FAQs

What is unique about NULISA Assay and how do you get to the sensitivity?

NULISA can achieve the attomolar limits of detection (LOD) which translates to single digit fg/ml analyte detection sensitivity. This is achieved by the proprietary assay background suppression methodology. Using the same antibodies NULISA can achieve a 10,000 fold sensitivity improvement over traditional PLA and ELISA technologies.

Why does sensitivity matter?

High sensitivity enables the detection of low-abundance proteins. Being able to detect low-abundance proteins reliably is critical in establishing baseline levels in healthy individuals or control conditions, supporting early diagnosis of diseases, disease monitoring, and biomarker discovery used in research and clinical applications.

What does dynamic range mean?

Dynamic range is the range of concentrations that an assay can quantitate with reliable accuracy and precision between the LLoQ (Lower Limit of Quantification) to the ULoQ (Upper Limit of Quantitation).

What is the dynamic range of the NULISA assay and what makes it so broad?

The broad dynamic range of the NULISA assay is due to the unique background suppression mechanism to significantly increase signal-to-noise ratio and the nucleic acid-based reporter read out either by qPCR or NGS.

What is the precision of NULISA Assay results?

Intra-assay precision measures the variation in results when a single sample is assayed multiple times within the same assay run.

Inter-assay precision measures the variation in results when the same sample is assayed multiple times in different assay runs or on different days.

For NULISAseq, the median intra/inter assay %CV across all targets is less than 15%.

For NULISAqpcr, the average intra/inter assay %CV is less than 15%.

For the most detailed and accurate information, please refer to the specific Data Sheet provided for each assay.

How many individual samples can be run on NULISA single-plex assays and on NULISAseq multi-plex panels?

NULISAqpcr Single-Plex Assay
74 samples in singlet

NULISAseq Multiplex Panel
86 samples in singlet

What is the minimum (uL) sample requirement to run the NULISA assay?

NULISAqpcr Single-Plex Assay
Minimum of 35uL (total loading volume) for one sample in singlet.

NULISAseq Multiplex Panel
Minimum of 25uL (total loading volume) for one sample in singlet.

What sample types can be used?

Plasma, Serum, and CSF. Other unique sample matrices/biofluids that have been tested with success include dried blood spots, saliva, urine, brain lysates, stool, tear fluid, and nasal swabs. For further details, please reach out to your local representative.

How do you ensure lot to lot reproducibility with the antibodies?

The vast majority of the antibodies used on our platform are monoclonal antibodies which can be produced in large quantities through hybridoma technology or recombinant DNA techniques, making them highly consistent. We also have established various QC criteria in our manufacturing processes to ensure lot-lot consistency.

How many runs can be loaded on the ARGO HT instrument?

3 runs can be loaded on the ARGO HT instrument at a time and ran sequentially. The runs can be a mixture of single-plex assays or multiplex assays.

How long are the runs on the Argo HT Instrument?

Each run (a single 96-well plate) takes about 8 hours on the ARGO HT instrument. This includes the entire workflow from sample preparation to pooled library, ready for NGS sequencing.

What additional equipment are required to run NULISA Assay?

The ARGO HT system is a fully automated instrument that requires only the basic equipment common to most labs e.g., centrifuge and pipettes.

To run the NULISA single-plex assay, the ARGO HT instrument comes with a real-time PCR machine incorporated into the system.

For multiplex assay, Alamar has validated Illumina and ElementBio sequencers for NGS readout. A Qubit fluorometer is required to quantify the concentration of the NULISAseq NGS library before sequencing.

What is the size of the ARGO HT instrument?

Width: 1143 mm (45 inches or 3.75 ft)

Height: 1016 mm (40 inches or 3.33 ft)

Does the ARGO HT instrument come with warranty?

With the purchase of the ARGO System, a 1-year warranty service warranty is included for parts and labor.

What comes included with purchase of the ARGO HT instrument and is training provided?

With the purchase of an ARGO System, Alamar will provide white glove delivery of the system to your site; 1 year of warranty for parts and labor.

A Field Service Engineer will perform a pre-site inspection, installation and training of the ARGO HT system.

A Field Application Scientist will provide applications training following completion of the installation.

Access to the Alamar's software is included with the purchase of the system.

What kind of analysis software is provided along with the purchase of the ARGO HT instrument?

The ARGOsystem comes with ARGO Command Center (ACC), a cloud-based software for instrument setup and results, and NULISA Analysis Software (NAS), a software for secondary analysis. Both primary and secondary analysis software are provided to the customer at no additional cost.

Can I perform my own analysis of the data without using Alamar software?

