CN102901823A - Leptin detection kit - Google Patents
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- CN102901823A CN102901823A CN2012104146056A CN201210414605A CN102901823A CN 102901823 A CN102901823 A CN 102901823A CN 2012104146056 A CN2012104146056 A CN 2012104146056A CN 201210414605 A CN201210414605 A CN 201210414605A CN 102901823 A CN102901823 A CN 102901823A
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Abstract
The invention relates to a leptin detection kit. The leptin detection kit comprises a leptin reference product, an antibody-coated microtiter plate, an enzyme-labeled anti-leptin, a washing solution, a color development solution and a stop solution, wherein the leptin reference product is of a recombinant human leptin polypeptide antigen, and the antibody-coated microtiter plate and the enzyme-labeled anti-leptin are of recombinant human leptin polypeptide monoclonal antibodies. The detection principle of the leptin detection kit disclosed by the invention is as follows: the recombinant human leptin polypeptide monoclonal antibody is taken as the antibody-coated microtiter plate, a serum sample to be detected is added, the corresponding enzyme-labeled antibody is further added, a compound of the coating antibody, the serum sample to be detected and the enzyme-labeled antibody is generated, the color development solution is added for color development, then the stop solution is further added for stopping, the residual solution is washed away by the washing solution, the light absorption value of the serum sample to be detected is determined, and the leptin content in the serum sample to be detected can be obtained by comparing with a standard curve.
Description
Technical field
The present invention relates to biology and field of medical examination, be specifically related to a kind of leptin detection kit.
Background technology
Along with human living standard's raising, a fat FAQs that has become the world today.Obesity is to ingest and the imbalance of energy consumption balancing, can cause various diseases, such as type ii diabetes, hypertension, high fat of blood and cancer etc.Leptin often is regarded as leptin, is the product of ob gene, and leptin acts on hypothalamus, and control metabolism, energy consumption and reproductive system are regulated body fat content and glycometabolism.Be all in the adipocyte and express, circulation leptin level and body fat amount are proportionate.Leptin works in hematopoiesis, adrenal cortex function, and the normal human serum leptin level is lower, and the variation of leptin can appear in, anorexia nervosa etc. unstable in artery sample sclerosis, growth hormone.
The leptin detection kit is a serodiagnostic conventional means of leptin, using the ultimate principle of euzymelinked immunosorbent assay (ELISA) carries out, described euzymelinked immunosorbent assay (ELISA) is also referred to as enzyme linked immunosorbent assay (ELISA) (enzyme-linked immunosorbent assay), be called for short ELLSA, the ELLSA process comprise antigen (antibody) be adsorbed on be called on the solid phase carrier coated, add antibody to be measured (antigen), add again corresponding enzyme labelled antibody (antigen), generate the compound of antigen (antibody)-antibody to be measured (antigen)-enzymic-labelled antibody, substrate reactions with this enzyme generates coloured product again, amount by spectrophotometric light absorption calculating antibody (antigen), quantitatively being directly proportional with coloured generation of antibody to be measured (antigen), the leptin detection kit is one of auxiliary diagnosis means that are used in the prior art clinical obesity and diabetes, but the sensitivity of existing leptin enzyme marking reagent and repeatability are relatively poor.
Summary of the invention
The purpose of this invention is to provide a kind of leptin detection kit, highly sensitive, the good reproducibility of this leptin detection kit.
For achieving the above object, the present invention is by the following technical solutions: a kind of leptin detection kit, comprise the leptin reference material, the coated antibody microwell plate, the anti-leptin of enzyme labeling, cleansing solution, nitrite ion, stop buffer, described leptin reference material is rh-Leptin's polypeptide antigen, described coated antibody microwell plate and the anti-leptin of enzyme labeling are rh-Leptin's polypeptide monoclonal antibody.
The detection principle of leptin detection kit of the present invention is as the coated antibody microwell plate with rh-Leptin's polypeptide monoclonal antibody, add the test serum sample, add again corresponding enzyme labelled antibody, generate the compound of coated antibody-test serum sample-enzyme labelled antibody, after adding the nitrite ion colour developing, add again stop buffer and stop, and with cleansing solution flush away Liquid Residue, measure the light absorption value of test serum sample, relatively can draw leptin content in the test serum sample with typical curve.
