Test and manufacture a device for rapid, cost
effective, non-invasive collection and ‘clean up’ of saliva (e.g.,
reduction of contaminants, matrix effects, etc) for early detection
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several advantages over blood; (1) No cultural / religious biases
associated with collection of blood, (2) reduced biohazard to
worker, (3) non-invasive, (4) easy
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Antibiotic-pathogen-biomarker screening by PCR must have SACIvan Brukner
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The current pandemic has generated the search for new reliable and economic alternatives for the detection of SARS-CoV-2, which produces the COVID-19 disease, one of the recommendations by the World Health Organization, is the detection of the virus by RT-qPCR methods from upper respiratory tract samples. The discomfort of the pharyngeal nasopharyngeal swab described by patients, the requirement of trained personnel, and the generation of aerosols, are factors that increase the risk of infections in this type of intake. It is known that the main means of transmission of SARS-CoV-2 is through aerosols or small droplets, which is why saliva is important as a relevant means of detecting COVID-19. In this study, a modified method based on SARS-CoV-2 RNA release from saliva is described, avoiding the isolation and purification of the genetic material and its quantification of viral copies; the results are compared with paired pharyngeal/nasopharyngeal swab samples (EF/EN). Results showed good agreement in saliva samples compared to EF/EN samples. On average, a sensitivity for virus detection of 80% was demonstrated in saliva samples competing with EF/EN samples. The use of saliva is a reliable alternative for the detection of SARS-CoV-2 by means of RT-PCR in the first days of infection, having important advantages over the conventional method. Saliva still needs to be studied completely to evaluate the detection capacity of the SARS-CoV-2 nucleic acid, however, the described process is viable, due to the decrease in materials and supplies, process times, the increment in the sampling and improvement of laboratory performance.
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Presenter: Corey D. Broeckling, Ph.D., Associate Director, Proteomics and Metabolomics Facility, Joint Assistant Professor, Colorado State University
Microfluidic technology offers multiple advantages including ease of use, robustness and sensitivity. Coupled with a tandem quadrupole mass spectrometer (such as the Xevo TQ-S) we can create an optimal and versatile “middle ground” platform in which these advantages can be exploited for both small molecule and peptide quantitative applications. For example, most small molecule applications are performed using standard flow chromatography (in the range of 600-100 L/min) consuming a high level of both solvent and sample which increases the cost (both fiscally and environmentally). The use of microfluidic technology for these small molecule applications can reduce solvent consumption by upwards of 150-fold and can significantly increase on-column sensitivity, thus reducing sample consumption. Conversely, quantitative peptide assays are almost exclusively performed using nanoscale chromatography (~400 nL/min) to achieve the required sensitivity for detection of these low abundance molecules within a complex matrix (e.g. serum, urine, etc.). We have found that the use of microfluidic technology for peptide quantitation yields the same or better sensitivity when compared to a nanoscale platform and has the additional, very significant advantages of ease of use, robustness, and improved chromatographic resolution (e.g. peak capacity). Thus, with a single analytical platform we can perform quantitative analysis for a wide range of compounds spanning from lipids/metabolites to peptides. One application in which the technology has struggled is the analysis of compounds in negative ionization mode. This limitation has been overcome in the development of a next generation microfluidic device that incorporates post-column addition of isopropanol to improve ionization and spray stability in negative mode applications. With this new capability we can now perform quantitative experiments in negative mode or with polarity switching.
This presentation was given at the 11th International Conference of the Metabolomics Society (Metabolomics 2015, #metsoc2015 on Twitter), June 29, 2015, in San Francisco.
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Presenter: Giuseppe Astarita, Ph.D., Principal Scientist, Waters Corp, Adjunct Professor, Georgetown University
A number of technological advancements have enhanced our ability to conduct metabolomics and lipidomics experiments. State-of-the-art chromatography, ionization sources, and MS technology combined with powerful informatics solutions provide a comprehensive set of tools to analyze complex mixtures of lipids and polar metabolites in biological samples. In this presentation, I will illustrate current workflows for metabolomics & lipidomics, including untargeted and targeted approaches, for discovery and routine applications.
