FAQ - General and Assay Development

Find answers to your questions about assay development, plates, buffers, and more.

What are typical detection limits?

  • Imperacer® assays are in average 1000fold more sensitive compared to standard fluorescence ELISAs. However, like for any immunoassay the choice of appropriate antibodies is extremely important for the performance of the assay. Using an optimal antibody-pair the detection limit of Imperacer® assays could be down to 1000 molecules per well, this correlates with the femtogram per ml range for a typical protein.

Is a specific type of IgG recommended for Imperacer® assay development?

  • No, Imperacer® assays could be developed using both polyclonal (pc) and monoclonal (mc) IgG. Generally, the use of a mc antibody for capture and a pc antibody for detection should be recommended. However, experience showed that this has to be evaluated for any assay separately.

Is a specific type of IgG recommended for Imperacer® conjugate synthesis?

  • No, Chimera Biotec's conjugate synthesis SOP is compatible with both polyclonal (pc) and monoclonal (mc) IgG. Also no specific subtype of IgG is recommended. But the antibodies must be available purified (ideally affinity purified) without any stabilizing proteins.

What amount of antibody do I need for a custom conjugate synthesis and assay development?

  • For the first steps in assay development and the conjugate synthesis typically 500 µg of antibody is sufficient. By using polyclonal (pc) antibodies be aware to have a batch of antibody large enough for the complete development (~ 1-5 mg) and sample measurement. Changing a pc antibody batch or substituting an antibody during assay development necessitates a new optimization and during kit production necessitates a new validation.

Are antibodies available from Chimera Biotec?

  • In general, Chimera Biotec uses either customers or commercial antibodies. Even though we do not produce antibodies by ourselves we have partners which can provide custom antibody development.

What kind of synthesis strategy is used for the linkage of the antibodies and DNA in the Imperacer® conjugates?

  • Chimera's unique synthesis technology couples several antibody and DNA molecules to an oligomeric Imperacer® conjugates with enhanced binding and signal-generating properties. The synthesis strategy, the stoichiometric ratio, the sequence and the probe design of the DNA marker are intellectual property of Chimera Biotec.

What kind of microplates can I use?

  • The use of Imperacer® requires Imperacer® ready microplate modules which are delivered with our assay development or customized kits.

What is the standard volume per well for Imperacer® assays?

  • The standard volume using Imperacer® microplate modules is 30 µl per well. This volume is recommended for antibody immobilization, protein incubation, detection and PCR. The typical volume for washing and blocking is 240 µl. However, by adding a pre-dilution step the samples volume could be reduced to less than 1 µl.

What are the differences of the Imperacer® development kits?

  • Imperacer® development kits are either designed for biotin labeled detection reagents (like the anti-biotin kit: cat. no 12-007) or a variety of unlabeled species specific detection antibodies (please inquire) for in house use. The species- or label-specific reagents can be combined with any antibody-pair, protein and matrix.

What kinds of controls are recommended?

  • It is recommended to perform an Imperacer® enabled Cycler & maintenance test before starting the assay development. Your calibration curve and samples should be measured in at least duplicates to identify outliers. A PCR negative control (clean well with PCR mastermix) and PCR positive control (clean well with PCR mastermix and diluted conjugate) should be added to test the functionality of the PCR reaction and cycler.

Do I need to expect unspecific background signals?

  • According to any immunoassay, unspecific background signals could not be eliminated completely. However, the combined performance of Imperacer® conjugates, buffers and PCR, combined with long-year experiences in assay development reduces unspecific background signals to a minimum.

Does DNA in the sample material have any negative effects on Imperacer® assays?

  • No, because the amplification of the DNA marker is separated from the immobilization of the sample material by several washing steps. Furthermore, the Imperacer® blocking buffer efficiently inhibits the unspecific binding of sample-endogen nucleic acids. Finally the DNA marker, primers and probes used for Imperacer® are completely artificial and not present in natural environments.

How many samples can be analyzed with an Imperacer® kit?

  • Assuming a standard protocol one optimized Imperacer® kit could be used to analyze approx. 35-40 samples in duplicate. Residual wells will be needed for the calibration curve and controls.