Technical Tips

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Tips and Troubleshooting

Hundreds of variables influence ELISA data. Below is a collection of general assay running tips and troubleshooting strategies that you may find helpful when trying to resolve ELISA issues. For Q-View Data Analysis tips, please see our Q-View Data Analysis Tips.

Troubleshooting

Q-Plex ELISA troubleshooting

When running a Q-Plex™ Array:

Do’s

  • DO be exact when calibrating shaker speed, being off by even 100 RPM can affect results.
  • DO dilute all sample types at least 1:2 (50%) with sample diluent (except cell culture media, which can be tested NEAT).
  • DO load all standards and samples into the microplate within 10 minutes of each other.
  • DO be exact with incubation times, particularly the SHRP incubation.
  • DO be exact when mixing Substrate A and B, being off by even 50 µL can affect results, and mix thoroughly.

Don’t

  • DON’T allow the plate to dry out between steps, particularly between washing SHRP and adding substrate
  • DON’T allow the SHRP, substrate, or IR dye to be exposed to UV light, as this may degrade it.
  • DON’T analyze from a color or jpeg image; save grayscale images using a lossless image file type such as Tiff or RAW

Q-Plex Experimental Troubleshooting

 

 

Image Troubleshooting

 

What can I do to get greater sensitivity from my Q-Plex Array?
If your antigen concentrations are very low, we first recommend that you select a High Sensitivity or SinglePlex Q-Plex™ Array. The assays in these kits have specifically been optimized to have the greatest sensitivity possible.

If you already have a normal kit, you can try the slightly modified protocol and data analysis tips below. Consider changes to the assay protocol carefully, however, as this can also have undesirable side effects. You might also consider requesting a Demo Kit to see if a modified protocol will work with your samples.

Modified Assay Protocol for Increased Sensitivity: Use at your own risk

To optimize for detection at the low end of the curve, extend the standard curve a few extra points, and use longer incubation times, for example:

This modified protocol sacrifices some quantitation, 1-2 points, at the top end. Also, some samples may have increased background with longer incubations, which will decrease sensitivity.

Data Analysis Tips

To optimize the curve fit for the low end, first, mask standard curve points if their % backfit is not 80%-120% (shown in the Report tab of Data Analysis). Next, mask the top 2-3 points of the standard curve if they haven’t already been masked. Finally, choose either Auto-Select or Log-Log as the regression model. This may provide a better fit at the low end of the curve.

Can I increase incubation temperatures?

This is not recommended. We find that increasing incubation temperatures can cause antigens to degrade and non-specific binding to increase.

Can I run my samples undiluted? Or less dilute?

This is not recommended for most sample types. Cell culture media is the only sample type that can be tested neat, but all other sample types should be diluted at least 1:2 (50%) for use in Q-Plex™ Arrays. This is because of the increased likelihood of getting false positives and high variability between replicates with neat samples – rheumatoid factors and heterophilic antibodies such as HAMA are usually the culprit in blood-derived samples, while high total protein or lipid content often causes well background problems with tissue-derived samples. Q-Plex™ Array diluents have additives to address these issues. If you are concerned that false positive signals may occur in your samples, test them at several dilutions, such as 1:2, 1:20, and 1:200, and check that the dose-response is linear. Signal from heterophilic antibodies will often not dilute out, while real signal should get dimmer as the sample is diluted.

What sample types are compatible with the Q-Plex Arrays?

Most Q-Plex™ Arrays are validated for use with serum and plasma, but for those who want to test other sample types, we offer:

  • FREE Demo Kit or FREE in-house sample testing of four samples: This provides the user with an opportunity to screen several sample preparation conditions and see how they work with standard Q-Plex™ assay conditions. In fact, the cytokine levels of a wide variety of tissue homogenates and cell lysates, such as mouse lung, liver, kidney, pancreas, kidney, and eye have been successfully analyzed using Q-Plex™ Arrays under standard assay conditions.
  • Custom development: Quansys can perform optimization and validation of assay conditions specific for the user’s sample type.
Preparing Tissue Homogenates and Cell Lysates for use in Q-Plex™ Arrays

The cytokine levels of a wide variety of tissue homogenates and cell lysates, including mouse lung, liver, kidney, pancreas, kidney, and eye, have been successfully measured using Q-Plex™ Arrays. Most homogenization or lysis protocols for an ELISA or western blot are fine to try with Q-Plex™ Arrays as long as the procedure is standardized across samples, the samples are kept cold at all times, and the diluents do not contain chemicals that interfere with the assay. Use the example protocols and reagent compatibility table below as a guideline in designing the optimal homogenization protocol for your sample type.

General Protocol for Tissue Homogenization/Cell Lysate Preparation for Q-Plex™ Arrays: KEEP SAMPLES COLD AT ALL TIMES
  1. Add ice-cold sterile non-denaturing medium to samples at 10-20% tissue weight/total volume. A typical medium is PBS containing a protease inhibitor cocktail (leupeptin, aprotinin, pepstatin, etc.). Cell lysates must have protease inhibitors, and may also benefit from low levels of chemicals that promote lysis (salts or sugars to create a hypotonic solution, detergents, etc.) as well as DNase (25-50 µg/mL) and RNase (50 µg/mL) to reduce the viscosity increase caused by the release of nucleic acids.
  2. Homogenize diluted tissues using pre-cooled equipment such as a manual or mechanical tissue homogenizer, mortar and pestle, sonication, etc. In cases where homogenization but not cell lysis is desired, e.g. only intercellular cytokines and not cytoplasmic cytokines are meant to be measured, use an extra gentle homogenization protocol, such as placing tissue and media in a sealable plastic bag and rolling a cylindrical object back and forth over the bag.
  3. Separate insoluble debris by centrifugation at 4oC. Spinning samples at 10,000 x g for 10-20 minutes is typical. For additional clarity, centrifuge again or filter using a low-protein-binding 4.5 micron filter.
  4. Keep clarified supernatant to be assayed right away at 4oC, or aliquot and store at -80oC. Minimize freeze/thaw cycles.
  5. Measure total protein levels of each sample before or after the ELISA so that cytokine data can be normalized to negate differences due to sample collection. The measurement can be done by spectrophotometric absorbance analysis at 280 nm, or by such methods as a Bradford or BCA assay. ELISA results are normalized by dividing the cytokine testing results (in pg/mL) by the total protein content (in micrograms or milligrams/mL) to obtain pg of cytokines/microgram of tissue.
  6. Assay clarified supernatants to determine cytokine levels. Dilute samples at least 1:2 (parts:total) in the Q-Plex™ Array Sample Diluent to avoid false positives.

