DNA/RNA dosage application using IMAplate
Contents
1. Wash the plate 4 times 200 uL well wash buffer Block the plate with 100 uL of Blocking solution to each well Incubate 2 hours at room temperature with agitation Wash the plate 4 times Prepare cTnI standards concentrations at 12 5 6 25 3 125 1 56 0 78 0 39 0 2 and 0 ng mL in PBS Add 50 uL well standard or sample Incubate 2 hours at room temperature with agitation nA A WwW N e Wash the plate 4 times 6 7 Add 50 uL well of detection antibody solution Incubate 2 hours at room temperature with agitation 8 Wash the plate 4 times 9 Add 100 uL of TMB substrate to each well Incubate 5 20 minutes at room temperature with agitation 10 Add 100 uL of stop solution to each well 11 Measure absorbance at 450 nm according to TMB solutions instructions Results Standard curves Figure 1 shows the cTnI standard curves obtained from both IMAPlate red and NUNC 96 well plate blue Two independent experiments have been performed for the two different assays IMAPlate and NUNC 96 well plate Samples were run in triplicate for IMAPlate assay and in duplicate for NUNC 96 well plate assay The standard curves from the IMAPlate and the 96 well plate are comparable The maximum absorbance background ratio is very close 41 31 fold background versus 42 31 for the IMAPlate and the 96 well Nunc plate respectively while the concentration of cTnI to reach half maximum absorbance background is around 30 lower for IMAPlate2. dilution the amount BSA in the reaction chamber from 0 04 to 0 4 ug shows a near linear relationship with the true absorbance values Absses Abssao 0 2000 4900 6000 8000 10000 BSA Concentration ug ml Conclusion The IMAPlate is an easy to use robust miniaturized analytical platform suitable for quantification and quality analysis of protein samples with 96 well plate readers It offers users a wide range of UV VIS IR spectrometric analysis of 5 ul of samples The measured samples can totally be recovered without cross contamination risk The flexible simple handling of the IMAPlate not only saves hands on time and material cost for the analysis but also enhances the lab productivity The method can be adapted with other protein dyes colorimetric fluorimetric luminometric Contact your local distributor Uptima powered by 213 Avenue J F Kennedy BP 1140 r i nte rco h i m 03103 Montlu on Cedex France T l 04 70 03 88 55 Fax 04 70 03 82 60 P 5 15 uptima interchim com Uptima Miniaturized Enzyme Linked ImmunoSorbent Assay by IMAplate FT DR961d Introduction Enzyme linked immunosorbent assay ELISA as a simple sensitive specific and economical method for qualification and quantification of a particular molecule e g protein peptide or small molecule in a sample is routinely used in life sciences health care and many other different industries Although it is developed in late 70 s the ELISA h
3. reader has a certain absorbance detection range Using a plate reader with a wide detection range may avoid the overflow for higher concentration standards However the missing peak absorbance data at 450nm can always be derived from the data set measured at a less sensitive off peak wavelength e g at 485nm by the linearity relationship between the data set at peak wavelength and the one at off peak wavelength Fig 2 Of course further shortening reaction time for the color development or reducing Streptavidin HRP concentration are possible alternative ways to avoid the overflow if the sensitivity does not need to be increased 0 02 04 06 08 1 12 14 16 ADS 485 Using the IMAPlate for the ELISA can dramatically reduce the consumption of the sample and reagents The time to results for a typical ELISA can be cut at least to half The sensitivity is also increased The unique touch loading and touch unloading procedure provides a parallel high throughput liquid transfer and especially eases the wash steps Besides the self dosed solution up taking may not need to use low volume precision pipette and can save the pipette tips The IMAPIlate is a user friendly robust miniaturized high throughput lab device for performing miniaturized ELISA Contact your local distributor Uptima powered by 213 Avenue J F Kennedy BP 1140 r i nte rco h i m 03103 Montlu on Cedex France T l 04 70 03 88 55 Fax 04 70 03 82 60 P 8 15 up
4. ELISA demonstrates better features over the conventional cTnI ELISA such as dramatically reducing the sample and reagents consumption and the time to results Materials and Methods Reagents and Materials Capture antibody clone 560 and detection antibody coupled with HRP clone MF4 for cardiac Troponin I Hytest Ref 4T21 Human cardiac Troponin I T C complex Hytest Ref 8T62 TMB Tetramethylbenzidine substrate Substrate Reagent Pack R amp D Systems Ref DY999 IMAPlateTM 5RC96 start kit Nunc 96 well Polysorp plates Perkin Elmer plate reader ENSPIRE 2300 MULTILABEL READER Reagents PBS pH 7 4 fat free powder milk Tween 20 H2SO4 Cardiac troponin I cTnI free serum Hytest Ref 8TFS Washing buffer PBS 0 05 Tween 20 Detection buffer PBS 0 05 Tween 20 0 2 fat free powder milk The capture antibody and detection antibodies have been diluted to 7 5 ug mL in PBS and to 1ug mL in detection buffer respectively IMAPlate protocol 1 Coat the IMAPlate 5RC96 plate by directly pipetting 5 uL of capture antibody solution into the reaction chamber from the bottom opening Incubate 30 minutes at room temperature in a high humidity environment to prevent from evaporation When working with very low sample volumes small interferences e g bubbles particles homogeneity of solution can have a large impact on the results Make sure to mix the sample solution thoroughly before pipetting Reverse pipetting
