Diagnostics Archives - Page 2 of 4

Does Herpes Zoster Increase the Risk of Stroke and Myocardial Infarction

INTRODUCTION:
Herpes zoster (HZ) reactivation is characterized as a vascular rash of unilateral distribution that can also cause complications such as post-herpetic neuralgia, ophthalmic zoster, and other neurological diseases. Emerging epidemiological and clinical data recognizes an association between HZ and subsequent acute strokes and myocardial infarction (MI). Read more

August 7, 2020/by Viro Research

Increased Risk of Herpes Zoster (HZ) with Immune-Mediated Diseases

Diagnostics

INTRODUCTION:
Patients have a higher risk of HZ with diseases such as rheumatoid arthritis (RA), psoriasis (PsO), and inflammatory bowel-related diseases (IBD) such as ulcerative colitis (UC) and Crohn’s disease (CD). Using immunosuppressive therapy, which treats these diseases, increases the risk of HZ.

DISCUSSION:
The most common risk factor for HZ is increasing age, presumably due to a weakening immune system as we age. In approximately 15% of the general population, varicella-zoster virus (VZV) reactivates after a latency period to cause HZ (shingles). Patients with autoimmune diseases, such as RA, IBD, UC, PsO, and CD diseases, have an increased risk of HZ compared to the general population. The risk of HZ increases by the use of immunosuppressive therapy to treat autoimmune diseases. One such drug for treatment is Janus kinase (JAK) inhibition. Tofacitinib, an oral JAK inhibitor for the treatment of RA and psoriatic arthritis, is under investigation for the treatment of UC and previously for PsO. Although there is a dose-dependent risk for HZ when taking tofacitinib, the majority of HZ cases reported are non-complicated, mild to moderate in severity, and manageable with standard antiviral therapy (acyclovir). Vaccination (SHINGRIX) should be considered before treating patients to reduce the risk of HZ patients receiving JAK inhibitors¹. Cullen, Baden, and Chiefetz² presented a review of publications describing VZV infections in inflammatory bowel disease (IBD) patients. They looked at 20 cases of primary VZV infection with IBD and 32 cases of HZ infections in patients with IBD. Fifteen of the 20 VZV cases had CD, which likely reflects the greater use of immunosuppression in this disease than UC. They identified various immunosuppressive drugs used in 20 patients, including anti-TNF (9 patients), corticosteroids (13), and either thiopurine or methotrexate (12). All 32 cases of HZ in IBD patients were on immunosuppression with corticosteroids, thiopurines, and anti-TNF. Combination therapy increased the risk of HZ even further. However, in a more comprehensive nationwide Veteran Administration study with 295 patients, Khan et al.³ found that the incidence and severity of HZ in patients on anti-TNF medications were found not to be associated with an increased risk of developing severe HZ among these IBD patients. They believed TNF-α to play an important role in viral clearance, so it was logical to think anti-TNF medications could impair host immune function. Still, the data suggest that IBD patients who develop HZ during anti-TNF therapy are not at increased risk of developing complications from the HZ infection.

CONCLUSION:
Despite the risk of a reactivating HZ infection in persons with autoimmune diseases, such as those described, there are several immunosuppressive drugs available to treat these diseases, while not increasing the further risk of HZ infections. Both tofacitinib and anti-TNF therapies, as referenced above, are two such drugs. There is growing support for patients with IBD to receive vaccination against HZ using the newly released vaccine, SHINGRIX, before immunosuppressive therapy treatment. SHINGRIX vaccination is recommended even if patients have received the previous live virus vaccine.