To view an example of a published protocol that has been successful for many of our customers homogenizing a variety of tissues, see: Clin Exp Immunol. 2004 July; 137(1): 65–73:

Compatibility of Common Medium Components: Avoid preparing samples using reagents that interfere with the assay.

NP40 No interference at 1%
Tween No interference at 1%
Triton No interference at 1%
Citrate No interference at 20%
EDTA No interference at 20 mM
Water No interference at 55 M
Heparin No interference at 30 mg/ml
Urea No interference at 1 M
DMEM No interference at 100%
HAMS No interference at 100%
RPMI No interference at 100%
SFM4 No interference at 100%
Q-View Imager installation troubleshooting

If your Q-View imager is connected to a computer but isn’t being recognized in the Q-View Software, please do the following:

  1. Refresh the connection: In the Q-View Software, go to Settings > Administration > Manage Imagers. Click Refresh in the dialog box that pops up.
  2. Ensure there is power: Is the Q-View Imager power cord plugged into a functioning power strip and/or wall outlet? Be sure to check alternate wall outlets that are on a different circuit. Is the power cord which connects into the back of the imager is plugged into the imager securely?
  3. Try alternate USB ports: Can you refresh the imager connection (as in step 1) when the Q-View Imager USB cable is plugged into a different port of the computer? Be sure to check all of your computer’s available ports.

If none of these fix the problem, please call 888-782-6797 for technical support.

If you are experiencing issues with your experiment or imaging, please see our tips on ELISA Troubleshooting above.

Assay Principles at Quansys

Serum and Plasma Preparation

Q-Plex kits are often developed with serum or plasma samples as the primary sample type. Here are some recommended procedures for the collection, processing, storage and shipment of serum and plasma samples. By definition, serum is the liquid fraction of whole blood that is collected after the blood is allowed to clot. The clot is removed by centrifugation and the resulting supernatant, designated serum, is carefully removed. Plasma is produced when whole blood is collected in tubes that are treated with an anticoagulant. The blood does not clot in the plasma tube. The cells are removed by centrifugation. The supernatant, designated plasma is carefully removed from the cell pellet using a pipette.

Serum Preparation

Collect whole blood in a covered test tube. After collection of the whole blood, allow the blood to clot by leaving it undisturbed at room temperature. This usually takes 30 minutes and no more than an hour. Remove the clot by centrifuging at 1,000-2,000 x g for 10 minutes.The resulting supernatant is designated serum. Following centrifugation, it is important to immediately transfer the liquid component (serum) into a clean polypropylene tube using a pipette. The samples should be maintained at 2-8°C while handling. If the serum is not analyzed immediately, the serum should be apportioned into aliquots, stored, and transported at -20°C or lower. It is important to avoid or at least minimize freeze-thaw cycles because this is detrimental to many serum components. Samples which are hemolyzed, icteric or lipemic can invalidate certain tests.

Plasma Preparation

Collect whole blood into tubes with the anticoagulant EDTA. Cells are removed from plasma by centrifugation for 10 minutes at 1,000-2,000 x g. Centrifugation for 15 minutes at 2,000 x g depletes platelets in the plasma sample.The resulting supernatant is designated plasma. Following centrifugation, it is important to immediately transfer the plasma into a clean polypropylene tube using a pipette. The samples should be maintained at 2-8°C while handling. If the plasma is not analyzed immediately, the plasma should be apportioned into aliquots, stored, and transported at –20°C or lower. It is important to avoid and/or minimize freeze-thaw cycles. Samples which are hemolyzed, icteric, or lipemic can invalidate certain tests. References

  1. Henry, J.B. (1979) Clinical Diagnosis and Management by Laboratory Methods, Volume 1, W.B Saunders Company, Philadelphia, PA, p. 60.
  2.  Thavasu, P.W., et al (1992) Measuring cytokine levels in blood. Importance of anticoagulants, processing, and storage conditions. J Immunol Methods 153:115-124.

 

Storage of Q-Plex Kit components for long-term studies
Using a freshly reconstituted Calibrator vial for each experiment is essential to obtaining consistent results over time. Let us know at the time of your initial purchase if you plan to utilize a portion of a Q‐Plex plate on a different day, so we can provide extra Calibrator vials from a single production Lot for each experiment, as well as extra plate seals. Our Stripwell Kits are manufactured specifically for this purpose, and so each kit comes with three calibrator vials from the same Lot, and extra plate seals. DO NOT use calibrator solution that has been frozen, as even a single freeze‐thaw cycle can have a significantly deleterious effect. Also, keep microplate wells to be used later dry; do not wash them along with the in‐use portion of the plate.

