Human 2,3-dinor-6-keto-PGF1a ELISA kit96 tests
INTENDED USE
The 2,3-dinor-6-K-PG F1a ELISA is to be used for the in-vitro quantitative determination of 2,3-dinor-6-K-PG F1a in serum plasmabuffered solubions or cell culture medium. The assay will recognize both naturalrecombinant 2,3-dinor-6-K-PG F1a. The kit has been configured for research use onlyis not to be used in diagnostic procedures.
PRINCIPLE OF THE METHOD
The 2,3-dinor-6-K-PG F1a kit is a solid phase phase sandwich enzyme linked immuno sorbent assayELISA. Samples , including standards of known 2,3-dinor-6-K-PG F1a concentrationsunknowns are pipetted into these wells. During the first incubation, the 2,3-dinor-6-K-PG F1a antigena biotinylated monoclonal antibody specific for 2,3-dinor-6-K-PG F1a are simultaneously incubated. After washing, the enzymestreptavidin-peroxydaseis added. After incubationwashing to remove all the unbound enzyme, a substrate solution which is acting on the bound enzyme is added to induce a coloured reaction product. The intensity of this coloured product is directly proportional to the concentration of 2,3-dinor-6-K-PG F1a present in the samples.
REAGENTS PROVIDED AND RECONSTTTUTION
MATERIAL REQUIRED BUT NOT PROVIDED
l Distilled water
l Pipettes:10ul、50ul、100ul、200ul、1000ul。
l Vortex mixermagnetic stirrer.
SAFETY
l For research use only
l The human blood components included in this KIT have been testedfound non reactive for HBSAGanti-HIV. Nevertheless, no known method can offer complete assurance that human blood derivatives will not transmit hepatitis , AIDS or other infections.there fore, handling of reagents, serum or plasma specimens should be in accordance with local safety procedures, e.g.CDC/NIH health manual.
l Avoid any skin contact with H2SO4TMB. In case of contact, wash thoroughly water.
l Do not eat, drink, smoke or apply cosmetics where kit reagents are used.
l Do not pipette by mouth.
PROCEDURAL NOTES/LAB.QUALITY CONTROL
l When not in use, kit components should be stored refrigerated or frozen as indicated on vials or bottles. All reagents should be warmed to room temperature before use. Lyophilized standards should be discarded after use.
l once the desired number of strips has been removed, immediately reseal the bag to protect the remaining strips from edterioration.
l Cover or cap all reagents when not in use.
l Do not mis or interchange reagents between different lots.
l Do not use reagents beyond the expiration date of the kit .
l Use a clean disposable plastic pipette tip for each reagent, standard, or specimen addition in order to avoid cross-contamination, for the dispensing of H2SO4substrate solution, avoid pipettes with metal parts.
l Use a clean plastic container to prepare the washing solution.
l Thoroughly mix the reagentssamples before use by agitation or swirling.
l All residual washing liquid must be drained from the wells by efficient aspiration or by decantation followed by tapping the plate forcefully on absorbent paper. Never absorbent paper directly into the wells.
l The TMB solution is light sensitive. Avoid prolonged exposure to light, also, avoid contact of the TMB solution with metal to prevent colour development. Warning TMB is toxic avoid direct contact with hands. Dispose off properly. If a dark blue colour develops within a few minutes after preparation, this indicates that the TMB solution has been contaminatedmust be discarede. Read absorbances within 1 hour after completion of the assay.
l When pipetting reagents, maintain a consistent order of addition from well-to-well. This will ensure equal incubation times for all wells.
l Respect incubation times described in the assay procedure.
l Dispense the TMB solution within 15 min. following the washing of the microtiter plate.
SPECIMEN COLLECTION PROCESSING AND STORAGE
l Serum－－-Avoid any inintentional stimulation of the cells by the procedure. Use pyrogenendotoxin free collecting tubes. Serum should be removed rapidlycarefully from the red cells after clothing. For that, after clothing, centrifuge at approximately 1000×g for 10 minremove serum.
l Plasma－－-EDTA citrateheparin plasma can be assayed. Spin samples at 1000×g for 30 min remove particulates. Harvest plasma.
l Cell culture supernatants－－-Remove particulatesaggregates by spinning at approximately 1000×g for 10 min.
l Storage－－-If not analyzed shortly after collection, samples should be aliquoted250-500ul to avoid freeze-thaw cyclesstored frozen at -70℃. Avoid multiple freeze-thaw cycles of frozen specimens. When possible, avoid use of badly hemolyzed or lipemic sera. If large amounts of particles are present, this should be removed prior to assay by centrifugation or filtration.
l Recommendation－－-Do not thaw by heating at 37℃ or 56℃. Thaw at room temperaturemake sure that sample is completely thawedhomogenous before assaying.
