user guideELISA Technical GuideGeneral information and guidelines for using Novex ELISA KitsRevision Date 10 April 2012Publication Part Number MAN0006706

ContentsProduct Information . 4About ELISA Kits . 4Methods . 6Before You Begin . 6Prepare Reagents and Samples . 9Perform the ELISA . 10Troubleshooting. 11Appendix A . 14Sample Preparation and Handling . 14Frequently Asked Questions (FAQs) . 18Ordering Information . 20Appendix B . 21Safety . 21Technical Support . 22Purchaser Notification . 23Explanation of Symbols . 243

Product InformationAbout ELISA KitsIntroductionThe Novex ELISA kits from Life Technologies are based on solid phasesandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA). The ELISA Kit isused for the quantitative determination of secreted proteins such as cytokinesand chemokines, as well as intracellular signaling proteins.ELISAs are adaptable to high-throughput screening because results are rapid,consistent and relatively easy to analyze. The best results are obtained with thesandwich format, utilizing highly purified, prematched capture and detectorantibodies. The resulting signal provides data which is consistent and highlyspecific.Shipping andstorageThe ELISA Kits are shipped at 2 to 8ºC. Upon receipt, store the kits at 2 to 8ºC.Some components may need storage at –20 C.ContentsThe ELISA Kits include all the reagents needed to perform a sandwich ELISA.This includes the capture antibody-coated 96-well plates, calibrated standard,Wash and Diluent buffers, detection antibody, secondary detection reagents,stabilized chromogen, stop solution, and plate covers.For detailed kit contents, refer to the product insert supplied with the product.For research use only. Not intended for any animal or human therapeutic ordiagnostic use.Types of ELISA kits(precoated plates) For extracellular proteins, ELISA kits are available for human, mouse, rat,monkey, bovine, and swine samples. These kits are available in thefollowing formats: Colorimetric ELISA kits that offer picogram detection range Ultrasensitive kits for low picogram detection range EASIA kits for use with clinical serum or plasma samples forpicogram detection range Chemiluminescence ELISA kits that offer low picogram detection rangeand an increased dynamic range over colorimetric ELISA kits.For intracellular proteins for signaling studies, phosphoELISA kits areavailable for measurement of total and phosphorylation, modification, orcleavage site-specific proteins.Continued on next page4

About ELISA Kits, ContinuedSystem overviewNovex ELISA kits are based on the solid phase sandwich ELISA technique. Forthis method, an antibody against the specific antigen is coated onto the wells ofthe microtiter strips provided in the ELISA kits.During the first incubation, standards of known content, controls, and unknownsamples are added to the coated wells to allow the antigen from the samples bindto the immobilized (capture) antibody.After washing, a detection antibody (antigen-specific rabbit antibody orbiotinylated antibody) is added that binds to the immobilized antigen capturedduring the first incubation. In some cases, the wash step is omitted and thedetection antibody is added at the same time as the samples.After removal of excess detection antibody, a horseradish peroxidase-labeledAnti-Rabbit IgG (Anti-Rabbit IgG HRP) or Streptavidin-HRP (enzyme) is added.This binds to the detection antibody to complete the four-member sandwich.After a second incubation and washing to remove all the unbound enzyme, astabilized substrate solution is added, which is acted upon by the bound enzymeto produce color. The intensity of this colored product is directly proportional tothe concentration of antigen present in the original specimen.The incubation times takes about 4 hours total and requires only 30 minuteshands-on time.For the EASIA kits, the technique is similar, but the detector antibody is directlyconjugated to HRP.For chemiluminescent sandwich ELISA kits, the protocol is similar with respect tothe capture antibody. The difference is that the detector antibody is conjugated toalkaline phosphatase (AP). After rinsing, a substrate, CSPD-Emerald II, is addedand a chemiluminescence readout is captured at the end. The incubation time is3.5 hours.Assay optimization5There is no need for optimization of precoated ELISA assays as each and everystep of the assay is optimized. This is one of the major benefits of using precoatedplates. However, it is very important to follow all steps of the protocol, especiallythe recommended washing steps and incubation times to obtain the best results.

MethodsBefore You BeginThe ELISA kits contain materials with small quantities of sodium azide.Sodium azide reacts with lead and copper plumbing to form explosive metalazides. Upon disposal, flush drains with a large volume of water to preventazide accumulation. Avoid ingestion and contact with eyes, skin and mucousmembranes. In case of contact, rinse affected area with plenty of water. Observeall federal, state, and local regulations for disposal.Materials requiredbut not provided Microtiter plate reader capable of measurement at or near 450 nm (forcolorimetric kits) Microtiter plate reader (luminometer) capable of chemiluminescencemeasurement (for chemiluminescent kits) Cell Extraction Buffer (Cat. no. FNN0011) for preparation of cell lysatesonly Calibrated adjustable precision pipettes, preferably with disposable plastictips. (A manifold multi-channel pipette is desirable for large assays.) Distilled or deionized water Plate washer: automated or manual (squirt bottle, manifold dispenser, etc.) Data analysis and graphing software Glass or plastic tubes for diluting and aliquoting Standard 37 C incubatorContinued on next page6

