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&&&INFO8>0?.$0180@A @ucytech.com www.ucytech.com An accompanying Addendum of the manual is available on our website: www.ucytech.com

2- or 5-plate format

Using Optimiser Technology

For research use only. Not for use in diagnostic or therapeutic procedures. This manual applies for the following U-CyTech CytoMax ELISA kits

Species

Old World New World Analyte Human Mouse Rat Monkey Monkey IFN-γ CT920 CT945 CT970 CT980 CT992 IL-2 CT947 CT981 IL-4 CT948 CT982 CT993 IL-6 CT925 CT950 CT974 CT984 IL-10 CT985 IL-13 CT954 CT971 TNF-α CT936 CT959 CT972

2 Contents Introduction 4 CytoMax ELISA plate 4 Principle of the Test 5 Hazard information 6 Warning and Precautions 6 Contents of the Kit 6 Storage and Stability 7 Materials and Equipment (required but not provided) 7 Assembling CytoMax ELISA plate on plate holder 8 Pipetting techniques 8 Reverse pipetting to avoid bubbles 9 Electronic 8-well multi-channel pipette setup 9 Reader setup 10 Preparation Solutions and Transfer plate 11 Assay procedure 13 Interpretation of Results 14 Technical assistance 16

Abbreviations ANSI American National Standards Institute ELISA Enzyme-linked immunosorbent assay min minute(s) HRP Horse Radish Peroxidase PMT Photomultiplier tube RFU Relative Fluorescence Units RT Room temperature SBS Society for Biomolecular Sciences SPP Streptavidin-HRP

3 Introduction U-CyTech’s CytoMax ELISA kits offer a rapid, sensitive and specific chemifluorescent-based immunoassay for the measurement of cytokines using exceptionally small ­sample volumes. The speed, sensitivity and small sample requirements are enabled by the ­unique microfluidic design of the CytoMax ELISA plate in which all reactions, including cytokine coating and detection, occur within a 4.5 µl microfluidic reaction chamber. The distinctive micro­channel geometry and small reaction volume favor rapid reaction kinetics. The CytoMax ELISA ­assay utilizes only 5 µl sample and each reaction step is completed in 5 to 20 min. A typical Cyto- Max ELISA assay can be completed within two hours and ten minutes, including wash time, substrate incubation and read time. The plate is compatible with all standard fluorescence plate readers, robotic sample processors and other equipment used in conducting traditional 96-well microplate-based ELISA assays.

CytoMax ELISA plate The CytoMax ELISA plate is a revolutionary new microplate format. With an ANSI/SBS ­compliant 96-well layout, the plate by using OptimiserTM Technology integrates the power of ­microfluidics for small volume and rapid immunoassay protocols. Figure 1 shows sche- matically the CytoMax ELISA plate with magnified view of one well. Each well of the plate contains a loading well (for the addition of reagents), which is connected to one microfluidic reaction chamber. Reagents and samples are pipetted in the loading well and via capillary action transported through the reaction chamber to an integrated absorbent pad. The unique design of the plate allows the well to be drained but each liquid is trapped in the reaction chamber by capillary forces. As the next liquid volume is added, the capillary ­barrier is broken and the liquid within the chamber is drawn out by the absorbent pad and replaced by the new reagent. All reactions occur on the inner surface of the microfluidic­ reaction chamber. Small flow rate variations (time to empty well) do not affect assay results.

Loading well

Microfluidic reaction chamber

Figure 1. The CytoMax ELISA plate and magnified view of one well.

4 Principle of the Test The CytoMax ELISA procedure is an immunoassay in which traditional ELISA reactions take place within the unique plate. Briefly, coating antibody is immobilized on the inner surface of the plate’s microfluidic reaction chamber. Next, the unreacted sites are blocked with a blocking buffer. Subsequently, standards and samples are dispended to the wells. The im- mobilized coating antibody on the surface of the reaction chamber will specifically bind cytokines present in standards and samples. Following another wash, a biotinylated detec- tion antibody is added to the wells. The biotinylated antibody will bind to the cytokine, thus ‘sandwiching’ the cytokine between the coating and detection antibody. Following another wash, streptavidin-HRP (SPP) conjugate is added to the wells. The SPP conjugate binds spe- cifically to the biotin moiety of the detection antibody in the complexes formed. Following two additional washes, a chemifluorescent substrate is applied. If HRP has been captured on the microchannel surface during the sequence of reactions cited above, the enzyme will react with the substrate solution and will yield a fluorescent signal when excited at the appropriate wavelength. The light signal emitted will be directly proportional to the concentration of cytokine in standards and samples and will be quantifiable when the plate is read using a fluorescence plate reader.

