ANP Technologies®, Inc.

824 Interchange Boulevard  Newark, Delaware 19711

302-283-1730

info@anptinc.com

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Copyright 2018

ANP Technologies®, Inc.

Diagnostics and Reagents Technology

 

The NIDS® technology enables lateral flow assays to increase sensitivity, improve precision, reduce false positive rates and Hook effect, as well as establish quantitation. To demonstrate the reach and versatility of its patented NIDS® technology, ANP has developed rapid lateral flow immunoassays for the quantitative measurement in a 5 µL sample of whole blood of myeloperoxidase (MPO)* and C-Reactive Protein (CRP)*. Assay test strips are measured using the NIDS® standalone handheld reader.

Myeloperoxidase (MPO) POCT Rapid Assay

By using a diluted fingerstick whole blood sample and ANP’s nanomanipulation technology based rapid assays, the NIDS® MPO rapid assay overcomes the challenges faced by MPO ELISAs of unreliable quantitation of the protein in whole blood and the instability of protein levels in serum and plasma due to the protein’s continued release from leukocytes over time. MPO is the earliest detectable biomarker released when a patient with chest pains is truly suffering a myocardial infarct (MI). Its levels are also predictive of a recurrent MI event 30 days and 6 months after the initial event. The assay requires only 5 µL of fingerstick whole blood from the patient.

 

Medical Diagnostic Assays

Figure 1. POCT rapid assays for Myeloperoxidase (MPO) and C-Reactive Protein (CRP)

NIDS MPO POCT Rapid Assay Standard Curve
NIDS MPO POCT Spike Recovery Data
NIDS MPO POCT Linearity of Dilution of Whole Bolld Samples
C-Reactive Protein (CRP) POCT Rapid Assay

CRP levels are being used to stratify a healthy person’s risk for future cardiovascular disease. It is also an efficient tool to distinguish between viral and bacterial infection in sick individuals. The assay requires only 5 µL of fingerstick whole blood from the patient. ANP offers diagnostic assay development services and technology licensing opportunities to potential partners to fully exploit and commercialize these assays and other emerging targets.

hsCRP Standard Curve
hsCRP Correlation with Instrument-based Method
Classification by Beckman Immage Values
 

Nanomanipulation-NIDS® High Sensitivity HyperBind ELISA Plates

Reference: Vallejo YR, Li J, Yin R. Enhancing assay performance using nanoscale detection. IVD Technology,Volume 16, May/June 2010

 

Please click here for a list of available HyperBind activated ELISA plates

 

High Sensitivity NIDS® HyperBind activated streptavidin or neutravidin-coated ELISA plates have proven to be the most sensitive ELISA plates for Serological Assays. These plates deliver dramatically enhanced sensitivity using significantly reduced levels of antigens for serological assays such as those for the detection of antibodies to proteins associated with HIV, HCV, the TORCH panel (toxoplasma, rubella, CMV, herpes simplex), various viral, bacterial and parasitical infectious diseases such as influenza, typhoid fever, lyme disease, allergy and a wide range of autoimmune diseases. Serological assays are also important in detecting the immunogenicity of biotherapeutic proteins. 

 

Advantages:

  • High Efficiency Coating

  • Low-cost

  • Ready to use: plates are pre-blocked with BSA

  • Sensitive: Detection of < 1 ng/mL of antibody or protein is easily attainable

  • Specific: High signal to noise ratios with low background

  • Stable: > 18 months at room temperature

 

Serologic methods have in common the use of a solid surface onto which is coated a bioreactive immunogenic protein. These biomolecules are typically complex long chain structures possessing optimal 3-dimensional conformation with specific loci for binding sites or epitopes. The conventional deposition of bioactive molecules onto solid surfaces is an inefficient and random process. For example, antibodies can easily be coated onto a plastic surface but only a subset of the deposited molecules will have the Fab binding regions available for reaction (Figure 1). This randomness limits both the binding capacity and sensitivity of assays that depend on conventional coating technology.

 

The Nano-Intelligent Detection System (NIDS®) technology has been designed to control the orientation of bioactive molecules at the nanoscale level by using a polymeric scaffold covalently conjugated to a binder such as an antibody1. Within this conjugate structure, the polymeric scaffold preferentially attaches itself to the solid surface, and extends the antigen away from the surface for most advantageous immunoreaction.The result of this innovation is a coated surface that exhibits greater binding efficiency.

Nanomanipulation-NIDS® High Sensitivity HyperBind ELISA Plates

Figure 2. Orientation of antibodies is random in conventional coating and optimally oriented with NIDS® nano-orientation technology

High Sensitivity NIDS® HyperBind plates deliver to users assays of greater sensitivity along with significantly reduced reagent usage, which is a critical consideration when these coating proteins are difficult to isolate and expensive to acquire.

 

High Sensitivity HyperBind plates are available in clear, black, and white formats for colorimetric, fluorometric and chemiluminescent assay formats, respectively. Our plates have been compared to many commercial plates. The nanoscale orientation achieved by conjugating Streptavidin or Neutravidin to the anchoring polymeric scaffold has led to impressive performance enhancements.