Leptin detection kit of the present invention mainly is to measure leptin content in human serum, blood plasma and other samples according to the enzyme linked immunological ratio juris, the auxiliary diagnosis that can be used for obesity and type ii diabetes, it is the leptin reference material that the present invention adopts rh-Leptin's polypeptide antigen, adopting rh-Leptin's polypeptide antigen monoclonal antibody is coated antibody microwell plate and the anti-leptin of enzyme labeling, the leptin detection kit that is consisted of by above-mentioned rh-Leptin's polypeptide antigen monoclonal antibody, highly sensitive, good reproducibility plays a significant role in the clinical detection of measuring leptin content.
Further, leptin detection kit of the present invention, wherein said coated antibody microwell plate is rh-Leptin's polypeptide antigen monoclonal antibody 6A5, the anti-leptin of described enzyme labeling is rh-Leptin's polypeptide antigen monoclonal antibody 3F7.Rh-Leptin's polypeptide antigen monoclonal antibody in coated antibody and the enzyme labelled antibody mainly is to determine according to the position of monoclonal antibody in 96 hole ELISA Plate.
Described cleansing solution is the Na by 28.8g
2HPO
412H
2The NaCl of O, 80g, the NaH of 3.9g
2PO
42H
2The Tween-20 of O, 50ml adds the purified water dissolving and is settled to 100000ml.
Described nitrite ion comprises nitrite ion A and nitrite ion B, nitrite ion A is to be 30% hydrogen peroxide is diluted to its volume with citrate buffer solution 1000 times of gained by concentration, and nitrite ion B is mixed with the 0.4mg/ml gained with the citrate buffer solution that contains 20% dimethyl sulfoxide (DMSO) with tetramethyl benzidine.Wherein said citrate buffer solution is the purified water gained that is dissolved in 5000ml by 3.4g sodium acetate and 5.2g citric acid.
Described stop buffer is the sulfuric acid solution of concentration 2mol/L.
Leptin detection kit of the present invention, described leptin reference material is rh-Leptin's polypeptide antigen, this rh-Leptin's polypeptide antigen adopts the synthetic method gained of the art routine, namely be to adopt the amplification leptin full-length gene of High fidelity PCR technology from people Cdna library, and be cloned into pUL, the dna sequence analysis result shows the in full accord of clone's leptin gene and reported in literature, subclone Leptin mature peptide coding section is to expression plasmid PJW2, construction expression plasmid pJL, Transformed E .coli DH5 α, the leptin reference material can be divided into the specification of 5 variable concentrations in the leptin detection kit of the present invention, be respectively 0ng/ml, 1ng/ml, 10ng/ml, 25ng/ml, 50ng/ml is in order to improve sensitivity and the repeatability of leptin detection kit of the present invention.
Described rh-Leptin's polypeptide antigen monoclonal antibody is prepared from by following steps:
(1) with female Balb/c mouse of leptin reference material immunity 6-8 week;
(2) getting immune spleen cell and the myeloma cell SP2/0 of above-mentioned mouse, is to merge to get hybridoma under 50% the polyglycol effect in concentration;
(3) hybridoma of above-mentioned fusion gained is cultivated through selective medium HAT, and filtered out the hybridoma that rh-Leptin's polypeptide antigen is had strong positive reaction, be the monoclonal antibody cell line;
(4) get that above-mentioned in good condition several suction pipe is blown down cell to monoclonal antibody cell line in growth period, centrifugal treating gained cell precipitation suspends take stroke-physiological saline solution and is adjusted to concentration as 2 * 10
7The cell suspending liquid of cells/ml;
(5) above-mentioned cell suspending liquid is seeded to the BALB/C mice abdominal cavity, 0.5ml/ only;
Observed afterwards in (6) 7 days and treat that mouse web portion expands blackout and can extract mouse ascites;
(7) with the mouse ascites that extracts through sad-ammonium sulfate precipitation, obtain rough monoclonal antibody after the dialysis;
(8) more above-mentioned rough monoclonal antibody being carried out purifying gets final product.
Leptin detection kit of the present invention, the reagent that uses all meets existing " Chinese pharmacopoeia and " the main raw and auxiliary material quality control standard of Products in China " requirement, do not include above-mentioned standardizing chemical reagent in, be chemical pure and above purity, employed external diagnosis reagent polyene hydroxyl drop bottle and other plastic products are produced by the prosperous space medicine equipment in Jintan City Ltd, all obtain State Food and Drug Administration (SFDA) registration, registration certificate number is state's cartridge bag word 20040041, used liquid component is by " Chinese Pharmacopoeia 2005 version biological products sterility test rules are carried out, therefore leptin detection kit of the present invention is safe and reliable.
