SIV p27 Elisa Assay
Contents
1. buffered saline pH 7 8 8 0 containing 2 Bottles 0 05 Tween 20 Must be diluted 60 mL before use 7 Substrate Solution Ready to use Tetramethylbenzidine 12 mL 8 Stop Solution 1 N H2S04 12 mL Additional Requirements for Manual Processing gt Disposable tip micropipettes to deliver volumes of 5 uL 10 uL 25 uL 100 uL and 200 uL multichannel pipette preferred for dispensing reagents into microtiter plates gt Distilled or deionized water gt 37 1 C incubator gt Clean disposable plastic glass test tubes approximate capacities 5 mL and 10 mL gt Range of standard clean volumetric laboratory glassware consisting of at least 15 mL and 100 mL beakers 1 L graduated cylinder 1 mL 5 mL and 10 mL glass pipettes gt Absorbent paper towels gt Automatic microtitration plate washer or laboratory wash bottle gt Microtitration plate reader with 450 nm filter Latex gloves safety glasses and other appropriate protective garments Biohazard infectious waste containers Safety pipetting devices for 1 mL or larger pipettes Timer vvvV Automatic or Semi automatic Processing The SIV p27 Assay may be used with a variety of automatic or semi automatic processors liquid handling systems It is essential that any such system is qualified before it is used routinely by demonstrating that the SIV p27 Assay results obtained using the automatic processor are equivalent to those obtained for the same specimens2. mL 0 000 0 000 0 000 Standard 2 125 pg mL 0 126 j 0 136 0 131 Standard 3 250 pg mL 0 293 0 317 0 305 Standard 4 500 pg mL 0 629 3 0 645 0 637 Standard 5 1000 pg mL 1 245 j 1 273 1 259 Standard 6 2000 pg mL 2 543 j 2 585 2 564 Specimen 1 0 328 0 338 0 333 271 Specimen 2 1 196 s 1 232 1 214 964 Example Standard Curve 3 E 25 O g 2 tw g 45 Cc a 1 5 2 05 2 o 0 0 500 1000 1500 2000 Concentration p27 pg ml Note This standard curve is only an example and should not be used to generate any results Computer Assisted Method Computer assisted data reduction may be used to create the standard curve Software providing a point to point curve fitting routine provides acceptable results Assay validation The SIV p27 assay should be considered valid if The negative control should be lt 0 10 The 1000 pg ml control should be 0 60 Procedure for Samples with SIV p27 assay values greater than the highest standard Many tissue culture samples will have p27 values greater than the highest standard In order to obtain accurate results for samples with SIV p27 assay values greater than the highest standard it is necessary to dilute and re test the sample Diluting the serum specimen 10 fold is recommended For example Make a 10 fold dilution by adding 0 1 mL of the initial specimen to 0 90 mL of tissue culture medium Mix thoroughly and repeat the assay according to the Assay Procedure Multiply the results
3. only 2 Caution All blood products should be treated as potentially infectious Essential precautions can be summarized as follows gt Do not pipette by mouth gt Wear disposable gloves during all specimen and assay manipulations gt Avoid use of sharp or pointed liquid handling devices which may puncture skin gt Do not smoke eat or drink in the laboratory work area gt Avoid splashing of liquid specimens and reagents and the formation of aerosols gt Wash hands thoroughly on completion of a manipulation gt The Centers for Disease Control amp Prevention and the National Institutes of Health recommend that potentially infectious agents be handled at the Bio safety Level 2 3 The SIV p27 kits contain reagent systems which are optimized and balanced for each kit lot Do not interchange reagents from kits with different lot numbers Do not interchange vial caps or stoppers either within or between kits 4 The Substrate Solution and Stop Solution in this kit contain ingredients that can irritate the skin and cause eye damage Handle them with care and wear suitable protective clothing and eye face protection In case of contact with skin or eyes immediately flush the affected area with plenty of water For eyes obtain medical attention Procedural 1 This kit should be used in strict accordance with the instructions in the Package Insert 2 Do not use SIV p27 Assay kits after the expiration date printed on the outer carton la
4. using the manual test method Subsequently the automatic processor should be periodically re qualified Storage and Stability All reagents should be stored at 2 8 C and should not be used beyond the expiration date on the label Once opened microtitration strips may be stored at 2 8 C until the expiration date on the label provided that desiccated conditions are maintained Unused strips should be returned to their original foil pouch along with the sachet of desiccant Opened pouches should be securely resealed by folding over the open end and securing it with adhesive tape The working strength Wash Buffer should not be stored for longer than 3 weeks at 2 8 C It is recommended that Wash Buffer be freshly diluted before each assay If the working strength buffer becomes visibly cloudy or develops precipitate during the 3 weeks do not use it Indications of Deterioration The SIV p27 Assay may be considered to have deteriorated if 1 The kit fails to meet the required criteria for a valid test see 6 Interpretation of Results 2 Reagents becoming visibly cloudy or develop precipitate Note Concentrated Wash buffer when cold normally develops crystalline precipitates which re dissolve on heating at 37 C 3 The Substrate Solution turns dark blue This is likely to be caused by chemical contamination of the Substrate Solution Warnings and Precaution Safety 1 The reagents supplied in this kit are for Research use
