ValitaTiter FAQs

What is the difference between ValitaTiter and ValitaTiter Plus plates?

The only differences are the detection range and the price. ValitaTiter detection range = 2.5-100 mg/L. ValitaTiter Plus detection range is 100-2000 mg/L. Ask for a quote to Valitacell support team to know about the pricing.

Which detection method / instrument do I need to measure IgG using ValitaTiter plates?

A standard microtiter plate reader with Fluorescence Polarisation (FP) configuration. If you do not know if your instrument has an FP detector, provide us the serial number and instrument type and we can determine this for you.

How do the technical specifications of my microtiter plate reader impact ValitaTiter measurement?

It is important to understand the optics of your microtiter plate reader (filter, monochromator or dual) and the adjustment and optimization (gain, G-factor, aperture, read Height, plate type, flash number).
Get in touch with our support team to receive full support for your microtiter plate reader setup.

How do I optimize ValitaTiter measurement?

We recommend running a human IgG standard curve on the plate in triplicate and look at three Quality Control criteria:
– mP Delta Shift = 100 mg/L – 0 mg/L ≥ 60 mP
– Standard Deviation < 2
– R^2 => 0.9
Get in touch with our support team to receive full support for your ValitaTiter assay optimization.

What is an acceptable Standard Deviation?

The acceptable Standard Deviation (StD) between replicate wells should be ≤ 2mP (millipolarization). What this translates to in mg/L varies depending on the range and total shift of the assay. We recommend verifying your StD is ≤ 2mP between replicates in your standard curve prior to proceeding to measuring test samples.
Get in touch with our support team to receive full support in your Valita®Titer assay optimization.

mP shift & assay resolution

The Fluorescence Polarization (FP) millipolarization (mP) shift can be defined in more general terms as the signal-to-noise ratio.
When the FP noise or background signal detected at 0 mg/L is too high, or when the highest detectable FP signal (100 mg/L for VAL003 and 2000 mg/L for VAL004) is too low, the signal and noise will be too close, leading to a smaller than recommended mP shift.
Measuring accurate results implies having high assay resolution, and for that it is necessary to have a signal that is easily distinguishable from noise – i.e. a high mP shift.
A higher mP shift will result in the best assay resolution.
For ValitaTiter and ValitaTiter Plus, the minimum acceptable mP shift was experimentally defined as 60 mP.
The mP shift is influenced by the molecular structure of the IgG target & by the IgG binding affinity for the Valita®Titer probe. The main factors to look at are: IgG MW, IgG format, species, subclass, light chain type, IgG CH2-CH3 Fc domain integrity, the presence of IgG aggregates, with IgG bispecific the presence of hetero or homodimer, and media composition (e.g. viscosity, feed, etc.).

My microtiter plate reader does not have a Fluorescence Polarisation module how can I test ValitaTiter plates?

We have close collaborations with the main microtiter plate reader companies and integrating a Fluorescence Polarisation module in your current microtiter plate reader is a simple machine upgrade which is low in cost and we can support you with. If you want to test ValitaTiter plates before the microtiter plate reader, just get in touch with our support team so we can arrange a microtiter plate reader free demo unit loan for you.

I do not have a microtiter plate reader, how can I test ValitaTiter plates?

Just get in touch with our support team so we can arrange a microtiter plate reader free demo unit loan for you to evaluate ValitaTITER plates.

How can I analyze ValitaTiter data?

Get in touch with our support team to receive the excel template for data analysis.

Can you describe ValitaTiter fluorescent probe?

ValitaTiter plates come pre-coated with ValitaTiter probe based on a FITC-labelled Fc-specific binding peptide. The Fc-specific binding peptide is a derivative of protein G, binding domain 1, aka GB1.

Can I use ValitaTiter to quantify mouse or rabbit IgG?