5. Push a new IMAPIlate down to the 96 well plate and the parafilm will automatically seal the bottom openings 4 Mix thoroughly with a plate shaker and incubate 6 Place the IMAPlate in an IMAPIlate adaptor and gently tap it in horizontal direction several times to ensure the assay solution filling up the lower compartment 8 Use true absorbance values Abspeak Absbase line to plot the standard curve and calculate the concentration of samples Uptima powered by PTT ea T Een E ian T l 04 70 03 88 55 Fax 04 70 03 82 60 ee Contact your local distributor uptima interchim com FT DR961d Uptima The assay volume should not be over the maximum volume which the IMAPIlate can hold to withstand each step of the assay procedure from dropping off Examples 1 0 8 06 T 04 J 0 2 0 0 0 1 02 03 O04 0 5 ALP concentration ug ml Figure 1 Concentration curves of Alkaline Phosphatase the line with solid circle is performed in IMAPlate with total 20 ul of assay solution and the line with open circle is performed in Nunc 96 well plate with 150 ul of total assay solution 2 5 Abs4o5 Abssoo 0 10 20 30 40 50 60 CALB concentration ug ml Figure 2 Concentration curves of Candida Anterctica Lipase B the line with solid circle is performed in IMAPlate with total 20 ul of assay solution and the line with open circle is performed in Nunc 96 well plate with 200 ul of total assay s
6. and measure absorbance at wavelength of 665 nm and base line absorbance at wavelength of 500 nm without acidic stop solution or at wavelength of 450 nm and base line absorbance at wavelength of 650nm with acidic stop solution For standards and samples having absorbance value over the upper detection limit of the reader pipetting 1 ul or 2 ul to IMAPlate for the measurement can make these standards and samples valid and may extend the upper detection limit of the ELISA 7 Use true absorbance values A665 A500 or A455 A650 to calculate the concentration of samples according to the standards Contact your local distributor Uptima powered by a 213 Avenue J F Kennedy BP 1140 AR gt _ 03103 Montl Cedex F ia interchim f 04 70 03 88 56 Fax 04 70 03 82 60 uptima interchim com P 9 15 _ Uptima Results and Discussion As predicted the detection sensitivity and the low detection limit can be significantly improved with the new ELISA setup Figure 1 shows the plot of two ELISA data sets for human TNF a standard curves with a series of 3 fold dilution The red curve is the plot of the data from the conventional ELISA setup and the green one is the plot of the data from the new ELISA setup Both ELISAs were performed in 96 well plate with flat bottom according to the protocol provided in the kit except that for the green curve 25 ul of TMB substrate solution was used instead of 100 ul for the incubation with vortexing and th
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8. is recommended in order to avoid bubble formation or incomplete dispensing from the tip 2 Wash the plate 4 times Empty the IMAPIlate by placing it on a filter paper and pushing slightly against filter paper for 10 seconds to let the solution completely absorbed by the filter paper Touch load the IMAPlate with wash buffer and empty the IMAPIlate four times Touch load procedure place IMAPIlate on a plate cover containing sufficient wash buffer and move up and down several times to ensure the capillarity reaction chambers fully loaded Instead of the plate cover a flat bottomed 96 well plate containing 100 uL of wash buffer in each well can also be used 3 Block IMAPIlate by touch loading Incubate 10 minutes in a high humidity environment at room temperature 4 Wash the plate 4 times as in step 2 Contact your local distributor Uptima powered by A 213 Avenue J F Kennedy BP 1140 i i nte rco h i m 03103 Montlu on Cedex France T l 04 70 03 88 55 Fax 04 70 03 82 60 P 11 15 uptima interchim com _ Uptima 5 Prepare cTnI standard concentrations at 12 5 6 25 3 125 1 56 0 78 0 39 0 2 and O ng mL in PBS Add standard or sample by directly pipetting 5 uL standard concentrations into the reaction chamber from the bottom opening 2 or 3 replicates are prepared Incubate 30 minutes at room temperature in a high humidity environment 6 Wash the plate 4 times as in step 2 7 Add detection antibody in IMAPI
9. of suitable buffer and derive the data from the linear fitting curve of the standards in both measurements Absorbance with 5 ul 0 0 5 1 1 5 2 Absorbance with 1 5 ul Figure 2 Conclusion Using the IMAPlate6 for the ELISA readout can dramatically increase the detection sensitivity of commercial ELISA kit with the described ELISA setup in which the ELISA is basically carried out according to user s protocol except the readout step So scientists can directly use commercial ELISA kits and perform the ELISA with their familiar routine procedure but the addition of several lower standards and the alternation of the readout The described approach provides scientists a very simple but very effective method to improve the detection sensitivity of ELISA and will accelerate the discovery and applications of low abundant proteins in research and diagnostics The IMAPlate6 is an easy to use robust miniaturized analytical platform and the multi utility of IMAPlate6 and the approach described will have a substantial impact not only on ELISA but also on other type of assays Contact your local distributor Uptima powered by 213 Avenue J F Kennedy BP 1140 AR a E 03103 Montl c F A interchim Bee MEPE uptima interchim com Uptima IMAPlate based rapid miniature ELISA for the quantification of Troponin using Perkin Elmer Enspire 2300 plate reader FT DR961d Introduction The cardiac Troponin I cTnI plays an important role
