSFDEIRKNAQLNIELEAAHH and KLH key azurin chelating, and this 26 peptide section is positioned the 467th to 491 plain amino acid of lactones, and biochemical (Shanghai) Co., Ltd. is synthetic by gill.
The preparation method of the plain polypeptide of standard lactones: according to the plain polypeptide of the biotin labeling lactones of Thermo Scientific company production.The plain polypeptide of lactones is placed bag filter, and dialysed overnight is removed micromolecular compound such as Tris, glycocoll in 1 * PBS.EZ-Link Sulfo-NHS-LC-Biotin (Thermo Cat#21435) is hatched half an hour with the plain polypeptide of equivalent lactones in the room temperature lucifuge; Place the 1 * PBS dialysed overnight that contains 0.1% Sodium azide immediately; Measure biotin labeling lactones plain peptide concentration, packing freeze-drying with BCA Protein Assay Kit (Pierce Cat#23225) next day.
The preparation method of the plain antibody of anti-lactones: the plain polypeptide 200 μ g immunity of the lactones that emulsification is good new zealand rabbit; Carried out the immunity second time with same dose again at a distance from 10 to 14 days; Again at a distance from 7 to 10 days with the dosage immunity that reduces by half; Adopt a small amount of serum from the new zealand rabbit auricular vein and carry out ELISA experiment detection antibody titer; As tire to reach and then carry out heart blood sampling more than the 1:100000, adopt GE affinity column HiTrap Protein A HP (Cat#17-0403-01) purified blood serum of connecting with HiTrap Protein G HP (Cat#17-0404-01) to process freeze-drying dry powder antibody.Certainly, other common technologies prepare the plain antibody of anti-lactones in this area with also adopting as required at other embodiment of the present invention.
Kind special two anti-AffiniPure Goat anti-rabbit IgG (H+L) are (Cat#111-005-003) available from Jackson Immuno company.
9, detect
The TMB One-Step substrate reactions liquid that adds 100 μ l was hatched under room temperature 0.5 hour, added the stop buffer of 50 μ l again.Carry out reading in ELIASA 450nm optical filter.With numerical value substitution bioanalysis software SigmaPlot, be standard items concentration with the X axle, the Y axle is obtained the plain concentration of lactones in the sample serum for light absorption value.
ELISA Plate OD450 testing result is shown in table one:
Table one
Blank Assay A dilution OD450 is 0.08 and 0.11, and the positive control biotin is 2.499 and 2.497.The calculating of the plain practical measurement amount of lactones is according to getting on average value of reading of mensuration and standard group OD450; Comprise positive and sample and the blank value of reading; With standard items concentration is the X axle, and absorbance number percent is the Y axle, uses Sigma Plot or Fourparameter Logistic regression model software; The drawing standard curve is determined the plain actual amount of lactones thus.Computing formula: the number percent of absorbance log=(B-blank OD value)/(BO-blank OD value)
B=sample or standard OD value BO=zero standard OD value (total bond)
Obtain a result shown in table two:
Table two
The result shows that the plain lowest detection dosage of lactones is 379pg/ml or 6.82pM in the sensitivity test, and the linearity is 1 to 100 ng/ml.
Substitution hepatocarcinoma patient serum data are the X axle with standard items concentration, and absorbance number percent is the Y axle, use Sigma Plot or Fourparameter Logistic regression model software, draw lactones cellulose content in the hepatocarcinoma patient serum.
Hepatocarcinoma patient serum OD450 data are shown in table three:
Table three
Change the back data through Sigma Plot or Fourparameter Logistic regression model software: measurement range is between 55.9 to 111.4, and mean value is 95.5.
Second batch of hepatocarcinoma patient serum OD450 data is shown in table four:
Table four
Change the back data through Sigma Plot or Fourparameter Logistic regression model software: measurement range is between 71.3 to 137.4, and mean value is 92.1, and the result is shown in table five:
Table five
The result show kit criticize in difference less than 10% (according to of the comparison of same patients serum's sample) at different microwell plates, differences between batches are (according to the comparison with a kind of disease institute's sample thief plain concentration mean value of lactones in the different batches kit) less than 15%.
Should be noted that; Above embodiment is only in order to technical scheme of the present invention to be described but not to the restriction of protection domain of the present invention; Although the present invention has been done detailed description with reference to preferred embodiment; Those of ordinary skill in the art should be appreciated that and can make amendment or be equal to replacement technical scheme of the present invention, and do not break away from the essence and the scope of technical scheme of the present invention.
< 110>Guangzhou Ray Biotechnology Co., Ltd.
