The present invention relates to a method for detecting Zika Virus. More particularly, the method and apparatus are directed to a method for making a temperature-independent paper test strip for detecting Zika virus.
The Zika virus (ZIKV) is an infectious disease from the virus family Flaviviridae which is spread from the Aedes mosquitoes which are active during the day. Zika was originally isolated in the Zika Forest of Uganda where it was found in monkeys, and then later identified in humans in 1952 (WHO, 2017). The first major outbreak of Zika infection was found in the Island of Yap in 2007 (WHO, 2017). As stated from an article published by Science Magazine, “For nearly 7 decades, the Zika virus would remain a virological curiosity, receiving little more attention than other obscure members of the Flaviviridae family that are transmitted by mosquitoes, such as Spondweni, Wesselsbron, and Ntaya. But now that it appears Zika might be causing serious harm to babies in Brazil, the World Health Organization has deemed it a ‘public health emergency of international concern.’ It's fast earning the reputation of the scariest virus on the planet.”
Zika virus is known to cause fever, rash, headache, and conjunctivitis, but Zika's biggest threat is to pregnant women, because the virus can pass onto the fetus. If Zika passes onto the fetus during pregnancy, the virus can cause a birth defect called microcephaly (CDC, 2017).
For most people, Zika is a very mild infection and is not harmful. An article reported by CNN, “In these areas, women who are pregnant need to protect themselves from mosquito bites by using repellents, permethrin-coated clothing, long sleeves and pants, and by staying indoors (ideally in places with air conditioning) as much as is practical. We advise pregnant women to postpone travel to areas where Zika is spreading” (CNN, 2016). Due to travel hazards, many people are canceling trips to Zika infested areas to avoid the risk of contracting the virus. However, many of the population of South America are not able to travel outside of South America to avoid contracting the virus, or are not able to use repellents, permethrin-coated clothing, etc. Some people may not be aware of special precautions whilst others are not aware that they are carrying the virus (New York Times, 2016).
IgM Antibody Capture Enzyme-Linked Immunosorbent Assay (MAC-ELISA) tests are currently one of the options for Zika detection and use an oxidation reaction caused by Horseradish peroxidase (HRP) onto 3,3′5,5′-Tetramethylbenzidine (TMB). HRP is an enzyme normally found in the roots of Horseradish, and TMB is an enzyme substrate. Initial inspiration was derived from journals about rapid HIV tests and MAC-ELISA antibody tests.
Current diagnostics for detecting Zika cost between $299 to $800 on the private market if patients do not fit the CDC's criteria (New York Times, 2016). Due to expensive prices, many pregnant women are resulting to abortions if infected, or they are deciding to not get pregnant. People must wait long periods of time to get tested if they don't meet the CDC's criteria. Many tests for Zika are complicated and take days up to weeks to get results back due to current diagnostics being lab tests. Zika tests are first come, first serve, and as stated from an article published by the New York Times, “Public health experts say the restrictions are necessary to ensure that people most at risk have access to testing. ‘We aren't interested in stimulating the testing of simply anxious people,’ according to an infectious diseases specialist at Vanderbilt University Medical Center. ‘We want health care providers to provide the appropriate counseling and to be selective in the use of this test, as they are in the use of any other test’” (New York Times, 2016). Due to lack of resources and time, many patients infected with Zika do not know they have the virus, and many cannot be tested due to lack of resources and time. In order to combat the problem in the field, a temperature-independent, low cost, efficient, diagnostic system needs to be made.
The present invention overcomes the deficiencies of the prior art by providing a method for making a low-cost paper test strip that can be used in remote locations, disaster areas, at home, or in the field of research, it will be possible to detect Zika in a more effective and faster way.
This summary is provided to introduce, in a simplified form, a selection of concepts that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
Disclosed herein is a method for making and using a resultant paper test strip product for detecting Zika antibodies indicating the presence of Zika virus in a patient sample. The paper test strip includes a strip of filter paper coated with a silk fibroin solution, wherein the silk fibroin solution is mixed with an enzyme solution in phosphate buffered saline buffer.
While the novel features of certain embodiments of the invention are set forth with particularity in the appended claims, the invention, both as to organization and content, will be better understood and appreciated, along with other objects and features thereof, from the following detailed description taken in conjunction with the drawings, in which:
In the drawings, identical reference numbers identify similar elements or components. The sizes and relative positions of elements in the drawings are not necessarily drawn to scale. For example, the shapes of various elements and angles are not drawn to scale, and some of these elements are arbitrarily enlarged and positioned to improve drawing legibility. Further, the particular shapes of the elements as drawn, are not intended to convey any information regarding the actual shape of the particular elements and have been solely selected for ease of recognition in the drawings.