This presentation was given at the 11th International Conference of the Metabolomics Society (Metabolomics 2015, #metsoc2015 on Twitter), June 29, 2015, in San Francisco.
Validation of bevacizumab elisa ich q2 ver3,0 dt14.03krishgen
This document presents a discussion of the characteristics of our KRIBIOLISA™
BEVACIZUMAB ELISA kit considered by us during the validation of this kit in accordance
with ICH Q2 (R1) guidelines. The document is prepared based on tests run in our laboratory
and does not necessarily seek to cover the testing that may be required at user’s end for
registration in, or regulatory submissions. The objective of this validation is to demonstrate
that it is suitable for its intended purpose – detection of Bevacizumab (Avastin)
IRJET- Study of Immunostimulatory Potential of Silver Nanoparticles
Similar to Rapid, cost effective and disposable device for saliva collection and processing for the in-the-field identification of malaria infection - mhsrs 2015
Antibiotic-pathogen-biomarker screening by PCR must have SACIvan Brukner
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Rapid, cost effective and disposable device for saliva collection and processing for the in-the-field identification of malaria infection - mhsrs 2015
1. Rapid, cost-effective and disposable device for saliva collection and
processing for the in-the-field identification of Malaria infection
Robert Gellibolian, Ph.D, Adam Markaryan, Ph.D*
*Presenting author: Dr. Adam Markaryan (Email: adam@cellectgen.com)
CellectGen, LLC., 2265 E. Foothill Blvd., Pasadena, CA 91107
Abstract ID
MHSRS-15-097
1. ABSTRACT
4. MATERIALS & METHODS
5. RESULTS
We propose to test and manufacture a device for rapid, cost
effective, non-invasive collection and ‘clean up’ of saliva (e.g.,
reduction of contaminants, matrix effects, etc) for early detection
of Malaria biomarkers (e.g., pfHRP2, etc). ‘Clean’ saliva offers
several advantages over blood; (1) No cultural / religious biases
associated with collection of blood, (2) reduced biohazard to
worker, (3) non-invasive, (4) easy and in-field usable, and (5)
requires minimal or no training
2. INTRODUCTION
Problem – Early, accurate diagnosis of Malaria at the point-of-care
(POC) is important because preventive action and treatment
should begin within 24 hours of fever onset.1 Delay in this process
can result in serious illness or death.2 Currently, blood is the gold
standard for definitive diagnosis of Malaria in suspected patients,
but it is invasive, requires trained personnel and raises the risk of
accidental transmission of infectious diseases. Difficulties with
patient compliance in communities with cultural objections and
signal background effects in blood limit its usefulness in in-field
settings. In contrast, saliva; (a) presents a non-invasive biofluid of
choice, (b) has been shown to harbor Malarial antigens (e.g.,
pfHRP2, etc),3-5 and (c) can be painlessly collected in relatively
large quantities by anyone. Like blood, best results can ONLY be
obtained when biomarker stability issues, contaminants (e.g., cells,
mucins, etc) and matrix effects (e.g., viscosity) in saliva are
eliminated through processing of the sample, which is currently
achieved using cold chain logistics and centrifugation, neither of
which are acceptable for in-field use. Fortunately, elimination of
these steps is easier to achieve in saliva than blood.
Solution – CellectGen has developed a patented technology that
within less than 10 seconds after collection significantly reduces
the viscosity and ‘cleans’ up whole saliva and readies the sample
for immediate testing. The innovation lies in elimination of both the
cold chain logistics and centrifugation steps in the workflow,
bringing sample testing to the point-of-care (POC).
Impact – We believe that CellectGen’s device will find broad
applicability in both military and civilian/community settings,
allowing for; (1) rapid in-field saliva ‘clean-up’, (2) reduction of time
to results, and (3) elimination of cultural taboos associated with
blood collection. Moreover, the device’s simplicity and practicality
could also facilitate disease surveillance and evaluation of control
interventions at the broader community level.