5 Warning and Precautions This kit is designed for research only, not for use in diagnostic or therapeutic procedures.

Hazard information

Warning: Substrate solutions (A and B) are classified as dangerous according to Regulation (EC) no.1272/2008 and Directive 67/548/EEC and their amendments. Other kit components are not classified as dangerous according to egulationR (EC) no.1272/2008 and Directive 67/548/EEC or 1999/45/EC and their amendments.

For additional information, please consult Material Safety Data Sheet on our website: www.ucytech.com/manuals.

Contents of the Kit

Items Quantity Quantity Storage (2-plate format) (5-plate format) conditions Coating antibody* 1 vial 1 vial 4 ºC Cytokine standard* 2 vials 5 vials 4 ºC Biotinylated detection antibody* 1 vial 1 vial 4 ºC Coating buffer (ready-to-use) 1 vial (5 ml) 1 vial (7.5 ml) 4 ºC Blocking buffer (ready-to-use) 1 vial (25 ml) 2 vials (2x 25 ml) 4 ºC Wash buffer (ready-to-use) 1 vial (30 ml) 2 vials (2x 30 ml) 4 ºC SPP conjugate* 1 vial 2 vials ≤-20 ºC Substrate solution A** 1 vial (2.5 ml) 1 vial (5 ml) 4 ºC Substrate solution B** 1 vial (2.5 ml) 1 vial (5 ml) 4 ºC Substrate solution C** 1 vial (0.5 ml) 1 vial (0.5 ml) 4 ºC CytoMax ELISA plates*** 2 5 RT CytoMax ELISA pads*** 4 10 RT 96-well U-bottom Transfer plates 2 none RT Adhesive cover slips 5 10 RT

* Lyophilized ** Substrate solutions are manufactured by Thermo Fisher Scientific, Inc. *** This item is manufactured by Siloam Biosciences, Inc.

A CytoMax plate holder*** can be ordered separately (Cat. No.: CT379).

6 Storage and Stability • The vials with lyophilized coating and biotinylated detection antibody can be safely stored at 4 ºC, until the expiry date (indicated on the vals). Ater reconstitution in sterile water, antibodies are stable for at least 12 months at 4 ºC when kept sterile. However, it is strongly recommended to divide the reconstituted antibody preparations into small alquots for single use. These aliquots should be stored at ≤ -20 ºC (stable for at least two years). • The vials with lyophilized cytokine standard can be safely stored in a refrigerator, until the expiry date (indicated on the vials). These vials are for single use only. • The vial(s) with lyophilized SPP conjugate can be stored best at ≤ -20 ºC, until the expiry date (indicated on the vials). After reconstitution in sterile water, it is recommended to divide the solution into small aliquots for single use. These aliquots should be stored at ≤-20 ºC in the dark (stable for at least one year). • The ready-to-use Coating, Blocking and Wash buffer and substrate solutions (A, B and C) should be stored at 4 ºC, until the expiry date (indicated on the vials). After opening, these solutions are stable for at least 6 months when kept sterile.

Materials and Equipment (required but not provided) Materials • Eppendorf or similar microtubes for centrifugation of reagent or sample dilutions. • Sterile distilled water for reconstitution of lyophilized cytokine standards, antibodies and SPP conjugate. • Reagent reservoirs (e.g. 96-well U-bottom Transfer plate) for the preparation of reagent working solutions to be transferred to the CytoMax ELISA plates (96-well U-bottom Transfer plates are supplied only with the 2-plate kit). • Pipette tips for the accurate delivery of volumes in the ranges of 1-10 µl, 10-100 µl, and 100-1000 µl. • KimwipeTM paper. • CytoMax plate holder (U-CyTech Cat. No.: CT379). This tool can be re-used. Equipment • (Electronic) multi-channel pipette for precise and accurate delivery of small volumes. • Compatible pipettes to transfer solutions to the wells. • Fluorescence or multi-mode plate reader with fluorescence reading capability for 96-well microtiter plates. • Microcentrifuge to remove particulates from samples capable to set at 13,000 x g. • Vortex mixer for adequate mixing of reagents. • Timer for exact timing of incubation steps. • Robotic sample processor (optional) for automation of ELISA procedure.