10. powered by 213 Avenue J F Kennedy BP 1140 a i nte rco h i m 03103 Montlu on Cedex France T l 04 70 03 88 55 Fax 04 70 03 82 60 P 6 15 uptima interchim com _ Uptima well plate by a pasture pipette or a pipette Immediately coat the IMAPlate plate s by touch loading Incubate 60 minutes in a humidity environment at room temperature 2 Touch unload the reaction chambers and wash with Wash Buffer by touch loading and touch unloading Wash Buffer three times 3 Block IMAPlate by touch loading of Reagent Diluent 1mg ml BSA in PBS to each reaction chamber Incubate 10 minutes in a humidity environment at room temperature 4 Wash three times as the step 2 The IMAPIlate plates are now ready for sample addition Sample amp Reagent Saving Protocol The plate preparation and the assay procedure in this Sample amp Reagent Saving Protocol basically follow the same protocol as the belox High throughput Protocol except of the way to load reaction chamber When it needs to save the samples or reagents the reaction chambers are loaded by a pipette instead of by the touch loading It is recommended to turn the IMAPlate upside down and to use the reverse pipetting technique to add 5ul of samples or reagents into the reaction chambers through the bottom openings Be careful for the sample and standard orientation In order to avoid the orientation mistakes mark the backside of the IMAPlate Assay Procedure High throughput 1 Tou
11. Distribution of 96 data from a known Fig 4 Relationship between the true IMAPlate 5RC96 concentration sample on an IMAPlate 5RC96 absorbance and the volume of DNA solutior added in the reaction chamber Figure 4 represents a diagram of the true absorbance against the volume of a DNA sample solution pipetted in the reaction chambers The true absorbance shows a linearity relation with the volume at the range from 1 ul to 5 ul Therefore the use of a pipette can provide an alternative to prepare a standard curve in which only one standard with an appropriate concentration can be added into the assigned reaction chambers on IMAPlate with a serial volume In such a way the preparation of a standard series of DNA solution is no longer necessary It may also eliminate the need for dilution of samples with high concentration just by adding 1 ul of samples to the reaction chambers for measurement Conclusion The IMAPlate is an easy to use robust miniaturized analytical platform suitable for quantification and quality analysis of DNA RNA samples with 96 well plate readers It offers users a wide range of UV VIS IR spectrometric analysis of 1 ul to 5 ul of samples and a variable light path from 1 mm to 5 mm The measured samples can totally be recovered without cross contamination risk The flexible simple handling of the IMAPlate not only saves hands on time and material cost for the analysis but also enhances the lab productivity Contact your loc
12. The absorbance of UV light at wavelength of 260 nm by a pure DNA or RNA provides a simple and accurate estimation of the concentration of nucleic acids in a sample For example the absorption of 1 OD A is approximately equivalent to 50 ug ml dsDNA 33 ug ml ssDNA 40 ug ml RNA or 30 ug ml for oligonucleotides Besides the quality of a DNA or RNA sample can also be checked by the spectroscopy The ratio of A260 A280 or A260 A230 is used to estimate the contamination of the sample by protein or by substances such as carbohydrates peptides phenols or aromatic compounds respectively The pure DNA should have an A260 A280 ratio of approximately 1 8 whereas pure RNA should give a value of approximately 2 0 The ratio A260 A230 should be approximately 2 2 for pure samples IMAPIlate is the world s first miniaturized analytical platform capable of manually performing high throughput liquid transfer analysis reaction and assay It comprises 96 identical funnel like reaction units positioned according to standard 96 well plate format and each reaction unit contains a 5 ul round reaction chamber with a light path of 5 mm long The IMAPIlate uses capillary force to confine samples in the bottomless reaction chambers and therefore the samples can spectroscopically be analyzed one by one in a microwell plate reader in any range of UV VIS IR spectra The use of IMAPlate combined with a microwell plate reader is an ideal routing method for quantification and qualit
13. Uptima FT DR961d IMAPlate Products Description The IMAPlate is a disposable Lab device to perform manually high throughput liquid transfer analysis and assay in a miniature format Features e 96 channel pipette for liquid transfer e 96 micro cuvette array for UV VIS or IR spectroscopy e 96 microwell plate for parallel reactions and assays IMAplates makes it possible pipette up to 96 individual samples simultaneously then to analyse them with your usual microplate reader then if needed to recover the samples Applications e UV VIS or IR spectroscopy e ELISA chromogenic fluorescent luminometric e 96 channel pipetting dispensing This document presents some key applications and technical tips DNA RNA quantification and quality analysis Protein quantification Bradford Protein Assay Miniaturized Enzyme Linked ImmunoSorbent Assay A simple solution to improve the detection sensitivity of ELISA A rapid miniature ELISA for the quantification of Troponin I using Perkin Elmer plate reader Miniaturize Homogenous Assays References Contact your local distributor Uptima powered by i n te rc h i m madenine pian Ph 1140 ont ucon Cedex France uptima interchim com 004 70 03 88 55 Fax 04 70 05 82 60 P 1 15 Uptima DNA RNA quantification and quality analysis by IMAplate FT DR961d Introduction The quantification of DNA or RNA by the spectroscopy is a well established routine method in many laboratories