< 120>plain competition inhibitory enzyme linked immune assay kit and the method for a kind of lactones
<160>?1
<210>?1
<211>?26
<212>?PRT
< 213>artificial sequence
<400>?1
CVTKSYSFDEIRKNAQLNIELEAAHH
Claims (10)
1. the plain competition inhibitory enzyme linked immune assay kit of a lactones is characterized in that comprising: the plain antibody of (1) anti-lactones with comprise the local or whole peptide section of the plain holoprotein of lactones and can with the plain polypeptide of lactones of the plain antibody generation of said anti-lactones specific anti antigen-antibody reaction; (2) reactant and detection agent are used for existing with not and confirm the material of its content through competition inhibition enzyme linked immunosorbent detection testing sample lactones is plain.
2. the competition inhibitory enzyme linked immune assay kit that lactones according to claim 1 is plain is characterized in that: the plain polypeptide of said lactones is the polypeptide of amino acid sequence shown in SEQ ID NO:1.
3. the competition inhibitory enzyme linked immune assay kit that lactones according to claim 2 is plain is characterized in that, this kit also comprises the plain polypeptide of standard lactones, and the plain polypeptide of said standard lactones is to be formed by plain polypeptide of lactones and KLH key azurin chelating.
4. the competition inhibitory enzyme linked immune assay kit that lactones according to claim 3 is plain; It is characterized in that; The plain polypeptide of said standard lactones prepares in the following manner: with the plain polypeptide of biotin labeling lactones, then biotin labeled lactones element polypeptide is placed bag filter, go out micromolecular compound wherein through dialysis; Hatch in the room temperature lucifuge in the KLH of equimolar amounts key azurin again; And place the 1 * PBS dialysed overnight that contains 0.1% Sodium azide, then quantitative its concentration, freeze-drying to preserve or prepare through doubling dilution in use the plain polypeptide of standard lactones of step concentration at once.
5. the competition inhibitory enzyme linked immune assay kit that lactones according to claim 1 is plain; It is characterized in that: the plain polypeptide marker of said lactones has biotin; Said reactant also comprises the biotin labeled Streptavidin of identification, and said marked by streptavidin has horseradish peroxidase.
6. the competition inhibitory enzyme linked immune assay kit that lactones according to claim 3 is plain; It is characterized in that; This kit also comprises and is coated with special two anti-ELISA Plates, said special two anti-can with the antigen-antibody reaction of the plain antibody generation of said anti-lactones specific anti.
7. the competition inhibitory enzyme linked immune detection method that lactones is plain is characterized in that, comprises the steps:
Step 1, the plain competition inhibitory enzyme linked immune assay kit of preparation lactones, this kit comprises: the plain antibody of (1) anti-lactones with comprise the local or whole peptide section of the plain holoprotein of lactones and can with the plain polypeptide of lactones of the plain antibody generation of said anti-lactones specific anti antigen-antibody reaction; (2) reactant and detection agent are used for existing with not and confirm the material of its content through competition inhibition enzyme linked immunosorbent detection testing sample lactones is plain;
Step 2, exist with not and confirm its content through lactones in the competition inhibition enzyme linked immunosorbent detection testing sample is plain.
8. the competition inhibitory enzyme linked immune detection method that lactones according to claim 7 is plain is characterized in that: the plain polypeptide of said lactones is the polypeptide of amino acid sequence shown in SEQ ID NO:1.
9. the competition inhibitory enzyme linked immune detection method that lactones according to claim 7 is plain; It is characterized in that; This kit also comprises the plain polypeptide of standard lactones, and the plain polypeptide of said standard lactones is to be formed by plain polypeptide of lactones and KLH key azurin chelating, comprises the steps: with the plain polypeptide of biotin labeling lactones; Then the plain polypeptide of biotin labeled lactones is placed bag filter; Go out micromolecular compound wherein through dialysis, hatch in the room temperature lucifuge in the KLH of equimolar amounts key azurin again, and place the 1 * PBS dialysed overnight that contains 0.1% Sodium azide at once; The plain polypeptide of standard lactones of step concentration is preserved or prepared through doubling dilution in use to then quantitative its concentration, freeze-drying.
10. the competition inhibitory enzyme linked immune assay kit that lactones according to claim 1 is plain; It is characterized in that: this kit also comprises and is coated with special two anti-ELISA Plates, said special two anti-can with the antigen-antibody reaction of the plain antibody generation of said anti-lactones specific anti; The plain polypeptide marker of said lactones has biotin, and said reactant also comprises the biotin labeled Streptavidin of identification, and said marked by streptavidin has horseradish peroxidase.
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