DRG Corticoprotein Releasing Factor (CRF) (mouse, rat, human
Contents
1. DIRE CG DRG Corticoprotein Releasing Factor CRF mouse rat human Florescent ELISA EIA 5082 USA INTRODUCTION This Fluorescent Immunoassay kit is designed to detect a specific peptide and its related peptides based on the principle of competitive enzyme immunoassay PRINCIPLE OF ENZYME IMMUNOASSAY WITH THIS KIT The immunoplate in this kit is pre coated with a secondary antibody and the nonspecific binding sites are blocked The secondary antibody can bind to the Fc fragment of the primary antibody peptide antibody whose Fab fragment will be competitively bound by both biotinylated peptide and peptide standard or targeted peptide in samples The biotinylated peptide interacts with streptavidin horseradish peroxidase SA HRP which catalyzes the substrate The fluorescence intensity is directly proportional to the amount of biotinylated peptide SA HRP complex but inversely proportional to the amount of the peptide in standard solutions or samples This is due to the competitive binding of the biotinylated peptide with the standard peptide or samples to the peptide antibody primary antibody A standard curve of known concentration can be established accordingly The unknown concentration in samples can be determined by extrapolation to this standard curve CAUTION INVESTIGATIONAL DEVICE LIMITED BY LAW TO INVESTIGATIONAL USE FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC PROCEDURES KIT MATERIALS 20x assay buffe2. the dynamic range of the standard curve e High levels of interfering proteins may cause variations within sample results therefore it is imperative to select the appropriate sample preparation procedure to obtain optimal results e Reagents of different lot numbers should not be mixed e Recheck the reagent labels when loading the plate to ensure that everything is added correctly e Manual washing may cause high duplicate coefficient variations To reduce this factor liquid from the plate should be removed by inverting and blotting the plate on an absorbent material e If the room temperature is not within the suggested range 20 23 C variations in results may occur e The same reservoir for the reagents may be reused if the reservoir is washed well with distilled water before each use e Each time a new tip is used make sure the tip is secure and free of air bubbles For better intra assay variation aspirate and expel a reagent or sample back into the container a few times prior to loading e Avoid submerging the whole tip into reagents because droplets can accumulate at the end of the tip causing an excess of reagent to be loaded into the well This can lead to poor results e For optimal results an orbital plate shaker capable of 300 400 rpm is recommended for all incubations e Modification of the existing protocol i e standard dilutions pipetting technique washing technique incubation time or temperature storage conditions
3. Absorbent material for blotting Lavender Vacutainer blood collection tubes optional Aprotinin 0 6TIU ml of blood optional ssesesesesesesos0 Catalog no RK APRO BUTTER A optional sessirnar esasan saaara aaea Catalog no RK BA 1 Buffer B optional sseseseseeceseseosocececcccceeoeoceceoecceceeeoeoseseseoeee Catalog no RK BB 1 C18 SEP COLUMN optional s seseceescsessosecoccoceseseososes ooo ooo Catalog no RK SEPCOL 1 ASSAY PROCEDURE Thoroughly read this protocol before performing an assay Please allow all kit components to return to room temperature before opening packages 25 45 minutes Dilute the 20x assay buffer concentrate with 950ml of distilled water This will be the 1X assay buffer solution used to dilute or reconstitute all other reagents in this kit and samples USA Note If crystals appear in the 20x assay buffer the buffer can be placed in a warm water bath for approximately 30 minutes or until no crystals are visible Mix thoroughly before use Centrifuge and dilute the standard peptide with 1ml of 1X assay buffer vortex The concentration of this stock solution is l ooong ml Allow the solution to sit at least 10 minutes at room temperature 20 23 C to completely dissolve in solution Centrifuge and vortex immediately before use Prepare peptide standard solutions as follows Stock 1000 ul 1 000 ng ml 1 1000 ul of stock 900 ul 100 ng ml 2 100 ul of 1 900 ul 10 000 pg ml 3 1
4. may occur Seal the immunoplate with the acetate plate sealer APS Incubate the immunoplate overnight approximately 16 24 hours at 4 C The next day rehydrate the biotinylated peptide with 1X assay buffer Refer to QC data sheet for instructions on rehydrating the biotinylated peptide Allow to sit for at least 5 minutes to completely dissolve Mix thoroughly Remove APS from immunoplate DO NOT WASH THE IMMUNOPLATE Add 25pl of rehydrated biotinylated peptide into each well except the Blank well Note A multi channel pipette is NOT recommended to load the biotinylated peptide because variations in results may occur Seal the immunoplate with acetate plate sealer APS Incubate the immunoplate for 1 5 hours at room temperature 20 23 C Orbital shaking at 300 400 rpm is recommended for the duration of the incubation Centrifuge the SA HRP vial provided in this kit 3 000 5 000 rpm 5 seconds and pipette 12 of SA HRP into 12ml of 1x assay buffer to make SA HRP solution vortex thoroughly Remove APS from the immunoplate Discard contents of the wells Wash each well with 350 ul of 1x assay buffer discard the buffer invert and blot dry plate Repeat 4 times Add 100 ul of SA HRP solution into each well Reseal the immunoplate with APS Incubate for 1 hour at room temperature 20 23 C Orbital shaking at 300 400 rpm is recom mended for the duration of the incubation Mix 9 parts of Substrate Solution to 1 part of Stable Peroxide Soluti