The following disclosure describes a method for making a temperature-independent test strip for detecting Zika virus. Several features of methods and products in accordance with example embodiments are set forth and described in the figures. It will be appreciated that methods and products in accordance with other example embodiments can include additional procedures or features different than those shown in the figures. Example embodiments are described herein with respect to a method and system directed to using silk fibroid and 2D paper networks for making the test strip. However, it will be understood that these examples are for illustrating the principles, and that the invention is not so limited.
Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
Reference throughout this specification to “one example,” “an example embodiment,” “one embodiment,” “an embodiment” or combinations and/or variations of these terms means that a particular feature, structure or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, the appearances of the phrases “in one example” or “in an example” in various places throughout this specification are not necessarily all referring to the same example embodiment or example. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments or examples.
Definitions
Generally, as used herein, the following terms have the following meanings when used within the context of diagnostic test strips:
The articles “a” or “an” and the phrase “at least one” as used herein refers to one or more.
Dimethyl sulfoxide (“DMSO”) is an organosulfur compound with the formula (CH3)2SO.
“HRP” refers to the enzyme horseradish peroxidase, found in the roots of horseradish.
As used herein, “plurality” is understood to mean more than one. For example, a plurality refers to at least two, three, four, five, ten, 25, 50, 75, 100, 1,000, 10,000 or more.
“Obtaining” is understood herein as manufacturing, purchasing, or otherwise coming into possession of.
“TMB” as used herein means 3,3′,5,5′-Tetramethylbenzidine that in one form can have the chemical formula C16H20N2.
“Zika virus” has its generally understood meaning as an infectious disease from the virus family Flaviviridae which is spread from the Aedes mosquitoes.
Referring now to
To make the test specific to Zika virus, an antibody conjugation to HRP caused a conjugation similarly found in MAC-ELISA tests. In MAC-ELISA tests, an antibody conjugation is used to differentiate between similar antigens and/or proteins. By having TMB and ZVNS1/HRP on opposite ends of the test strip, it will cause the reagents to not react unless the ZVNS1 antigen or protein is present. In MAC-ELISA tests, a pile of reagents is used to cause a reaction.
To make the test strip temperature-independent, different films and biomaterials were researched using journals and literature. Silk fibroin was found to stabilize temperature-dependent reagents and create a film to encase reagents. In one example, Cocoons of Bombyx mori silkworm silk, HRP (Type VI-A lyophilized powder), 3,3′,5,5′-tetramethylbenzidine (TMB) solutions were used. Lithium Bromide for the fabrication of silk fibroin film was purchased from Sigma-Aldrich. 102 filter paper 2 mm, EZ-Link Peroxidase Conjugation Kit, and Zika Virus NS1 Antibodies were purchased by ThermoFisher. Zika NS1 (ZVNS1), Dengue NS1 (DVNS1), and West Nile NS1 (WNVNS1) proteins were cultured from Human T-Cells and were supplied from MyBioSource and The Gale Laboratory at UW Medicine.
In one example, silk fibroin protein extraction was accomplished using the following method and the materials as listed above:
Referring now to
As indicated above, MAC-ELISA tests for Zika virus cost around $300-$800 and use Immunoglobin M (IgM) antibody. IgM is the capture antibody and attaches onto the target antigen. MAC-ELISA tests are used for the qualitative detection of diseases using antibodies from patient-matched serum samples. As opposed to expensive MAC-ELISA testing, the new test strip 120, disclosed for the first time herein, costs less than $1 to make and is about 99.9% cheaper to produce versus MAC-ELISA tests for Zika virus.
In known MAC-ELISA tests, a color change will occur using a microplate reader. The paper test strip to detect Zika virus uses technology that is in MAC-ELISA tests, but puts it on a 2D paper network. The test strip delivers a response in under 2 minutes in contrast to MAC-ELISA tests which typically take hours to run and a few days to analyze. As indicated by the patterned indication mark 130, a positive result for Zika virus will result in, for example, a green colorimetric change using Bromophenol Blue on the bottom of the strip. Using an identical test strip, a negative result will make the strip stay blue.
In one example, strip 120A represents a test strip 120 that has been inserted in a sample 150 held in a container 155. Reference arrow 144 indicates the flow of reagents as they move down the strip from the indicator dot 122 to the indication mark 130.
Referring now to
applying a ZVNS1/HRP conjugation onto a sample region 202;
applying TMB to an indication region distally located with respect to the sample region 204;
applying a specimen sample to the sample region 206;
then applying a color marker to the indication region 208;
observing the color of the indication region after applying the specimen sample 210; and
classifying the specimen sample as positive or negative depending upon the color.
In one example, the act of applying a color marker included applying a pH indicator. The pH indicator may include Bromophenol Blue, or the like, for example, wherein the color green indicates a positive result.