Note: We recommend the Biohit eLINE® 8-channel Electronic pipette (volume range 5-120 µl), Model 730340, In combination with the Biohit Optifit tips (volume range 0.5-300 µl) (Biohit Cat. No.: 790300/790302).

7 Assembling CytoMax ELISA plate on plate holder Before starting an experiment, the CytoMax ELISA plate needs to be assembled as illustrated schematically in Figure 2. 1. Position the CytoMax ELISA pad on the CytoMax plate holder. 2. Align the CytoMax ELISA plate on the pad. 3. Press down the plate gently to click-lock in the holder.

CytoMax plate

Pad Pad

Holder Holder

Figure 2. Assembly of the CytoMax ELISA plate.

Note: The pad must be oriented correctly with the tape side facing the holder and absor- bent surface touching the microplate.

Pipetting techniques Assays on CytoMax ELISA plates require the accurate and precise delivery of 5 μl ­volumes. The following guidance is offered to users. 1. Use pipette for which the upper limit of their operating range is ≤10 µl. 2. Use pipette tips appropriate for 5 µl pipetting. 3. To aspirate liquid, hold the pipette near vertical and immerse the pipette tip to a depth of approximately 2 mm in the liquid. Release the operating button steadily. Wait one second. Withdraw the tip from the liquid. 4. To dispense liquid, hold the pipette nearly vertical. With the pipette tips touching the surface of the well, press the operating button steadily until the liquid is dispensed.

Note: The pipette tip must make contact with the well surface for proper dispensing (see “RIGHT” frame in Figure 3). Do not pipet directly into the hole at the bottom of the well (see “WRONG” frame in Figure 3).

RIGHT WRONG

Figure 3. Pipette tip positioning for loading the reaction chambers.

8 Reverse pipetting to avoid bubbles • Bubbles will compromise the performance of the CytoMax ELISA plate by interfering with the flow of liquid within the microchannels. • Reagents may form bubbles readily with standard pipetting techniques. • To avoid complications due to bubbles, U-CyTech recommends the use of the “Reverse Pipetting” technique during all pipetting steps (see Figure 4). 1. To aspirate liquid, press the operating button of the pipette to the second stop (position 1). 2. Immerse the pipette tip in the liquid to a depth of about 2 mm and steadily release the operating button completely (position 2). 3. Withdraw the tip from the liquid, touching it against the edge of the reservoir to remove excess liquid. 4. Dispense the liquid into the loading well of the plate by gently and steadily pressing the pipette’s operating button to the first stop (position 3). Briefly hold the operating ­button in this position. 5. With the button in this position, move the tip from the loading well to the reagent reservoir, immerse the tip in the liquid and aspirate (position 4).

Figure 4. Reverse pipetting technique.

Note: An electronic 8-well multi-channel pipette is ideally suited for use with CytoMax ELISA plates since (a) it eliminates possibility of injecting bubbles and (b) can be used for convenient repetitive loads with a single aspiration step for rapid reagent transfer.

Electronic 8-well multi-channel pipette setup 1. Use a pipette that can handle volumes of 5 μl and 30 μl (e.g. with volume range of 5-120 μl). 2. Choose “Reverse Pipetting” in function setting. 3. Use “Multiple Dispensing” mode to transfer the solution into the CytoMax ELISA plate. 4. For example, to transfer coating antibody solution to a full plate, set the program for 12 times dispensing, 5 μl per dispensing step. In this way, the pipette will first automatically aspirate more than 60 μl of solution (a few µl extra will be aspirated to avoid dispensing air) and dispense the preselected volume 12 times.