5. on next page LD RS DRG Corticoprotein Releasing Factor CRF mouse ra t human Florescen t ELISA EIA 5082 Loam wana P OOOOO0OKO OOOO 5 USA
6. 00 ul of 2 900 ul 1 000 pg ml 4 100 ul of 3 900 ul 100 pg ml 5 100 ul of 4 900 ul 10 pg ml 6 100 ul of 5 900 ul 1 pg ml 14 15 16 17 18 19 20 21 22 Rehydrate primary antibody with 1 X assay buffer Refer to QC data sheet for instructions on rehydrating the primary 0ul WOul 100ul WOpl WO 0p Blank SNONONONONEOEN Total Binding C 1 pg ml 10 pg ml 10pm a rt 1 000 pgm QO 10 000 pg ml 2 Positive Control Fc STOCK 1 2 5 6 1000 100 10 000 1000 100 10 ngm ngimi pgm pgm pgm pgm pgimi antibody Allow to sit for at least 5 minutes to completely dissolve Mix thoroughly Rehydrate the positive contol with 1X assay buffer Refer to QC data sheet for instructions on rehydrating the positive control Leave wells A 1 and A 2 empty as Blank Add 50 ul of 1X assay buffer into wells B 1 and B 2 as Total Binding Add 50 ul of prepared peptide standards from 6 to 2 in reverse order of serial dilution into wells from C 1 and C 2 to G 1 and G 2 respectively Note Peptide standards should be assayed in duplicate Add 50 ul of positive control solutions in wells H1 H2 Add 50 ul of prepared samples in their designated wells in duplicate Add 25 ul of rehydrated primary antibody into each well except the B lank well Gently tap the plate to ensure thorough mixing Note A multi channel pipette is NOT recommended to load the primary antibody because variations in results
7. and kit expiration may affect the sensitivity and specificity of the test e Fluorometric units are typically defined as relative fluorescene units RFU because the integrated signal is dependent on instrument settings Consult the fluorometer s user manual for specific instrument capabilities and settings Calculation of results Plot the standard curve on semi log graph paper A standard curve is constructed by plotting the known concentrations of standard peptide on the log scale X axis and its corresponding fluorescence intensity reading on the linear scale Y axis It is strongly recommended to use curve fitting software capable of 4 parameter logistics or log logit to quantify the concentration of standard peptide The standard curve shows an inverse relationship between peptide concentrations and the corresponding fluorescence intensity As the standard concentration increases the fluorescence intensity decreases The concentration of peptide in a sample is determined by locating the sample s O D on the Y axis then drawing a horizontal line to intersect with the standard curve A vertical line drawn from this point will intersect the X axis at a coordinate corresponding to the peptide concentration in the sample If samples have been diluted prior to the assay the measured concentration must be multiplied by their respective dilution factors The standard curve will be a reverse sigmoidal shape Refer to QC Data Sheet for acceptable valu
8. coagulation Transfer the blood from the Lavender Vacutainer tubes to the centrifuge tubes containing aprotinin 0 6 TIU ml of blood and gently rock several times to inhibit the activity of proteinases Centrifuge the blood at 1 600 x g for 15 minutes at 4 C and collect the plasma Plasma kept at 70 C may be stable for one month If Lavender Vacutainer tubes are centrifuge safe the aprotinin may be added into the initial collection step Extraction of Peptides from Plasma 1 Acidify the plasma with an equal amount of buffer A For example if you are using 1ml of plasma add 1ml of buffer A Mix and centrifuge at 6 000 to 17 000 x g for 20 minutes at 4 C 2 Equilibrate a SEP COLUMN containing 200mg of C18 Cat No RK SEPCOL 1 by washing with buffer B 1ml once followed by buffer A 3ml 3 times Note From steps 3 5 no pressure should be applied to the column 3 Load the acidified plasma solution onto the pre equilibrated C 18 SEP Column 4 Slowly wash the column with buffer A 3ml twice and discard the wash 5 Elute the peptide slowly with buffer B 3ml once and collect the eluant into a polystyrene tube 6 Evaporate eluant to dryness in a centrifugal concentrator or by a suitable substitute method 7 Keep the dried extract at 20 C and perform the assay as soon as possible Use assay buffer to reconstitute the dried extract If the peptide value does not fall within the range of detection dilute or concentrate the sample ac