14. al distributor Uptima powered by 213 Avenue J F Kennedy BP 1140 Oo gt Y 03103 Monti Cedex F M interchim PAAA uptima interchim com P 3 15 Uptima Protein quantification Bradford Protein Assay by IMAplate FT DR961d Introduction The Bradford protein assay is a simple and rapid method to determine the total protein concentration in a sample The assay uses Coomassie G 250 Dye as a colorimetric reagent for the quantification of protein In the acidic environment of the reagent mixture the absorbance peak of the dye will change from 465 nm to 595 nm when the dye binds to protein Within the linear range the absorbance value at 595 nm is proportion to the total amount of protein existing in the reagent mixture IMAPlate is the world s first miniaturized analytical platform capable of manually performing high throughput liquid transfer analysis reaction and assay It comprises 96 identical funnel like reaction units positioned according to standard 96 well plate format and each reaction unit contains a 5 ul round reaction chamber with a light path of 5 mm The bottomless reaction chamber uses capillary force to confine sample solution inside it therefore up to 96 samples can be analyzed one by one in a microwell plate reader The use of IMAPlate for the Bradford protein assay would provide scientists an easy to use miniaturized analytical tool for protein quantification It offers benefits such as m minimize the consumpt
15. as almost kept its original assay format the assay is performed in a 96 well plate with a working volume of 100ul or 200ul Attempt to reduce the reaction volume of the ELISA by using 384 well plate with a working volume of 20ul or 50ul has been reported successful But technical difficulties in liquid handling hampered such kind of approaches being used IMAPIlate is the world s first miniaturized analytical platform capable of manually performing high throughput liquid transfer analysis reaction and assay Its unique liquid handling concept has resolved the technical difficulties for transferring tiny amount of solution Up to 96 individual samples or solution can simultaneously be transferred in and out of the 5 ul reaction chambers of the IMAPlate by touch loading or touch unloading procedure Therefore the processing of each step for ELISA is in parallel and high throughput This unique liquid handling concept especially eases the tedious washing steps of the ELISA The use of IMAPlate for ELISA would bring scientists many benefits such as m minimizing the consumption of delicate samples and reagents 5 ul m reducing time to result by at least half m high productivity m cost effectiveness m user friendly m benefit environment producing less biological and chemical wasters ELISA benefits IMAplate microplates have unique bottom free chambers instead of wells that are designed to have a slightly larger surface for immun
16. cTnI No difference could be observed between IMAPlate and 96 wells plate assays for concentrations around 5 ng mL Discussion The IMAPIlate technology is the world s first miniaturized analytical platform that also supports manual high throughput liquid handling Due to the very small dimension of the capillarity reaction chamber using high quality plate readers such as from Perkin Elmer are recommended for the measurement of IMAPlate The rapid auto new plate definition function of the Perkin Elmer Enspire 2300 multilabel reader makes it much more attractive for the measurement of IMAPIlate to obtain high quality data The IMAPlate based miniature cTnI ELISA assay showed comparable results to conventional 96 wells plate for cTnI ELISA assay The shape of two standard curves was quite similar except that the one from IMAPlate was shifted in parallels towards low concentration Combined the observation of lower limit of detection in IMAPIlate the IMAPlate based cTnI ELISA should have a better sensitivity Besides the requirement for samples and reagents was greatly decreased The time to results is reduced from overnight coating plus 7 hours to less than 3 hours including coating procedure The unique self dosed liquid taking up feature also leads to very simple washing steps However the touch loading procedure may require some technical practices for a new user In conclusion the Perkin Elmer Enspire multilabel reader is compatible with the IMAP
