ELISA Amp Sys 19589 14Jul08.qxd
Contents
1. invitrogen User Manual ELISA Amplification System Cat No 19589 019 EEE Table of Contents 1 Notices to Customer Important Information Precautions ceee 2 Overview 3 Methods Components SR re pe 0 DOM Advance Preparations 1 1 1 2 3 5 3 6 3 1 3 8 3 9 Recommendations for Optimal ResUllS init ELISA Amplification System Protocol Arsen Color Measurement AssayS Use of the Positive Control Factors Which May Affect Assay Results Performance Standards 4 Troubleshooting Guide 5 References Table of Contents Figures 1 Schematic of Alkaline Phosphatase Signal Amplification enn 3 2 Rate of Absorbance Increase as a Function of Total Incubation Time 11 Table 1 ELISA Amplification Versus pNPP in an Indirect ELISA nn 12 Notices to Customer 1 1 Important Information This product is authorized for laboratory research use only The product has not been qualified or found safe and effective for any human or animal diagnostic or therapeutic application Uses for other than the labeled intended use may be a vio lation of applicable law 1 2 Precautions PRECAUTIONS FOR USE Warning This product contains hazardous reagents It is the end user s responsibility to consult the applicable MSDS s before using this product Disposal of waste organ ics acids bases and radioactive mate2. Upon receipt the reagents in the ELISA Amplification System should be stored at 4 C The substrate and amplifier are supplied lyophilized After reconstitution the amplifier is stable for 1 week at 20 C and the substrate is stable for 8 days at 2 8 C A slight pink color in the amplifier solution may develop during storage This will not affect the interpretation of results 3 3 Advance Preparations The following reagents are required but they are notprovided with the system Prepare them as fol lows Final washing buffer positive control diluent Tris buffered saline TBS 0 05 M Tris HCI pH 7 5 and 0 15 M NaCl This amount of buffer is sufficient for two 96 well plate assays To prepare add 1 51 g of Tris base MW 121 14 and 2 19 g of sodium chloride MW 58 5 to 230 ml of deionized water Stir until dissolved Adjust the pH to 7 5 with 2 M HCI Adjust the volume to 250 ml with deionized water Stop Solution 0 3 M H S0 To 59 ml of deion ized water slowly add 1 ml of concen trated sulfuric acid MW 98 specific gravity 1 84 purity 96 to 98 Caution When using sulfuric acid exercise caution and follow manufacturer s safety recommendations When making dilutions always add acid to water 1 Bring all reagents except the positive control to room temperature before use 2 Reconstitute the substrate at room temperature 10 min before use by adding 12 ml of substrate diluent directly to the substrate vial
3. Gently mix until completely dissolved using clean pipets to avoid contamination Do not leave uncovered Reconstituted substrate is stable for 8 days at 2 8 C Methods Reconstitute the amplifier 10 min before use by adding 12 ml of amplifier diluent Gently mix until completely dissolved using clean pipets to avoid contamination Do not leave uncovered Reconsituted amplifier is stable for 1 week at 20 C Dilute the positive control before use Please read Section 3 7 Use of the Positive Control for instructions Selecta color measurement assay as described in the Section 3 6 Color Measurement Assays 3 4 Recommendations for Optimal Results 1 Keep pipets and laboratory vessels used to dispense alkaline phosphatase conjugate and the positive control away from those used for other components especially the substrate Minimize any nonspecific binding of antibody or conjugate by carefully washing the microtiter plate wells Amplification reagents will amplify any nonspecific signal Avoid using phosphate in any wash buffer It strongly inhibits the substrate reaction The substrate and amplifier reactions occur quite rapidly and require precise timing To ensure equal incubation times in each well add the substrate amplifier and stop solution in the same well to well sequence Note Please read the protocol thoroughly before beginning Some of the steps in this procedure need to
4. 8 Notes invitrogen United States Headquarters Invitrogen Corporation 5791 Van Allen Way Carlsbad California 92008 Tel 1 760 603 7200 Fax 1 760 602 6500 Email tech supportQinvitrogen com For country specific contact information visit our website at www invitrogen com Part No 19589 Rev Date 14 July 2008
5. Kinetic assay Read the absorbance at 495 nm of each well every 3 min for 15 min 6 To maintain the effectiveness of reconstituted substrate and amplifier for future use store them immediately at4 C 3 6 Color Measurement Assays The following descriptions apply to measurement of color obtained in microtiter plate based assays However the general principle is applicable to many systems employing a variety of reaction ves sels End Point or Stopped Assays In this type of assay color development is allowed to proceed for a predetermined time so that sufficient color develops in the test wells Stopping the reaction in effect halting any further cycling activity is achieved by adding dilute acid The stop solution must be added to the wells in a timed sequence identical to that used for the addition of substrate and amplifier Assuming a reaction volume of 50 ul of substrate plus 50 ul of amplifier 50 ul of acid will stop the reaction Once all the wells have been stopped the color can be quantified by reading with a suitable colorimeter at 495 nm Kinetic Assays In a kinetic assay color devel opment is not stopped but read repeatedly over a period of time and the rate of color develop ment calculated Since cycling reactions show linear kinetics rates of reaction can be determined by plotting a regression line through the series of optical density measurements The rates on the unknowns are then compared with the rate