9. cordingly Tips for extraction of plasma When using a C 18 SEP COLUMN for the first time use a bulb not supplied to apply pressure to the column after the addition of 1ml of buffer B to facilitate the flow through the column From steps 3 5 no pressure should be applied DRE G DRG Corticoprotein Releasing Factor CRF mouse rat human Florescent ELISA EIA 50872 USA Ensure there is a constant flow for all solutions during the extaction procedure Do not allow air bubbles to enter the C 18 matrix for optimal sample processing and recovery Drying Sample After Extraction A combination of a centrifugal concentrator i e Speedvac and a lyophilizer freeze dryer produces the best results for drying the sample after extraction First use a Speedvac to dry sample for approximately 15 minutes to remove the organic layer Then snap freeze the remaining sample and freeze dry overnight using a lyophilizer This two step procedure produces a more consistent fluffy powder that is easier to rehydrate than a sample dried only with a centrifugal concentrator However if a centrifugal concentrator is not accessible freeze drying overnight using a lyophilizer will be sufficient REFERENCES 1 Porstmann T and Kiessig S T Enzyme Immunoassay Techniques An Overview Journal of Immunological Methods 150 5 21 1992 2 Avrameas S Amplification Systems in Immunoenzymatic Techniques Journal of 12 1992 ASSAY DIAGRAM follows
10. es of the positive controls STORAGE 1 Store the kit at 4 C upon receipt 2 Itis highly recommended that solutions be used as soon as possible after rehydration 3 Store 1x assay buffer at 4 C 4 Keep rehydrated solution of Standard Biotinylated peptide Antibody and HRP at 4 C DRE G DRG Corticoprotein Releasing Factor CRF mouse rat human Florescent ELISA EIA 50872 USA SUMMARY OF ASSAY PROTOCOL Add 50 ul well of standard positive control or sample and 25 ul primary antibody y Incubate overnight approximately 16 24 hours at 4 C y Add 25 ul well of biotinylated peptide y Incubate at room temperature 20 23 C for 1 5 hours Wash immunoplate 4 times with 350 ul well of 1X assay buffer Add 100 I well of SA HRP solution vy Incubate at room temperature 20 23 C for 1 hour y Wash immunoplate 4 times with 350 ul well of 1x assay buffer vy Add 100 I well of prepared substrate solution Incubate at room temperature 20 23 C for 15 20 minutes Terminate reaction with 100 ul well of stop solution y M eas urere l ative flu ore sce n c e i nte n s ity of ea ch well and calculate results SUGGESTED METHOD FOR THE EXTRACTION OF PEPTIDES FROM PLASMA Blood Withdrawal Collect blood samples into the Lavender Vacutainer tubes which contain anti coagulant and can collect up to 7ml of blood Gently rock the Lavender Vacutainer tubes several times immediately after collection of blood for anti
11. on This working solution is stable for 24 hours at room tempera ture 20 23 C and no protection from light is required To reduce variability equilibrate the working solution to room temperature 20 23 C before adding to the wells Remove APS from the immunoplate Wash and blot dry the immunoplate 4 times with 1x assay buffer as described above in step 17 DRE DRG Corticoprotein Releasing Factor CRF mouse rat human Florescent ELISA EIA 50872 USA 23 Add 100 l of prepared Substrate Solution into each well Gently tap the immunoplate to ensure thorough mixing 24 Reseal the immunoplate with APS Incubate for 15 20 minutes at room temparature 20 23 C Orbital shaking at 300 400 rpm is recommended for the duration of the incubation 25 Remove APS from the immunoplate Add 100 of stop solution into each well to stop the reaction Gently tap the plate to ensure thorough mixing Proceed to the next step within 20 minutes 26 Load the immunoplate onto a fluorescence microplate reader and measure relative fluorescence units RFU of each well The excitation and emission maxima for the Substrate Solution are 325 and 420 nm respectively Wavelengths between 315 and 340 nm for excitation and 370 and 470 nm for emission also can be used for detection Additional Recommended Procedural Notes e Each laboratory must determine the appropriate dilution factors for the samples to be measured to ensure that the samples are within
12. r concentrate 50ml 96 well immunoplate 1 plate Acetate plate sealer APS 3 pieces Primary antibody rabbit anti peptide IgG 1 vial Standard peptide 1 ug Biotinylated peptide 1 vial Streptavidin horseradish peroxidase SA HRP 30 pl Substrate solution 13ml Positive control 1 vial O Stable peroxide solution 1 5ml 11 Stop solution 13ml 12 Assay diagram 1 sheet General protocol 1 book NOTE DRG International Inc guarantees that its products conform to the information contained in this publication The purchaser must determine the suitability of the product for their particular needs and establish optimum sample concentration DRE DRG Corticoprotein Releasing Factor CRF mouse rat human Florescent ELISA EIA 5082 MATERIALS REQUIRED BUT NOT SUPPLIED Fluorescence microplate reader 325nm to 420nm O ON AW RW N O 11 NOTE The kit should be equilibrated to room temperature 20 23 C before opening any vials and starting the assay It is highly recommended that the solutions be used as soon as possible after rehydration Recommended blood collection protocol is provided on page 9 Each kit contains sufficient reagents for 96 wells and is capable of assaying 40 duplicate samples Orbital plate shaker capable of 300 400rpm recommended Microtiter plate washer recommended Multi channel pipette capable of dispensing 50 100 ul recommended Solution reservoir recommended
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