During testing, there were two different testing phases to test for the detection of Zika virus and to test if the strip could differentiate between virus types. Phase 1 tests for ZVNS1 detection used biological cross-reactivity with an unrelated protein sample, and non-biological cross-reactivity with a lysate buffer. Phase 2 tests for differentiation between Zika and NS1 protein types used Dengue NS1 (DVNS1) protein and West Nile NS1 (WNVNS1) protein samples.
Zika Antigen Detection via Silk Fibroin Encasement
In one example, Zika Virus NS1 Antibody was conjugated to Horseradish peroxidase (HRP) using the EZ-Link Peroxidase Kit according to the following procedure.
ZVNS1/HRP Conjugation
Subsequently, 3,3′5,5′-Tetramethylbenzidine (TMB) substrate solution was diluted in 1 mL of Dimethyl Sulfoxide (DMSO) and 9 mL of 0.05 M Phosphate Citrate Buffer was added to the solution.
5 μL of TMB substrate solution was pipetted on the left side of the filter paper and 5 μL of the ZVNS1/HRP conjugate solution was pipetted on the right side of the filter paper. The filter paper dried for 10 min until being soaked in silk fibroin solution for 5 sec and dried for 3 hours before use.
Detection of ZVNS1
The following procedure was used in conducting tests for detection of ZVNS1.
A 5 μL of sample was added on the red insert dot on a test strip.
In one example, 102 mm filter paper strips were cut into 2×6 mm strips. Using thin paper strips allows for an easy flow-through of reagents to pass through the flow channels in the filter paper. Following, 1 mL of ZVNS1 monoclonal antibody was conjugated to the enzyme Horse Radish Peroxidase (HRP) per EZ-Link HRP Conjugation kit and on the top of the strip, 2×1 mm of the strip was dipped into the ZVNS1/HRP conjugate. On the bottom of the strip, 2×1 mm of the filter paper was dipped into the 3,3′5,5′-Tetramethylbenzidine (TMB) solution. The test strip was then encased with the silk fibroin solution and left to dry for 3 hours.
Methodology: Prototype #2
In another example, 102 mm filter paper strips were cut into 2×6 mm strips. Following, 1 mL of ZVNS1 monoclonal antibody was conjugated to the enzyme Horse Radish Peroxidase (HRP) per EZ-Link HRP Conjugation kit and 5 μL of ZVNS1/NS1 conjugate was pipetted on the top of the strip. 100 μL of Congo Red was diluted in Phosphate Buffered Saline (PBS) was added to a total volume of 2 mL of the ZVNS1/HRP conjugation before being placed on the filter paper. 5 μL of TMB solution was pipetted on the bottom of the strip. By pipetting the reagents instead of dipping, it allows for the reagents to not unintentionally react, reduces the waste of materials, and allows for a controlled amount of reagent. The test strip was then soaked in the silk fibroin solution for 5 seconds and then allowed to dry for 3 hours.
Protein Concentrations
In a study published by Fourcade, viral RNA was isolated from 140 μL samples of Zika infected patients and cell culture supernatant using a RT-PCR (Fourcade, 2016). In plasma, the viral load of ZVNS1 antigens is between 100 copies/mL to 630 copies/mL. In urine, the viral load is between 260 copies/mL to 74,000 copies/mL. To determine the concentration of the protein samples used, the following calculation was made:
By converting from copies/mL to International Units (IU), the conversion to ug/mL could be made. The concentrations used were 0.01 ug/mL to 0.13 ug/mL. The protein calculations were within range of human serum and urine samples.
Discussion & Results
Using an antibody conjugate to HRP, when the virus was present, the reaction between HRP and TMB was activated and produced a colorimetric change using protein dilutions that realistically represent that for human blood serum and urine samples.
In prototype #1, the success rate was 53% due to pipetting the sample in the center of the strip. Originally, the sample was hypothesized to pull the reagents to the center to cause an oxidation reaction to produce a pH change in response to the ZVNS1 protein. By having a low success rate, the test could not accurately nor precisely detect ZVNS1 proteins and/or antigens. The false positive rates were under 17% for prototype #1 which led to a refining in the prototype to create prototype #2.
In prototype #2, the sample was placed on the ZVNS1/HRP conjugate, also known as the sample insert dot, and then hung vertically for 1 to 2 minutes. Following, HRP and TMB reacted and produced a 3.24 pH change in response to ZVNS1 proteins and/or antigens. The overall success rate for the detection of ZVNS1 was 99% and the response time was under 2 minutes. Thus, the test could detect ZVNS1 proteins and/or antigens.
Testing for biological and non-biological cross-reactivity using non-related samples, the false positive rate was under 3% overall. The GFP biological control false positive rate was 1% whereas the mock biological control false positive rate was 3%. This suggests that the test strip has a low false positive rate when introduced to other biological and non-biological agents, suggesting that the strip only reacts to ZVNS1 proteins and/or antigens.