PREPARATION OF REAGENTS
l Standards: Standard have to be reconstituled with the volume of standard buffer diluent indicated on the vial. This reconstitution produces a stock solution of 240 ng/ml 2,3-dinor-6-K-PG F1a. Allow standard to stand for 5 minutes with gentle swirling prior to making dilutions. Serial dilutions of standard must be made before each assyscannot be stored.
l
l Washing buffer 20×concentrate: Dilute 20 times in distilled water.
ASSAY METHOD
l Before use, mix all reagents thoroughly without making foam.
l Determine the number of microwell strips required to test the desired number of samples,plus appropriate number of wells needed for running blanks standards. Each sample, standardblank should be assayed in duplicate. Remove sufficient microwell strips from the pouch.
l Add 50ul of standard diluent to standard wells B1,B2, C1,C2, D1,D2, E1,E2, F1,F2. Reconstitute standard vial with the appropriate volume as described in the chapter reagents preparation. Preparation. Pipet 100ul of standard into wells A1A2 see plate scheme below. Transfer 50ul from A1A2 to B1B2 wells. Mix the contents by repeated aspirationsejections. Take care not to scratch the inner surface of microwells. Repat this procedure from the wells B1,B2 to wells C1,C2from wells C1,C2 to D1,D2so on creating two parallel rows of 2,3-dinor-6-K-PG F1a standard dilutions ranging，Add 50ul of standard diluent to the bland wells.
l Add 50ul of sample to sample wells.
l Add 50ul of diluted biotinylated anti-2,3-dinor-6-K-PG F1a to all wells.
l Cover with a plate voverincubate for 1 hour at 37℃.
l Remove the coverwash the plate as follows: ⑴ aspirate the liquid from each well, ⑵ dispensse 0.3ml of washing solution into each well. ⑶ Aspirate again the contet of each well after 0.5 minute. ⑷ Repeat steps ⑵⑶ three times.
l Distribute 80ul of streptavidin-HRP solution to all wells, including blank wells.
l Coverincubate 30 min at 37℃.
l Remove the coverempty wells, Wash microwell strips according to step, Proceed immediately to the next step.
l Add 50ul Substrate ASubstrate B to each well。Incubate for 15 min at 37℃。
l The enzyme-substrate reaction is stopped by quickly pipetting 50ul of H2SO4. stop reagent into each well, including the blank wells, to completelyuniformly inactivate the enzyme. Results must be red immediately after the addition of H2SO4.
l Read absorbance of each well on a spectrophotometer using 450nm as the primary wavelengthoptionally 620nm 610nm to 650nm is acceptable as the reference wavelength.
SUGGESTED PLATE SCHEME
LIMITATIONS OF THE PROCEDURE
Do not extrapolate the standard curve beyond the max standard curve point. The dose-response is non-linear in this regiongood accruacy is difficult to obtain.
INTENDED USE
The 2,3-dinor-6-K-PG F1a ELISA is to be used for the in-vitro quantitative determination of 2,3-dinor-6-K-PG F1a in serum plasmabuffered solubions or cell culture medium. The assay will recognize both naturalrecombinant 2,3-dinor-6-K-PG F1a. The kit has been configured for research use onlyis not to be used in diagnostic procedures.
PRINCIPLE OF THE METHOD
The 2,3-dinor-6-K-PG F1a kit is a solid phase phase sandwich enzyme linked immuno sorbent assayELISA. Samples , including standards of known 2,3-dinor-6-K-PG F1a concentrationsunknowns are pipetted into these wells. During the first incubation, the 2,3-dinor-6-K-PG F1a antigena biotinylated monoclonal antibody specific for 2,3-dinor-6-K-PG F1a are simultaneously incubated. After washing, the enzymestreptavidin-peroxydaseis added. After incubationwashing to remove all the unbound enzyme, a substrate solution which is acting on the bound enzyme is added to induce a coloured reaction product. The intensity of this coloured product is directly proportional to the concentration of 2,3-dinor-6-K-PG F1a present in the samples.