6. NADH in the presence of diaphorase reduces a tetrazolium salt iodonitrote trazolium violet or INT violet to form an intensely colored formazan dye and is itself oxidized to form NAD NAD is then reduced to NADH and ethanol is oxidized to acetaldehyde in the presence of alcohol dehydrogenase The rate of reduction of the tetrazolium salt is directly proportional to the concentration of NADH origin ally formed by the enzyme in the bound conjugate A schematic of the reaction is shown in figure 1 NAD Formazan Ethanol Diaphorase Alcohol Dehydrogenase INT Violet Acetaldehyde NADH Alkaline Phosphatase Pi NADPH Colored end product Figure 1 Schematic of alkaline phosphatase signal amplification Methods 3 1 Components The components of the ELISA Amplification System are as follows Each vial of substrate sub strate diluent amplifier and amplifier diluent contains enough reagent to perform two 96 well microtiter plate assays with the volumes suggested in the instructions Each system provides reagents sufficient to perform 10 microtiter plate assays Component Amount Substrate lyophilized NADPH 5 vials Substrate diluent 1X buffer for substrate 60 ml 5 x 12 ml Amplifier lyophilized amplifying enzymes 5 vials alcohol dehydrogenase and diaphorase Amplifier diluent 1X buffer for amplifier 60 ml 5 x 12 ml Positive control streptavidin alkaline 100 ul phosphatase conjugate 1 mg ml 3 2 Storage
7. ated with optimal dilutions of Biotinylated Goat Anti rabbit IgG followed by Streptavidin Alkaline Phosphatase Conjugate The substrate pNPP was used at 1 mg ml in 10 diethanolamine pH 9 8 0 5 mM MgCl and incubated for 60 min Temperature Because the amplification steps are enzymatic reactions they are temperature dependent For consistent results we recom mend that the amplification reagents be used at a constant temperature between 20 C and 25 C Reagent Volumes and Concentrations The sig nal may be strengthened by increasing the final assay volumes of the substrate and amplifier and to some extent by using a more concentrated amplifier However the reagents supplied with this system are paired and we do not ensure the stability or performance of nonstandard volumes or concentrations 12 3 9 Performance Standards The strength of signal and sensitivity of the ELISA Amplification System are routinely analyzed to ensure performance We require the ELISA Amplification System to give maximal absorbance readings versus 10 ng of purified antigen within 30 min during the substrate and amplifier incuba tions In addition the system must be able to detect as little as 100 pg of purified antigen within the same timeframe We measure these parame ters in an indirect ELISA using optimal dilutions of biotinylated antibodies and Streptavidin Alkaline Phosphatase Conjugate 13 Problem High background Random high ab
8. be performed simultane ously 3 5 ELISA Amplification System Protocol 1 Perform a standard ELISA using alkaline phosphatase Leave the last row of the microtiter plate empty at this stage for addition of the positive control 2 Remove excess alkaline phosphatase by washing each well four times with 0 25 ml of TBS buffer see Section 3 3 Advance Preparations Wet the positive control wells with this buffer Remove the fourth wash from all wells just before you proceed with the next step 3 Add the positive control dilutions see Section 3 7 Use of the Positive Control to the last row of the microtiter plate 4 Substrate Add 50 ul of reconstituted substrate prepared in advance to each well including the wells you have reserved for the positive control Incubate for 15 min at 25 C To increase sensitivity extend the length of incuba tion as described in Section 3 8 Factors Which May Affect Results Amplifier Add 50 ul of reconstituted amplifier prepared in advance to each well Incubate for at least 15 min at 25 C being careful not to cross contaminate wells Add amplifier and substrate in same sequence 5 Measure the color development using the assay method you have selected in advance End point or stopped assay Stop color development after 15 min with 50 ul of 0 3 M H S0 per well Add the stop reagent in Methods the same time sequence as the amplifier Record the absorbance at 495 nm
9. re 2 Rate of absorbance increase as a function of total incubation time In line 1 substrate and amplifier incubation times are equal In line 2 substrate incubation times were increased while amplifier incubation times were held constant at 15 min In line 3 substrate incubation times were held constant at 15 min while amplifier incubation times were increased sensitivity over the traditionally used substrate pNPP as demonstrated in table 1 If more or less sensitivity is required incubation of both reagents for longer or shorter time periods can achieve the sensitivity required It is important to maintain equivalent substrate and amplifier incubation times for optimal signal generation If incubation times are increased it is critical to minimize any nonspecific reactions during the ELISA procedure 11 Methods Table 1 ELISA amplification versus pNPP in an indirect ELISA Total Incubation Time of Approximate Fold Increase in Amplification Reagents min Sensitivity over pNPP 30 15 for substrate 10 15 for amplifier 45 22 5 for substrate 15 22 5 for amplifier 60 30 for substrate 20 30 for amplifier 90 45 for substrate 30 45 for amplifier Fold increase was determined by comparing the quantity of antigen that gave equivalent absorbances within the linear range of the detector Antigen purified rabbit IgG concentrations ranged from 50 ng to 0 1 pg well All wells were incub
10. rials must comply with all appropriate federal state and local regulations lf you have any questions concerning the hazards associated with this product please call the Invitrogen Environmental Health and Safety Chemical Emergency hotline at 301 431 8585 Overview The ELISA Amplification System is designed to amplify the amount of color generated by a given quantity of immobilized alkaline phosphatase in a soluble substrate enzyme linked immunosorbent assay ELISA In conventional detection systems bound enzyme acts directly on the substrate to produce a colored end product The signal gener ated by this type of system is limited by the linear nature of the reaction kinetics In the ELISA Amplification System the bound enzyme acts on a substrate whose product initiates a secondary cyclic enzyme reaction resulting in a colored prod uct Each molecule of product from the first reac tion takes part in many cycles of the second reac tion thus the signal generated by the first enzyme reaction is amp ed by participation of its end product in the cycles ofthe second reaction The substrate in this system is the reduced form of nicotinamide adenine dinucleotide phosphate NADPH which is dephosphorylated by bound alkaline phosphatase to reduced nicotinamide ade nine dinucleotide NADH NADH activates a sec ondary enzyme system which comprises a redox cycle driven by diaphorase and alcohol dehydroge nase In this cycle
11. s of standard samples This method of reading also reduces problems which can occur with some multioptic readers from channel to channel variation in optical density 3 7 Use ofthe Positive Control The positive control included with this system ensures that the components provided are performing properly The positive control is alkaline phosphatase chemically coupled to streptavidin a reagent used in many ELISA systems When dilutions of the alkaline phosphatase conjugate are mixed with the amplifications reagents color development occurs The expected absorbance from each dilution will vary for each detector however the optical densities should decrease linearly as the control is diluted The approximate concentration of alkaline phos phatase in the conjugate before dilution is 1 mg ml Therefore final dilution in each well ranges from 1 4 000 250 ng ml to 1 512 000 2 ng ml in two fold increments per well We recommend you reserve the last row of each assay plate for the positive control reaction using 25 ul per well of serially diluted positive control 1 Dilute the Positive Control 1 4 000 with TBS see Section 3 3 Advance Preparations 2 Transfer 50 ul of the diluted positive control to the first well of the last row in the microtiter plate Methods 10 3 Add 25 ul TBS to the remaining wells in the same row 4 Transfer 25 ul from the well containing the positive control to the adjacent
12. sorbing wells Troubleshooting Guide Probable Causes Substrate not fresh Substrate amplifier incubated too long Stop Solution omitted Insufficient washing Wash buffer too dilute Contamination of substrate with phosphatase Reagent too old or improperly stored High level of nonspecific binding Positive control splashed into other wells P ipet tips contaminated with positive control Insufficient washing Substrate amplifier not properly dissolved or thoroughly mixed Completely red plate Substrate mixed in a container which previously contained alkaline phosphatase Weak color development in positive control Incubation temperature of the assay too high or low Substrate amplifier incubation time too short Incorrect preparation of substrate amplifier Substrate amplifier not fully dissolved Positive control omitted Unexpectedly high low or variable absorbance readings Plate reader at wrong wavelength Debris on bottom of wells Bubbles in wells caused by inaccurate pipetting or incorrect preparation of substrate amplifier Gross variation in signal 14 Inefficient removal of wash buffer Inaccurate pipetting Contaminated pipet tips Incorrect preparation of substrate amplifier References 1 Self C H 1985 J Immunol Method 76 389 2 Stanley C J Johannsson A and Self C H 1985 J Immunol Method 83 89 15 16 Notes Notes 1
13. well in that row and mix Transfer 25 ul from this well to the next adjacent well Continue transferring 25 ul to adjacent wells until the last well in the row is used Discard 25 ul from the last well 3 8 Factors Which May Affect Assay Results Timing of Substrate and Amplifier Incubation of Steps In the two step amplifications process both steps have linear reaction kinetics In the first step substrate incubation the dephospho rylation of NADPH to produce NADH is termi nated by adding the amplifier which contains phosphate in addition to the amplifying enzymes and effectively inhibits the activity of alkaline phosphatase In the second step amplifier incubation the rate of color production is proportional to the concentration of NADH produced in the first step For a given amount of alkaline phosphatase maximum sensitivity is achieved when substrate and amplifier incubations are of equal length figure 2 Increasing the length of either incubation alone will increase the signal but to a lesser degree than equal length incubations Length of Incubation Incubation times for the amplification reagents can be varied depending on the sensitivity required We recommend that you incubate each reagent for 15 min This gives approximately a 10 fold increase in 1 4 12 1 0 0 8 0 6 Absorbance 495 nm 0 4 3 0 2 10 20 30 40 50 60 70 80 90 100 Total Incubation Time min Substrate Amplifier Figu
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