Description of drawings
Fig. 1 is the canonical plotting of OD value of the present invention.
Embodiment
The composition of leptin detection kit:
The leptin reference material of (1) 5 bottle of variable concentrations specification: 0ng/ml, 1ng/ml, 10ng/ml, 25ng/ml, 50ng/ml, 1-3ml/ bottle;
(2) coated antibody microwell plate;
(3) the anti-leptin of enzyme labeling;
(4) cleansing solution, the 20ml/ bottle;
(5) nitrite ion A, nitrite ion B are the 10-15ml/ bottle;
(6) stop buffer, the 15-20ml/ bottle.
The preparation of rh-Leptin's polypeptide antigen monoclonal antibody may further comprise the steps:
(1) with female Balb/c mouse of rh-Leptin's polypeptide antigen immunity 6-8 week;
(2) getting immune spleen cell and the myeloma cell SP2/0 of above-mentioned mouse, is to merge to get hybridoma under 50% the polyglycol effect in concentration; Polyglycol is produced by the Sigma of the U.S., and molecular weight is 4000;
(3) hybridoma of above-mentioned fusion gained is cultivated through selective medium HAT, and filtered out the hybridoma that rh-Leptin's polypeptide antigen is had strong positive reaction, be the monoclonal antibody cell line; Selective medium HAT is produced by the Sigma of the U.S.;
(4) get that in good condition several suction pipe is blown down cell to monoclonal antibody cell line in growth period, centrifugal treating gained cell precipitation suspends take stroke-physiological saline solution and is adjusted to concentration as 2 * 10
7The cell suspending liquid of cells/ml;
(5) with syringe above-mentioned cell suspending liquid is seeded to the BALB/C mice abdominal cavity, 0.5ml/ only;
Observed afterwards in (6) 7 days and treat that mouse web portion expands blackout and can extract mouse ascites, usually adopt No. 12 injection needles to thrust mouse web portion, and with ascites drainage to centrifuge tube, the extraction amount of general every mouse is 2-4ml/, then the ascites that extracts in being the centrifuge tube of 3000rpm, was processed 5 minutes rotating speed, place in the clean saline bottle with washing pipe absorption supernatant, leave subzero 20 ℃ in, the shelf-life is 2 years.
(7) mouse ascites that extracts obtains rough monoclonal antibody through sad-ammonium sulfate precipitation after the dialysis;
(8) more above-mentioned rough monoclonal antibody being carried out purifying gets final product.
The purifying of concrete rh-Leptin's polypeptide antigen monoclonal antibody may further comprise the steps:
(1) getting rough monoclonal antibody flowing water melts;
(2) centrifugal removal precipitation in hydro-extractor, supernatant adds 2 times of volumes of acetic acid sodium damping fluid dilutions, and centrifugal treating got final product in 10 minutes when rotating speed is 8000rpm usually;
(3) drip 33 μ l by every milliliter of monoclonal antibody sad, and the continuous agitating solution muddiness that is creamy white, usually select magnetic stirring apparatus under room temperature environment, to stir and got final product in 1 hour;
(4) with the centrifugal removal of mentioned solution precipitation, centrifugal treating 15 minutes when rotating speed is 15000rpm normally, supernatant drips the cold saturated ammonium sulfate solution of equal volume, is reaction 2 hours under 2-8 ℃ the condition in temperature;
(5) again with above-mentioned reactant liquor centrifugal treating, centrifugal treating 15 minutes when 8000rpm normally, the gained precipitation PBS damping fluid dissolving identical with the volume of former monoclonal antibody, and the PBS damping fluid carried out dialysis treatment is reaction 12-14 hour under 2-8 ℃ the condition in temperature;
(6) namely get the monoclonal antibody of purifying after the centrifugal removal of the above-mentioned dialysate precipitation, the monoclonal antibody behind the purifying is to preserve under 2-8 ℃ the condition in temperature usually, and the shelf-life is 6 months; If the monoclonal antibody behind the purifying is preserved under temperature is subzero 20 ℃ condition, the shelf-life is 2 years.
