Diaclone - Editos
- Patients’ Immune Battle Against COVID-19
- Covid-19 Latest News
- The Dark Side of CAR-T Therapy: CRS
- Biologically Active Antibodies
- What is the KD of my Antibody?
- Diaclone ELISPOT - It was 30 years ago today...
- Diaclone - The CAM family
- CART poster Diaclone
- Poster IL-8 Phage display
- Diaclone IL-17 Products
My stepfather entered the hallway, he seemed troubled by something. He isn’t the kind of man who usually shows his feelings. He used to pass by our house at least twice a week to discuss a while and to barter. I buy fresh eggs from my neighbour and trade them against fresh vegetables my stepfather grows.
But that morning, Luc wasn’t himself. His gaze averted, not the slightest of smile on his lips. He leaned gently towards me and whispered a few words.
His mother had just passed away. It all happened so suddenly. In the space of just a few days.
A brief review of the expected complications
1. Primary battle zone: From fever to lung infection
On the first day it starts with a fever for 94% of us. We feel exhausted in some cases (23%), and face muscle pain like the day after a big workout (15%). And we start coughing after 5 days. A lot, in fact (79% of reported cases) (1).
At this point, severe cases (15%, according to the Chinese CDC) start experiencing polypnea; an increase in the number of breathing cycles per minute and a decrease in the amplitude of breathing movements. In short, you are progressively running out of air.
Doctors are accustomed to polypnea (first described in 1889) and know to search the causes in lung or heart diseases. At the beginning of the pandemic, polypnea was the first symptom observed to attest worsening infection. On January 7th 2020, Chinese scientists released the first X-ray images and sample bronchoalveolar-lavage fluids to finally isolate and sequence viral RNA. This coronavirus was not like the others. Scientists just stumbled upon the newest member of the coronavirus family, the 7th one (2): COVID-19.
And curiously enough, they started observing something else that was occurring in patients.
2. Multiple battlefields: Beyond the lung infection
By mid-January 2020, in the hospital of Wuhan, the dialysis machines began to run out.
In the emergency rooms treating COVID-19, renal distress was raging: 23 patients out of a total of 85 exhibited acute renal failure (3).
In February, another hospital reported heart-related complications: 23% of the patients were suffering arrythmia, and 10% of them faced acute cardiac injury (4).
Lungs. Kidneys. Heart. Where does the list end?
These abnormalities followed on from one another to finally reach the neurological system. Acute cerebrovascular events spread among severely infected patients (36% of a total of 214 patients). Loss of smell, headaches, ataxia and nerve pains were proliferating (5).
But this time, the scientific community had a suspect.
A familiar suspect we have already encountered in many virus investigations.
3. Your immune system is fighting against you
Cytokine Release Syndrome (CRS) had just made its appearance at the top of the list of suspects.
Knowing where to look, scientists started to search for some clues. Tracking elevated concentrations of inflammatory cytokines and chemokines, they found that IL-6, IL-2, IL-1β, IL-8, IL-17, IFN-γ, TNF-α, IP10, MCP-1, IL-10 and IL-4 were all present in COVID-19 patients. There was no doubt, CRS was involved (6).
This disproportionate immune response from the host becomes unfavourable, deleterious, and leads to the failure of several vital organs and death.
But something even more surprising was recently revealed at the end of April.
During any viral infection, our immune response produces the first type of interferon - IFNa. This cytokine has the potential to protect patients and has consequently been used to treat hepatitis B and C.
In the particular case of COVID-19, scientists found that interferon alpha may induce ACE2 gene expression. Since ACE2 is the receptor of the virus on cells, it would mean that your immune system creates new entries for the virus to get into cells. This discovery is still awaiting confirmation.
More your immune system responds to the threat, more it helps the virus to destroy you.
If lungs were the first organs targeted by COVID-19, due to the high expression of ACE2 on the surface of epithelial cells, it appears that the viral infection triggers Cytokine Release Syndrome.
This immune reaction overdrive leads inexorably to the fall, one after the other, of all the patients’ organs.
Worse than that and due to the overexpression of the ACE2 gene, the cytokine IFNa seems to offer more cell portals to the virus, so accelerating the process of a highly probable death.
The best trick of the virus is being able to turn our proper immune system against us.
Diaclone offers a specific range of Antibodies, ELISA and ELISpot kits for COVID-19 research that can be used to monitor cytokine levels in samples.
Are you working on infectious diseases and want to monitor humoral immune response?
Better try DIACLONE’s products to succeed!
(1) Retrospective study on the first 191 patients in Wuhan, China. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study – Fei Zhou, Ting Yu, Ronghui Du.
(2) A Novel Coronavirus from Patients with Pneumonia in China, 2019 - Na Zhu, Ph.D., Dingyu Zhang, M.D., Wenling Wang, Ph.D., Xingwang Li, M.D., Bo Yang, M.S., Jingdong Song, Ph.D., Xiang Zhao, Ph.D., Baoying Huang, Ph.D., Weifeng Shi, Ph.D., Roujian Lu, M.D., Peihua Niu, Ph.D., Faxian Zhan, Ph.D., et al.
(3) Human Kidney is a Target for Novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection – Bo Diao, Chenhui Wang, Rongshuai Wang, Zeqing Feng, Yingjun Tan, Huiming Wang, Changsong Wang, Liang Liu
(4) Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus–Infected Pneumonia in Wuhan, China - Dawei Wang, MD; Bo Hu, MD; Chang Hu, MD; et al
(5) Neurologic Manifestations of Hospitalized Patients With Coronavirus Disease 2019 in Wuhan, China- Ling Mao; Huijuan Jin; Mengdie Wang; et al
(7) SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues - Carly G.K. Ziegler, Samuel J. Allon, Sarah K. Nyquist, Alex K. Shalek, Jose Ordovas-Montanes
The obstacle course of serological tests, viral reinfection, and reactivation.