Part Description Reagent Preparation Storage of Opened Material
Q-Plex Array Microplate Spotted and blocked 96-well polystyrene microtiter plate Ready for use. If the plate contains un-wetted wells to be used later, return the plate to 4°C in its sealed foil pouch. Good until kit expiration.
Wash Buffer Concentrate (20X) Liquid, 50 mL/vial of a concentrated solution of buffered surfactant Place 50 mL of the 20X concentrate into 950 mL deionized water, mix thoroughly. 4°C until kit expiration.
Calibrator Lyophilized, recombinant calibrators in a buffered protein base with preservatives, see the Product Card for high points When ready to begin the assay, reconstitute in Sample Diluent according to the Product Card which accompanies the kit, mix gently until fully reconstituted. Differences Between ELISA Kits ExplainedDiscard unused reconstituted calibrator. Do not freeze/thaw. Good for 6 hrs on ice.*
Sample Diluent Liquid, 6 mL/vial of a buffered protein solution with heterophilic antibody and rheumatoid factor blockers, and preservatives Ready for use. 4°C until kit expiration
Detection Mix Liquid, 6 mL/vial of biotinylated antibodies in a buffered protein solution with preservatives Ready for use. 4°C until kit expiration
Streptavidin-HRP 1X Liquid, 6 mL/vial of streptavidin-conjugated horse radish peroxidase Ready for use. Do not expose to UV light. Do not expose to UV light. 4°C until kit expiration.
Substrate A Liquid, 3 mL/vial of stabilized hydrogen peroxide Do not expose to UV light. Do not cool after mixing. During the assay, mix 3 mL of Substrate A with 3 mL of Substrate B, and mix gently. Do not expose to UV light. Do not cool after mixing. Store mixed substrate solution at room temperature for up to 1 week. Store unmixed solution at 4°C until kit expiration.
Substrate B Liquid, 3 mL/vial of stabilized signal enhancer Do not expose to UV light. Do not cool after mixing. During the assay, mix 3 mL of Substrate A with 3 mL of Substrate B, and mix gently. Do not expose to UV light. Do not cool after mixing. Store mixed substrate solution at room temperature for up to 1 week. Store unmixed solution at 4°C until kit expiration.
Plate Seals (3) Adhesive strips None Non-perishable

*DO NOT re-use reconstituted calibrator solution that has been frozen, as even a single freeze-thaw cycle will have a deleterious effect on the calibrators.

How are Q-Plex Arrays validated?

Every Quansys product is built such that each assay in the array passes precision, specificity, recovery, linearity, drift, and stability criteria. Custom products may have specialized criteria depending on customer needs. General validation criteria for our non-custom quantitative products include the following:

Precision

Edge Effects: The % difference between replicates of a mid-level positive control run in the outer wells and inner wells of a 96-well plate must be below 10%,

Intra-Assay: The %CV for 20 replicates of a high-level, mid-level, and low-level positive control over 4 plates from the same Lot must be below 15%,

Inter-Assay: The % CV for 4 replicates on 20 plates from 3 different Lots run by 3 users must also be below 15%.

Specificity

The % cross reactivity must be below 1% between all assays in an array Standard Curve: %Backfit obtained using a 5PL regression model must be between 80%-120% for all points of the standard curve

Recovery

The % recovery of a high-level, mid-level, and low-level positive control should preferably be between 80%-120% in serum and plasma (or other sample types as arranged). If this is not feasible, then % recovery must replicate over multiple tests.

Linearity

The % linearity must be between 80%-120% in serum and plasma (or other sample types as arranged) for at least 5 points of a 1:2 dilution series of a high-level positive control.

Drift

The % difference between samples added to the plate at 0 minutes and 10 minutes must not exceed 15%.

Stability

Overall Stability: The standard curve and a high-level, mid-level, and low-level positive control must perform to within 2 standard deviations of Day 0 data for at least 1 year when stored at 4oC.

Shipping Stability: The standard curve and a high-level, mid-level, and low-level positive control must perform to within 2 standard deviations of Day 0 data after kits are exposed to either 4 hours of either -20oC or 45oC followed by 16 hours at 25oC, then stored at 4oC for 9 weeks.

Opened Kit Stability: The standard curve and a high-level, mid-level, and low-level positive control must perform to within 2 standard deviations of Day 0 data after kits are opened, all regents reconstituted, and kits stored at 4oC, -20oC, or -80oC for 4 weeks.

An example of a validation report that includes real data for each of the tests specified above is available upon request. Once a product is validated in the Quansys R&D department, each future Lot of reagent and plates is tested using similar criteria by our Quality Control department before being released for sale.

An explanation of recovery and linearity

In ELISA development, recovery and linearity experiments are used to assess the compatibility of a particular sample diluent to be used for assaying analytes from a particular sample type such as serum, plasma, saliva, urine, etc. Specifically, recovery tests are used to determine if the assay is affected by the difference between the diluent used to prepare the standard curve and the sample matrix. Linearity tests determine the extent to which the dose-response of the analyte is linear in a particular diluent. Calculations for both rely on dividing the Observed values by the Expected values.

It is preferable to do recovery and linearity testing with samples that have known high concentrations of each analyte. However, if such natural samples are unavailable, testing may also be done by adding (spiking) known amounts of the analyte into samples.

Example Spike Recovery Protocol
  1. Thaw samples to be tested.
  2. Follow the assay protocol to reconstitute the calibrator and make the standard curve.
  3. Prepare a 7-point 1:2 dilution series of the calibrator plus one blank – make 110 uL per sample and diluent.
  4. Prepare eight 100 uL aliquots of the kit diluent and each endogenous sample to be tested.
  5. Dilute the sample and diluent aliquots 1:2 (50%) with 100 uL of the 1:2 calibrator series.
    Calculate the %Recovery for each spiked aliquot: 

    • %Recovery = ((Observed Concentration – Endogenous Concentration)/ Spiked Diluent Concentration)*100.
    • The mean percent recovery for any sample type should meet design specifications, which are typically 80-120%.
Example Linearity Protocol
  1. Thaw samples to be tested.
  2. Follow the assay protocol to reconstitute the calibrator and make the standard curve.
  3. If samples are expected to have high levels of the analyte, spiking is not necessary. Make a 1:2 serial dilution curve of the samples to be tested in sample diluent.
  4. If samples are expected to have very low levels of the analyte being tested, spike a 1:8 dilution of the calibrator into samples. Then perform a 1:2 serial dilution curve of the spiked samples in sample diluent.
  5. Calculate the %Linearity for each dilution of the samples:
    • %Linearity = (Observed Concentration / (Previous observed value in the dilution series / Dilution Factor))*100.
    • The mean percent linearity for each should be 80-120%, preferably 90-110%.
Cross reactivity testing at Quansy Biosciences

All assays to be multi-plexed in an array must be tested for various types of cross-reactivity to determine if the reagents are specific enough to give true positive signal. For example, the following types of cross-reactivity can occur:

Antigen-Capture Antibody Cross-Reactivity: A capture antibody binds the wrong antigen, which was then either specifically detected by its own detection antibody or nonspecifically detected by another detection antibody. If Ag-Cab cross-reactivity is truly present, then the two systems cannot be multiplexed under the conditions tested and different capture antibodies or assay conditions should be screened.