Step 3
The concentration of coated antibody is definite in the coated antibody microwell plate, specifically may further comprise the steps:
(1) be that the concentration of 9.6 coated damping fluid dilution rh-Leptin polypeptide antigen monoclonal antibody 6A5 is respectively 1,2,5,10,20,40,100 μ l/ml with the pH value, and it is added respectively 96 hole ELISA Plate, the addition in every hole is 100 μ l, in temperature is to place 12-14 hour under 2-8 ℃ the condition;
(2) wash ELISA Plate with cleansing solution, it is 1% bovine serum albumin(BSA) that every hole adds 200 μ l concentration, and is to place 30 minutes under 37 ℃ the condition in temperature;
(3) wash ELISA Plate with cleansing solution, every hole adds the HRP-sheep anti mouse of 100 μ l, in temperature is to hatch 1 hour under 37 ℃ the condition;
(4) wash ELISA Plate with cleansing solution, every Kong Jun adds each 50 μ l of nitrite ion A, B, lucifuge reaction 10 minutes;
(5) every hole adds one of stop buffer, the rearmounted microplate reader 450nm of mixing wavelength is measured each hole optical density value, choosing the saturated least concentration of colour developing is coated antibody concentration, generally coated antibody concentration is 10 μ/ml, the coated antibody of this concentration is high in leptin detection kit medium sensitivity of the present invention, good reproducibility.
Wherein the pH value is that 9.6 coated damping fluids are to be settled to the 12000ml gained with the NaHCO3 of 32.3g and the Na2CO3 adding purified water dissolving of 17.5g.Concentration is that 1% bovine serum albumin(BSA) is that NaH2PO42H2O by the Na2HPO412H2O of Nacl, the 60.5g of the bovine serum albumin(BSA) of 210g, 168g and 8.2g adds the purified water dissolving and is settled to the 21000ml gained.
Step 4
The concentration of enzyme labelled antibody is definite in the anti-leptin of enzyme labeling, specifically may further comprise the steps:
(1) be that the concentration of 9.6 coated damping fluid dilution rh-Leptin polypeptide antigen monoclonal antibody 3F7 is 10 μ l/ml with the pH value, and it is added 96 hole ELISA Plate, the addition in every hole is 100 μ l, in temperature is to place 12-14 hour under 2-8 ℃ the condition;
(2) wash ELISA Plate with cleansing solution, it is 1% bovine serum albumin(BSA) that every hole adds 200 μ l concentration, in temperature is to place 30 minutes under 37 ℃ the condition;
(3) wash ELISA Plate with cleansing solution, it is the leptin reference material 100 μ l of 50ng/ml that every hole adds concentration, in temperature is to hatch 1 hour under 37 ℃ the condition;
(4) wash ELISA Plate with cleansing solution, every hole adds the enzyme labelled antibody solution of 100ul variable concentrations, in temperature is to hatch 1 hour under 37 ℃ the condition; The enzyme labelled antibody solution of described variable concentrations specifically mixes with damping fluid enzyme labelled antibody according to following proportioning, be respectively 1:200,1:400,1:800,1:1000,1:1500,1:2000;
(5) wash ELISA Plate with cleansing solution, every hole adds respectively nitrite ion A, the B of 50ul, lucifuge reaction 10 minutes;
(6) every hole adds one of stop buffer, the rearmounted microplate reader 450nm of mixing wavelength is measured each hole optical density value, choosing the saturated high dilution of colour developing is the concentration of enzyme labelled antibody, normally according to 1/1000 times of dilution of enzyme labelled antibody stoste, this concentration is fit to leptin detection kit of the present invention, good reproducibility when reality is used, highly sensitive.
Step 5
The Stability Determination of coated antibody microwell plate specifically may further comprise the steps:
(1) the coated antibody microwell plate being placed temperature is to place 3 days under 37 ℃ the condition;
(2) be that 50ng/ml rh-Leptin polypeptide antigen adds ELISA Plate with concentration, the addition in every hole is 100 μ l, does simultaneously 10 holes, hatches under 37 ℃ condition 1 hour in temperature;
(3) wash ELISA Plate with cleansing solution, every hole adds enzyme labelled antibody liquid 100 μ l, in temperature is to hatch 1 hour under 37 ℃ the condition;
(4) wash ELISA Plate with cleansing solution, every Kong Jun adds each 50 μ l of 50ul nitrite ion A, B, lucifuge reaction 10 minutes;
(5) then every hole adds stop buffer 50 μ l, puts microplate reader 450nm and measures optical density (OD) value, and the coefficient of variation is not higher than 10%.