ValitaTiter probe is a derivative of protein G binding domain 1 (aka GB1) conjugated with a FITC fluorescent label. GB1 binds to the Fc domain of an IgG so it will bind to any construct with an exposed intact Fc domain including mouse, Rabbit and other species but with a different affinity than human IgG. ValitaTiter has been fully validated for human IgG and this is the target we have been mainly working with so far. To suit different IgG species the ValitaTiter protocol needs to be optimized.
Get in touch with our support team to receive full support for your IgG species optimization.

Is it possible to use only part of Valita®Titer plates? How long is ValitaTiter stable for, once opened?

ValitaTiter after opening the foil package, when the liquid from the used wells is removed, stored in the resealed foil package at 4ºC, is stable for 7-days. 
So, you can use a plate in different experiment over a week time. It is not recommended to freeze ValitaTiter plates or to store them in any other way different as recommended.

What IgG should I use for my standard curve?

This will depend on your test samples. To ensure the production of accurate data, it is important to use a standard that is as homogenous as possible to your test samples (i.e. same HC and same LC isotype). For example, if your test samples are human IgG1 kappa LC, you should use a human IgG1 kappa LC construct as your standard.
Ideally, if available, you would use a purified version of your test sample. If not a hybrid control (in-house generated generic IgG of known HC and LC isotype) or commercially available standard would suffice.
When working with ValitaTiter Plus, due to the high concentrations required for the standard curve (highest concentration = 2000mg/L), it is important to use a high stock concentration sample as your standard. Using a low stock concentration IgG standard for your standard curve can impact the accuracy of the output data. The reason for this is that the actual buffer that the IgG is diluted in can affect the assay output if not diluted out enough.

Do you need to carry out analysis of standard or test samples in duplicate/triplicate?

We would recommend running the standard curve in duplicate. Test samples do not need to be run in duplicate/triplicate however this is really a company policy/decision.

In the Valita®Titer assay, the standards are prepared in fresh media. Considering the test samples will be in conditioned or spent media, will this impact the assay results and reduce accuracy?

Typically, no, conditioned or spent media does not impact the accuracy of the output data, however, due to the inherent fluorescent nature of cell culture media and sometimes feed, it is important to ensure the standard is prepared in the same fresh media that the test samples are cultured in. This could mean preparing a number of standards in a range of different media types if you have a range of test samples in different media. If heavily conditioned media or specific feeds appear to be impacting the output data (this would be observed from the total fluorescence), the introduction of a dilution with fresh media to the test samples will mitigate this issue. Alternatively, if the issue remains due to a usual feed etc., you could prepare your standard in conditioned media containing that specific additive.

Do I need to measure a standard curve for every test sample?

Yes, for best practice, you would need to include a standard curve in every plate, however, this is user dependent. To save time, provided there are no major process changes you can also reuse the standard curve data previously acquired. We advise to prepare and run a fresh standard curve for every new test sample set to ensure quality output data.

Any pipetting tips to prepare the assay correctly?

It is important to avoid generating bubbles when prepping or mixing your samples in the ValitaTiter plate. Bubbles not only impact the read of the plate but can also alter volume in the well which has a significant impact on the quality of the output data. This can sometimes be observed in the raw fluorescence output data. To avoid bubble formation, carefully add 60 µL of media to each well using a multichannel pipette. Following this, add 60 µL of your test sample using a single or multichannel pipette, depending on the vessel you are transferring from, which will provide you with 120 µL of liquid per well. For mixing, set the multichannel pipette to 60 µL, place the pipette tips in the liquid of the wells and aspirate and dispense slowly 3-5 times.
Take your time with plate preparation.

Are there any additives that may impact the performance of the assay?

Yes, supplements or additives that alter the pH or viscosity of the test samples will impact the quality and accuracy of the output data since the standard curve is typically prepared in fresh media. You can overcome significant impacts by simply diluting the test samples in fresh media to decrease the concentration of these additives in the test samples and therefore reduce the impact observed in the output data. The impact from additives or viscous samples is usually observed when you look at the the raw fluorescence output data.

After the incubation time how precise do I need to be in relation to the protocol i.e. if I delay reading the plate by 15 minutes is the experiment compromised?