The purpose of this study is to test the viability and
feasibility of a predicate device (not in-field ready) that
functionally mimics the prototype of our patented device
for rapid, non-invasive collection and ‘clean up’ of saliva.
Due to lack of access to saliva samples from Malaria
patients, we tested the validity of our objective using two
common salivary biomarkers; (a) cortisol (a steroid), and
(b) interleukin 1β (a protein).
3. OBJECTIVE
Saliva collection and processing
Saliva samples were collected by voluntary consent from one of the CellectGen team members.
For proof-of-concept, all ‘clean-up’ was performed using our predicate device (PD, not in-field
ready) (see left panel). Prior to collection, donor rinsed his mouth with water and refrained from
eating/drinking for at least 1 hour. Saliva was then collected by passive drooling and transferred
into the vial of PD using a straw and the filter tip carefully assembled at the bottom of the vial to
avoid contamination. For each experiment, ~5 ml of saliva was collected in a span of ~10 min
(flow rate ~0.5 ml/min). Samples were processed for the following analyses:
1. Human and bacterial DNA purification, and quantitation by RT-PCR
‘Clean-up’ was measured by quantitating levels of human and bacterial DNA in whole as well as filtered saliva. DNA from saliva samples
were purified by using prepIT-C2D genomic DNA MiniPrep Kit from DNA Genotek (Canada, ON) as per manufacturer’s protocol.
Quantitative duplex real time PCR (RT-PCR) was performed to determine the amounts of human and bacterial DNA in each sample using
TaqMan® human RNAse P (VIC/TAMRA) and bacterial 16S ribosomal RNA (FAM/MGB) primer probe sets and 20ng of input DNA. The
assay was run on the CFX 96 RT-PCR System (Bio-Rad, Hercules, CA) and the saliva filtration efficiency of the device was determined
by comparing Ct numbers for bacterial and human DNA in whole saliva and filtrate by using the ΔΔCt method. E.coli DNA from
Sigma and human genomic DNA provided in the TaqMan RNase P kit were used as standards.
2. Biomarker assay; Cortisol and Il-1β ELISA
The levels of biomarkers (IL-1β and cortisol) in ‘cleaned’ versus whole saliva was measured as a function of time. We used the
Salimetrics (Carlsbad,CA) salivary cortisol ELISA Kit for quantitative determination of free cortisol in saliva samples based on competitive
assay. For the quantitative determination of IL-1β in saliva, we used the Boster Bio (Pleasanton,CA) ELISA Kit based on standard
sandwich ELISA technology.
vial
saliva
filter
Tip
squeeze
cleaned
saliva
debris
2. REFERENCES
1) Expanded use of malaria test. The Innovation Countdown 2030 Report, p. 22.
2) Kain, K.C, et al. Malaria deaths in visitors to Canada and in Canadian
travellers: a case series. Canadian Med. Assoc. J. 164, 654–659 (2001)
3) Fung, A.O. et al. Quantitative detection of PfHRP2 in saliva of malaria
patients in the Philippines. Malaria J. 11, 175 (2012).
4) Wilson, N.O. et al. Detection of Plasmodium falciparum histidine-rich protein II
in saliva of malaria patients. Am. J. Tropic. Med. and Hyg. 78, 733-735 (2008).
5) Gbotosho, G.O. et al. Rapid detection of lactate dehydrogenase and
genotyping of Plasmodium falciparum in saliva of children with acute
uncomplicated malaria. Am. J. Tropic. Med. and Hyg. 83, 496-501 (2010).
6) Bossaller, L. et al. Cutting edge: FAS (CD95) mediates noncanonical IL-1β and
IL-18 maturation via caspase-8 in an RIP3-independent manner. J. Immunol.
189(12),5508-12 (2012)
100%
80%
60%
40%
20
Whole
Saliva
Whole
Saliva
%
0%
RNAseP
(human)
16S rDNA
(bacteria)
Fig.1 - Percentage of bacterial and human cells before
and after ‘clean up’ of whole saliva through a 1.0µ or
5.0µ pore size filter.