Step 6
The monoclonal antibody cell line also passes through sterility test and detection of mycoplasma:
Used calibrating equipment comprises constant incubator, mold incubator, super-clean bench, test tube, nutrient culture media etc. during the sterility test of monoclonal antibody cell line; Adopt direct inoculation, concrete operation step is as follows: get 4ml cell suspension inoculation to be measured and increase bacterium in 200ml THIOGLYCOLLIC ACID salt fluid nutrient culture media; In temperature be cultivate under 20-25 ℃ the condition after 3-4 days that culture transferring to THIOGLYCOLLIC ACID salt fluid is cultivated, nutrient agar inclined-plane and each 2 pipe of improvement Martin nutrient culture media, every pipe 10ml culture medium inoculated 0.5ml; With THIOGLYCOLLIC ACID salt fluid cultivate, to put temperature be to cultivate under 30-35 ℃ the condition to each 1 pipe of nutrient agar inclined-plane, it is to cultivate under 20-25 ℃ the condition that all the other each pipes are put temperature; Simultaneously make negative control with 0.9% aseptic sodium chloride; Increase the overall process of tube and culture transferring pipe incubation time more than or equal to 14 days, concrete operation is carried out according to the regulation of " biological products sterility test " by these rules appendix method of inspection.
Calibrating equipment used during monoclonal antibody cell line detection of mycoplasma comprises constant incubator, super-clean bench, test tube, nutrient culture media etc.; Adopt cultivation, concrete operation step is as follows: get each 4 of the mycoplasma semifluid nutrient culture media of every loading amount 10ml and mycoplasma broth bouillons; Every inoculation monoclonal antibody cell line test sample 0.5-1.0ml puts temperature and is under 36 ± 1 ℃ the condition and cultivated 21 days; From 4, get 2 on the 7th day and carry out time culture, each 2 of every transferred species mycoplasma semifluid nutrient culture media and mycoplasma broth bouillons, put temperature and be under 36 ± 1 ℃ the condition and cultivated 21 days, observed once every 3 days, the relevant regulations that nutrient culture media variable color situation meets conventional detection of mycoplasma gets final product.
Wherein the composition of above-mentioned mycoplasma broth bouillon is as follows: the porcine stomach digestant of 500ml, and the sodium chloride of 2.5g, the beef infusion broth of 500ml (1:2), the glucose of 5.0g, the yeast of 5.0g soaks powder, and 0.02g's is phenol red; The pH value of described mycoplasma broth bouillon is 7.6 ± 0.2, is 121 ℃ in temperature and sterilized 15 minutes when reality is used, add the fire extinguishing calf serum before using and fully shake up, and the mycoplasma broth bouillon: serum is according to the ratio mixing of 8:2.
The composition of mycoplasma semifluid nutrient culture media does not add phenol red by mycoplasma broth bouillon prescription, other adds agar 2.5-3.0g; Be sterilization 15 minutes under 121 ℃ the condition in temperature, boiled before the use 10-15 minute, be cooled to about 56 ℃, add the fire extinguishing calf serum before using and fully shake up, wherein mycoplasma semifluid nutrient culture media: serum mixes according to the ratio of 8:2.
The monoclonal antibody cell line all adopts conventional sterility test and detection of mycoplasma method, can guarantee like this leptin detection kit of the present invention when reality is used, and is safe and reliable.
Step 7
The titre of monoclonal antibody, the mensuration of protein concentration:
The mensuration of the titre of monoclonal antibody may further comprise the steps:
(1) be 9.6 coated damping fluids dilution leptin reference material to 10 μ l/ml with the pH value, and it is added 96 hole ELISA Plate, the addition in every hole is 100 μ l, in temperature is to place 12-14 hour under 2-8 ℃ the condition;
(2) wash ELISA Plate with cleansing solution, every hole adds 1% bovine serum albumin(BSA) that concentration is 200 μ l, in temperature is to place 30 minutes under 37 ℃ the condition;
(3) wash ELISA Plate with cleansing solution, every hole adds 100 μ l monoclonal antibodies, with lavation buffer solution 2 times of gradient dilutions, is to hatch 1 hour under 37 ℃ the condition in temperature;
(4) wash ELISA Plate with cleansing solution, every hole adds the HRP-sheep anti mouse of 100 μ l, is 37 ℃ in temperature and hatches 1 hour;
(5) wash ELISA Plate with cleansing solution, every Kong Jun adds 50 μ l nitrite ion A, B, lucifuge reaction 10 minutes;
(6) every hole adds one of stop buffer, and mixing is put microplate reader 450nm wavelength and measured each hole optical density value, and the titre of gained monoclonal antibody is more than or equal to 1:10000.