Detection-Capture Antibody Cross-Reactivity: A capture antibody spot binds a detection antibody directly, lighting up in wells where the appropriate antigen was not present. Cab-Dab cross-reactivity can often be minimized via reagent or diluent optimization.

Antigen-Detection Antibody Cross-Reactivity: An antigen is captured by the appropriate capture antibody, but then detected by an antibody for another system. Ag-Dab cross-reactivity is not necessarily a problem and can even be used to one’s advantage, as the cross-reacting detection antibody can be used as an additional detection for the targeted antigen.

Capture Antibody-Conjugate Cross-Reactivity: The conjugate, such as SHRP, nonspecifically binds directly to a capture antibody. This type of cross-reactivity is rare, and is more often due to biotin contamination of the capture antibody. Although rare, Capture-Conjugate cross-reactivity must be tested for, as this type of cross-reactivity is unacceptable and must be resolved for the assay to be validated.

Antigen-Conjugate Cross-Reactivity: The conjugate, such as SHRP, nonspecifically binds to an antigen bound by its respective capture antibody. This type of cross-reactivity is rare, and is more often due to biotin contamination of the antigen. Although rare, Antigen-Conjugate crossing must be tested for, as this type of cross-reactivity is unacceptable and must be resolved for the assay to be validated.

Example of a Cross-Reactivity Experiment

Method

Individual antigens, individual detection antibodies, and negative controls for both were run on a Q-PlexTM microplate in duplicate in a grid pattern under normal assay conditions. Percent cross-reactivity was calculated by dividing the calculated concentration of a particular antigen run with a particular matched pair by the calculated concentration of the antigen with its intended matched pair.

Spot Order

Experimental Setup and Results

Discussion of Results

Note: Because the CRP assay is a competitive ELISA, the chemiluminescence in the absence of CRP is expected to be bright, and a decrease in chemiluminescence means that something was detected. All other systems are sandwich ELISA.

No significant cross-reactivity (> 1%) was observed. For example:

  • No non-specific binding was observed across rows or columns or in negative control wells.
  • GM-CSF antigen was detected only in the presence of GM-CSF detection (wells A1and A2) on the GM-CSF capture spot (spot 14)
  • G-CSF antigen was detected only in the presence of G-CSF detection (wells B3 and B4) on the G-CSF capture spot (spot 15)
  • Unlabeled CRP was detected only in the presence of labeled CRP (wells C5 and C6) on the CRP capture spot (spot 13)
  • Systems in the Quansys Hu Chemokine or Hu Cytokine arrays were detected only in the presence of the appropriate Dab Mix (wells D7:E10, note that IL-8 (spot 5) is in both products.)
Crosstalk in multiplex ELISA
Crosstalk in chemiluminescent or infrared fluorescent arrays is any phenomenon by which a signal from one spot creates an undesired effect on another. For example, if the signal from one well or microspot is so bright that it causes falsely high background or signal on one or more wells or assays next to it. Crosstalk is different from cross-reactivity in that the former accounts for light effects seen during image capture and analysis, while the latter deals specifically with chemical interferences between systems.

Well-to-Well Crosstalk

Crosstalk between wells is a potential issue in clear-walled microplates. Using microplates with opaque black walls will minimize the amount of light that can travel between wells. Such microplates are used for all non-stripwell Q-PlexTM products.

Spot to Spot Crosstalk

Crosstalk between spots within a well is also a potential issue in any microarray. To minimize the amount of light detected outside spot boundaries:

  1. Ensure that samples are diluted correctly according to the kit protocol.
  2. Ensure that the imager is suitable for use with Q-Plex Technology
    • Q-View Imager Pro
    • Q-View Imager LS
Crosstalk Testing at Quansys Biosciences

A Human Cytokine Screen Kit, consisting of 16 assay spots per well, was run using individual antigens to create signal on one select assay per well. Concentrations in pg/mL were calculated in Q-ViewTM 2.14 using a 5PL regression model with the LLOQ set as the lower limit for each standard curve. Percent crosstalk for each assay spot was calculated as: (spot signal / calibrator high-point signal for that assay) * 100. Values for each assay spot location were then averaged over all 16 test conditions.

Results

Spot Position Analyte Average Crosstalk Spot Position
Spot 1 IL-1α 0.00% Spot 1
Spot 2 IL-1β 0.06% Spot 2
Spot 3 IL-2 0.03% Spot 3
Spot 4 IL-4 0.01% Spot 4
Spot 5 IL-5 0.00% Spot 5
Spot 6 IL-6 0.00% Spot 6
Spot 7 IL-8 0.00% Spot 7
Spot 8 IL-10 0.00% Spot 8
Spot 9 IL-12p70 0.00% Spot 9
Spot 10 IL-13 0.00% Spot 10
Spot 11 IL-15 0.00% Spot 11
Spot 12 IL-17 0.09% Spot 12
Spot 13 IL-23 0.03% Spot 13
Spot 14 IFNy 0.00% Spot 14
Spot 15 TNFα 0.04% Spot 15
Spot 16 TNFβ 0.00% Spot 16
An Explanation of Sensitivity and the LLD, LLOQ, and ULOQ of a Multiplex ELISA
Assay sensitivity refers to the ability of a method or instrument to detect an analyte at a specified concentration and is often defined by a detection limit. There are a number of different “detection limits” that are commonly used in scientific literature and government agencies, but even when the same terminology is used, there can be differences according to what type of noise contributes to the measurement, calibration methods, instrument precision, etc.