‘Clean’ Saliva
DNAContent(%)
1.0
µm
5.0
µm
1.0
µm
5.0
µm
‘Clean’ Saliva
6. CONCLUSIONS
7. FUTURE DIRECTIONS
The efficiency of clean up as measured
by reduction in the amount of cells in
the filtrate from whole saliva was as
follows:
Observation: Filtration of whole saliva led to
drastic reduction in its viscosity, facilitating
handling in downstream assays.
0.4
0.2
0.3
0.1
Cortisol(µg/dl)
Whole Saliva
0
Centrifuged
Saliva
Filtered
Saliva
Time (days): 0 7 30 0 7 30 0 7 30
Fig.2 - Time course study for detection of cortisol (a steroid hormone) in saliva.
Data represent; (a) whole saliva, (b) centrifuged saliva, and (3) filtered saliva. Time
course study of salivary cortisol levels was done at 0, 7, and 30 days post-collection.
Efficiency of saliva clean-up Salivary cortisol levels
200
100
150
50
IL-1β(pg/ml)
0
Centrifuged
Saliva
Filtered
Saliva
Time (hrs): 0 0.5 1 2 0 0.5 1 2
Fig.3 - Tme course study for detection of IL-1β (a protein) in
saliva. Data represent; (a) centrifuged saliva, and (b) filtered saliva.
Time course study of salivary IL-1β levels was done at 0, 0.5, 1,
and 2 hrs post-collection.
Salivary IL-1β levels
Figures 2 and 3 show the comparison in levels of two different salivary analytes (cortisol and
IL-1β) using centrifugation (the gold standard for saliva processing) and our technology.
• Cortisol levels are stable when comparing centrifuged and filtered saliva samples (Fig. 2).
• The trend between centrifuged saliva and filtered saliva remain the same, confirming the
merits of our technology as a viable replacement for traditional methods using centrifugation
and cold chain logistics for in-field use.
• A similar trend is observed when analyzing IL-1β (a protein) levels in centrifuged and filtered
saliva (Fig. 3).
• Observation: The upward trend in levels of IL-1β between 0.5 and 1 hr may be the result of
activation of IL-1β pecursor by the inflammasome pathway involving caspase-1.6
humanpore size
70.52,500
10,000 713
fold reduction
1.0 µm
5.0 µm
bacteria
CortisolIL-1β
♦ ‘Clean-up’ of saliva using filtration is as effective as
centrifugation-based methods in removing cells, debris and
reducing viscosity (see results, Figs. 1-3).
♦ CellectGen‘s technology is non-invasive and amenable for
in-field use.
♦ CellectGen‘s technology allows for rapid, field-based collection
and processing of saliva, readying the sample for immediate
downstream testing using Rapid Diagnostic Tests (RDTs).
♦ CellectGen‘s filtration works with various analytes, including
steroid hormones (cortisol) and proteins (IL-1β) (see results,
Figs. 1-3).
♦ We believe that the proof of concept study outlined here will
pave the way to applying our technology towards detection of
salivary markers for Malaria using existing RDTs.
1) Phase 1 - Viability for detecting salivary Malaria biomarkers:
In this phase, we intend to extend the use of our predicate
device (not in-field ready) to assess whether the ‘clean up’ of
saliva leads to effective detection of the typical biomarkers of
Malaria in saliva (e.g., pfHRP2, etc) with field testable samples.
Collaborator: Noguchi Memorial Institute for Medical
Research (NMIMR) in Ghana, Africa.
2) Phase 2 - Design and manufacture of field-ready prototype:
Once Phase 1 objectives have been established, we will begin
the design and ‘limited-scope’ manufacture of prototype
versions of a field-ready device.
Adam Markaryan, Ph.D
co-founder / Senior Scientist
CellectGen, LLC
2265 E. Foothill Blvd.Pasadena, CA 91107
Email | adam@cellectgen.com
Contact information: