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Section 1. Technology Based Questions:

Question 1. What specimen types are compatible for use in the AIM based assays?
Assays have been developed using serum, plasma (with various anticoagulants such as K2 and K3 EDTA), fecal extracts, cell lysates and cell culture supernatant. Although the technology can utilize urine and whole blood, no assays have been developed in-house using these matrices.

Question 2. Can you run pharmacokinetic (PK) based assays on the AIM platform?
If the customer’s current PK assay is in the form of an ELISA, it can be converted to run on the AIM platform. The AIM platform does not measure chemical reactions and is based on color change (colorimetric) not fluorescence or luminescence.

Question 3. What substances are compatible with the AIM technology?
The AIM technology works with protein based immunoassays including protein arrays, multiplexed immunoassays, and ELISAs detected on the AQ 1000 Instrument System. The SilverQuant® Detection Reagent can be used with immunoassays or DNA and other nucleic acid based arrays and can be detected using the AQ 1000 Instrument System.

Question 4. Can you attach whole cells direct to the slides or plates used in the AIM platform?
It is possible to attach whole cells but may require a different surface on the slide or plate other than what is used in the AIM assays.

Question 5. Do you need to use the AQ 1000 to read the AIM based assays or can another plate reader be used?
It is possible to use another plate reader if it has a 96-well format, reads colorimetric reactions and has spot analysis software. It is not advised to use another reader as each assay has been designed and validated using the AQ 1000 before transition to the customer. Intuitive only supports applications using the AQ 1000 reader.

Question 6. Does the AIM technology have any data comparisons to competitive systems available on market?
Intuitive Biosciences has data comparisons to a bead based assay and to ELISA that can be shared. We are in the process of generating comparison data to major competitors using outside laboratories and will make the data available when the studies are complete.

Question 7. How many replicates were used to evaluate performance in the AIM comparison to ELISA? What about the comparison to the bead based assay?
In the evaluation of the AIM versus ELISA, triplicate wells were run to make the comparison. For the bead based assay comparison, 7 replicates were run for each analyte.

Question 8. Is there a list of examples of the types of assays that Intuitive Biosciences has built?
Yes, Intuitive Biosciences has experience building antibody or antigen sandwich assays and antigen capture assays. The technology can perform competitive antigen immunoassays and DNA hybridization assays but Intuitive Biosciences has not developed these assays to date.

Question 9. Why are the percent CVs better in the AIM assay versus the ELISA?
The AIM assays are optimized for performance including percent CVs. There is enhanced technical precision in a multiplex AIM assay over an ELISA.

Question 10. What is the dynamic range provided by assays developed on the AIM platform?
The dynamic range is stated as being 3.5 logs or greater. Actual dynamic range is measured independently for each assay developed and is based on several factors including antibodies used in development, and what is being measured by the assay.

Section 2. Assay Based Questions:

Question 1. What is the stability of the SilverQuant Reagents?
The SilverQuant reagents have one-year stability from the manufacturing date The SilverQuant Reagent A is sensitive to light. The SilverQuant reagent B is sensitive to oxidation and must be used within 24 hours of being opened. Once the SilverQuant Reagent B is mixed with Reagent A it must be used immediately.

Question 2. How long can the assay plate sit prior to being read?
The assay plate can be read immediately following the end of the assay or it can be read at a later time. The assay plate is stabile indefinitely and can be read multiple times. There is no reduction in the reaction signal as is common with fluorescence or luminescence based assays. Actual time for reading the plate is built into the assay design.

Question 3. What makes the assay plate stabile indefinitely?
The silver deposition onto the gold nanoparticles creates a bond based on the reduction of silver. The bond is permanent and cannot be broken unless you do so physically or mechanically.

Question 4. What is the LOD or LLOD of the AIM assays?
The LOD or LLOD varies by assay, is based on the affinity of the antibodies used and final design of the assay. The AIM assay development team builds assays with a goal of providing performance CVs at very low levels of detection. In most cases the percent CVs are less than 10%.

Question 5. How is the LOD or LLOD of the AIM assays calculated?
To quantify unknown samples from a standard curve of known target protein concentrations, a regression analysis is performed. For the multiplex AIM analysis, the same standard dilutions series as a typical ELISA results in a nonlinear curve. This requires a 4 or 5-point regression curve fit analysis.