At Quansys Biosciences, we often use three terms to define the range and sensitivity of our multiplex ELISA assays: Lower Limit of Quantification (LLOQ), Lower Limit of Detection (LLD), and Upper Limit of Quantification. The definitions of these terms as used at Quansys Biosciences and in science are discussed below:

Upper and Lower Limit of Quantification (ULOQ and LLOQ)

The ULOQ and LLOQ are the highest and lowest standard curve points that can still be used for quantification; they are the values below and above which, respectively, quantitative results may be obtained with a specified degree of confidence or the highest/lowest concentration of an analyte that can be accurately measured. Together, the ULOQ and LLOQ define the range of quantification for the assay. Limits of quantitation are matrix, method, and analyte-specific, and can be calculated as follows:

Equation 1. (Calculation used in Q-View): ULOQ & LLOQ = Highest or Lowest Standard, respectively, with a %backfit of 120%-80%, a %CV of < 30%, and a positive mean pixel intensity difference between it and the negative control.

Equation 2. (Commonly used in science to estimate the LLOQ): LLOQ = (Mean negative control pixel intensity) + 10 * (StDev of negative control pixel intensities).

Lower Limit of Detection (LLD)

The LLD is the lowest concentration level that can be determined to be statistically different from a blank at a 99% confidence level. In other words, it is the lowest quantity of a substance that can be distinguished from the absence of that substance (a blank value) within a stated confidence limit, generally 1%. The Limit of detection is matrix, method, and analyte-specific, and can be calculated as follows:

Equation 1. (Calculation used in Q-View): LLD = 2*(StDev of negative control pixel intensities before ‘Negative Well Subtraction’)*LLOQ/ (Difference between pixel intensity of lowest standard and negative control)

Equation 2. (Commonly used in science to estimate the LLD, which estimate is typically deemed acceptable if it falls within a region where the signal to noise ratio is greater than 5): LLD = (Mean negative control pixel intensity) + 2 * (StDev of negative control pixel intensities).

We recommend using the lot defined ULOQ/LLOQ, which are selected in “Assay Limits” in the “Data Analysis” tab in Q-View. If those same data limits are used, data from all of your plates can be compared. The assay determined ULOQ/LLOQ/LLD can vary some from plate to plate because only two replicates of the standard curve are tested.

Reporting

When reporting assay data, it is conventional to include values such as the ULOQ, LLOQ, and LLD. Data that falls outside the limits should be reported as > ULOQ, < LLOQ, or < LLD. Data extrapolated beyond the limits are typically not included in published results.

Dilutions: Explanations and examples of common methods
There are many ways of expressing concentrations and dilution. The following is a brief explanation of some ways of calculating dilutions that are common in biological science and often used at Quansys Biosciences.

Using C1V1 = C2V2

To make a fixed amount of a dilute solution from a stock solution, you can use the formula: C1V1 = C2V2 where:

  • V1 = Volume of stock solution needed to make the new solution
  • C1 = Concentration of stock solution
  • V2 = Final volume of new solution
  • C2 = Final concentration of new solution
  • Example: Make 5 mL of a 0.25 M solution from a 1 M solution
  • Formula: C1V1 = C2V2
  • Plug values in: (V1)(1 M) = (5 mL)(0.25 M)
  • Rearrange: V1 = [(5 mL)(0.25 M)] / (1 M)V1 = 1.25 mL
  • Answer: Place 1.25 mL of the 1 M solution into V1-V2 = 5 mL – 1.25 mL = 3.75 mL of diluent
Using Dilution Factors

To make a dilute solution without calculating concentrations, you can rely on a derivation of the above formula:
(Final Volume / Solute Volume) = Dilution Factor (can also be used with mass)

This way of expressing a dilution as a ratio of the parts of solute to the total number of parts is common in biology. The dilution factor (DF) can be used alone or as the denominator of the fraction, for example, a DF of 10 means a 1:10 dilution, or 1 part solute + 9 parts diluent, for a total of 10 parts. This is different than a “dilution ratio,” which typically refers to a ratio of the parts of solute to the parts of solvent, for example, a 1:9 using the previous example. Dilution factors are related to dilution ratios in that the DF equals the parts of solvent + 1 part.

  • Example: Make 300 μL of a 1:250 dilution
  • Formula: Final Volume / Solute Volume = DF
  • Plug values in: (300 μL) / Solute Volume = 250
  • Rearrange: Solute Volume = 300 μL / 250 = 1.2 μL
  • Answer: Place 1.2 μL of the stock solution into 300 μL – 1.2 μL = 298.8 μL diluent
Step Dilutions

If the dilution factor is larger than the final volume needed, or the amount of stock is too small to be pipetted, one or more intermediary dilutions may be required. Use the formula: Final DF = DF1 * DF2 * DF3 etc., to choose your step dilutions such that their product is the final dilution.