The mensuration of the protein concentration of monoclonal antibody may further comprise the steps: 30 times of the PBS damping fluid dilutions that get monoclonal anti body and function Ph value and be 7.2, concentration is 0.01m, and measure absorbance log, by formula protein concentration=OD at 280nm and 260nm respectively
280nm÷ 1.4 calculates, and the protein concentration of the monoclonal antibody behind the purifying is more than or equal to 3mg/ml.
Step 8
The sensitivity of leptin detection kit, specificity, batch in and batch between imprecision and sensing range test:
(1) sensitivity test:
Calibrating equipment: constant temperature oven, microplate reader etc.; Wherein the leptin reference material comprises PBS, 0.5ng/ml, 1ng/ml, 10ng/ml; Leptin detection kit in 10 different lot number steps 1; Calibration method: leptin detection kit method of operating routinely, the 0-50ng/ml standard items are added ELISA Plate 100 μ l/ holes, add respectively reference product 100ul, all do multiple sky, hatched 1 hour for 37 ℃; Wash plate 3 times, every hole adds enzyme labelled antibody liquid 100ul, hatches 1 hour for 37 ℃; Wash plate 5 times, every hole adds nitrite ion A50ul, nitrite ion B50ul, lucifuge reaction 10 minutes; Every hole adds stop buffer 50ul, and mixing is put microplate reader 450nm and surveyed the OD value, draws shown in typical curve Fig. 1, and the testing result of 10 batches of different lot number leptin detection kit is as shown in table 1, shown in Fig. 1 and table 1, the sensitivity of leptin detection kit of the present invention is 1ng/ml, and is highly sensitive.
Table 1:10 criticizes different lot number leptin reagent testing results
(2) specific test:
The same sensitivity test of calibrating equipment, reference product: recombinant rat leptin (5ng/ml), rat blood serum, rh-Leptin (5ng/ml), human serum,
Calibration method: method of operating routinely, the 0-50ng/ml standard items are added ELISA Plate 100 μ l/ holes, add respectively the recombinant rat leptin, rat blood serum, the rh-Leptin, human serum 100ul all does multiple sky, hatches 1 hour for 37 ℃; Wash plate 3 times, every hole adds enzyme labelled antibody liquid 100ul, hatches 1 hour for 37 ℃; Wash plate 5 times, every hole adds nitrite ion A50ul, nitrite ion B50ul, lucifuge reaction 10 minutes; Every hole adds stop buffer 50ul, mixing is put microplate reader 450nm and is surveyed the OD value, drawing typical curve shows, recombinant rat leptin (5ng/ml) 0.11, rat blood serum 0.12, rh-Leptin (5ng/ml) 0.56, human serum 0.42, leptin detection kit of the present invention and rat leptin no cross reaction are described, specificity is good.
(3) criticize in and batch between imprecision test:
Calibrating equipment same sensitivity test, same lot number reagent, calibration method is done respectively 10 times continuously with the method for operating of above-mentioned routine, shown in table 2 and table 3, leptin detection kit of the present invention batch in and batch between imprecision all less than 15%.
Table 2: imprecision test findings table 3 between batch: imprecision test findings in batch
(4) sensing range test:
The same sensitivity test of calibrating equipment, reference product PBS, 0.5ng/ml, 1ng/ml, 10ng/ml, 25ng/ml, 50ng/ml, 100ng/ml; Calibration method is according to the assay method of routine, and test findings shows that the sensing range of leptin detection kit of the present invention is 0-50ng/ml.