Data can be exported directly from either the ARGO Command Center or NULISA Analysis Software. For those familiar with the R programming language, we also provide an R library called NULISAseqR. This provides functions for reading, writing, and advanced analysis of NULISAseq data.

Does Alamar offer both single-plex and multi-plex assays? How are they quantified?

Alamar offers single-plex assays, a 5-plex assay, and multiplex panels. The single-plex assays and the 5-plex assay provide absolute quantification with a qPCR readout. Multiplex panels primarily provide relative quantification with an NGS readout, with the exception of the NULISAseq Inflammation Panel AQ, which provides absolute quantification.
 
For the full product catalog, visit our Products webpage or contact your local Alamar representative.

How many individual samples can be run on NULISA single-plex assays and on NULISAseq multi-plex panels?

NULISAqpcr Single-Plex Assay: 74 samples run in singlet
NULISAqpcr 5-plex Assay: 84 samples in singlet
NULISAseq Multiplex Panel: 86 samples in singlet

What is the minimum (μL) sample required to run the NULISA assay?

NULISAqpcr Single-Plex and 5-plex Assays: minimum of 35 μL total loading volume per sample in singlet.
NULISAseq Multiplex Panel: minimum of 25 μL total loading volume per sample in singlet.

How were the targets for the NULISAseq multiplex panels selected?

Targets are carefully selected and validated through deep reviews of the literature and working directly with KOLs in the disease areas in which the panels are designed to address.

Can multiple NULISAseq libraries be pooled and can they be pooled with other library types to be sequenced in the same lane?

No - NULISAseq libraries do not contain an index sequence that allows them to be pooled in a single sequencing lane. Because the libraries require a specific sequencing recipe, this prevents them from being sequenced with libraries of other types either in the same lane or on the same flow cell.

How many sequencing reads are required per plate?

The target is 400 million single end reads per NULISAseq plate. After filtering unmatched reads, the minimum reads per plate to pass QC is 100 million.

What is the required sequencing read length?

NULISAseq libraries require 34 bp of sequencing and must be sequenced using custom recipes that can be provided by Alamar support.

Is a custom sequencing protocol required?

Yes. We have different custom NULISAseq sequencing protocols which are required for running on the different sequencing platforms.

What sequencing platforms are compatible with NULISAseq?

Illumina NextSeq 550
Kit: TG NextSeq™ 500/550 High Output Kit v2.5 (75 Cycles)

Illumina NextSeq 1000/2000
Kit: NextSeq 1000/2000 P2 Reagents (100 Cycles) v3 or NextSeq™ 1000/2000 P2 XLEAP-SBS™ Reagent Kit (100 Cycles)

Illumina NovaSeq 6000
Kit: NovaSeq SP Reagent Kit (100 Cycles) v1.5

Illumina NovaSeq X & X Plus
Kit: NovaSeq X Series 1.5B Reagent Kit (100 Cycle) or NovaSeq X Series 10B Reagent Kit (100 Cycle)

Element Biosciences AVITI
Kit: AVITI 2x75 Sequencing Kit Cloudbreak FS High Output (Note: AVITI has two independent flow cells)

How many samples can be run per plate?

84 wells of a 96 well plate can be dedicated to samples.

What type of samples can be run?

The panel has been validated on plasma.

Is it recommended to run samples in singles, duplicate or triplicate?

The panel has been validated as single sample runs and duplicate runs where necessary such as precision studies.

Which targets are quantifiable?

BD-pTau217, NfL, GFAP and Aβ42 are reported as absolute quantitative values, while ApoE4 is reports carrier status.

What is the readout for APOE4?

APOE4 is a genotyping test and the readout is for the presence/absence of the gene. It does not distinguish between heterozygosity and homozygosity.

What is the minimum sample volume requirement?

35 μl is the minimum required volume.

How long does it take to run a plate?

~7.5 hours is the time from sample to data.

Is this product CE marked / IVD approved?

No. This product is for research use only (RUO).

Can this assay be used to support clinical decision making?

No. This product is for research use only (RUO).

Are there any special requirements for running the panel?

Yes. The qPCR instrument needs to be calibrated by an Alamar Field Service Engineer.

Can more than 1 bay be run?

Yes, more than one bay can be run.

How does does this panel compare to running the markers individually?

Single-plex assays correlate very well with the NULISAqpcr AD 5-plex assay. Data is available to review upon request.

How was specificity confirmed for each target?

Specificity of each target was confirmed by testing cross-reactivity with other analytes in the panel. Data is available in the datasheet.

Can custom assays be created for NULISA?

