ࡱ> %` R zbjbjٕB=#')FFF8FTFGf.NHdH"HHHhKhKhKeeeeeee$gh-jZeN(K@hKNNeHHQfXXXN<8HHeXNeXX[[HBH !]FSV[6^\gf0f[,jqUj[j[XhKvKTX2LDvLihKhKhKeecX^hKhKhKfNNNN$&++  SHAPE \* MERGEFORMAT  Goat anti- Rat Collagen Type IV Collect Sample serum or blood plasma Storage: 2 - 8 C Package size: 96 determinations PRINCIPLE OF THE METHOD The Co IV kit is a solid phase phase sandwich enzyme linked immuno sorbent assay(ELISA). Samples , including standards of known Co IV concentrations and unknowns are pipetted into these wells. During the first incubation, the Co IV antigen and a biotinylated monoclonal antibody specific for Co IV are simultaneously incubated. After washing, the enzyme(streptavidin-peroxydase)is added. After incubation and washing 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 Co IV present in the samples. REAGENTS PROVIDED AND RECONSTTTUTION REAGENTSStore at 2-8! 196 WELLS0.596 WELLSRECONSTTTUTION96/48-wells microtiter plates10.5Ready-to-usePlastiv cover21Ready-to-useStandard: 800ng/ml1Vials (0.6ml)0.5Vials (0.3ml)See reagents preparation on page 3Blank control1Vials (1.0ml)1Vials (0.5ml)Ready-to-useStandard Diluent1Vials (5ml)1Vials (2.5ml)Ready-to-useBiotinylated anti-Co IV1Vials (6ml)1Vials (3.0ml)Ready-to-useStreptavidin-HRP1Vials (10ml)1Vials (5.0ml)Ready-to-useWashing Buffer1Vials (20ml)1Vials (10ml)50 concentrateSubstrate A1Vials (6.0ml)1Vials (3.0ml)Ready-to-useSubstrate B1Vials 6.0ml)1Vials (3.0ml)Ready-to-useStopping Solution1Vials (6.0ml)1Vials (3.0ml)Ready-to-useSample Diluent 1Vials (12ml)1Vials (6.0ml)Ready-to-use MATERIAL REQUIRED BUT NOT PROVIDED Distilled water Pipettes:10ul050ul0100ul0200ul01000ul0 Vortex mixer and magnetic stirrer. SAFETY For research use only Avoid any skin contact with H2SO4 and TMB. In case of contact, wash thoroughly water. Do not eat, drink, smoke or apply cosmetics where kit reagents are used. Do not pipette by mouth. PROCEDURAL NOTES/LAB.QUALITY CONTROL 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. Once the desired number of strips has been removed, immediately reseal the bag to protect the remaining strips from edterioration. Cover or cap all reagents when not in use. Do not mis or interchange reagents between different lots. Do not use reagents beyond the expiration date of the kit . Use a clean disposable plastic pipette tip for each reagent, standard, or specimen addition in order to avoid cross-contamination, for the dispensing of H2SO4 and substrate solution, avoid pipettes with metal parts. Use a clean plastic container to prepare the washing solution. Thoroughly mix the reagents and samples before use by agitation or swirling. 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 insert absorbent paper directly into the wells. 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 contaminated and must be discarede. Read absorbances within 1 hour after completion of the assay. When pipetting reagents, maintain a consistent order of addition from well-to-well. This will ensure equal incubation times for all wells. Respect incubation times described in the assay procedure. SPECIMEN COLLECTION\ PROCESSING AND STORAGE Serum---Avoid any inintentional stimulation of the cells by the procedure. Use pyrogen\endotoxin free collecting tubes. Serum should be removed rapidly and carefully from the red cells after clothing. For that, after clothing, centrifuge at approximately 1000g for 10 min and remove serum. Plasma---EDTA\ citrate and heparin plasma can be assayed. Spin samples at 1000g for 30 min remove particulates. Harvest plasma. Cell culture supernatants---Remove particulates and aggregates by spinning at approximately 1000g for 10 min. Storage---If not analyzed shortly after collection, samples should be aliquoted(250-500ul) to avoid freeze-thaw cycles and stored 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. Recommendation---Do not thaw by heating at 37! or 56!. Thaw at room temperature and make sure that sample is completely thawed and homogenous before assaying. PREPARATION OF REAGENTS Standards: Standard have to be reconstituled with the volume of standard buffer diluent indicated on the vial. This reconstitution produces a stock solution of 800ng/ml Co IV. Allow standard to stand for 5 minutes with gentle swirling prior to making dilutions. Serial dilutions of standard must be made before each assys and cannot be stored. 800 ng/ml6 Standard Original density 50ul0400 ng/ml5 Standard 100ul 6 Standard +100ul diludent200 ng/ml4 Standard 100ul 5 Standard +100ul diludent100 ng/ml3 Standard 100ul 4 Standard +100ul diludent50 ng/ml2 Standard 100ul 3 Standard +100ul diludent25 ng/ml1 Standard 100ul 2 Standard +100ul diludent0 ng/mlBlank Control50ul0 Washing buffer 50concentrate: Dilute 50 times in distilled water. ASSAY METHOD Before use, mix all reagents thoroughly without making foam. 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, standard and blank should be assayed in duplicate. Remove sufficient microwell strips from the pouch. 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 A1 and A2 (see plate scheme below). Transfer 50ul from A1 and A2 to B1 and B2 wells. Mix the contents by repeated aspirations and ejections. Take care not to scratch the inner surface of microwells. 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