  • Example: Make only 300 μL of a 1:1000 dilution, assuming the smallest volume you can pipette is 2 μL
  • Choose step DFs: Need a total dilution factor of 1000. Let’s do a 1:10 followed by a 1:100 (10 * 100 = 1000)
  • Formula: Final Volume / Solute Volume = DF
  • Plug values in: (300 μL) / Solute Volume = 10
  • Rearrange: Solute Volume = 300 μL / 10 = 30 μL
    Answer: Perform a 1:10 dilution that makes at least 30 μL (e.g. 4 μL solute into 36 μL diluent), then move 30 μL of the mixed 1:10 into 300 μL – 3 μL = 297 μL diluent to perform the 1:100 dilution
Serial Dilutions

A dilution series is a succession of step dilutions, each with the same dilution factor, where the diluted material of the previous step is used to make the subsequent dilution. This is how standard curves for ELISA can be made. To make a dilution series, use the following formulas:

  • Move Volume = Final Volume / (DF -1)
  • Diluent Volume = Final Volume – Move Volume
  • Total Mixing Volume = Diluent Volume + Move Volume
  • Example 1: Make a 7-point 1:3 standard curve, starting Neat, such that you can pipette duplicates of 50 μL per well
  • Calculations:
    • Calculate the minimum diluent volume per step: 50 μL per well * 2 for duplicates = 100 μL minimum. Add extra volume to compensate for pipetting error, for example, 20 μL, which brings our desired Diluent Volume to 120 μL
    • Calculate Move Volume: Move Volume = 120 μL / (3-1) = 60 μL
    • Calculate Total Mixing Volume: Total Mixing Volume = 120 μL + 60 μL = 180 μL
  • Answer:
    • Prepare the first point of the standard curve, which is 180 μL of Neat standard
    • Prepare the diluent for the rest of the points, or six aliquots of 120 μL of diluent
    • Move 60 μL of the first point into the second and mix thoroughly, move 60 μL of that into the next, and so on

  • Example 2: Make a 7-point 1:2 standard curve, starting at a 1:5, such that you can pipette duplicates of 50 μL per well
  • Calculations:
    • Calculate minimum diluent volume per step: 50 μL per well * 2 for duplicates = 100 μL minimum. Add extra volume to compensate for pipetting error, for example, 20 μL, which brings our desired Diluent Volume to 120 μL
    • Calculate Move Volume: Move Volume = 120 μL / (2-1) = 120 μL
    • Calculate Total Mixing volume: Total Mixing Volume = 120 μL + 120 μL = 240 μL
    • Calculate first point dilution volumes: you need 240 μL of a 1:5
  • Answer:
    • Prepare the first point of the standard curve, which is a 1:5, so pipette (240 μL /5) = 48 μL solute into 192 μL diluent
    • Prepare the diluent for the rest of the points, or six aliquots of 120 μL of diluent
    • Move 120 μL of the first point into the second and mix thoroughly, move 60 μL of that into the next, and so on

Immunoassay Signal and Evaluating Standard Curves – Common Q-Plex™ FAQ
What is chemiluminescent signal?

In general, chemiluminescence is the production of light by a chemical reaction. Quansys chemiluminescent multiplex ELISA kits utilize a secondary antibody, or other molecule as applicable, labeled with the enzyme horseradish peroxidase (HRP) as the reporter molecule and a modified form of luminol as the substrate. In this reaction, the immobilized HRP catalyzes the oxidation of luminol, and this reaction is accompanied by the emission of light at 428 nm. Chemiluminescent signal decreases over time, which is why it is recommended to wait no longer than 10 minutes to image a chemiluminescent Q-Plex plate.

What is IR fluorescent signal?

Fluorescence is the emission of light by a material that has absorbed electromagnetic radiation. Quansys IR fluorescent multiplex ELISA kits utilize an immobilized secondary antibody, or other molecule as applicable, labeled with a fluorescent dye as the reporter molecule. This dye has an optimal excitation wavelength of 774 nm and an emission wavelength of 789 nm. The IR fluorescent signal can be imaged up to for 24 hours if the plate is stored in a dry and dark environment.

Why doesn’t the signal diffuse during imaging?

The reporter molecule is immobilized onto to a specific capture spot during the assay process. Light is only produced at the site of the immobilized reporter molecule.

How does the Q-View Software determine pixel intensity and concentration?

In the plate layout view of Q-View software, the pixels within each overlay circle are averaged using a proprietary weighting algorithm which is designed to prevent potential well background from having a large impact on the calculation. The Q-View Software allows the user to choose one of seven curve fit options: 5PL, 4PL, Log-Log, Linear, Point to Point, Qualitative, and Auto-Select (this option fits standard curves for each system individually based on the lowest AIC value). The concentrations of the unknown samples are then assigned using the regression model applied to the standard curve data. The user can also choose to have limit values such as ULOQ, LLOQ, and LLD, applied to the calculated concentrations, or to mask values so they are not included in any calculations.

I only see a few points of my standard curve in the image, why can’t I see the entire standard curve?

Many computer monitors cannot display as many levels of gray as are present in high-resolution Q-Plex images. To visualize dim or bright spots in Q-View, go to Image Processing > Image Options > Adjust Gamma. To evaluate curve quality properly, see below.

What does adjusting gamma in Q-View do? Why does my imported image appear dimmer in Q-View?

Gamma settings in many imaging programs, including Q-View, control how image brightness and contrast are displayed but do not affect underlying data. Gamma options are useful for visualizing very bright or dim spots but are generally not saved outside that program. Thus, an imported image may appear dimmer because only the original image is brought into the new program without any adjustments to gamma settings.

How can I evaluate the quality of my standard curves?

The criteria in the Q-View Software for a standard curve point to be used for quantitation is that the %backfit must be 120%-80%, replicates must have a %CV of < 30%, and the pixel intensities must be above that of the average of the Negative Control wells. To see if your standard curve points fit the first two criteria, view the %Backfit and %CV of your standard curves points in Data Analysis > Report tab. You can simplify the report using the View button. Pixel intensities can be viewed in either the Report or Data tabs. Additionally, you can check the curve statistics listed at the top of each chart in Data Analysis > Charts tab. If a standard curve point did not perform as expected, first ensure that all plate overlay spots are aligned and dilution factors have been assigned. Then, consider masking points that may be outliers if the %CV between replicates is greater than 30%, or the %Backfit is outside 80%–120%.

Equipment Validation Recommendations from Quansys Biosciences

Prior to running a multiplex ELISA kit, ensure all equipment is functioning properly to obtain optimal results.