The clinical trial report:
Blood station, center, 300 Hefei Cities Voluntary Blood Donors, in age 18-60 year, the height and weight index is in normal range, aseptic venous blood samples 3ml, centrifuging serum is to be measured under 4 ℃ the condition in temperature;
Concrete experimental technique is as follows:
(1) get coated microwell plate, every hole increase serum sample 100ul, each concentration leptin reference material 100ul all establishes multiple hole, in temperature is to place 1 hour under 37 ℃ the condition;
(2) with the coated microwell plate of cleansing solution flushing 3 times, pat dry, every hole adds the anti-leptin 100ul of enzyme labeling, in temperature is to place 1 hour under 37 ℃ the condition;
(3) be coated with microwell plate 5 times with the cleansing solution flushing, pat dry, every hole adds each one of nitrite ion A, B, lucifuge reaction 10 minutes;
(4) one of stop buffer is added in every hole again, and mixing is put microplate reader 450nm wavelength and measured each hole optical density value.
Take the A value of leptin reference material as ordinate, take the concentration of leptin reference material as horizontal ordinate, drawing standard response curve on coordinate paper, find leptin in the sample to be checked at curve, measure again after suitably diluting with dilution for the sample that exceeds 50ng/ml, experimental result is as shown in table 4, is shown by experiment, and the normal leptin reference range of the male sex is 1.56-2.52ng/ml; Female normal leptin reference range is 4.75-5.99ng/ml.
Table 4: the normal leptin term of reference that detects of leptin detection kit of the present invention
| The male sex | The women | |
| Ｘ | 2.04（ng/ml） | 5.37（ng/ml） |
| ＳＤ | 0.24（ng/ml） | 0.31（ng/ml） |
| Term of reference (X ± 2SD) | 2.04±0.48（ng/ml） | 5.37±0.62（ng/ml） |
Claims (8)
1. leptin detection kit, comprise the leptin reference material, the coated antibody microwell plate, the anti-leptin of enzyme labeling, cleansing solution, nitrite ion, stop buffer, it is characterized in that: described leptin reference material is rh-Leptin's polypeptide antigen, and described coated antibody microwell plate and the anti-leptin of enzyme labeling are rh-Leptin's polypeptide monoclonal antibody.
2. leptin detection kit according to claim 1, it is characterized in that: described coated antibody microwell plate is rh-Leptin's polypeptide antigen monoclonal antibody 6A5, the anti-leptin of described enzyme labeling is rh-Leptin's polypeptide antigen monoclonal antibody 3F7.
3. leptin detection kit according to claim 1, it is characterized in that: described cleansing solution is the Na by 28.8g
2HPO
412H
2The NaCl of O, 80g, the NaH of 3.9g
2PO
42H
2The Tween-20 of O, 50ml adds the purified water dissolving and is settled to 100000ml.
4. leptin detection kit according to claim 1, it is characterized in that: described nitrite ion comprises nitrite ion A and nitrite ion B.
5. leptin detection kit according to claim 1, it is characterized in that: described stop buffer is the sulfuric acid solution of concentration 2mol/L.
6. leptin detection kit according to claim 3 is characterized in that, described rh-Leptin's polypeptide antigen monoclonal antibody is prepared from by following steps:
(1) with female Balb/c mouse of leptin reference material immunity 6-8 week;
(2) getting immune spleen cell and the myeloma cell SP2/0 of above-mentioned mouse, is to merge to get hybridoma under 50% the polyglycol effect in concentration;
(3) hybridoma of above-mentioned fusion gained is cultivated through selective medium HAT, and filtered out the hybridoma that rh-Leptin's polypeptide antigen is had strong positive reaction, be the monoclonal antibody cell line;
(4) get that above-mentioned in good condition several suction pipe is blown down cell to monoclonal antibody cell line in growth period, centrifugal treating gained cell precipitation suspends take stroke-physiological saline solution and is adjusted to concentration as 2 * 10
7The cell suspending liquid of cells/ml;
(5) above-mentioned cell suspending liquid is seeded to the BALB/C mice abdominal cavity, 0.5ml/ only;
Observed afterwards in (6) 7 days and treat that mouse web portion expands blackout and can extract mouse ascites;
(7) with the mouse ascites that extracts through sad-ammonium sulfate precipitation, obtain rough monoclonal antibody after the dialysis;
(8) more above-mentioned rough monoclonal antibody being carried out purifying gets final product.
7. leptin detection kit according to claim 4 is characterized in that: described nitrite ion A is to be 30% hydrogen peroxide is diluted to its volume with citrate buffer solution 1000 times of gained by concentration; Described nitrite ion B is mixed with the 0.4mg/ml gained with the citrate buffer solution that contains 20% dimethyl sulfoxide (DMSO) with tetramethyl benzidine.