5. For Research Use Only Not for Diagnostic Use XpressBio Life Science Products SIV p27 Elisa Assay Catalog SK845 Introduction Principle of the Assay Microtitration wells coated with murine anti SIV p27 capture antibody are exposed to test specimens which may contain p27 reactive determinants After an incubation period unbound components in the test sample are washed away Specifically bound p27 reactive determinants react with a mouse anti SIV p27 conjugated with biotin during a second incubation period Following a second wash cycle the biotinylated antibody is detected by the addition of a streptavidin HRP conjugate Following a third wash cycle specifically bound enzyme conjugate is detected by reaction with the substrate solution tetramethylbenzidine TMB The assay is measured spectrophotometrically to indicate the level of p27 reactive determinants present in a sample Kit Presentation Materials Supplied The reagents supplied in this pack are for Research use only 1 Coated microwell strips 1 plate Plastic microtitration wells coated with 96 wells anti SIV p27 murine monoclonal antibody in foil pouch with desiccant 2 Positive p27 Control 100 ng ml 0 1 mL 3 Lysis Buffer 6 mL 4 Detector antibody anti SIV p27 12 mL conjugated to biotin 5 Conjugate Streptavidin conjugated to horseradish peroxidase enzyme 12 mL containing 0 01 Bromonitrodioxane as preservative 6 Wash Buffer 20x concentrated Tris
6. bel 3 Do not cross contaminate reagents Always use fresh pipette tips when drawing from stock reagent bottles 4 Always use clean preferably disposable glassware for all reagent preparation 5 Allow foil bags to warm to room temperature before opening This avoids condensation on the inner surface of the bag which may contribute to a deterioration of coated strips intended for future use 6 Reagents should be dispensed with the tip of the micropipettes touching the side of the well at a point about mid section Follow manufacturer s recommendations for automatic processors 7 Always keep the upper surface of the microtitration strips free from excess fluid droplets Reagents and buffer over spill should be blotted dry on completion of the manipulation 8 Do not allow the wells to completely dry during an assay 9 Disposal or decontamination of fluid in the waste reservoir from either the plate washer or trap for vacuum line in the manual system should be in accordance with guidelines set forth in the Department of Labor Occupational Safety and Health Administration occupational exposure to blood borne pathogens final rule 29 CFR 1910 1030 FEDERAL REGISTER pp 64176 84177 12 6 91 10 Automatic or semi automatic EIA processors or liquid handling systems should be qualified specifically for use with SIV p27 Assay by demonstration of equivalence to the manual processing methods 11 Consistent with good laboratory practice it i
7. by 10 to determine the correct SIV p27 assay values in the sample Limitations of Use 1 Assay values determined using assays from different manufacturers or different methods may not be used interchangeably 2 Samples with very high p27 assay values levels may exhibit in a prozone effect For this assay antigen levels must be greater than 50 000 pg mL before the assay gives erroneous results of less than 2000 pg mL 3 The assay cannot be used to quantitate samples with SIV p27 assay values greater than the highest standard without further serial dilution of the samples See the Interpretation of Results section for directions on testing such samples 4 The performance characteristics have not been established for any matrices other than tissue culture media Performance Characteristics Analytical Sensitivity To determine the sensitivity of the assay the 0 standard was assayed 20 times The minimal detectable level was calculated by adding two standard deviations to the mean absorbance for the 0 standard The minimal detectable level is 25 pg mL Linearity Four strongly reactive samples were serially two fold diluted and run on the assay The values obtained were compared to the expected values by standard linear regression The r values obtained ranged from 0 998 to 1 00 Precision Four samples with different levels of activity were assayed ten times each on three different assays The results are summarized in the foll
8. n every assay If a standard curve is to be run the quantitative protocol should be used 3 Select sufficient microtitration well strips to accommodate all test specimens controls and reagent blank Fit the strips into the holding frame Label wells according to specimen identity using the letter number cross reference system moulded into the plastic frame 4 Dispense 20ul of lysis buffer to each well 5 Dispense 200 uL of each control and specimen into appropriate wells Note All controls and samples should be tested in duplicate 6 Incubate at 37 1 C for 60 5 minutes 7 Aspirate the contents of the wells and wash the microtitration plate as described in the Rinse Cycle section 8 Pipette 100 uL of detector antibody into each well and incubate at 37 1 C for 60 5 minutes 9 Aspirate the detector antibody from the wells and wash the microtitration plate as described in the Rinse Cycle section 10 Pipette 100ul of Streptavidin HRP conjugate into each well and incubate at room temperature 18 25 C for 30 5 minutes 11 Aspirate the conjugate from the wells and wash the microtitration plate as described in the Rinse Cycle section 12 Without delay dispense 100 uL Substrate Solution into each well A multichannel pipette should be used for best results Leave at room temperature 18 25 C protected from direct sunlight for 30 2 minutes 13 Stop the reaction by adding 100 uL of Stop Solution to each well including the reage