In prototype #2, the overall success rate increased through trials 1 to 7 due to an optimization of the prototype. Limiting TMB's exposure to oxygen and light by aliquoting 100 μL samples of TMB increased the shelf life of the test strip. Using fresh samples of the ZVNS1/HRP conjugate allowed for a longer shelf life of the test strip due to the intrinsic nature of the ZVNS1 antibody. By using fresh samples of the silk fibroin protein, the encasement of the reagents allowed for a better stabilization of the reagents under temperatures as high as 45° C. and humidity of 90%.
During phase 2 of testing for differentiation of NS1 protein types, failure rates were under 4%. The DVNS1 failure rate was 3% whereas the WNVNS1 failure rate was 4% for preliminary trials. With failure rates being low, the test strip was determined to differentiate between protein types, did not react to tested biological agents, and could only detect the ZVNS1 protein.
Because the test strip uses an antibody conjugation, the antibody could be replaced and engineered towards other diseases: Ebola, HIV/AIDS, MERS-COV, Malaria, pancreatic cancer, lung cancer, colon cancer, etc.
Test results are summarized in Table I.
Each trial in table 1 included a sample size of 100 samples. Success was interpreted as correctly classifying sample as positive or negative. Fallis classified as incorrectly classifying a sample, as for example a false positive or a false negative.
The invention has been described herein in considerable detail in order to comply with the Patent Statutes and to provide those skilled in the art with the information needed to apply the novel principles of the present invention, and to construct and use such exemplary and specialized components as are required. However, it is to be understood that the invention may be carried out by different equipment, and devices, and that various modifications, both as to the equipment details and operating procedures, may be accomplished without departing from the true spirit and scope of the present invention.
The teachings of the following publication are incorporated herein in their entirety by this reference.
1. BIOTREND Cheikalien GmbH. General Elisa Kit [Material Safety Data Sheet]. Kaufman, P. et al., Visualization and Measurement of Flow in Two-dimensional Paper Networks. Lab Chip. 2010, 10, No. 19. 2614-2617
2. Centers for Disease Control. (2017). Retrieved from www.cdc.gov
3. Centers for Disease Control. (2017). Symptoms. Retrieved from www.cdc.gov
4. Cohen, J. (2016). Science Magazine. Zika's Long, Strange Trip Into Limelight. Retrieved from www.sciencemag.org.
5. CNN. (2016). What We're Doing About The Zika Virus. Retrieved from www.cnn.com.
6. Lu, Q., Wang, X., Hu, X., Cebe, P., Omenetto, F. and Kaplan, D. L. (2010), Stabilization and Release of Enzymes from Silk Films. Macromol. Biosci., 10: 359-368. doi: 10.1002/mabi.200900388.
7. New York Times. (2016). Want a Zika Test? It's Not So Easy. Retrieved from www.nytimes.com.
8. Rockwood, Danielle N., Preda, Rucsanda C., Yucel, T., Wang, X., Lovett, M., & Kaplan, D. L., Materials Fabrication from Bombyx mori silk fibroin. Nature Protocols. 2011, Vol 6, No. 10. 1612-1631.
9. World Health Organization. (2017). Retrieved from www.who.int.
10. Vepari, C. & Kaplan, D. L. Silk as a biomaterial. Prog. Poly. Sci. 2007, 32, 991-1007.
11. Viral load kinetics of Zika virus in plasma, urine and saliva in a couple returning from Martinique, French West Indies. Fourcade, Camille et al. Journal of Clinical Virology, Volume 82, 1-4.
Entry |
---|
Qi et al. International J. Molecular Sciences 2017 vol. 18, 237 (Year: 2017). |
Kaufman et al., “Visualization and Measurement of Flow in Two-dimensional Paper Networks,” Lab Chip. Oct. 7, 2010; 10(19): 2614-2617. |
Lu et al., “Stabilization and Release of Enzymes from Silk Films,” Biomacromolecules May 11, 2009, 10(5), 1032-1042. |
Rockwood et al., “Materials Fabrication from Bombyx mori Silk Fibroin,” Nat Protoc. Sep. 22, 2011; 6(10): 10.1038/nprot.2011.379. |
Vepari et al., “Silk as a biomaterial,” Prog. Poly Sci. 2007, 32, 991-1007. |
Fourcade et al., “Viral Load Kinetics of Zika Virus in Plasma, Urine and Saliva in a Couple Returning from Martinique, French West Indies,” Journal of Clinical Virology, vol. 82, 1-4, 2016. |
Number | Date | Country | |
---|---|---|---|
20190234949 A1 | Aug 2019 | US |
Number | Date | Country | |
---|---|---|---|
62622665 | Jan 2018 | US |