REAGENTS PROVIDED AND RECONSTTTUTION
| REAGENTS（Store at 2-8℃） | 1×96 WELLS | 0.5×96 WELLS | RECONSTTTUTION |
| 96/48-wells microtiter plates | 1 | 0.5 | Ready-to-use |
| Plastiv cover | 2 | 1 | Ready-to-use |
| Standard: 240ng/ml | 1Vials 0.6ml | 0.5Vials 0.3ml | See reagents preparation on page 3 |
| Blank control | 1Vials 1.0ml | 1Vials 0.5ml | Ready-to-use |
| Standard Diluent | 1Vials 5ml | 1Vials 2.5ml | Ready-to-use |
| Biotinylated anti-2,3-dinor-6-K-PG F1a | 1Vials 6ml | 1Vials 3.0ml | Ready-to-use |
| Streptavidin-HRP | 1Vials 10ml | 1Vials 5.0ml | Ready-to-use |
| Washing Buffer | 1Vials 60ml | 1Vials 30ml | 20× concentrate |
| Substrate A | 1Vials 6.0ml | 1Vials 3.0ml | Ready-to-use |
| Substrate B | 1Vials 6.0ml | 1Vials 3.0ml | Ready-to-use |
| Stopping Solution | 1Vials 6.0ml | 1Vials 3.0ml | Ready-to-use |
MATERIAL REQUIRED BUT NOT PROVIDED
l Distilled water
l Pipettes:10ul、50ul、100ul、200ul、1000ul。
l Vortex mixermagnetic stirrer.
SAFETY
l For research use only
l The human blood components included in this KIT have been testedfound non reactive for HBSAGanti-HIV. Nevertheless, no known method can offer complete assurance that human blood derivatives will not transmit hepatitis , AIDS or other infections.there fore, handling of reagents, serum or plasma specimens should be in accordance with local safety procedures, e.g.CDC/NIH health manual.
l Avoid any skin contact with H2SO4TMB. In case of contact, wash thoroughly water.
l Do not eat, drink, smoke or apply cosmetics where kit reagents are used.
l Do not pipette by mouth.
PROCEDURAL NOTES/LAB.QUALITY CONTROL
l When not in use, kit components should be stored refrigerated or frozen as indicated on vials or bottles. All reagents should be warmed to room temperature before use. Lyophilized standards should be discarded after use.
l once the desired number of strips has been removed, immediately reseal the bag to protect the remaining strips from edterioration.
l Cover or cap all reagents when not in use.
l Do not mis or interchange reagents between different lots.
l Do not use reagents beyond the expiration date of the kit .
l Use a clean disposable plastic pipette tip for each reagent, standard, or specimen addition in order to avoid cross-contamination, for the dispensing of H2SO4substrate solution, avoid pipettes with metal parts.
l Use a clean plastic container to prepare the washing solution.
l Thoroughly mix the reagentssamples before use by agitation or swirling.
l All residual washing liquid must be drained from the wells by efficient aspiration or by decantation followed by tapping the plate forcefully on absorbent paper. Never absorbent paper directly into the wells.
l The TMB solution is light sensitive. Avoid prolonged exposure to light, also, avoid contact of the TMB solution with metal to prevent colour development. Warning TMB is toxic avoid direct contact with hands. Dispose off properly. If a dark blue colour develops within a few minutes after preparation, this indicates that the TMB solution has been contaminatedmust be discarede. Read absorbances within 1 hour after completion of the assay.
l When pipetting reagents, maintain a consistent order of addition from well-to-well. This will ensure equal incubation times for all wells.
l Respect incubation times described in the assay procedure.
l Dispense the TMB solution within 15 min. following the washing of the microtiter plate.
SPECIMEN COLLECTION PROCESSING AND STORAGE
l Serum－－-Avoid any inintentional stimulation of the cells by the procedure. Use pyrogenendotoxin free collecting tubes. Serum should be removed rapidlycarefully from the red cells after clothing. For that, after clothing, centrifuge at approximately 1000×g for 10 minremove serum.
l Plasma－－-EDTA citrateheparin plasma can be assayed. Spin samples at 1000×g for 30 min remove particulates. Harvest plasma.
l Cell culture supernatants－－-Remove particulatesaggregates by spinning at approximately 1000×g for 10 min.
l Storage－－-If not analyzed shortly after collection, samples should be aliquoted250-500ul to avoid freeze-thaw cyclesstored frozen at -70℃. Avoid multiple freeze-thaw cycles of frozen specimens. When possible, avoid use of badly hemolyzed or lipemic sera. If large amounts of particles are present, this should be removed prior to assay by centrifugation or filtration.
l Recommendation－－-Do not thaw by heating at 37℃ or 56℃. Thaw at room temperaturemake sure that sample is completely thawedhomogenous before assaying.