9 Reader setup The CytoMax ELISA is compatible with standard fluorescence plate readers and multi-mode plate readers with fluorescence reading capability (e.g. Synergy HT from BioTek Instruments, Inc.). In this section, we present a general guidance for setting up the plate readers. For further assistance, please contact U-CyTech’s technical support ­([email protected]). Step 1: Selecting the wavelength for excitation and emission The CytoMax ELISA includes a substrate, which can be detected using 530-575 nm for ex- citation and 585-630 nm for emission (Figure 5). Quantitation does not require filters that precisely match the excitation/emission maxima. However, a non-overlapping filter set with a band-pass that includes the excitation/emission spectra is required. Below are examples for different types of readers: • Filter-based readers: install 528/20 nm (or similar) filter for excitation and 590/35 nm (or similar) filter for emission. • Monochromator-based readers: set excitation at 544 nm and emission at 590 nm. • Readers with pre-configured optical set: select the wavelength setting for Rhodamine or Cy3.

450 500 550 600 650 700 Wavelength (nm) Figure 5. Normalized absorption (left) and emission (right) spectra of substrate.

Step 2: Selecting the plate type CytoMax ELISA plate fits 96-well SBS standard in all specifications. Please use ­“96-well stan- dard” or similar in plate type setting. Step 3: Selecting the probe direction Select “top reading” for probe direction and when possible the most optimal probe height. Step 4: Selecting the sensitivity/gain When defining reading parameters for fluorescence analysis, setting the PMT sensitivity (or “gain”) is important for obtaining useful measurements. The procedure below describes a manual sensitivity/gain setting that is recommended before reading the plates. 1. In a clean plastic tube, add 50 µl of substrate solution A, 50 µl substrate solution B, ­ 1 µl of substrate solution C and 1 µl of undiluted SPP conjugate. Mix well the components. After 2 min, the substrate is fully developed and will stay stable for hours. 2. Add 4 µl of the mixture into one loading well of a CytoMax ELISA plate and wait until the well is completely empty. 10 3. Read the plate in the plate reader with various gain setting (do not use plate holder and pad). 4. Select the gain, which gives the RFU closest to 11,000. 5. Use the same gain setting, read one blank well (the read out should be less than 50). Save or record this gain setting. 6. This gain defines the maximum reading (RFUmax) that CytoMax ELISA based assays can reach with this reader gain/sensitivity setting.

Note: The gain will be valid for all following CytoMax ELISA experiments. Repeat step 1 to 5 if: a) changing the plate reader or b) changing the optical unit such as light bulb, filters, etc.

Preparation Solutions and Transfer plate For rapid, accurate and precise transfer of solutions to the CytoMax ELISA plate, it is recom- mended to use a transfer plate in which the standards, samples and assay reagents can be prepared before their use in the assay procedure. The 2-plate kit contains such a transfer plate, while for the 5-plate kit we recommend the use of a 96-well U-bottom plate. Coating antibody The contents of the vial with coating antibody should be reconstituted in 200 µl (2-plate format kit) or 300 µl (5-plate format kit) sterile water and allowed to stand for 5 min at RT. To coat all wells of one plate, 30 µl is pipetted out of the vial (or use a frozen aliquot) and added to 720 µl coating buffer (ready-to-use). Mix the solution gently but thoroughly for ap- proximately 15 seconds and allow it to stand for 1 min at RT. Distribute this mix over eight wells (90 µl/well) of a 96-well U-bottom Transfer plate (Figure 6). Now, all wells of one plate can be coated with 5 µl coating solution/well with use of a multi-channel pipette. Blocking The Blocking buffer is ready-to-use and wells are blocked with 5 µl Blocking buffer. For blocking all wells of one CytoMax ELISA plate, a total of 750 μl Blocking buffer is ­required. ­Distribute this volume over eight wells (90 µl/well) of a 96-well ­U-bottom Transfer plate (Figure 6). Standard For maximum recovery, the contents of the vial should be reconstituted in 0.5 ml distilled water and allowed to stand for 5 min at RT and thereafter placed on ice. The reconstituted stock solution should be diluted in Blocking buffer to the highest cytokine concentration to be used in the standard range and used within 1 hour. For example, if the standard concentrations range from 2.5 to 160 pg/ml, the stock solution should first be diluted to 160 pg/ml. The standards are now obtainable in a series of two-fold dilutions in Blocking buffer starting from 160 to 2.5 pg/ml. Always, include a blank (Blocking buffer only) in the standard range. Prepare at least 50 µl of each standard dilution using one column of the 96-well U-bottom Transfer plate (Figure 6).