17. ch load sample or standard in Reagent Diluent from the U bottomed 96 well plate which is prepared in advance by adding a drop of the samples or the standards to the appropriate wells Incubate 60 minutes in the humidity chamber at room temperature 2 Wash three times as the step 2 of Plate Preparation 3 Touch load the Detection Antibody diluted in Reagent Diluent from a U bottomed 96 well plate Incubate 60 minutes in the humidity chamber at room temperature 4 Wash three times as the step 2 of Plate Preparation 5 Touch load 1 200 diluted Streptavidin HRP solution from a U bottomed 96 well plate Incubate 15 minutes in the humidity chamber at room temperature 6 Wash five times as the step 2 of Plate Preparation 7 Touch load Substrate Solution Incubate 5 10 minutes at room temperature 8 Place the IMAPlate on a U bottomed 96 well plate containing 15ul of stop solution 2 5M H SO in each well Make sure the bottom openings are contacted with the stop solution After touch exchange 30 seconds slowly remove the IMAPlate from the U bottomed 96 well plate and gently invert several times until the blue colored TMB substrate solution turning to yellow 9 Immediately determine the true absorbance of each reaction chamber by a two wavelength measurement measure both peak absorbance at 450nm and the baseline absorbance at 650nm any wavelength between 550nm to 700nm can be used and subtract the peak absorbance from the corresponden
18. e result was read out using the IMAPlate with the volume of 5 ul Absorbance The detection sensitivity the slope of the initial increase was increased about 7 fold and the low detection limit also went down correspondently If a 96 well plate with V or U bottom is used and the substrate solution wareduced to 15 ul the detection sensitivity is expected to increase more than 10 fold and the low detection limit can go down 0 fold as well The detectable rate for those low abundant analytes can be increased further when an increased sample volume is applied It has to be pointed out that to use higher concentration substrate solution would be preferred in order to avoid quickly 0 200 A00 600 800 1000 running out of the substrate for standards of high concentration defined in the provided human TNF alpha pg ml rotocol Figure 1 Since the absorbance value of two standards with the highest concentration was over the upper detection limit of the plate reader a second measurement using 1 5 ul of the reaction solution was performed Because the obtained absorbance value with 1 5 ul of the reaction solution decreased about 3 3 fold and matched the calculation based on the decrease of the light path the linear fitting curve of standards in Figure 2 is used to derive the absorbance data for the two tandards with 5 ul Alternatively it is also ractical to measure the absorbance value irectly from the 96 well ELISA plate after adition of 100 ul
19. g 50 ul for flat bottom or 40 ul for V and U bottom may reduce the background 2 Block remaining protein binding sites of the well with blocking reagent Note above preparation steps are only for ELISA reagent kit 3 Perform the ELISA according to the protocol provided by manufacturer In order to increase the detectable rate an increased volume of sample may be preferred for low abundant molecule measurement 4 Add reduced volume of substrate solution e g TMB solution and vortex for 15 to 30 minutes The volume of substrate solution used depends on the demands of the sensitivity and the bottom format of the 96 well plate 25 ul would be the low limit for a flat bottom plate otherwise not all immobilized enzymes are able to make contact with the substrate solution If the volume of the substrate solution is the same as that for coating the vortex is not necessary 5 After incubation transfer 5 ul of the substrate solution with or without the addition of acidic stop solution from 96 well ELISA plate to IMAPlate by pipette or aspirate by capillary force if the volume is large enough e g 75 ul for flat bottom or 20 ul for V and U bottom It is suggested to measure the substrate solution without addition of acidic stop solution because it may cause the precipitation for standards and samples with high concentration and make the absorbance value of these standards and samples invalid 6 Place the IMAPlate in the reader with the adaptor
20. in the regulation of cardiac muscle contraction It does not ordinarily exist in peripheral circulation but will be released into circulation in myocardial necrosis Therefore the cTnI is a reliable cardiac biomarker for the cardiac muscle injury The amount of cTnI in peripheral circulation is usually quantified by Enzyme linked immunosorbent assay ELISA which is performed in a 96 microwell plate The conventional cTnI ELISA suffers from a number of limitations e g long assay ae 5 incubation times large sample volumes and laborious liquid handling l The IMAPlateTM 5RC96 is a miniaturized analytical platform which can replace the 96 well 7 a plate for carrying out reactions and assays It comprises 96 identical funnel like reaction units and each reaction unit contains a 5 uL capillarity reaction chamber with a light path length of 5 mm Besides being economic sensitive and flexible for liquid handling by pipetting the unique self dosed liquid taking up feature of IMAPlate also supports manual high throughput in which up to 96 individual samples can simultaneously be loaded in a second by simply dipping the open bottom of the capillarity reaction chambers and emptied by applying them to a filter paper Loaded solution can then be measured fluorimetrically or colorimetrically by plate readers In this application we use Perkin Elmer Enspire 2300 plate reader to measure the cTnI ELISA performed in IMAPIate This IMAPlate based miniature