PREPARATION OF REAGENTS
l Standards: Standard have to be reconstituled with the volume of standard buffer diluent indicated on the vial. This reconstitution produces a stock solution of 240 ng/ml 2,3-dinor-6-K-PG F1a. Allow standard to stand for 5 minutes with gentle swirling prior to making dilutions. Serial dilutions of standard must be made before each assyscannot be stored.
l
| 240 ng/ml | （6 Standard） | Original density 50ul。 |
| 120 ng/ml | （5 Standard） | 100ul 6 Standard +100ul diludent |
| 60 ng/ml | （4 Standard） | 100ul 5 Standard +100ul diludent |
| 30 ng/ml | （3 Standard） | 100ul 4 Standard +100ul diludent |
| 15 ng/ml | （2 Standard） | 100ul 3 Standard +100ul diludent |
| 7.5 ng/ml | （1 Standard） | 100ul 2 Standard +100ul diludent |
| 0 ng/ml | Blank Control | 50ul。 |
l Washing buffer 20×concentrate: Dilute 20 times in distilled water.
ASSAY METHOD
l Before use, mix all reagents thoroughly without making foam.
l Determine the number of microwell strips required to test the desired number of samples,plus appropriate number of wells needed for running blanks standards. Each sample, standardblank should be assayed in duplicate. Remove sufficient microwell strips from the pouch.
l Add 50ul of standard diluent to standard wells B1,B2, C1,C2, D1,D2, E1,E2, F1,F2. Reconstitute standard vial with the appropriate volume as described in the chapter reagents preparation. Preparation. Pipet 100ul of standard into wells A1A2 see plate scheme below. Transfer 50ul from A1A2 to B1B2 wells. Mix the contents by repeated aspirationsejections. Take care not to scratch the inner surface of microwells. Repat this procedure from the wells B1,B2 to wells C1,C2from wells C1,C2 to D1,D2so on creating two parallel rows of 2,3-dinor-6-K-PG F1a standard dilutions ranging，Add 50ul of standard diluent to the bland wells.
l Add 50ul of sample to sample wells.
l Add 50ul of diluted biotinylated anti-2,3-dinor-6-K-PG F1a to all wells.
l Cover with a plate voverincubate for 1 hour at 37℃.
l Remove the coverwash the plate as follows: ⑴ aspirate the liquid from each well, ⑵ dispensse 0.3ml of washing solution into each well. ⑶ Aspirate again the contet of each well after 0.5 minute. ⑷ Repeat steps ⑵⑶ three times.
l Distribute 80ul of streptavidin-HRP solution to all wells, including blank wells.
l Coverincubate 30 min at 37℃.
l Remove the coverempty wells, Wash microwell strips according to step, Proceed immediately to the next step.
l Add 50ul Substrate ASubstrate B to each well。Incubate for 15 min at 37℃。
l The enzyme-substrate reaction is stopped by quickly pipetting 50ul of H2SO4. stop reagent into each well, including the blank wells, to completelyuniformly inactivate the enzyme. Results must be red immediately after the addition of H2SO4.
l Read absorbance of each well on a spectrophotometer using 450nm as the primary wavelengthoptionally 620nm 610nm to 650nm is acceptable as the reference wavelength.
SUGGESTED PLATE SCHEME
| | Standard concentration（ng/ml） | | ||||||||||
| A | 240 | 240 | sample | sample | sample | sample | sample | sample | sample | sample | sample | sample |
| B | 120 | 120 | sample | sample | sample | sample | sample | sample | sample | sample | sample | sample |
| C | 60 | 60 | sample | sample | sample | sample | sample | sample | sample | sample | sample | sample |
| D | 30 | 30 | sample | sample | sample | sample | sample | sample | sample | sample | sample | sample |
| E | 15 | 15 | sample | sample | sample | sample | sample | sample | sample | sample | sample | sample |
| F | 7.5 | 7.5 | sample | sample | sample | sample | sample | sample | sample | sample | sample | sample |
| G | 0 | 0 | sample | sample | sample | sample | sample | sample | sample | sample | sample | sample |
| H | sample | sample | sample | sample | sample | sample | sample | sample | sample | sample | sample | sample |
LIMITATIONS OF THE PROCEDURE
Do not extrapolate the standard curve beyond the max standard curve point. The dose-response is non-linear in this regiongood accruacy is difficult to obtain.