Yes. Custom assays can be developed using our Custom Assay Development Kit or through our TAP (Technology Access Program) services. For TAP-based development, please contact your local Alamar representative.

What types of antibodies can be conjugated?

The NULISAqpcr Antibody Conjugation Kit has been validated with Immunoglobulin G (IgG) antibodies from mouse, rabbit, goat, sheep, and donkey. IgG antibodies from different species may exhibit slight differences in conjugation efficiency. For compatibility with other antibody types, contact Alamar Support.

How much antibody is required for NULISAqpcr custom conjugation?

Each conjugation requires 100 µg of antibody.

How many samples can I run using the custom conjugated antibodies?

A single conjugation provides enough material to analyze more than 3,500 samples.

Should antibody solution be carrier free?

Yes, carrier proteins, such as BSA and gelatin, will interfere with NULISAqpcr antibody conjugation. If a carrier-free antibody formulation is not available, the antibodies must be purified to remove carrier proteins.

What chemicals interfere with NULISAqpcr antibody conjugation?

Primary amines and strong nucleophiles can interfere with antibody-oligo coupling. Detergents in antibody formulations may also cause column leakage by disrupting surface tension.

Antibodies should be desalted or buffer-exchanged into the Conjugation Buffer provided in the NULISAqpcr Antibody Conjugation Kit.

Using incompatible buffers or agents outside the recommended concentrations may lead to lower conjugation yield.

Can I run my custom assay on more than 1 bay?

You can perform a 3-bay run using the Custom Target plate with your conjugated antibodies.

What does NPQ stand for?

NPQ refers to NULISA Protein Quantification, which is an Alamar-specific relative quantification unit for NULISAseq assays. NPQ is calculated by normalizing NGS read counts by an internal control, normalizing using interplate control samples, rescaling the data, and then performing a log2 transformation. A one unit increase in NPQ represents a doubling of protein target level.

Can we compare the NPQ values across different targets?

NPQ values for different targets should not be compared. Only NPQ values for the same target should be compared across samples.

What is detectability? What is a “detectable” target?

The detectability for each target is defined as the percentage of samples with results above the Limit of Detection (LOD). A target is considered detectable when more than 50% of samples are above the LOD.

How is the Limit of Detection (LoD) calculated for NULISA assays?

For NULISAseq multiplex assays, LOD is calculated as the mean plus 3 times the standard deviation of the normalized counts of the negative controls. For NULISAqpcr single-plex assays, LOD is calculated as the mean plus 2.5 times the standard deviation of the negative controls

Why is detectability different in different sample types?

NULISAseq may be used to measure proteins in different sample types or sample matrices, such as blood plasma, serum, or cerebrospinal fluid for example.

Different sample types represent different tissue and cellular sources of proteins thus can have drastically different abundances for some or many proteins. Other considerations affecting detectability include sample quality, sample treatment, sample processing, and sample preparation.

Still have questions? Get in touch.

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Steve Williams, MD, PhD

CSO

Dr. Willams serves as the company’s Chief Scientific Officer. He was previously Chief Medical Officer at Standard Biotools and at SomaLogic where he pioneered the discipline for discovery and validation of predictive, diagnostic and prognostic models using machine-learning applied to large-plex proteomics. 20 such tests were used for drug characterization, safety and efficacy when incorporated in clinical drug trials at Pharma/Biotech and 17 different multivariate tests were validated and translated into regulated healthcare uses. Prior to SomaLogic, Dr. Williams was at Pfizer in the UK and the USA as a clinical triallist in Translational Medicine, and subsequently as VP, Global Clinical Technology. He sat on the National Advisory Council for the National Institute of Biomedical Imaging and Bioengineering, the Executive Committee for the FNIH Biomarkers Consortium, and worked with FDA and PhRMA on developing evidentiary standards for biomarker qualification. Dr. William’s medical training was in London, at Charing Cross and Westminster Medical School, followed by a PhD in medicine/physiology at the same institution and training in Radiology at the University of Newcastle Upon Tyne. Steve is co-inventor on 26 proteomics patents and author/coauthor on multiple foundational proteomics manuscripts.

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Justin McAnear

CFO

Mr. McAnear serves as the company’s Chief Financial Officer. He brings over 25 years of operational and financial leadership experience across various sectors and was instrumental in taking 10x Genomics public in 2019, serving as its CFO for over five years. Mr. McAnear served for over 3 years as Tesla’s VP of Worldwide Finance and Operations, supporting landmark initiatives such as the Model X and Model 3 launches and Solar City acquisition.  He also held various roles at Apple and J&J earlier in his career and served as a naval officer and aviator for over 9 years.