Before You Begin, ContinuedProceduralguidelines Refrigerate kit when not in use. Warm all reagents to room temperaturebefore use. Allow the microtiter plates to equilibrate to room temperature beforeopening the foil bags. Once the desired number of strips are removed,immediately reseal the bag and store the plate at 2 to 8 C to maintain plateintegrity. Collect samples in pyrogen/endotoxin-free tubes. Samples should be frozen if not analyzed shortly after collection. Avoidmultiple freeze-thaw cycles of frozen samples. Thaw completely and mixwell prior to analysis. When possible, avoid use of badly hemolyzed or lipemic sera. If largeamounts of particulate matter are present, centrifuge or filter sample priorto analysis. Run all standards, controls, and samples in duplicate. When pipetting reagents, maintain a consistent order of addition from wellto-well to ensure equal incubation times for all wells. Cover or cap all reagents when not in use. Do not mix or interchange different reagent lots from various kit lots. Do not use reagents after the kit expiration date. For best results, read absorbances within 30 minutes of assay completion.Plates can be read up to 2 hours after assay completion when kept in thedark. The controls provided should be run with every assay. If control values falloutside pre-established ranges, the accuracy of the assay is suspect. Drain residual wash liquid from the wells by efficient aspiration or bydecantation followed by tapping the plate forcefully on absorbent paper.Never insert absorbent paper directly into the wells. Because Stabilized Chromogen is light sensitive, avoid prolonged exposureto light. Avoid contact between chromogen and metal (e.g., aluminum foil), or colormay develop. Protect CSPD-Emerald Substrate from prolonged exposure to light.Continued on next page7

Before You Begin, ContinuedPlate washingdirectionsPerform all washing with the Wash Buffer Concentrate (25X) supplied with thekit. Dilute the 25X stock (see page 9) before use as described below. Incompletewashing adversely affects the results. Manual WashingCompletely aspirate the liquid from all wells by gently lowering anaspiration tip into the bottom of each well. Take care not to scratch theinside of the well. After aspiration, fill the wells with at least 0.4 mL dilutedWash Buffer. Allow the buffer to stand for 15 to 30 seconds, and thenaspirate the liquid. Repeat as directed under Assay Procedure. After thewashing procedure, invert the plate and tap dry on an absorbent tissue. Washing with a Squirt BottlePlace the diluted Wash Buffer into a squirt bottle. Flood the plate with thediluted Wash Buffer, completely filling all wells. After the washingprocedure, invert the plate and tap dry on an absorbent tissue. Automated Plate WasherIf using an automated washer, follow the manufacturer’s instructions forplate washing.Procedurelimitations Do not extrapolate the standard curve beyond the top point of the standard;the dose-response is non-linear in this region and accuracy is difficult toobtain. Dilute samples with concentrations exceeding the linear portion ofthe standard curve with the Standard Diluent Buffer; reanalyze these andmultiply results by the appropriate dilution factor. The influence of various drugs and the use of biological fluids in place oftissue culture media have not been thoroughly investigated. The rate ofdegradation of native protein in various matrices has not been investigated.The immunoassay literature contains frequent references to aberrant signalsseen with some sera, attributed to heterophilic antibodies. Though suchsamples have not been seen to date, the possibility of this occurrence cannotbe excluded.8

Prepare Reagents and SamplesDilute wash bufferDilute the standards1.Allow the Wash Buffer Concentrate (25X) to reach room temperature andmix gently to ensure that any precipitated salts have redissolved beforediluting.2.Dilute 1 volume of the Wash Buffer Concentrate (25X) with 24 volumes ofdeionized water (e.g., 50 mL may be diluted up to 1.25 liters, 100 mL maybe diluted up to 2.5 liters). Label as Working Wash Buffer.3.Store both the concentrate and the Working Wash Buffer in therefrigerator. Use the diluted buffer within 14 days.Note: Use glass or plastic tubes for standard dilutions.Reconstitute standard with Standard Diluent Buffer. Refer to the standard viallabel for instructions.Make serial dilutions of the standard as described in the product insertsupplied with the product.Prepare secondaryantibodyPrepare samples9Note: The anti-rabbit HRP or Streptavidin-HRP (100X) is in 50% glycerol. Thissolution is viscous. To ensure accurate dilution,1.Allow the reagent to reach room temperature. Gently mix.2.Pipette 10 μL (100X) solution. Wipe the pipette tip with clean absorbentpaper to remove any excess solution and transfer the solution to a tubecontaining 990 μL Diluent for each 8-well strip used in the assay, and mixwell. Label appropriately.3.Return the unused reagent to the refrigerator.4.Use the diluted antibody within 15 minutes.See Sample Preparation and Handling in Appendix for some methods toprepare samples.

Perform the ELISAELISA procedureReview Procedural Notes section before starting.1.Allow all reagents to reach room temperature before use. Gently mix allliquid reagents prior to use.Important: Run a standard curve with each assay.Read the plate andgenerate thestandard curve2.Perform the ELISA procedure as described in the product insert suppliedwith the product.1.For colorimetric kits, read the absorbance of each well at 450 nm. Read theplate within the recommended time after adding the Stop Solution.2.For chemiluminescent kits, read the luminescence 30 minutes after theaddition of the substrate with a 1000 msec integration time.3.Use curve-fitting software to generate the standard curve. A four parameteralgorithm provides the best standard curve fit.4.Read the concentrations for unknown samples and controls from thestandard curve. Multiply value(s) obtained for sample(s) by the appropriatefactor to correct for the sample dilution.Note: Dilute samples producing signals greater than that of the higheststandard in Standard Dil

2. Pipette 10 μL (100X) solution. Wipe the pipette tip with clean absorbent paper to remove any excess solution and transfer the solution to a tube containing 990 μL Diluent for each 8-well strip used in the assay, and mix well. Label appropriately. 3. Return the unused reagent to the refrigerator. 4. Use the diluted antibody within 15 minutes.