9. nt blank The blue solution should change to a uniform yellow color Ensure that the undersides of the wells are dry and that there are no air bubbles in the well contents 14 Immediately after adding the Stop solution read the absorbance values at 450 nm using a microtitration plate reader blanked on the negative control well Quantitative Assay Procedure To test quantitatively a standard curve should be prepared using tissue culture media as the diluent First prepare the 2000pg ml standard as above in step 1 Prepare four serial two fold dilutions to prepare 1000 pg ml 500pg ml 250 pg ml and 125 pg ml standards using the tissue culture media as diluent Each standard plus an inoculated tissue culture control should be run in duplicate Interpretation of Results Qualitative Analysis The following criteria should be met for a valid assay The negative control should be lt 0 10 The 1000 pg ml control should be 0 60 Quantitative Analysis Manual Method The calibration curve can be constructed manually on linear graph paper by plotting the mean absorbance for each standard on the y axis versus the concentration of the standard value printed on vial on the x axis Connect the points to produce a point to point curve Do not force the line to be linear The concentration of the specimens can be found directly from the standard curve Table 1 Example Data at 450nm Sample 450 nm abs Mean abs pg mL Standard 1 0 pg
10. owing table Precision Data Sample 1 Sample 2 ample 3 Sample 4 Assay 1 Mean mL 1606 527 1110 308 n 10 SD 2 3 41 0 50 4 29 8 CV 5 13 7 79 54 9 70 Assay 2 Mean mL 1749 586 1207 347 n 10 SD 4 5 41 1 1 4 26 7 CV 2 54 7 01 3 43 6 97 Assay 3 Mean mL 1761 577 1309 450 n 10 SD 9 7 54 8 135 1 41 8 CV 3 97 9 50 10 3 9 28 Inter Mean mL 1706 563 1209 368 Assay SD 6 8 51 7 117 6 69 2 n 30 CV 5 67 9 18 9 73 18 8 Contact Information Express Biotech International P O BOX 458 Thurmont MD 21788 USA Tel 301 228 2444 Fax 301 560 6570 www expressbiotech com info expressbiotech com
11. s recommended that all pipetting devices manual or automatic timers and thermometers are regularly calibrated according to the manufacturer s instructions 12 Care must be taken to ensure that specimens are dispensed correctly to each test well If a specimen is inadvertently not added to a well the result for that well will be non reactive regardless of the actual result of the specimen Method of Use Specimen Collection and Storage SIV p27 Assay is intended for use with tissue culture supernatants The specimen should be tested as soon as possible However if the specimen needs storage the specimens should be stored frozen at 20 C or below Do not use self defrosting freezers Specimens that have been frozen and thawed should be thoroughly mixed before testing Rinse Cycle Efficient rinsing to remove un complexed components is a fundamental requirement of enzyme immunoassay procedures The SIV p27 assay utilises three standard six rinse cycles Automatic plate washers may be used provided they meet the following criteria 1 All wells are completely aspirated 2 All wells are filled to the rim 350 uL during the rinse cycle 3 Wash Buffer is dispensed at a good flow rate 4 The microtitration plate washer must be well maintained to prevent contamination from previous use Manufacturer s cleaning procedures must be followed diligently For each rinse cycle the machine should be set to six consecutive washes On comple
12. tion of the cycle invert the microtitration plate and tap firmly on absorbent paper towels Check for any residual Wash Buffer in the wells and blot dry the upper surface of the wells with a paper towel Alternatively the following manual system may be employed 1 Aspirate well contents using a vacuum line fitted with a trap 2 Fill all wells to the brim with Wash Buffer dispensed from a squeeze type laboratory wash bottle 3 Aspirate all wells 4 Repeat steps 2 and 3 five times 5 Invert the microtitration plate and tap firmly on absorbent paper towels Preparation for the Assay 1 Kit Positive Control 100 ng ml Prepare working strength Positive Control by diluting 20ul of positive control into 980 ul 1 50 dilution of uninoculated tissue culture media This will give a final concentration of 2000 pg ml 2 Wash Buffer Prepare working strength Wash Buffer by diluting 1 part concentrate with 19 parts of distilled or de ionized water If a kit is likely to be utilized over a period in excess of 4 weeks then it is recommended that only enough stock concentrate be diluted sufficient for immediate needs Each row of 8 wells may be adequately washed with 150 mL of working strength buffer Qualitative Assay Procedure 1 Allow all reagents to reach room temperature 18 25 C 2 The diluted positive control 2000pg ml and uninnoculated cell culture media for use as a negative control should be tested at least in duplicate i
Download Pdf Manuals
Related Search