Question 6. What is the variability at the low end of the AIM based assays?
The variability at the low end varies from assay to assay. Each assay is designed to maximize the limit of detection minimizing the variability at the low end. Part of the performance at the low end is also dependent on the antibodies chosen in the assay design.

Question 7. If the customer provides antibodies to build an assay with the AIM technology, how much is required?
The best amount of antibody to provide for assay development work is 1mg. If 1mg is not available, the customer should provide no less than 250µg of antibody. For antigen assays, 250µg or more is suitable.

Question 8. Are there washing steps required in the assay procedure?
Yes, washing steps are required in steps 2-4 of the 4-step standard assay procedure.

Question 9. Does the assay use a single dilution per sample or do you have multiple dilutions per sample to achieve antibody detection?
The AIM based assays are designed to use one dilution per sample. The antibody pairs are specifically chosen to work together in the assay and at different levels but using a single dilution per sample for detection.

Question 10. What is the impact to assay performance when using the shorter incubation times in the AIM assays?
The AIM assays are built to maximize performance based on customer required specifications. Each assay varies based on type of assay, number of plex and the antibodies or antigens chosen for the assay. When using shorter incubation times, there can be a slight loss in sensitivities, Actual amount of loss of performance needs to be determined and is under evaluation.

Question 11. What is the matrix used for the QC controls used in the AIM assays?
The matrix used for the AIM assay varies from assay to assay and is dependent on assay design. We always try to use real sample (plasma, serum, etc.) diluted at the same concentration as the test sample so the matrix is the same. The AIM assay positive QC controls are built in sample matrix with targets spiked into the matrix at a determined concentration. For the negative QC control, it is typically a normal sample.

Section 3. Instrument Based Questions

Question 1. What LIMS systems are compatible with the AQ 1000?
The AQ 1000 is compatible with several LIMS systems. Each lab has their own LIMS. Intuitive Biosciences works with the customer to create a customized link and data format to upload into the lab owned LIMS system.

Question 2. How is the reaction read in the AQ 1000?
The AQ 1000 uses a CCD (Charge-coupled device) camera and an LED light source to capture the image. The reaction is colorimetric, not fluorescent, or luminescent so the CCD camera is capturing the precipitation of silver that has occurred because of the assay reaction. This results in a grey scale image, where darkness of the spots corresponds to the amount of captured target, and is measured in Relative Intensity Units (RIU).

Question 3. Does the AQ 1000 read every spot in every well?
Yes, the AQ 1000 software calculates the relative intensity of each pixel in an image to differentiate every spot in each well and reports the average spot intensity along with the background intensity surrounding the spot in a raw data format.

Question 4. Does Intuitive offer IQ/OQ/PQ for the instrument?
The AQ 1000 is an OEM instrument purchased by Intuitive Biosciences. The instrument company maintains a FDA 21CFR 820 quality system. The OEM company follows UL/CSA conformance for North American customers and CE conformance per EU low voltage and EMC directives. The OEM company is also ISO 9001 and ISO 13485 certified.

Question 5. How does the AQ 1000 get calibrated?
The AQ 1000 has an internal calibration built into the system that automatically calibrates the instrument upon start up. It calibrates the CCD camera and light source.

Question 6. What maintenance is required for the AQ 1000?
The AQ1000 does not require maintenance other than to turn it on and to shut it off. The only moving part is the tray which moves in and out. The CCD camera and LED light do not move and has automated calibration internal to the system.

Question 7. Can the AQ 1000 be integrated with automated pipetting systems?
It is possible to integrate the AQ 1000 reader into automated pipetting systems.

Question 8. Does the AIM system have a bar code reader?
The AQ 1000 does not have an integrated bar code reader.

Section 4. Manufacturing Based Questions:

Question 1. What validation is done to ensure the product is manufactured correctly?
Manufacturing consistency validation is done with spot to spot, well to well within assay, day to day and person to person testing to ensure the plate is manufactured correctly and will perform to the validated specifications. If QC does not pass at the established acceptable rate, the product is scrapped and remanufactured. The actual acceptable rate varies by product and are based on customer required specifications determined as part of the assay design.

Question 2. What is the quality certification status of the lab or manufacturing facility (GLP, GMP, ISO)?
Intuitive Biosciences functions under “GLP LIKE” and GMP procedures per the guidelines.