Note: After reconstitution of the standard, the concentration (ng/ml) will become twice the amount on the label (e.g. 4.8 ng/vial will become 9.6 ng/ml). It is recommended to test each standard dilution in triplicate.

11 Samples For measuring cytokines in cell culture supernatant, serum or plasma, samples should be diluted at least 1:1 in Blocking buffer. Particulates in samples may block liquid flow through the microchannels. It is therefore important to centrifuge (10 min at 13,000 x g) or filtrate (0.2 µm) samples containing serum or plasma prior to testing. Diluted samples should be distributed over appropriate wells of one 96-well U-bottom ­Transfer plate (Figure 6). When the assay is performed in triplicate, a total volume of at least 20 µl (using a single-channel pipette) or 45 µl (using a multi-channel pipette) is required for each sample concentration (5 µl sample per well). Coating antibody Blocking buffer Standard Samples Detection antibody conjugate SPP Substrate

1 2 3 4 5 6 7 8 9 10 11 12 A S1 S9 S17 St1 B S2 S10 S18 St2 C S3 S11 S19 St3 D S4 S12 S20 St4 E S5 S13 S21 St5 F S6 S14 S22 St6 G S7 S15 S23 St7 H S8 S16 S24 St8

Figure 6. Example of layout of the 96-well U-bottom Transfer plate (for testing in triplicate).

St1 to St8: serial dilutions of standard [160 (St1) to 2.5 (St7) pg/ml and blank (St8)]; Sx: sample x.

Biotinylated detection antibody The contents of the vial with biotinylated detection antibody should be reconstituted in 200 µl (2-plate format kit) or 300 µl (5-plate format kit) sterile water and stand for 5 min at RT. For one CytoMax ELISA plate, 30 µl is pipetted out of the vial (or use a frozen aliquot; see “Storage and Stability”) and added to 720 µl Blocking buffer. Mix the solution gently but thoroughly for approximately 15 seconds and allow it to stand for 1 min at RT. Distribute the solution over eight wells (90 µl/well) of one column of the Transfer plate (Figure 6). SPP conjugate The contents of the vial with lyophilized SPP should be reconstituted in distilled water (200 µl per vial) and allowed to stand for 5 min at RT. For one CytoMax ELISA plate, 30 µl is pipetted out of the vial (or use a frozen ­aliquot) and added to 720 µl Blocking buffer. Mix the solution gently but thoroughly for approximately 15 seconds and allows it to stand for 1 min at RT. Distribute over eight wells (90 µl/well) of one column of the Transfer plate (Figure 6).

12 Substrate The substrate solution should be prepared no more than 30 min before loading­ into the ­CytoMax ELISA plate. For one plate, mix 700 µl substrate solution A, 700 µl substrate solution­ B and 14 µl substrate solution C. Mix thoroughly by gently ­vortexing. Distribute over eight wells (150 µl/well) of one column of the Transfer plate (Figure 6).

Note: Substrate solution C should be completely thawed before mixing.

Washing The Wash buffer is ready-to-use and wells are washed three times with 5 µl and twice with 30 µl buffer. For washing all wells of one CytoMax ELISA plate a total of 8.5 ml Wash buffer is required. This volume can be added to a separate reagent reservoir (or part of the Washing buffer can be added to 24 wells in the Transfer plate and filled up during the procedure).

Assay procedure

Note: All following incubation steps are performed at RT.

1. Add 5 µl of the diluted coating antibody solution to the required number of wells of the CytoMax ELISA plate and incubate for 20 min at RT. 2. Wash the wells by adding 5 µl of Wash buffer to each well and incubate for 5 min. 3. Add 5 µl of Blocking buffer incubate for 10 min. 4. Add 5 µl of diluted standards and samples to appropriate wells and incubate for 20 min.