Pipettes

Pipettes should be regularly calibrated and maintained according to manufacturer’s specifications. Check pipettes between calibrations using gravimetric methods, such as weighing the maximum and minimum volumes of water four times each, using a new pre-rinsed tip for each volume setting, and ensuring that the mean volume and standard deviation for each volume setting meet manufacturer’s specifications.

Imager

Q-View Imagers can be calibrated by going to Settings > Administration > Manage Imagers. Qualified customers can receive a FREE Calibration Kit specifically for imager validation purposes by contacting info@quansysbio.com.

Automatic Plate Washer

First, ensure that the plate washer has a program that will not scratch the bottom of the microplate and will preferably leave a small, uniform amount behind to prevent plate drying.

For example:

Next, ensure that all washer pins are functioning: connect the prepared wash buffer to your automatic plate washer, run 1-2 priming cycles, and then separately dispense and aspirate 100µL wash buffer into a spare microtiter plate, and ensure that all pins dispensed or aspirated uniformly.

Plate Shaker

For all our multiplex ELISA kits, we recommend using a rotational microplate shaker capable of 300-1,100 rotations per minute (RPM), such as a Barnstead/Labline 4625 titer plate shaker, IKA MTS 2/4, or equivalent. Prior to running the assay, determine the shaker’s actual RPM using the manufacturer’s instructions. If no instructions are available, use one of the following protocols:

  1. To digitally determine the shaker’s RPM, secure an accelerometer (such as a smartphone with an accelerometer app) onto the platform, turn the shaker on for 10 seconds, and record the number of peaks read by the accelerometer and multiply by 6 to calculate the RPM at that setting. Repeat this procedure at least 3 times to ensure accuracy.
  2. To manually determine the shaker’s RPM, fasten a pen or marker to the side of your shaker, and set a timer for 10 seconds. Pull a sheet of paper horizontally under the pen for the 10-second interval, and drop the paper once the alarm sounds. Count the number of oscillations and multiply by 6 to calculate the RPM at that setting. Repeat this procedure at least 3 times to ensure accuracy.
Performing Western and Dot Blots on the Q-View™ Imager

While the Q-View Imager was exclusively designed and developed to be used with Q-Plex Arrays, some customers have been successful in using the device for western and dot blot imaging. Due to the numerous methods and reagents available, we are not able to provide specific steps on how to do this. Keep in mind, customers will have to optimize their methods to the imager. Notwithstanding, we can provide a few tips so if you do choose to use the Q-View Imager for this purpose, you’ll have a better chance of success.

Substrate

There are numerous chemiluminescent substrates available on the market of a variety of quality and performance. The substrate used in the Q-Plex kits is very sensitive and stable. The customers that have had good success with the Q-View Imager have used this substrate. From what we have observed, some of the less sensitive substrates are not acceptable for this application. Please contact your sales representative to learn more about where you can purchase this substrate.

Exposure

The camera used in the Q-View Imager is able to do long exposures. We recommend doing multiple exposures, some short and some longer. It may be necessary to do exposures up to five minutes or longer.

Streptavidin HRP (SHRP)

While every experiment is different we recommend maximizing the concentration of SHRP to achieve the desired sensitivity.

Examples of Customers Images
Tips for Pipetting
Using a pipette the correct way can lead to better data and more reliable results. Here are a few of the ways you can control variation between wells.

Pre-wet your pipette

For all volumes over 10μL, aspirate and dispense at least 3 times to increase humidity within the tip and reduce the amount of variation by evaporation.

Visually look at amounts of liquid in your pipette

You may be able to tell visually if there is any variation between volumes of liquid in each tip of your multichannel pipette.

Secure tips

Loose tips can cause variation between the volumes of each well and lead to skewed data.

Touch down once

Avoid sliding pipette tips down the side of a well. This can lead to residue clinging to the sides of the wells which leads to background in the well. Instead, just touch your tips down one time in the bottom corner of the well and dispense liquid. Sliding tips in the bottom of the wells can also smear spots leading to unclear results.

Minimize the number of times you have to pipette

Pipette the fewest number of times possible. Choose a pipette volume that will minimize the number of times pipetting into a well. The fewer times you have to pipette, the less chance there is for error.

Minimize bubbles as much as possible

Try not to blow any air into your wells when dispensing the liquid. This is especially critical when adding substrate before imaging.

Pipette large volumes over small

Large volumes are easier to pipette accurately. Pipetting 50μL is more likely to be accurate than pipetting 5μL.

10% rule for accuracy of volumes

Pipettes are less accurate at minimum and maximum settings. Avoid using pipettes for volumes less than 10% of the maximum volume.

Stick to one way of pipetting and do not switch methods during an assay. There are 2 popular ways to pipette:
  • Standard or forward pipetting
    • Depress plunger to first stop and aspirate. Then dispense to first stop, discard excess.
  • Reverse pipetting
    • Depress plunger to second stop and aspirate. Then dispense to first stop/ discard excess (this is only if you have enough sample that discarding some liquid is not troublesome).

Q-View Software

Getting the most from Q-View Software

Q-View Basics

New look and touch screen compatible, but still three easy steps in the software to obtain concentrations:

  • Image Processing: Acquire/import images and align a plate overlay.
  • Well Assignment: Assign wells a type, name (optional), and dilution factor.
  • Data Analysis: Customize and copy or export the automatically generated concentrations, statistics, and charts.

Let’s Practice

– You’ve just finished the last step of the assay:

  1. Capture the image(s): Acquire on a Q-View Imager or Import a grayscale 16-bit TIFF. New Image Processing produces lower %CVs at the low end of the curve and eliminates stacking.
  2. Input the Software Product Code: Found on the kit product card.
  3. Place the overlay: Use Image Options to optimize image gamma, zoom, and orientation for your convenience. Use the Overlay Options tools to align the plate overlay. Always finish with Auto-Adjust Spots. (Also Blot Tools)
  4. Assign Wells: Use Sequential Naming and Templates to quickly assign repeat layouts. Sample Controls can be assigned if they are part of the product definition.
  5. Analyze Data: Click Perform Analysis to fit using the new optimal regression settings or modify via dialog, default assay limits. Then choose Statistics & optimal dilution, mask outliers, compare curve fit and weighting options. Example use of new Help button. Copy or Export Charts or data out as needed.

Data Analysis Troubleshooting

If a curve or sample did not perform as expected, first ensure that all plate overlay spots are aligned, the image has white spots on a black background, and dilution factors have been assigned. Then check the actual data, not just visual cues in the image.

  1. Evaluate the Standard Curve
    1. Check the standard curve charts, statistics for outliers to mask. Also consider masking a standard curve point if: %CV > 20%, or %Backfit ≠ 80% – 120%.
    2. Check for signs of compression: Are there saturated points? Are there repeating pixel intensities?
  2. Evaluate Sample Signals
    1. Are the assay limits set to the ULOQ & LLOQ and does the signal fall on the linear part of the curve?
    2. Is there high well or Negative Control background? Toggle Negative Well Subtraction setting.
    3. Did sample controls, if present, pass?
  3. Re-optimize the Curve Fit if Needed: Compare various regression models, weighting, and masking options.
Tips for data analysis in the Q-View Software
The Q-View™ Software is a tool for the quantitative analysis of multiplexed chemiluminescent or infrared fluorescent assays, such as those found in Q-Plex™ planar-based arrays. The software enables users to:

  • Acquire images of microarrays and stack images optimized for bright and dim reactions, obtaining a single high dynamic range image.
  • Easily locate spots for each assay using the software’s Auto-Set Plate Overlay and Auto-Adjust Spots features.
  • Assign wells as samples, controls, standards, or negatives, and specify their dilution factors.
  • Fit curves using any of six curve-fitting models, including 4 and 5 Parameter Logistic (PL), and customize or export the automatically generated charts and reports.
How does Q-View calculate concentrations?

Once the plate overlay has been placed, foreground pixels within each overlay circle are averaged using a proprietary weighting algorithm which is designed to prevent well background from having a large impact on the calculation.

How do I know what curve fitting model to choose?

We recommend the 4PL or 5PL regression models for Q-Plex data, as these are generally considered to provide the best overall fit for immunoassay curves. Use the Auto-Select option to have the Q-View Software automatically fit each curve using the model with the lowest AIC statistic. If your data is abnormal or you are trying to optimize the fit in a particular range (see below), one of the other Q-View regression models may better fit your needs.

When should I mask a standard curve point?

Consider masking a standard curve point if the %CV between the standard curve replicates is greater than 20%, or if the average %Backfit is outside 80% – 120%. You might also consider masking if you are trying to optimize the fit in a particular range (see below).

Can I optimize the curve fit for the low or high end of the curve?

Yes, in Q-View, try masking wells at the other end of the curve and choosing either the Auto-Select or Log-Log option. For example, masking the top 2-3 points of the standard curve and using Log-Log may better fit the low end of the curve.

Can I report pixel intensities?

Maybe. There are some issues with reporting pixel intensities, including:

  • Data is no longer “normalized” between plates such that small differences in things like the SHRP incubation time or time from when substrate is added until the plate is imaged can cause significant differences between experiments.
  • The fact that the signal is non-linear (due to both kinetic and camera effects) is no longer taken into account.
  • Some reviewers will not accept data below the assay Lower Limit of Quantification (LLOQ) because it is considered likely to have unacceptably high variability, recovery, linearity, etc.

Some reviewers will accept Pixel Intensities if the data is presented as qualitative rather than quantitative. For example, if you could establish that the Pixel Intensities of your samples were statistically different than your negative controls, then you may be allowed to report Pixel Intensities relatively. One convention for doing this is to use “the sum of 6-10X the standard deviation of the negative controls plus the average negative control value” as the cutoff point.

Creating custom overlays and standards in Q-View
  1. To enter antigen names and high-point concentrations for a custom calibrator, go to Settings > Administration > Lot Definitions.
    1. To enter new analytes, type the analyte name, for example, “Hu IL-2,” in the Antigen Name field in the top part of the Lot window. To view a list of existing systems, press the down arrow while the cursor is in the field. Once the desired name is entered, select New. The antigen name you enter will be automatically completed with “.custom“.
    2. Once all systems have been entered, enter a name for the Lot of calibrator, for example, “mm-dd-yy,” and enter the number of analytes that are in the calibrator mix.
    3. Rows of fields will appear where you can enter the calibrator names you just created above with their corresponding concentration and unit.
    4. Click Save. The Lot name you entered will be automatically completed with “.custom”.
  2. Once the custom Antigen Lot is saved, create a Product Definition by going to Settings > Administration > Product Definitions.
    1. Enter a name for the product, for example, “mm-dd-yy,” and the plex. To view a list of existing plexes, press the down arrow while the cursor is in the field. An image of how the spots are arranged in a well for that plex appears at the bottom of the window.
    2. Rows of fields will appear where you can enter any lot number you created in the Lot Definitions window, the antigen names you created, and the spot diameter (0.6 mm is typical). The concentration will be filled in automatically.
    3. Click Save. The Product name you enter will be automatically completed with “.custom”.

You can now use the new product definition to analyze images by entering the product name in the Product field on the project screen. All user-defined product and lot definitions will be saved within the project. If saved on a local network or flash drive, the project that utilizes user-defined product and lot files can be opened on any computer. User-defined product and lot files will not be overridden when software updates are released.

As researchers and problem solvers ourselves, we understand the value of sound data. We are proud to be a part of research that can better the world. We value the relationships, partnerships, and friendships that we have built with the people who trust and use our technology. We are committed to building these relationships. You can count on us to answer the phone and take time to thoroughly address questions or concerns about any of our products. In an industry that is reputed for grandiose claims, we trust that our quality standards and our customer service set us apart from the competition. If you’re happy with our products or if you think we can do something better, we hope you will let us know.

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info@quansysbio.com

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