21. ion of delicate protein samples only requiring 0 1 ul to 4 ul sample m no need for time consuming sample dilutions flexible sample volume m large linear measurement range up to 4000 ug ml m high throughput obtaining up to 96 individual data in one measurement m save reagent and produce less chemical waster Experimental Reagents and Materials Bradford Reagent Uptima CooAssay UPF86400 Protein standards Pipettes can accurately transfer 1 and 4 ul IMAPlate start kit Microwell plate reader e g BioTek PowerWave Microplate Spectrophotometer Procedure A high concentration of protein sample 1 Pipette 4ul of 1 4 diluted Bradford reagent fresh prepared mixture of one part of the reagent plus 3 part of distilled water to the reaction chambers 2 Pipette 1 ul of protein standards and sample mixing very well before use to the assigned reaction chambers 3 Invert the IMAPlate several times to mix the solution 4 Place the IMAPlate in the reader with the adaptor 5 Measure the peak absorbance at wavelength of 595 nm and base line absorbance at wavelength of 800 nm If desired the peak absorbance and base line absorbance can also be measured at other wavelengths between 575 nm to 615 nm and 750 nm to 850 nm respectively 6 Use true absorbance values A595 A800 to plot the standard curve and calculate the concentration of samples according to the standards Procedure B low concen
22. late by directly pipetting 5 uL of antibody solution into the reaction chamber through the bottom opening Incubate 30 minutes at room temperature in a high humidity environment 8 Wash the plate 4 times as in step 2 9 Add TMB substrate by touch loading of TMB solution from a plate cover or a 96 well plate Incubate 5 20 minutes at room temperature For a U or V bottomed 96 well plate 25 uL TMB solution in the wells is enough and for a flat bottomed 96 well plate 80 100 is needed 10 Pipette 0 5 uL of 3M H2SO4 stop solution directly into the reaction chamber from the bottom opening Slowly inverse the IMAPlate several times to make sure the solutions are mixed well Alternatively place the IMAPlate on a 96 well plate that contains the stop solution 15 uL for a U or V bottomed 96 wll plate and 80 uL for a flat bottomed 96 well plate and wait for several seconds to allow the solution in the reaction chamber to partially exchange with the stop solution through the bottom openings Slowly lift the IMAPlate and inverse several times to make sure the solutions are mixed well 11 Measure absorbance at both peak wavelength 450 nm for TMB and base line wavelength e g 650 nm for TMB by using the IMAPlate adaptor according to the IMAPlate user manual Calculate the true absorbance Abstrue Abspeak Absbaseline 96 well plate protocol Coat the plate with 50 uwL well of capture antibody solution Incubate overnight at 4 C
23. late technology The combination of these two technologies provides a very useful miniature lab tool for routinely performing assays as well as analyzing samples Contact your local distributor Uptima powered by a 213 Avenue J F Kennedy BP 1140 y i n te rco h i m 03103 Montlu on Cedex France T l 04 70 03 88 55 Fax 04 70 03 82 60 P 13 15 uptima interchim com Uptima Homogenous assays Miniature high sensitive homogeneous assays Introduction Due to relatively low cost of absorbance plate readers cheap and uncomplicated reagents preparation low sample consumption and simple add mix measure procedure 96 well plate based homogeneous colorimetric assays are still popularly used in the labs for routine analysis research application and compound screening Attempting to miniature the assays in 384 or 1536 well plate seems not practicable for manual pipetting and remains a challenge even for automations Although the add mix measure assay procedure is straightforward false results can easily be generated due to the air gap between the solutions caused by multiple pipetting into a small well IMAPlate comprises 96 identical funnel like bottomless reaction units in which the diameter of the upper compartment is larger than the well of 384 well plate while the diameter of the lower compartment is smaller than the well of 1536 well plate The unique design of the IMAPlate enables each reaction unit to hold more
24. ological reactions a smaller volume of reagent a slightly longer path lenght be able to load samples and reagent quickly capillary force m A a result ELISA assays performed in IMAplate show quiker kinetic of immunological reaction and higher signal increase absorbance values up to 7 folds while using only 5uL of samples and reagents and gaining considerable time in handling m The miniaturazation of the method is critical when analysing rare or precious samples when using expensive antibodies primary antibodies will be used and eventuelly more diluted or other costly reagents such a ECL substrates or saturating agents The method also reduce consumption of the washing buffer furthermore that ther is no need of washer that need large volume for setting up the process m The benefits are combined in in High Throught Put analysis HTS and when combining several analysis on each samples Experimental Reagents and Materials IMAPIlate start kit Pastuer pipette and or pipettes Human IL 6 DuoSet ELISA Development kit Microwell plate reader U bottomed 96 well plates Miniaturized ELISA High throughput Protocol Plate Preparation coating 1 Dilute the Capture Antibody to the working concentration 2 ug ml in PBS according to the protocol provided by the manufacturer Add one drop between 20ul to 50u1 of the diluted Capture Antibody to each well of the U bottomed 96 Contact your local distributor Uptima
