Description: Lyo NZYSupreme qPCR Probe Master Mix (2x) is an optimized and highly efficient freeze-dried reaction mixture developed for realtime PCR. This master mix was engineered with a dual hot-start enzyme control mechanism to provide the highest detection sensitivity. In addition, the latest developments in PCR enhancers have been incorporated in the Lyo NZYSupreme qPCR Probe Master Mix, including buffer chemistry and incorporation of highly robust engineered enzymes. This master mix does not contain ROX and it was specifically developed for probe-detection technology, including molecular beacons. For qPCR instruments that require ROX reference dye, please add ROX (Cat. No. MB406) according to the table presented in the section “ROX reference dye”. Lyo NZYSupreme qPCR Probe Master Mix (2x) is provided as a simple-to-use, stabilized 2x reaction mixture that includes all components for quantitative PCR, except sample DNA, primers, probe and water.
Features:
– Eco-friendly room temperature shipment
– Stable at room temperature for 1 month
– Dual hot-start mode
– Ultra-sensitive: detects low-copy number targets
– Batch-to-batch reproducibility
– Intra-batch reproducibility
– Simple and reproducible
– Compatible with multiple real-time platforms
Applications:
– Real-time qPCR
– Two-step RT-qPCR
– Developed for probe-detection technology
New Lyo qPCR Probe Master Mix (2x)
Lyo NZYSupreme qPCR Probe Master Mix (2x)
Description: Lyo NZYSupreme qPCR Probe Master Mix (2x) is an optimized and highly efficient freeze-dried reaction mixture developed for realtime PCR. This master mix was engineered with a dual hot-start enzyme control mechanism to provide the highest detection sensitivity. In addition, the latest developments in PCR enhancers have been incorporated in the Lyo NZYSupreme qPCR Probe Master Mix, including buffer chemistry and incorporation of highly robust engineered enzymes. This master mix does not contain ROX and it was specifically developed for probe-detection technology, including molecular beacons. For qPCR instruments that require ROX reference dye, please add ROX (Cat. No. MB406) according to the table presented in the section “ROX reference dye”. Lyo NZYSupreme qPCR Probe Master Mix (2x) is provided as a simple-to-use, stabilized 2x reaction mixture that includes all components for quantitative PCR, except sample DNA, primers, probe and water.
lyo NZYSupreme qPCR Probe Master Mix
Features:
– Eco-friendly room temperature shipment
– Stable at room temperature for 1 month
– Dual hot-start mode
– Ultra-sensitive: detects low-copy number targets
– Batch-to-batch reproducibility
– Intra-batch reproducibility
– Simple and reproducible
– Compatible with multiple real-time platforms
TSPOT.COVID is an ELISpot interferon gamma-release assay for SARS-CoV-2
•
TSPOT.COVID identifies a T cell response to SARS-CoV-2 spike S1 and N peptides
•
2–8 weeks post SARS-CoV-2 diagnosis TSPOT.COVID detected 98% of infections
•
In comparison, immunoglobulin G (IgG) serology detected 83% of infections in the same period
•
Cellular immune response activated sooner and lasted longer than antibodies
Abstract
Objective
To evaluate the performance of the T-SPOT.COVID test for identifying SARS-CoV-2-responsive T-cells in participants with SARS-CoV-2 infection.
Methods
The T-SPOT.COVID test uses ELISpot interferon-gamma release assay (IGRA) methodology to measure T cell responses to SARS-CoV-2 spike S1 and nucleocapsid peptides. T-SPOT.COVID and anti-N immunoglobulin (Ig) G serology tests were performed on blood from 186 patients with nucleic acid amplification test (NAAT)-confirmed-SARS-CoV-2 infection and 100 control group participants.
Results
In the 2–8 weeks after NAAT-diagnosed SARS-CoV-2 infection, the T-SPOT.COVID test detected 98.4% (63 of 64) of infected participants, while anti-N IgG serology detected 82.8%. In the first 2 weeks after diagnosis, during adaptive immune response activation, there were less reactive T-SPOT.COVID responses (75.7%, 28 of 37 infected participants) and many less seropositive responses (32.4%). Response numbers tapered after 8 weeks; however, T-SPOT.COVID test continued to detect most participants with confirmed infection (83.6%, 56 of 67) and continued to out-perform serology (52.2%). T-SPOT.COVID response due to cross-reactive T cells was ruled out by demonstrating that, of 44 control group participants with T cells responsive to 4 human common cold coronavirus peptides, only 1 was T-SPOT.COVID reactive.
Conclusion
The T-SPOT.COVID test performed well in detecting SARS-CoV-2-sensitized T-cells over many months
Introduction
Long-term protection from infectious agents, such as the SARS-CoV-2 virus, is mediated by T cells and antibody-mediated immunity of the adaptive immune system (
Sette and Crotty, 2021
). The T-SPOT.COVID test was developed to identify the presence of SARS-CoV-2-responsive T cells.
T cells contribute to the understanding of SARS-CoV-2 infections in many ways. T cells can identify past SARS-CoV-2 infections at a time when PCR tests would be negative and antibodies levels may be waning (
Dan et al., 2021
;
Gudbjartsson et al., 2020
;
Poland et al., 2020
). T cells can provide immune memory lasting for months (
Dan et al., 2021
) and perhaps years, as suggested by the discovery of T cells to the SARS-CoV-1 coronavirus 17 years after infection (
Le Bert et al., 2020
). T cells may act independently of antibodies to control a SARS-CoV-2 infection, as shown by the recovery of COVID-19 patients who lack detectable antibodies but have SARS-CoV-2-responsive T cells (
Gallais et al., 2021
;
Sekine et al., 2020
). T cells also show reactivity to numerous SARS-CoV-2 epitopes, so have the potential to protect against many SARS-CoV-2 variants (
Grifoni et al., 2020
;
Tarke et al., 2021
). T cell-based assays can probe the longevity of an immune response following a SARS-CoV-2 infection or vaccination (
Goletti et al., 2021
;
Liu et al., 2021
;
Reynolds et al., 2021
). These various roles suggest that a T cell assay can be a key contributor to SARS-CoV-2 investigations.
The T-SPOT.COVID test, an enzyme-linked immunospot (ELISpot) assay, identifies T cells in peripheral blood that release interferon-gamma (IFN-γ) in response to stimulation with SARS-CoV-2 peptides. The T-SPOT.COVID test builds on the T-SPOT platform (Oxford Immunotec) used worldwide for tuberculosis and cytomegalovirus testing and the research version, the T-SPOT Discovery SARS-CoV-2 test (
Liu et al., 2021
;
t.spot covid
Wyllie et al., 2021
). The T-SPOT.COVID ELISpot methodology is performed in many laboratories and offers a standardized comparison of T cell immunity among participants. In addition, ELISpot assays normalize the number of peripheral blood mononuclear cells (PBMCs), thus maintaining test effectiveness in participants with lymphopenia, a commonly reported condition in many COVID-19 patients (
Altmann and Boyton, 2020
) and immunosuppressed people.
The objective of this study was to evaluate the ability of the T-SPOT.COVID test to detect T cell responses in participants with or without a history of SARS-CoV-2 infection and to compare the T-SPOT.COVID test results with anti-N immunoglobulin (Ig)G serology results in the first several months after infection.
Materials and Methods
2.1 Participant recruitment
Participants for this single-center, cross-sectional study were recruited from patients who had attended the outpatient Primacare medical center in Fall River, Massachusetts, USA, between November 30, 2020, and March 24, 2021, a time of high demand for COVID-19 testing. Among other healthcare services, Primacare provided COVID-19 testing to anyone wanting or required to be tested. The New England Center for Clinical Research (NECCR) invited participants to join the study if they had received a positive SARS-CoV-2 nucleic acid amplification test (NAAT) at Primacare or if NECCR deemed them to be at low risk of SARS-CoV-2 infection. As this study was run independently from the participants’ healthcare providers, clinical data such as chest x-rays and hospitalizations records were not obtained. Informed consent and study approval were obtained from the Advarra institutional review board by NECCR at Primacare.
Confirmed-infection group: A NAAT, which detects the presence of the SARS-CoV-2 virus, was used to identify people infected with SARS-CoV-2 at the time of testing (
Rai et al., 2021
). Participants in the confirmed-infection group were recruited from asymptomatic and symptomatic patients who had had a positive SARS-CoV-2 NAAT result within the past 9 months. The date of the first positive NAAT result was considered the date of diagnosis of SARS-CoV-2 infection. Blood was drawn for Abbott SARS-CoV-2 chemiluminescent microparticle immunoassay (CMIA) anti-N IgG serology and T-SPOT.COVID tests between 0 to 249 days after diagnosis.
The analysis of responses was divided into 3 time periods: 0 to 2 weeks after diagnosis (0 to 14 days); 2+ to 8 weeks after diagnosis (15 to 56 days); and 8+ weeks after diagnosis (57+ days).
Control group: Many SARS-CoV-2 studies use frozen pre-pandemic blood for control samples; however, the T-SPOT platform requires fresh blood to ensure consistent results. Therefore fresh blood was obtained from control group participants prospectively recruited from individuals with low risk of prior SARS-CoV-2 infection. Requirements for enrollment included no current or prior signs or symptoms of COVID-19, no known contact with a confirmed SARS-CoV-2-infected individual, no prior history of a positive SARS-CoV-2 NAAT, no SARS-CoV-2 vaccination, and no prior diagnosis with SARS-CoV-1 or Middle Eastern Respiratory Syndrome (MERS). In addition, the BIOHIT HealthCare SARS-CoV-2 lateral flow anti-N IgM/IgG serology test was performed at enrollment, and the 1 person with a positive BIOHIT result was not enrolled. Blood was drawn at enrollment for testing with T-SPOT.COVID and the Abbott CMIA anti-N IgG serology test and anyone with a positive serology result was excluded from the control group.
2.2 T-SPOT.COVID test
The T-SPOT.COVID test includes over 250 SARS-CoV-2 peptides (15-mer peptides overlapping by 11 amino acids) in 2 antigen peptide pools; one pool contains peptides from the spike S1 protein, including the receptor-binding domain, and the other contains peptides from the nucleocapsid protein.
Blood samples for the T-SPOT.COVID test were processed and analyzed according to the manufacturer’s instructions. Briefly, blood samples were drawn into lithium heparin tubes which were shipped overnight to Oxford Immunotec (Abingdon, UK) in temperature-controlled shipping boxes. Next, the T-Cell Xtend reagent (Oxford Immunotec) was added to the samples, and PBMCs were isolated by density gradient centrifugation, washed, counted, and 250 000 cells/well were plated into 4 wells of a 96-well plate.
Description: SARS-CoV-2 Spike (S) glycoprotein:Spike (S) glycoprotein corresponds to one of the leading targets for COVID-19 disease. Present on the surface of Sars-CoV-2 virus, Spike S protein in a class I fusion protein that allows the virus to enter host cells.Variants package available:Some proteins of Sars-CoV-2 are identified as leading targets for COVID-19 therapies. SB-PEPTIDE offers a special pack pre-made peptide libraries of Spike protein including peptides from COVID-19 variants: UK COVID-19 variant B.1.1.7, South Africa COVID-19 variant B.1.351, Brazil COVID-19 variant B.1.1.248. SB-PEPTIDE offers additional plate containing only peptides with Spike S protein mutation of COVID-19 B.1.1.7, B.1.3.5.1 and B.1.1.248 (cited below).Variants package and Spike (S) glycoprotein peptide library can be used for T-cell assays, immune monitoring, antigen specific T-cell stimulation, T-cell expansion and cellular immune response.
Description: COVID-19 Spike protein (S) Virus Like Particle, packaged with GFP genomic material. Particles were concentrated and provided in PBS solution
During allergy skin tests, your skin is exposed to suspected allergy-causing substances (allergens) and is then observed for signs of an allergic reaction.
Along with your medical history, allergy tests may be able to confirm whether a particular substance you touch, breathe, or eat is causing symptoms.
Allergy skin tests are widely used to help diagnose allergic conditions, including:
Hay fever (allergic rhinitis)
Allergic asthma
Dermatitis (eczema)
Food allergies
Penicillin allergy
Bee venom allergy
Skin tests are generally safe for adults and children of all ages, including infants. In certain circumstances, though, skin tests aren’t recommended. Your doctor may advise against skin testing if you:
Have ever had a severe allergic reaction. You may be so sensitive to certain substances that even the tiny amounts used in skin tests could trigger a life-threatening reaction (anaphylaxis).
Take medications that could interfere with test results. These include antihistamines, many antidepressants and some heartburn medications. Your doctor may determine that it’s better for you to continue taking these medications than to temporarily discontinue them in preparation for a skin test.
Have certain skin conditions. If severe eczema or psoriasis affects large areas of skin on your arms and back — the usual testing sites — there may not be enough clear, uninvolved skin to do an effective test. Other skin conditions, such as dermatographism, can cause unreliable test results.
Blood tests (in vitro immunoglobulin E antibody tests) can be useful for those who shouldn’t or can’t undergo skin tests. Blood tests aren’t used for penicillin allergy.
In general, allergy skin tests are reliable for diagnosing allergies to airborne substances, such as pollen, pet dander and dust mites. Skin testing may help diagnose food allergies. But because food allergies can be complex, you may need additional tests or procedures.
Types of allergens
Allergens are substances that can cause an allergic reaction. There are three primary types of allergens:
Inhaled allergens affect the body when they come in contact with the lungs or membranes of the nostrils or throat. Pollen is the most common inhaled allergen.
Ingested allergens are present in certain foods, such as peanuts, soy, and seafood.
Contact allergens must come in contact with your skin to produce a reaction. An example of a reaction from a contact allergen is the rash and itching caused by poison ivy.
Allergy tests involve exposing you to a very small amount of a particular allergen and recording the reaction.
Insect sting allergy tests
Why allergy testing is performed
Allergies affect more than 50 million people living in the USA, according to the American College of Allergy, Asthma, and Immunology. Inhaled allergens are by far the most common type. Seasonal allergies and hay fever, which is an allergic response to pollen, affect more than 40 million Americans.
The World Allergy Organization estimates that asthma is responsible for 250,000 deaths annually. These deaths can be avoided with proper allergy care, as asthma is considered an allergic disease process.
How allergy testing is performed
An allergy test may involve either a skin test or a blood test. You may have to go on an elimination diet if your doctor thinks you might have a food allergy.
AIRBORNE ALLERGY SCREEN KIT
Skin tests
Skin tests are used to identify numerous potential allergens. This includes airborne, food-related, and contact allergens. The three types of skin tests are scratch, intradermal, and patch tests.
Your doctor will typically try a scratch test first. During this test, an allergen is placed in liquid, then that liquid is placed on a section of your skin with a special tool that lightly punctures the allergen into the skin’s surface. You’ll be closely monitored to see how your skin reacts to the foreign substance. If there’s localized redness, swelling, elevation, or itchiness of the skin over the test site, you’re allergic to that specific allergen.
If the scratch test is inconclusive, your doctor may order an intradermal skin test. This test requires injecting a tiny amount of allergen into the dermis layer of your skin. Again, your doctor will monitor your reaction.
Another form of skin test is the patch test (T.R.U.E. TESTTrusted Source). This involves using adhesive patches loaded with suspected allergens and placing these patches on your skin. The patches will remain on your body after you leave your doctor’s office. The patches are then reviewed at 48 hours after application and again at 72 to 96 hours after application.
Blood tests
If there’s a chance you’ll have a severe allergic reaction to a skin test, your doctor may call for a blood test. The blood is tested in a laboratory for the presence of antibodies that fight specific allergens. This test, called ImmunoCAP, is very successful in detecting IgE antibodies to major allergens.
Elimination diet
An elimination diet may help your doctor determine which foods are causing you to have an allergic reaction. It entails removing certain foods from your diet and later adding them back in. Your reactions will help determine which foods cause problems.
Diagnostic accuracy of rapid diagnostic tests for the early detection of leptospirosis
Abstract
Background
Leptospirosis is often misdiagnosed with several other tropical febrile illnesses in Malaysia due to similarities in clinical manifestations. Although treatment regimens could be started based on clinical judgments, early diagnosis has become paramount as a guide to chemotherapeutic interventions. Confirmed laboratory diagnosis through MAT or PCR is time consuming and usually available only in reference laboratories and not practical in healthcare settings. Rapid and easy to perform diagnostic tests are widely used in these settings as the point of care diagnosis. The present study was undertaken to compare the diagnostic performance of two IgM based immunodiagnostic assay kits for acute leptospirosis.
Methods
A total of 50 serum samples were collected from patients clinically suspected for acute leptospirosis on admission in the Hospital Serdang, from June 2016 to June 2017. All the samples were subjected to MAT, lipL32 PCR and the two rapid tests (Leptocheck-WB and ImmuneMed Leptospira IgM Duo Rapid test).
Results
Out of the 50 clinically suspected patients sampled, 19 were confirmed positive for leptospirosis. Six (12%) were confirmed by MAT and 13 (26%) by PCR. Similarly, of the 50 clinically suspected cases, 17 (34%) showed positivity for Leptocheck-WB and 7 (14%) for ImmuneMed Leptospira IgM Duo Rapid test. The overall sensitivity and specificity was 47.37% and 80.65% for Leptocheck-WB, and 21.05% and 90.32% for ImmuneMed Leptospira IgM Duo Rapid test. In another set of previously confirmed MAT positive samples (1:400–1:3600) obtained from a reference laboratory, Leptocheck-WB showed higher sensitivity (90.72%) than ImmuneMed Leptospira IgM Duo Rapid test (40.21%), and comparable specificity for ImmuneMed Leptospira IgM Duo Rapid test (88.89%) and Leptocheck-WB (82.86%).
immunemed leptospira rapid
Conclusion
The sensitivity was higher for Leptocheck-WB and had a comparable specificity with ImmuneMed Leptospira IgM Duo Rapid test. Therefore, based on the present study, Leptocheck-WB is found to be a more sensitive rapid immunodiagnostic test for acute leptospirosis screening in hospital settings.
Introduction
The neglected tropical illness leptospirosis caused by the spirochete Leptospira, is now an alarming re-emerging zoonosis with a worldwide distribution. In Malaysia, leptospirosis is gazetted as a notifiable disease since December 2010. The number of cases according to the data from the Ministry of Health Malaysia (MOH) showed an increase from 3665 in 2012 to 5284 in 2016. Leptospirosis is a biphasic infection, the first phase (acute or septic phase) commences from 3 to 10 days of disease onset and the second phase (immune phase) ranges from 7 to 14 days .
During the acute phase, the bacteria can be found in the blood and then migrate and reside in the kidney where it continues to be shed in the urine. While in the second phase, a detectable number of antibodies develops and this stage coincides with the disappearance of the bacteria in the blood. Confirmatory laboratory diagnosis for leptospirosis involves testing for antigen (bacteria by culture or PCR of Leptospira pathogenic genes) in the first phase and antibodies in the second phase through the gold standard microscopic agglutination test (MAT).
Culture is not suitable for early diagnosis as Leptospira takes two weeks to four months to grow. On the other hand, PCR based detection, although gives a confirmatory diagnosis, it involves DNA extraction, technical expertise, expensive PCR machines and reagents, which limits the feasibility in many health care facilities. MAT is technically tedious and interpretations are very subjective and most importantly requires a greater panel of live leptospiral cultures to serve as antigens with the regular incorporation of new local and international serovars.
In Malaysia, MAT is available only at the National Leptospirosis Reference Centres such as the Institute for Medical Research located at capital Kuala Lumpur as well as the Zonal Public Health Laboratories. Considering all these shortcomings and challenges, a rapid test is highly desired and mostly preferred in hospitals in Malaysia, as it is a point of care test that can be performed in-house, and it is fast, technically simple and can be easily interpreted. It is pertinent to note that, there are several rapid tests commercially available which detect IgM antibodies produced against Leptospira antigens in the human serum. However, the sensitivity and the specificity vary for different kits in different geographical regions. To date, only two studies have evaluated commercially available rapid diagnostic kits for acute leptospirosis in Malaysia.
One of these studies, evaluated two commonly used rapid leptospirosis serological tests in Malaysia, Leptorapide(Linnodee, Northern Ireland) and VISITECT-LEPTO (Omega Diagnostics, Scotland, UK) reported limited diagnostic value in detecting acute leptospirosis as they showed lower sensitivities and specificities [11]. However, a more recent evaluation study on the IgM Duo Rapid test kit from Korea (immunochromatographic assay), showed a diagnostic sensitivity of 73% and specificity of 90%.
Nonetheless, a point of note regarding the aforementioned studies is that, both of them were performed on previously confirmed MAT/PCR positive samples rather than a prospective clinical evaluation of samples from patients in a hospital setting. As elsewhere, in Malaysia few serovars isolated locally are frequently observed among patients. Hence, it is also important to determine the diagnostic efficacy of any RDT against the locally isolated serovars as well.
Oxford Immunotec Global PLC is a global, high-growth diagnostics company. We bring energy and invention to a world in need of diagnostic truth.
Our leading product, the T-SPOT®.TB test, is used for diagnosing infection with Tuberculosis, the world’s largest cause of death from infectious disease.
The Human Immune System
The human immune system is composed of three principle branches: Innate Immunity, Cellular (or T cell) Immunity and Humoral (or B cell) Immunity.
Despite these three branches and the multitude of immune cell types, the diagnostic world has only largely embraced antibodies. Leveraging our T-SPOT technology, Oxford Immunotec is one of the few companies to focus on standardising T cell and innate immunity measurements. Our T-SPOT technology gives us a unique toolbox to develop proprietary tests based on prosecuting the previously unexploited T cell and innate immunity branches of the immune system.
T-SPOT Technology
Our proprietary T-SPOT technology measures immune cell responses at a single cell level. Our technology studies the responses of living T cells and innate immune cells, which gives us a direct window into the immune system.
Simplistically, the technology starts with a blood sample obtained through a standard blood collection tube from which white blood cells, or WBC’s, are separated and purified. The cells are quantified and placed into specially designed plates where they are challenged with antigens specific to the disease under study. Disease-specific cells responding to these antigens will release immune messenger molecules, called cytokines. We then use chemistry to allow us to visualise those WBCs releasing cytokines (and hence those which react to the antigen), resulting in a spot on the bottom of the plate, corresponding to the footprint of an individual reacting WBC. Finally, we use an automated image analysis system to identify and count each of these spots to give a quantitative readout. That quantitative readout gives us the frequency of responsive disease-specific cells.
The exquisite sensitivity afforded by single-cell resolution is key to the platform’s applicability across different areas of medicine. This is because, for many conditions, the frequency of T cells or innate immune cells can be very low. Our technology has the ability to detect an individual reacting cell in a population of 250,000 white blood cells.
t spot tb oxford immunotec
Because we always standardise the number of cells used in each assay, we can compare responses over time to a common standard. It is this normalisation of the sample that makes longitudinal measurement valid and the platform well-suited for monitoring applications.
The sensitivity of the T-SPOT platform, as well as its use for monitoring applications, are two of many reasons why this technique has key advantages over other technologies, such as ELISA.
About the T-SPOT.TB Test
The T-SPOT.TB test is a single-visit blood test for TB screening and is one of only two recommended alternatives to the tuberculin skin test (TST) – otherwise known as purified protein derivative (PPD) test, or the Mantoux test. The T-SPOT.TB test is available in over 50 countries including Europe, China, Japan, and the US, and is recognized by the WHO as one of the 100 essential diagnostic tests that should be available in every country. The T-SPOT.TB test uses a standardized sample, reducing the influence of factors in the blood which may affect performance, and normalizing for cell number variation.
The test has been shown to have a sensitivity of 95.6% and a specificity in excess of 97.1% and is able to maintain performance even in samples otherwise difficult to test, such as samples from immunosuppressed patients. The T-SPOT.TB test does not cross-react with the BCG vaccine. Automation of the T-SPOT.TB test is possible using the T-Cell Select reagent kit. Automation solutions are available for the low, medium, and high throughput settings2. The T-Cell Select reagent kit also enables samples to be stored at ambient temperature for up to 54 hours after blood is collected.
About Oxford Immunotec
Oxford Immunotec Global PLC is a global, high-growth diagnostics company. We bring energy and invention to a world in need of diagnostic truth. Our leading product, the T-SPOT.TB test is used for diagnosing infection with Tuberculosis, the world’s largest cause of death from infectious disease. The T-SPOT.TB test has been approved for sale in over 50 countries, including the United States, where it has received pre-market approval from the Food and Drug Administration, Europe, where it has obtained a CE mark, as well as Japan and China.