By David Kilpatrick, PhD and Abbas Vafai, PhD

MKTG 1047 Rev A

July 9, 2020/by Viro Research

Using Lateral Flow Assays to Detect Herpes Viruses in Saliva

Diagnostics

INTRODUCTION:
Oral fluids have been used to detect Herpes virus antibodies, including secretory IgA, IgM, and IgG. Herpes virus particles have also been identified in saliva. Several Herpes viruses, such as Epstein–Barr virus (EBV), varicella-zoster virus (VZV), and herpes-simplex-1 (HSV-1), have even been detected in the saliva of Astronauts from shuttle-flights and ISS missions¹ ². The ease of sample collection, along with the cost-effective use of lateral flow assays for detection, opens a wide range of opportunities for easily detecting Herpes viruses in point-of-care settings.

DISCUSSION:
A recent review by Miočević et al.³ discusses the strengths and weaknesses of using lateral flow assays (LFAs) for detecting viruses in saliva. The collection of saliva allows for a repeated collection, if needed, without the stress of drawing blood. Even with the advent of LFAs for diagnostic assays in recent years, there are relatively few such assays for viral detection in saliva. LFAs can work either as an immunoassay (LFIA) to detect viral-specific antibodies in the collected sample or to directly detect the virus particle present in the sample. The assay works based on liquid movement (containing the analyte to be detected) across a strip of polymeric material containing dry reagents that activate by the lateral movement of a liquid sample up the strip membrane. The specific detection area on the strip can contain either (1) viral-specific recombinant proteins, to which the viral antibodies in the saliva will recognize by binding to the recombinant viral protein; or (2) viral-specific antibodies on the test strip, to which the virus particle in the saliva will be recognized and bound. Despite the simplicity of this assay description, extensive development of these assays is required by the manufacturer to overcome assay limitations, such as lower analyte concentrations in the sample. Developers are utilizing various approaches such as using colloidal gold or carbon, fluorescent or luminescent materials, or colored latex beads. As an example, colloidal nanoparticles generate direct signals, whereas the use of other materials may require additional steps to derive analytical results, such as upconverting phosphor technology (UPT). UPT is based on sub-micron sized ceramic particles coated with lanthanides that absorb infrared light (excitation) and emit visible light (response signal). The particles functionalize with antibodies and antigens for use as labels on a lateral flow strip. There can be many steps in the assay development to consider including, sample composition and how the sample will flow along the strip, as well as the concentration of the analyte to be detected in the sample. Manufacturers must ensure that only the molecules of interest bind to the antigens or antibodies coated on the test strip.

CONCLUSION:
The use of lateral flow assays for detecting virus particles or virus-specific antibodies is a promising approach when applied to saliva-based assays. There are many advantages to both of these sample collection and detection assays. Although there are several commercial assays to detect Herpes viral nucleic acid in saliva, at present, there are few if any such assays available for detecting Herpes virus analytes (antibodies or virions) in saliva using an LFA.

By David Kilpatrick, PhD and Abbas Vafai, PhD

MKTG 1046 Rev A

June 16, 2020/by Viro Research

A Lateral Flow Assay for Detecting IgG/IgM for COVID-19

Diagnostics

INTRODUCTION:
Numerous labs are developing antibody assays to detect COVID-19. There are presently (as of May 1, 2020) four FDA approved assays for detecting IgG/IgM and three for detecting IgG only. Four of these assays use a lateral flow assay (LFA) architecture. All of the assays use the viral S1 glycoprotein as the antibody target. This short note will describe a typical LFA for COVID-19.

DISCUSSION:
A rapid point-of-care assay that detects either/or both IgG/IgM is critical for detecting spread on the infection through the population. A recent infection (<7days) is usually seen with the production of IgM, while older infections (>8 days since infection) detect the generation of IgG. Li et al. (2020) developed an assay that detects both IgG and IgM, detecting antibodies to the SARS-CoV-2 S1 spike protein. They purified the recombinant S1 antigen (MK201027) by protein A affinity chromatography and size-exclusion chromatography. They based the design of the S1 antigen on the published SARS-CoV-2 sequence (MK201027). Antibodies obtained from Sigma include bovine serum albumin (BSA), goat anti-human IgG and IgM antibodies, rabbit IgG, and goat anti-rabbit IgG antibodies. Shanghai KinBio Inc. provided 40Nm gold nanoparticle (AuNP) colloids, NC membrane, and plastic pad, and Whatman provided the glass fiber conjugate (GFC). Sigma produced the PBS. Hunan CDC, China supplied inactivated COVID-19 serum and negative serum samples of patients.

To prepare the AuNP conjugate, they added SARS-CoV-2 recombinant protein dissolved in PBS (1mg/ml) to the mixture of 1ml AuNP colloid (40nm in diameter, OD=1) and 0.1ml of borate buffer (0.1M, pH 8.5). After incubation for 30 minutes at room temperature, the mixture was centrifuged at 10,000 rpm at 4oC for 20 minutes. Next, and 1ml of BSA in PBS was added to the AuNP conjugate to be re-suspended after discarding the supernatant. They repeated the centrifugation and suspension twice, and the final suspension was in PBS. The AuNP-rabbit IgG conjugates were prepared/purified by the same procedure. The main body of the test strip consists of five parts, including plastic backing, sample pad, conjugate pad, absorbent pad, and NC membrane. Each component of the strip is pretreated as follows: the NC membrane was attached to a plastic backing layer for cutting/handling. Researchers immobilized the anti-human-IgM, anti-human-IgG, and anti-rabbit-IgG at test M, G, and control line C. Then, they sprayed conjugate pad with a mixture of AuNP-COVID-19 recombinant antigen conjugate and AuNP-rabbit-IgG. Sample pad was pretreated with BSA (3%, w/v) and Tween-20 (0.5% w/v) before use. To run the assay (at room temperature), researchers pipetted 20 ul whole blood sample (or 10 ul of serum/plasma samples) into the sample port, followed by adding 2-3 drops (70-100ul) of dilution buffer (10mM PBS) to drive capillary action along the strip. The test takes approximately 15 minutes to complete. If only the C line shows red, the sample is negative. Either M or G line or both lines turning red indicates the presence of anti-SARS-CoV-2-IgM or IgG, or both if IgG and IgM are in the specimen.

CONCLUSION:
Of the 397 blood samples (vein blood) from SARS-CoV-2 infected patients, 352 tested positive, for a sensitivity of 88.66%. Twelve of the blood samples from the 128 non-infected patients were positive, for a specificity of 90.63%. Also, 256 out of 397 (64.48%) were positive for both IgG and IgM. Patient finger stick blood was tested and showed that all positive/negative results matched with 100% consistency between vein and finger stick blood, indicating the use of this assay as a point-of-care test using fingerstick blood.

By David Kilpatrick, PhD and Abbas Vafai, PhD

Li, Z., Yi, Y., Luo, X., Xiong, N., Liu, Y., Li, S., Sun, R., Wang, Y., Hu, B., Chen, W., Zhang, Y., Wang, J., Huang, B., Lin, Y., Yang, J., Cai, W., Wang, X., Cheng, J., Chen, Z., Sun, K., Pan, W., Zhan, Z., Chen, L., & Zhang, Y. (2020). Development and clinical application of a rapid IgM‐IgG combined antibody test for SARS-CoV-2 infection diagnosis.

Journal of Medical Virology. https://doi.org/10.1002/jmv.25727

May 12, 2020/by Viro Research

Possible HHV-8 Protein Epitopes for Serological Detection

Diagnostics

INTRODUCTION:
Positive and negative Kaposi sarcoma-associated (KSA) individuals need an improved diagnostic assay for human herpesvirus-8 (HHV-8) detection. The selection of one or more viral epitopes that elicit strong immune responses is essential in developing this assay.
Read more

March 24, 2020/by Viro Research

Detection of HHV-8 Associated with Kaposi’s Sarcoma

Diagnostics

INTRODUCTION:
Human herpesvirus-8 (HHV-8) is a γ-herpesvirus which is related to the Epstein-Barr virus and is etiologically associated with Kaposi’s sarcoma (KS). The AIDS epidemic over the last 40 years has resulted in a high rate of KS due to immunosuppression. The seroprevalence for HHV-8 varies from 1-5% in the US to up to 80% in sub-Saharan Africa. A rapid, inexpensive diagnostic assay to detect HHV-8 would be very beneficial to screen those who may be potentially at risk for developing KS. Several immunological assays are available which require advanced instrumentation or technical staff to perform and analyze the results. At present, there is no lateral flow assay (LFA) available for HHV-8. The future development of an LFA for HHV-8 would be ideal in terms of patient confidentiality, analytical speed, and low cost. Read more

March 9, 2020/by Viro Research

Nanophosphor Lateral Flow Assay for Detecting HSV-2

Diagnostics

INTRODUCTION:
Human Herpes Simplex Type 2 (HSV-2) causes a life-long infection and occurs in over 10% of adult individuals. This report details the approach by Goux et al. (2019) to identify HSV-2 infections using a Smartphone to detect the nanophosphor signal of a lateral flow device.

DISCUSSION:
Lateral flow assays (LFAs) are needed to improve the detection of HSV-2 without the time, cost, and lack of privacy associated with a laboratory setting. As of December 2019, there are no commercially available gold nanoparticle LFAs for HSV-2. Laderman et al. (2008) described the development of a gold nanoparticle-based immunoblot test for HSV-2. This assay, called Sure-Vue HSV-2 Rapid test, had a sensitivity of 94% and a specificity of 98%. Goux et al. (2019) developed the assay to improve the clinical sensitivity and specificity.

To do this, Paterson et al. (2004) noted they used strontium aluminate persistent luminescent nanoparticles, which they had previously developed (PLNPs, nanophosphors) as the LFA reporters. These PLNPs have a long-lasting, bright glow excitation, which allows for a delay of emission measurement, reduced background autofluorescence, and eliminates the need for precision optical filters. The strontium aluminate doped with europium and dysprosium has a bright, long-lasting light emission, which is inexpensive and widely used in “glow-in-the-dark” signs and toys. Paterson et al. (2014) stated that strontium aluminate PLNP LFAs showed to have a higher analytical sensitivity than traditional LFAs. Goux et al. (2019) tested a panel of 21 human plasma and serum samples ranging from negative to strongly positive for HSV-1 and HSV-2 (PTH2020; SeraCare Life Science). Then, they mixed 20μl of human plasma or serum samples (10μl of sample + 25μl of buffer) with 15μl of anti-human IgG-PLNP conjugate. Finally, they dispensed the sample/nanophosphor mixture (35 μl) onto the LFA pad. Goat anti-human IgG nanophosphors bearing anti-HSV human IgG (if present in the sample) migrated up the membrane. A recombinant HSV-2 antigen immobilized at the test line captured the
nanophosphors. Unbound anti-human nanophosphors bearing human IgG migrated further up the strip until captured by the goat anti-human IgGs immobilized at the control line.

A custom LFA iPhone app on an iPhone 7 Plus Smartphone (Apple Inc.) in combination with a 3D-printed attachment imaged the LFA strips. The PLNPs were excited by turning on the iPhone light (4s, maximum intensity) and then cycling the camera’s flash. After a delay (~100ms after excitation), the phone camera acquired an image of phosphor emission. Researchers repeated the excitation/imaging cycle four times, and the four images were stacked together to reduce background noise and increase reproducibility. They were able to detect between 5.7 to 23.3 mg/ml of human IgG. They detected no crossreactivity to HSV-1 in 10 tested samples. For comparison, the LFA test strips were also imaged on a FluorChembased imaging platform with two 10W ultraviolet LED lights (395-400nm) and a CoolSNAP K4 CCD 2,048 x 2,048-pixel camera. PLNP were excited with the LEDs for 1 min and imaged with an exposure time of 1s and pixel binning of 4.

CONCLUSION:
The nanophosphor HSV-2 LFA had a sensitivity of 96.7%, with 100% specificity for detecting HSV-2 in the tested samples. This sensitivity was higher than that of commercially available rapid HSV-2 assays tested with the same panel. This smartphone-based nanophosphor LFA technology shows promise for private self-testing for sexually-transmitted infections.

By David Kilpatrick, PhD and Abbas Vafai, PhD

Goux, H. J., Raja, B., Kourentzi, K., Trabuco, J. R. C., Vu, B. V., Paterson, A. S., Blane, T., Lee, M., Truong, V. T. T., & Pedroza, C. (2019). Evaluation of a nanophosphor lateral-flow assay for self-testing for herpes simplex virus type 2 seropositivity. PloS One, 14(12).
https://doi.org/10.1371/journal.pone.0225365

Laderman, E. I., Whitworth, E., Dumaual, E., Jones, M., Hudak, A., Hogrefe, W., Carney, J., & Groen, J. (2008). Rapid, sensitive, and specific lateral-flow immunochromatographic point-of-care device for detection of herpes simplex virus type 2-specific immunoglobulin G antibodies in serum and whole blood. Clin. Vaccine Immunol., 15(1), 159-163.
https://doi.org/10.1128/cvi.00218-07

Paterson, A. S., Raja, B., Garvey, G., Kolhatkar, A., Hagström, A. E., Kourentzi, K., Lee, T. R., & Willson, R. C. (2014). Persistent luminescence strontium aluminate nanoparticles as reporters in lateral flow assays. Analytical Chemistry, 86(19), 9481-9488.
https://doi.org/10.1021/ac5012624

January 29, 2020/by Viro Research

Multiplexed Lateral Flow Device using the Laser Direct-Write Technique

Diagnostics

INTRODUCTION:
The detection of a single analyte in a lateral flow assay or lateral flow device (LFD) is a common, low cost, rapid diagnostic assay. There is, however, a need to detect more than one analyte in a single assay to further reduce the costs and time involved in the detection of multiple analytes. A problem with multiplexed assays is that cross-reactivity may sometimes occur with the detection of more than one analyte. He, Katis, Eason, and Sones (2018) presented an innovative method to reduce the potential cross-reactivity of multiple analytes.

DISCUSSION:
The authors used a novel approach for detecting multiple analytes by using a laser direct-write (LDW) method. These laser-created multiple channels allowed for the simultaneous detection of different analytes—in this case, C-reactive protein (CRP) and serum amyloid A-1 (SAA1), which aid in the diagnosis of bacterial infections—individually within each of the parallel channels without any cross-reactivity. As illustrated in Figure 1, from their report, shows the general layout of the reaction pad for this assay.

This multiple test method required neither multiple inlets nor increased sample volumes. The laser-etched channels completely removed any interference between individual detection sites positioned within the sample channel. The laser used for the LDW process was a 405 continuouswave diode laser (MLDTM 405 nm, Cobolt AB, Stockholm, Sweden) with a maximum output power of 110 mW. The photopolymer used for creating the boundary walls between individual channels was DeSolite® 3471-3-14 (DSM Desotech, Inc, Elgin, IL, USA). The dispenser platform used for the local deposition of the photopolymer onto the substrate was a PICO® Pμlse™ dispensing system (Nordson EFD, UK). A different reagent-dispensing system, the XYZ3210 dispense platform (Biodot, Irvine, CA, USA), was used for the local deposition of antibodies onto the reaction pad for the reaction of test lines and the control line. He et al. (2018) locally deposited the capture antibodies for both test lines (CRP & SAA-1) into individual channels. The appearance or absence of a test line signifies the presence or absence of either marker in the sample. The test results detected both CRP and SAA-1 at a concentration of 50ng/mL.

CONCLUSION:
The results show the laser-patterned LFDs performed equally well as the single LFDs and did not need increased device dimensions or additional sample volumes. The multiple isolated parallel flow-paths allowed for the individual detection of different analytes in each of the separated channels without interference or cross-reaction. Although they only used two channels, there was space on the assay for additional channels (for a total of six). Their proof of concept using laser direct-write channels for LFDs shows that this may be a viable approach for multiplexing lateral flow assays.

He, P., Katis, I., Eason, R., & Sones, C. (2018). Rapid multiplexed detection on lateral-flow devices using a laser direct-write technique. Biosensors, 8(4), 97.

December 17, 2019/by Viro Research

Three Color, Multiplex LFA

Diagnostics

INTRODUCTION:
Lateral flow assays (LFA) have been used for several decades to detect chemicals, biologically relevant proteins, toxins, metals, and specific disease-related analytes (either bacterial or viral). The low cost and ease of use make these assays ideal for point-of-care diagnostics. One of the disadvantages of LFAs has been that they are prone to false positive when used for detecting more than one analyte on a strip, e.g., multiplexing the assay. Nanomaterials are being used to overcome the disadvantages of LFAs. A review on multiplexing nanoparticle-based LFAs, Yahaya, Zakaria, Noordin, and Abdul Razak (2018)¹ detailed several nanomaterials which have been used as labels, such as colloidal gold (AuNPs), silver (AgNPs), carbon (CNPs), selenium (SNPs), quantum dots (QDs), up-converting phosphors (UCPs), dye-doped and magnetic nanoparticles (NPs). This report will detail the use of a three color multiplex assay using AgNPs.

DISCUSSION:
Detecting more than one analyte in a single assay reduces the time and cost for detecting multiple biological agents. Yen et al. (2015)2 designed such an assay for detecting dengue, Yellow Fever, and Ebola viruses in a single assay. The researchers used the size-dependent optical properties of silver nanoparticles (AgNPs) to develop a multiplexed LFA. Researchers conjugated triangular plateshaped AgNPs of varying sizes to antibodies that bind to specific biomarkers. Triangular plate-shaped AgNPs have narrow absorbances that are “tunable” through the visible spectrum. The growth of large AgNPs resulted in color changes based on the morphology change from spherical particles to triangular nanoplates. The AgNP colors were evident and distinguishable from one another when applied to paper and dried. These AgNPs were prepared for LFA by conjugating monoclonal antibodies to the NPs. It is combining antibodies with AgNP in a solution that results in antibody binding to the AgNP by electrostatic adsorption. The virus-specific antibodies recognized the dengue virus (DENV) NS1 protein (green), Yellow Fever virus (YFV) NS1 protein (orange), and Ebola virus, Zaire strain (ZEBOV) glycoprotein GP (red). A sandwich assay formed to capture the viral protein ligand (NS1 or GP) bound by both the antibody conjugated to the AgNP and the capture antibody loaded onto the test areas of the nitrocellulose strip. In a typical run, you load into the sample pad, the sample solution containing the antigen (NS1 protein of either DENV or YFV; GP of ZEBOV). The liquid migrates through the conjugate pad, where the antigen binds to the AgNP-Ab. The AgNP-Ab/ antigen complex then wicks through the strip, and they are captured by the antibodies (specific for each viral protein) “printed” at each test line. The reaction creates a colored band at the test detection area. For a positive result, the single test area was orange if YFV NS1 was present, green for DENV NS1, and red if ZEBOV GP was present. A positive control detection area, which is brown when all three AgNPs are present, is essential to demonstrate a complete test run and that the reagents worked as expected. In the absence of antigen, the test line was blank, indicating that the AgNP-Abantigen binding is specific, and the non-specific adsorption of the AgNP-Ab to the test line was undetectable.

CONCLUSION:
Yen et al. (2015)² demonstrated the ability to utilize the optical properties of AgNPs for multiplexing point of care diagnostics for infectious disease using their size-tunable absorption spectra. Results showed a capacity for three test lines, each with a different color based on which AgNP-Ab bound to a specific viral protein. The limit of detection of the biomarkers for each virus was 150ng/mL. A three test line approach may apply to multiplexing other biological analytes for developing new, improved multiplexed LFAs.

November 19, 2019/by Viro Research

Optimization of Lateral Flow Assays

Diagnostics

INTRODUCTION:
Lateral flow assays (LFAs) are a widely used diagnostic tool for point-of-care diagnostics. LFAs require optimization of the component assembly, sample pad, conjugate pad, nitrocellulose membrane (NCM), and absorbent pad on the plastic backing in preparation for a specific assay.

DISCUSSION:
Components to be optimized include analytical membranes (typically NCM), conjugate and sample pads, target analyte (e.g., recombinant protein), antibodies such as goat anti-human IgG if clinical human sera is being used to recognize the target analyte, blocking reagents/buffers, and the design of delivery geometry. Hsieh, Dantzler, and Weigl (2017) presented a helpful flow chart for the optimization procedures for LFAs¹. Once you set the goals for the assay, as outlined in Figure 2 of their paper, you will prepare three components for the LFA.

The first component is to capture the antibody test line and strip onto the NCM. Next, the detector antibody, such as a goat anti-human IgG Ab, is attached to a nanoparticle (NP), like gold, which generates the test signal. The last component is a running buffer (RB) that allows for the flow of the detector antibody-NP through the NCM. Multiple types of NCM (pore size, for instance) and various preparations of the Ab-NP and RB require titration to identify the best initial conditions. You must re-evaluate the Ab-NP concentrations If there is significant nonspecific binding (NSB), blocking reagents, or a revision of the analyte and or detection. For instance, Kim et al. (2016) showed to detect hepatitis B surface antigens, gold nanoparticles (GNP) 42nm in size showed superior performance in detecting the hepatitis B antigen².

However, Zhan et al. (2017) indicated they could increase the sensitivity of their LFA 256-fold by using GNPs 100nm in size³. Additional optimization can include the capture Ab striping conditions, such as the Ab concentration and buffer pH. Kim et al. (2016) used pH values of between 6-10 and Ab concentrations of 1-20 μg/ml and found optimum pH and concentrations were nine and 10μg/ml, respectively, with the 42nm GNP⁴. Analyte concentration on the test strip depends on the protein/detection Ab affinity. Kim et al. (2016) tested concentrations of between 1ng to 100μg, with between 1-10μg being optimum for their assay⁵.

CONCLUSION:
Determining necessary protocol parameters, assembly, and pretreatment is essential for any new LFA. As Borse and Srivastava (2019) point out, overlapping the sample pad and conjugate pad is vital for proper wicking⁶. The conjugate pad material needs to pull and hold the fluid better than the material used for the sample pad. The conjugate pad improves the fluid flow and its movement from the sample pad to NCM. Some LFA assays require special pretreatment for the strip, such as buffer cure or the use of a blocking agent. Borse and Srivastava (2019) noted heat treatment (55ºC for 20 min) removed residual moisture present on the NCM and facilitated the fluid flow⁷. They also found that the minimal concentration of capture antibodies was 1.2μg/ml for the appearance of the control line⁸. Finally, as shown in these references, each assay is unique and requires optimization in order to find the correct concentrations for the analyte and detection antibodies.

November 12, 2019/by Viro Research

Does Herpes Zoster Increase the Risk of Stroke and Myocardial Infarction

Increased Risk of Herpes Zoster (HZ) with Immune-Mediated Diseases

Using Lateral Flow Assays to Detect Herpes Viruses in Saliva

A Lateral Flow Assay for Detecting IgG/IgM for COVID-19

Possible HHV-8 Protein Epitopes for Serological Detection

Detection of HHV-8 Associated with Kaposi’s Sarcoma

Nanophosphor Lateral Flow Assay for Detecting HSV-2

Multiplexed Lateral Flow Device using the Laser Direct-Write Technique

Three Color, Multiplex LFA

Optimization of Lateral Flow Assays

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