In the IFU we recommend measuring the plate after 5 minutes incubation. In practice you have a window of 45 minutes to measure the plate, after which the signal fall-off is approximately 10% within the first hour. The key factor to remember is to measure your test samples at the same time as your standard curve. If your standard curve is in the same plate as your test samples, then changing the incubation is not an issue, however, if you measure your standard curve samples after 5 minutes and test samples after 30 minutes, the accuracy of the data will be significantly reduced.

What is the shelf life of the ValitaTiter plates?

18-month shelf-life at 2-8°C from manufacturing date.

Do I need to remove cells from my samples before ValitaTiter measurement?

No, unlike other IgG quantification screening tools, ValitaTiter is robust to cellular contamination up to 15 million cells/mL. This means that there is no sample prep, pre-purification or centrifugation to remove cells required prior to analysis. Samples can be analysed neat straight from culture to ValitaTiter plates once they fall within the functional range of the assay.

Can the ValitaTiter assay measure higher than the noted functional range?

Whilst there can be variances in the absolute limit of the ValitaTiter and ValitaTiter Plus assays depending on the molecule being measured, appropriate dilutions are recommended to measure samples above the detection ranges. In our experience companies have adopted fairly standard approaches to diluting their samples depending on whether the sample is from an early stage cloning plate, late stage fed-batch sample, stability study sample, etc.

Do I need to cover the plate during incubation to avoid evaporation?

No, due to the minimal incubation time, evaporation is not a concern. However, as this is a fluorescence-based assay, it is good practice to limit light exposure throughout incubation.

How long does it take to read a plate?

This depends on the hardware used, plate type (96 or 384), in addition to, the measurement parameters. Typically, a standard plate reader will read a full 96-well plate in 3-8 minutes. Some instrument parameters, such as the flash number, increase the length of time taken to read a plate. You can save time by reducing flash number; however, this may reduce the quality of the output data. This is tradeoff between speed to results and good quality data production.

Can ValitaTiter be used for fragment antibodies or non-standard Fc containing IgGs?

ValitaTiter has been validated to accurately quantify and measure standard human IgG1,2,3 and 4. If working with unusual or non-standard Fc containing IgGs, we can work with you to alter or adjust the protocol to identify the optimum method for screening these molecules. These products cannot quantify non-Fc containing antibodies or antibody fragments. We are actively working on the development of a fragment antibody screening tool.

What type of microplates are used to manufacture ValitaTiter plates?

Valitacell uses Corning/Costar 96-well Half Area Black Flat Bottom (# 3694) to manufacture ValitaTiter plates.

ValitaTiter User Manual Says to use 60 µL of media to reconstitute the probe and then 60 µL of test sample. can I just add 120 µL of test sample and use this for reconstitution?

In theory, yes, you could use 120 µL of test sample to reconstitute the probe, however, this will cut the functional range of the assay in half. The 1:1 dilution of cell culture media for probe reconstitution and test sample allows each assay to cover the stated functional range. Additionally, Valitacell have validated the specified workflow of using 60 µL media for reconstitution and 60 µL test sample.

Can I use varying volumes in each well?

No, the total volume in every well needs to be the same (120 µL). Fluorescence polarization detection is very sensitive to volume discrepancies. Varying volume from well-to-well will impact the quality of the output data.

How does ValitaTiter assay compare to standard IgG quantification techniques?

ValitaTiter compares very well, not only to other relative quantification techniques, but also to absolute quantification methods, such as HPLC ProA. The advantageous characteristics associated with ValitaTiter for IgG quantification are the low cost, rapid assay prep and analysis time, robustness to cellular contamination and accuracy.

Are ValitaTiter plates easy to integrate into automated workflows?

Yes, our products are brand agnostic and automation friendly, meaning they will easily integrate into your automated workflow to provide a fully automated IgG quantification platform.

What are the main applications ValitaTiter plates are used for?

Clone ranking, transfection pool selection, lead clone selection for expansion, process optimization & development.

Still have questions?

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