8. leptin detection kit according to claim 7, it is characterized in that: described citrate buffer solution is the purified water gained that is dissolved in 5000ml by 3.4g sodium acetate and 5.2g citric acid.
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| CN104459153A (en) * | 2014-12-05 | 2015-03-25 | 重庆中元生物技术有限公司 | Soluble leptin receptor (sOB-R) latex reinforced immunonephelometry assay kit by utilizing surface functional group |
| CN106290920A (en) * | 2016-08-30 | 2017-01-04 | 安徽安科生物工程（集团）股份有限公司 | Exenatide detection kit |
| CN106405117A (en) * | 2016-08-25 | 2017-02-15 | 杨凌波 | Plate type magnetic microparticle chemiluminescence analysis kit for detecting leptin antigen |
| CN113447661A (en) * | 2021-07-01 | 2021-09-28 | 安徽大千生物工程有限公司 | Leptin latex enhanced turbidimetry detection kit and preparation and use method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1378648A (en) * | 1999-10-10 | 2002-11-06 | 耶达研究发展有限公司 | Method for determining leptin |
| CN1814794A (en) * | 2005-11-22 | 2006-08-09 | 浙江大学 | Leptin and leptin receptor gene polymorphism detecting chip, and its preparing method and use |
| JP2006225312A (en) * | 2005-02-17 | 2006-08-31 | Fuji Oil Co Ltd | Leptin secretion-inhibiting composition |
| JP2006316020A (en) * | 2005-05-16 | 2006-11-24 | Nicca Chemical Co Ltd | Leptin production inhibitor |
-
2012
- 2012-10-26 CN CN2012104146056A patent/CN102901823A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1378648A (en) * | 1999-10-10 | 2002-11-06 | 耶达研究发展有限公司 | Method for determining leptin |
| JP2006225312A (en) * | 2005-02-17 | 2006-08-31 | Fuji Oil Co Ltd | Leptin secretion-inhibiting composition |
| JP2006316020A (en) * | 2005-05-16 | 2006-11-24 | Nicca Chemical Co Ltd | Leptin production inhibitor |
| CN1814794A (en) * | 2005-11-22 | 2006-08-09 | 浙江大学 | Leptin and leptin receptor gene polymorphism detecting chip, and its preparing method and use |
Non-Patent Citations (1)
| Title |
|---|
| 谢小强: "重组人瘦素蛋白单克隆抗体制备及乳腺癌患者血清瘦素水平检测方法建立", 《中国优秀硕士学位论文全文数据库 医药卫生辑》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104391120A (en) * | 2014-12-05 | 2015-03-04 | 重庆中元生物技术有限公司 | Latex enhanced turbidimetric immunoassay kit for detecting leptin by utilizing surface functional groups |
| CN104407157A (en) * | 2014-12-05 | 2015-03-11 | 重庆中元生物技术有限公司 | Latex enhanced turbidimetric immunoassay kit for detecting leptin by using ion stabilizer cooperatively with suspension stabilizer |
| CN104459105A (en) * | 2014-12-05 | 2015-03-25 | 重庆中元生物技术有限公司 | Latex-enhanced immunoturbidimetric assay kit for detecting leptin (LP) |
| CN104459153A (en) * | 2014-12-05 | 2015-03-25 | 重庆中元生物技术有限公司 | Soluble leptin receptor (sOB-R) latex reinforced immunonephelometry assay kit by utilizing surface functional group |
| CN104407157B (en) * | 2014-12-05 | 2016-03-30 | 重庆中元生物技术有限公司 | A kind of ion stabilizer and suspension stabilizer with the use of the latex enhancing immune of detection leptin than turbid kit |
| CN104459153B (en) * | 2014-12-05 | 2016-03-30 | 重庆中元生物技术有限公司 | A kind of sOB-R latex of surface functional group that utilizes strengthens immunoturbidimetry detection kit |
| CN106405117A (en) * | 2016-08-25 | 2017-02-15 | 杨凌波 | Plate type magnetic microparticle chemiluminescence analysis kit for detecting leptin antigen |
| CN106290920A (en) * | 2016-08-30 | 2017-01-04 | 安徽安科生物工程（集团）股份有限公司 | Exenatide detection kit |
| CN113447661A (en) * | 2021-07-01 | 2021-09-28 | 安徽大千生物工程有限公司 | Leptin latex enhanced turbidimetry detection kit and preparation and use method thereof |
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