25. olution References 1 Spies P et al Establishment of a miniaturized enzyme linked immunosorbent assay for human transferrin quantification using an intelligent multifunctional analytical plate Analytical Biochemistry 382 2008 35 39 Article 2 Spies P et al A simple approach to improve the sensitivity of ELISA using IMAPlate 5RC96 for result readout Analytical Biochemistry 397 2010 48 55 Articlepdf 3 Sciotti M A et al IMAPlate Based Miniature High Sensitive Rapid Screening Method for Detecting Bioengineered Secreted Lipase Activities in Yeast Expression Systems CHIMIA 64 2010 789 792 Articlepdf Ordering information Designation Product number Quantity IMAplate Start Kit DR9601 1 Kit Technical sheet and price IMAplate 96 ucuves Whites DR9611 Sea IMAplate 96 ucuves Blacks DT5431 5 ea 5 ea Product Highlight IMAplate 96 ucuves Yellows DT5441 5 ea 5 ea the starter kit contains 5 white IMAplates and Reader adapter White plate are recommended for luminescence measurments Black for fluorescence measurements and yellow plates for UV vis spectrometry and sample handling Catalog size quantities and prices may be found at http www interchim com Please inquire for higher quantities availability shipment conditions For any information please ask Uptima Interchim Hotline 33 0 4 70 03 73 06 Rev L7E J11E Uptima powered by 213 Avenue J F Kennedy BP 1140 pi
26. t baseline absorbance to get true absorbance Abstrue Abs450nm Abs650nm The true absorbance should be used for the calculation and plotting If the reader has spectral scan mode it is recommended to use the spectral measurement setting for the two wavelength measurement e g starting at 450nm and ending at 650nm with a step of 200nm Results and Discussion Figure 1 shows a typical plot of the data set of human IL 6 B standards from an ELISA that was performed in IMAPIlate with a high throughput protocol and a fitting curve of the standards with one site binding mode The standards were very well distributed around the fitting curve and the CV of the IL 6 standards was usually less than 10 in triplicates typically around 5 When the same concentration of all the reagents was used to perform ELISA on conventional 96 well plate and IMAPIlate the slope of the standard curve markedly increased with IMAPlate Therefore the sensitivity was correspondently increased even that the time for reactions in IMAPIlate was reduced to half 0 Contact your local distributor Uptima power 0 200 400 600 800 1000 213 Avenu ad or l z I i i i i interchim EUN human IL 5 ae mi Abs 450 Abs 650 uptima interchim com Abs 450 Uptima FT DR961d It has to be pointed out that when the sensitivity increases the detection range may be narrowed due to the overflow of the absorbance for higher concentration standards each plate
27. tandards Contact your local distributor uptima interchim com FT DR961d Results and Discussion Figure 1 shows spectra of calf thymus DNA solution and water in the reaction chambers of an IMAPlate measured by a 0g microwell plate reader The obtained spectrum is a typical spectrum of a pure DNA sample measured by a spectrophotometer and can be used for both quantitative and quality analysis The data of calf thymus DNA standards generated with the Procedure A can very well be fitted with linear regression equation Figure 2 The coefficient of variation from 96 0 2 DMA solution reaction chambers of an IMAPIlate is 5 8 for measuring a known concentration of calf thymus DNA solution in the low rl i E detection range Figure 3 According to the results the 740 270 300 330 360 2390 concentration of a DNA sample within 5 ug ml to 250 ug ml Wavelength nm could accurately be determined by this procedure The Procedure A is an easy fast robust and effective means for the 0 6 0 4 Absomance Heo Fig 1 Spectra of pure DONA solution and water analysis of a large number of samples measured using an IMAPlate SRC 96 1 2 Std curve A 23 2ug mi calf thymus DNA Std curve B _ 0 8 8 2 p _ am Si a lt lt E a U2 t 0 50 100 150 200 250 10 ne 30 AQ 40 0 1 2 3 4 5 E DNA concentration ug ml DNA concentration ug ml volume Ul Fig 2 DNA standard curves generated from an Fig 3
28. than 50 ul of solution When the lower bottom opening is sealed the solution pipetted into the upper compartment does not flow into the lower compartment For example by using a parafilm sheet to temperately seal the bottom reagent and sample can stay in the upper compartment for thoroughly mixing and incubation Once the parafilm sheet removed assay solutions will flow and fill up the lower compartment and immediately increase total liquid thickness about 5 mm Therefore for the absorbance measurement the light path length is increased ultimately Using IMAPlate can overcome the incomplete mix and reaction observed in 384 and 1536 well plate based assays It not only reduces the assay volume but also keeps high or increases the sensitivity of the assay The IMAPlate based miniature homogeneous assay can fit for both manual operation and automated liquid handling workstation IMAPlate Macro Volume Miniature Assay Procedure 1 Prepare a piece of parafilm e g 100 x 150 mm and place it on the top of an empty 96 well plate without the wax paper 3 Add assay components one by one into the upper compartment Total assay volume is recommended between 15 ul to 25 ul 5 Before measurement peel off the parafilm and the assay solution will flow into the lower compartment 7 Load the IMAPlate adaptor with the assay IMAPIlate into the reader and measure both the peak absorbance and base line absorbance 2