Note: See example on the following page (Figure 7).

5. Wash the wells by adding 5 µl of Wash buffer to each well and incubate for 5 min. 6. Add 5 µl of the diluted detection antibody solution to each well and incubate for 10 min. 7. Wash the wells by adding 5 µl of Wash buffer to each well and incubate for 5 min. 8. Dispense 5 µl of diluted SPP conjugate solution to each well and incubate for 15 min. 9. Wash the wells by adding 30 µl of Wash buffer and incubate for 10 min. 10. Repeat washing step 9.

Note: Within this 10 min incubation step prepare the substrate solution.

11. Dispense 10 µl substrate solution into each well. 12. Incubate for 20-30 min. 13. Remove plate and pad from the holder. 14. Discard the pad. 15. Wipe the bottom of the plate with a KimwipeTM paper. 16. Place the CytoMax ELISA plate in the reader chamber of the fluorescence plate reader.

13 1 2 3 4 5 6 7 8 9 10 11 12

A S1 S1 S1 S9 S9 S9 S17 S17 S17 St1 St1 St1

B S2 S2 S2 S10 S10 S10 S18 S18 S18 St2 St2 St2

C S3 S3 S3 S11 S11 S11 S19 S19 S19 St3 St3 St3

D S4 S4 S4 S12 S12 S12 S20 S20 S20 St4 St4 St4

E S5 S5 S5 S13 S13 S13 S21 S21 S21 St5 St5 St5

F S6 S6 S6 S14 S14 S14 S22 S22 S22 St6 St6 St6

G S7 S7 S7 S15 S15 S15 S23 S23 S23 St7 St7 St7

H S8 S8 S8 S16 S16 S16 S24 S24 S24 St8 St8 St8

Figure 7. Example of layout of the CytoMax ELISA plate (for testing in triplicate).

St1 to St8: serial dilutions of standard [160 (St1) to 2.5 (St7) pg/ml and blank (St8)]; Sx: sample x.

Interpretation of Results Calculate the mean of the RFUx values for the standards (e.g. 0.625 to 40 pg/ml) and the negative control (blank). Subtract mean blank from all mean RFUx values (= RFU40, RFU20, etc.). Plot these values (mean RFUx minus mean blank) versus the cytokine concentration (in pg/ml). Draw the standard curve, using a linear or polynomial regression curve.

Example of monkey IL-13 CytoMax ELISA

RFUx values Mean RFUx minus IL-13 (pg/ml) 1 2 3 Mean SD CV (%) Mean blank 40 2910 2915 2906 2910 5 0.2 2732 20 1949 2062 2235 2082 144 6.9 1904 10 1149 1048 1098 1098 51 4.6 920 5 584 603 610 599 13 2.2 421 2.5 360 361 375 365 8 2.3 187 1.25 282 295 290 289 7 2.3 111 0.625 242 256 250 249 7 2.8 71 blank 176 171 187 178 8 4.6 0

See an example of a polynomial regression curve in Figure 8. On www.ucytech.com/manuals, you can find the specific standard curve of different CytoMax ELISA kits.

14 Determination of cytokine concentration in samples Calculate the mean of the RFUx values recorded for your samples. Subtract the mean ­blank from all mean RFU values. Plot the calculated values on the standard curve. The cytokine level can now be read from the abscissa of the standard curve (or cal- culated using the equation of the standard curve). The results should be multiplied by the dilution factor to obtain the final concentration in the sample.

Standard curve of the monkey IL-13 CytoMax ELISA

3000

2500

2000

RFU-blank 1500

1000

500

0 5 10 15 20 25 30 35 40 45 recombinant IL-13 (pg/ml)

Figure 8. Example of a standard curve for the monkey IL-13 CytoMax ELISA. This standard curve is for demonstration only. A standard curve should be run with each assay.

15 Technical assistance If you require assistance, information or have any questions, please contact our company: U-CyTech biosciences Phone: +31.30.253 5960 E-mail: [email protected]

On our website (www.ucytech.com) you can find: the Manual, Typical data, MSDS and Addendum of our CytoMax ELISA kits.

CytoMax ELISA kit version 140102