Comparison of Direct Binding of Biotinylated Proteins for Serological Assays

High Sensitivity HyperBind strreptavidin (SA) coatedplates exhibit greater binding efficiency of biotinylated proteins than leading commercial SA plates (Figure 2). In a representative model serological assay, 100 μL of increasing concentrations of biotinylated mouse IgG antibody are added to the wells of a High Sensitivity HyperBind, a Nunc Immobilizer™, a Pierce HBC Reacti-Bind™, and an R&D Systems EvenCoat™ plate and incubated for an hour on a platform shaker. After washing, 100 μL of rabbit anti-mouse IgG conjugated to horseradish peroxidase (HRP) are added to the wells and allowed to incubate for an hour and a half on a platform shaker. Following a wash step, the substrate/chromogen reagent consisting of hydrogen peroxide and tetramethylbenzidine(TMB) is added to the wells and color allowed to develop for 20 minutes, stopped with 2N sulfuric acid and the absorbance measured at 450 nm.

 

Binding efficiency: HyperBind plates vs. commercial plates

Figure 3. The binding efficiency of HyperBind clear plates is significantly greater than leading commercial plates.

White plates were also treated with NIDS®-activated Streptavidin for use in chemiluminescence-based assays. The binding efficiency of the HyperBind™ plates was shown to be superior to that of the Nunc Immobilizer™ white plate using the test procedure with biotinylated mouse IgG previously described and a chemiluminescent substrate reagent.

 

Binding efficiency: HyperBind plates vs. commercial black plates

Figure 4. Binding efficiency of HyperBind™ black Streptavidin (SA) and Neutravidin (NA) plates is superior to that of commercial plates.

White plates were also treated with NIDS®-activated Streptavidin for use in chemiluminescence-based assays. The binding efficiency of the HyperBind™ plates was shown to be superior to that of the Nunc Immobilizer™ white plate using the test procedure with biotinylated mouse IgG previously described and a chemiluminescent substrate reagent.

 

Binding efficiency: HyperBind white plates vs. commercial plates

Figure 5. Binding efficiency of HyperBind white Streptavidin (SA) plates is superior to that of a commercial plate.

 

The conclusion derived from the above experiments is that for a given concentration of biotinylated protein added to the microwells, more is bound by the High Sensitivity NIDS® HyperBind clear, black, and white plates than the Pierce, R&D Systems, and Nunc plates. This finding translates to significantly better assay performance and major reductions in reagent use and cost when designing Serological Assays.

 

In addition, High Sensitivity NIDS® HyperBind plates can also be used for competitive assays and direct detection analytical methods which require immobilization of antigens directly on a solid surface.

 

Endogenous Protein Extraction Using NIDS® Nanoparticles

Reference: A Lateral Flow Immunochromatographic Method for Anti-Drug Antibody Detection in Human Serum. Jian Li, Nathan Cheadle, Allen Schantz, Gopi Shankar, poster, AAPS NBC Meeting, May 2013 (Janssen Research & Development LLC)

 

Development of a clinical assay for measuring anti-drug antibodies against a monoclonal antibody drug: Overcoming soluble target interference. Qiang Qu, Alok Rathi, Boris Gorovits, Deborah Finco, Rosalin Arends, Chun-Hua Cai, Jim McNally, poster, AAPS NBC Meeting, May 2013 (Pfizer Research and Development)

 

Methods for measuring analytes in biological matrices are susceptible to endogenous interfering proteins which can significantly affect the accuracy and validity of test results. Interfering substances such as endogenous binding proteins, free drug molecules, blockers, immune complexes, and receptors can be removed from the sample using a binding agent (such as an antibody) attached to our NIDS® extracting nanoparticles. ANP is the only company that offers a simple and effective solution for a broad range of customers that need to remove such interfering substances in their bioanalytical assays.

Our NIDS® nanoparticle based universal protein extraction kits exhibit the following advantages:

1. Smaller diameter, greater surface area compared to micron-size magnetic particles which are the current particles of choice. Extraction efficiency and capacity are significantly enhanced. Up to 1 µg/mL of endogenous interfering protein can be removed.

2. Binding agents are optimally oriented. The binding agents are attached using our NIDS® chemistry which employs proprietary linkers that orient the binding sites outwardly to capture their targets most efficiently. Other particle-coating chemistries will not work as well.

3. Less binding agent used. As much as 100-fold less of scavenging agents such as antibodies is required.

4. Easy to use procedure. Optimal loading and particle usage are easily calculated.

5. Flexible universal platform. Any protein or nucleic acid can be coated onto our nanoparticles. The customer can choose from a list of binding agents for 140 different endogenous proteins currently available or provide their own binding agents for attachment to the extraction nanoparticles.

 

Bioanalytical methods such as diagnostic, pharmacokinetic, pharmacodynamic, and immunogenicity assays have shown improved accuracy and sensitivity after sample pretreatment using these innovative extraction nanoparticles linked to target-specific off-the-shelf binding reagents such as antibodies. Our NIDS® nanoparticle based protein extraction kits are currently being used by leading biopharmaceutical companies in various stages of drug development.

NIDS® High Throughput Screening (HTS) System for Antibody Drugs

ANP has developed a high throughput system for high volume screening of test samples on rapid immunogenicity assay test strips. A multistrip carrier, a tray that can hold test strips for 12 patient samples, is the basic test device in this system. This carrier has the same footprint and layout of a 96-well ELISA microplate so that it is fully compatible with available ELISA robotic systems. Sample incubation and transfer to test strips can therefore be automated, with the finished strips being read in their trays by the NIDS® HTS reader. With antigen usage substantially reduced, the HTS provides a significantly faster (less than 15 min) and simpler (one-step) low-cost alternative to multi-step ELISA-based screening for the development of antibody drugs.

Figure 6. A Multistrip Carrier holding 12 rapid assay test strips

Figure 7. The ANP NIDS® High Throughput Screening reader. Two multistrip carriers are ready to be read on the two trays on the right. Results are displayed on the top screen as shown.