29. than 96 well Nunc plate 4 2 vs 5 9 ng ml for the IMAPlate and the 96 well Nunc plate respectively Therefore the IMAPlate should have a better resolution towards the low concentration Figure 1 4 PL fit of the absorbance background ratio versus Sensitivity Limit of Detection LOD log of cTnI concentration using the Graphpad Prism Software Limit of detection LOD was calculated as Average of Mean SD N 1 the results are representative of the two i independent experiments background 2 5 SD of background and are shown in Table 1 below The IMAPlate offers a slightly better sensitivity compared to conventional 96 well plate assay Table 1 LOD values mean of 2 independent experiments Contact your local distributor Uptima powered by AF t H mogron pleco Ph 1140 ee Fii 0470035 se Fax 04 70 03 02 60 P 12 15 uptima interchim com Uptima FT DR961d for each assay N 2 IMAPLATE 96 WELL PLATE 0 993 0 991 LOD 0 74 ng ml 0 94 ng ml oh a MAPLATE 96 VVELLS PLATE 5 Absorbance Background S E 1 0 0 5 0 0 0 5 1 0 1 5 Log Troponin Concentration ng mL Intra assay precision The CV of the cTnI standards was usually less than 20 in triplicates for IMAPlate assay and in duplicates for 96 well plate assay from cTnI concentrations from 1 56 to 12 5 ng mL Recovery in serum Additional samples have been prepared by spiking pure Troponin free serum with known concentrations of
30. tima interchim com Uptima IMAplate simple solution to improve the detection sensitivity of ELISA FT DR961d Introduction ELISA usually uses the same 96 well microplate for the reaction and the readout typically 100uL The following method takes advantage of unique IMAplate feature to increas the detection sensitivity A standard ELISA procedure is performed in conventionnal microplates taking advantage of the large surface of the 96 wells but the final staining of immobilized enzyme is done with only 25uL instead of 100uL Hence the concentration of the colored product formed is about 4 times higher if all the immobilized enzyme is able to react Then the colored solution is pipetted by IMAplate 5uL funnel like chambers SuL and made available for reading in a standard microplate reader with a higher lenght pass so higher signal can be recorded All together up 7 fold higher signal can be achieved Alternatively all the ELISA assay procedure can be performed directly in IMAplate with additionnal benefits See above applications Miniaturized ELISA assay Experimental Reagents and Materials Ready to use ELISA kit or ELISA reagent kit 96 well ELISA plate Pipette IMAPIlate start kit Microwell plate reader Procedure for ELISA using the IMAPIlate for result readout 1 Coat 96 well ELISA plate according to the protocol provided in the reagent kit with capture antibody Note A reduced volume for coating e
31. tration of protein sample 1 Pipette 1ul of un diluted Bradford reagent to the reaction chambers 2 Pipette 4ul of protein standards and sample mixing very well before use to the assigned reaction chambers 3 Invert the IMAPlate several times to mix the solution 4 Place the IMAPIlate in the reader with the adaptor 5 Measure the peak absorbance at wavelength of 595 nm and base line absorbance at wavelength of 800 nm 6 Use true absorbance values A595 A800 to plot the standard curve and calculate the concentration of samples according to the standards Contact your local distributor Uptima powered by A 213 Avenue J F Kennedy BP 1140 AR a _ 03103 Montl Cedex F A interchim Bee uptima interchim com P 4 15 FT DR961d Results and Discussion The plot of the true absorbance values against BSA concentration gives a typical binding curve as expected see left figure If the data are treated by a graph fitting software with binding mode the detection range of the BSA can be from 0 to 10000 ug ml But a straight line can be obtained in a narrow range with low concentration of protein For example in Procedure A the concentration of BSA is between 0 to 400 ug ml or in Procedure B the concentration of BSA is between 0 to 100 ug ml the linear range also can be extended up to 4000 ug ml if 0 1 ul of sample is mixed with 5 ul of 1 5 diluted Bradford reagent To the current setup the final reagent concentration is 1 5
32. y analysis of DNA or RNA samples especially with limited amount of sample volume Experimental to determine the concentration of samples Reagents and Materials DNA standards IMAPlate and reader adaptor 96 well V bottom plate Microwell plate reader with UV range Pipette Procedure A using capillary force based liquid transfer high throughput 1 Transfer 15 to 20 ul of a standard series of DNA solution to the assigned wells of a 96 well V bottom plate 2 Transfer 15 to 20 ul of sample solutions to the rest well of the 96 well V bottom plate 3 Aspirate the standard and sample solutions to IMAPIlate by capillary force 4 Place the IMAPIlate in the reader with the adaptor 5 Measure absorbance at wavelength of 260 nm and base line absorbance at wavelength of 350 nm 6 Use true absorbance values A260 A350 to calculate DNA concentration of samples according to the standards Procedure B using pipette loading 1 Pipette 1 2 3 4 and 5 ul of a standard DNA solution to the assigned reaction chambers on IMAPIate 2 Pipette appropriate amount of sample solutions 1 to 5 ul to the rest reaction chambers on IMAPlate according to the expected concentration 3 Place the IMAPIlate in the reader with the adaptor 4 Measure absorbance at wavelength of 260 nm and base line absorbance at wavelength of 350 nm 5 Use true absorbance values A260 A350 to calculate DNA concentration of samples according to the s
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