With more than 3,000,000 cases and 200,000 deaths, and after three months of worldwide outbreak, scientists keep on learning about COVID-19 every day.
How can people be positively diagnosed twice to COVID? Are serological diagnostic tests 100% reliable? Which part of the population is more likely to be infected?
Let’s review the latest updates on what we know about this challenging virus, and the on-going assay developments.
1. The obstacle course of serological tests
The last few months have seen the fast emergence of rapid molecular tests to test presence of virus RNA in patients. Today, it’s the turn of serological tests to take centre stage. With serological tests, the aim is now to measure host immunity to the virus. But the game isn't won yet, and many challenges stand in the way of success:
- Specificity: Many seasonal coronavirus (HCoV-OC43, HCoV-HKU1, HCoV-229E, and HCoV-NL63) that cause colds circulate around the globe. They are not genetically identical to COVID-19, but sufficiently enough to give a false positive in case of low specificity tests.
- Sensitivity: A test with low sensitivity - despite a great specificity for the COVID-19 strain - could miss a weak immune response, and so provides a false negative result. A Chinese cohort of 175 patients demonstrated that immune response was highly variable and undetectable in 6% of the cases (Neutralizing antibody responses to SARS-CoV-2 in a COVID-19 recovered patient cohort and their implications - Fan Wu, Aojie Wang, Mei Liu, Qimin Wang, Jun Chen).
- Timing: If people have been infected less than 12 days ago, the seroconversion mechanism hasn’t occurred yet. You’re just measuring no biological material at all during the incubation phase.
- Individual targeted epitopes: It’s also possible that some individuals are developing antibodies against a different viral protein than the one of the test. Nevertheless, it has been reported that antibodies targets are mainly full-length S protein and its receptor-binding domain (A serological assay to detect SARS-CoV-2 seroconversion in humans, Fatima Amanat, Daniel Stadlbauer, Shirin Strohmeier).
- And finally: you need to show the relationship between seroconversion and seroneutralization. If patients are producing specific antibodies against COVID-19, in sufficient amounts, against the tested epitope, how can we be sure these antibodies are protective?
2. All men are created equal. Are you sure?
- Hypertension, cardiovascular diseases, diabetes mellitus, chronic obstructive pulmonary disease (COPD), and chronic kidney disease are aggravating factors of COVID-infected patients’ conditions (Emami A, Javanmardi F, Pirbonyeh N, Akbari A. Prevalence of Underlying Diseases in Hospitalized Patients with COVID-19: a Systematic Review and Meta-Analysis. Arch Acad Emerg Med. 2020;8(1):e35).
- Men are more sensitive than women (Based upon the data of the WHO website, 63% of all deaths were men). Oestrogen concentration? X chromosome? For the moment, we don’t have a clue about the root cause of such a difference, but this question should be addressed in the context of vaccine development.
- Children seem to be less affected by COVID-19 than adults. In a large report gathering 72 314 cases in China, only 2% were under 19 years old. Another report confirms that no ICU admissions or deaths were reported among persons aged ≤19 years in the USA (Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China. Severe Outcomes Among Patients with Coronavirus Disease 2019 (COVID-19) - United States, February 12-March 16, 2020). How can we explain this fact? One hypothesis is that children present less ACE-2 receptors and so less potential cell entries for the virus. We may also suggest that a young immune system doesn’t present the same features as a mature one. The level of antibodies could be lower and the risk of cytokine storm syndrome decreased.
3. Positively diagnosed to COVID-19. Not once, but twice!
Recently, South Korea reported some cases of cured patients tested positive to Covid-19. The Korea Centers for Disease Control and Prevention explained that of 7,829 people who have recovered from coronavirus, 2.1% were positively retested (163 cases).
How is this possible?
- One hypothesis is reactivation. The coronavirus could remain latent in the body and attack the respiratory organs again once it is reactivated. So far, this hypothesis has not been proven, and experts of HIV and HPV are not really in favour of such an option.
- Another explanation lies in the accuracy of diagnostic kits:
- Poor quality of the components of the test can be blamed. Nevertheless, regarding the offer on the market for such tests, and the level of expertise in South Korea in biotechnology field, we can be confident that the country has access to a large number of high-quality grade tests.
- Mutation of the virus may be an option. In fact, it’s unlikely that COVID-19 has already mutated in a drastic way. What’s in it for COVID at the moment if it mutates? It is spreading so easily among the worldwide population, there’s no necessity to change what already works pretty well.
- We might also suggest that fragments of the virus remain in the body. In fact, despite the seroconversion and the cured state of the patient, some residual virus RNA can persist in the bloodstream.
- Reinfection is another possibility. Some people with underlying weak immune system conditions, and presenting lower antibody levels, could face a second infection to the virus. Nevertheless, regarding the antibody immune response of patients, the probability of such event is estimated as being low.
It is clear that we are still in the early stages of our coronavirus discoveries. The obstacles of accuracy in serological tests are numerous, some subset populations present higher risks and the viral reactivation is not so clear for the moment.
However, we need to make decisions without full knowledge of the immunity of COVID-19. The stakes are too high to sit back and do nothing…
To effectively monitor Covid-19 infection & cytokine storm syndrome, DIACLONE has developed highly specific ELISA, Multiplex and ELISpot kits, already validated in a large number of studies.
CD? Did you say CD?
In immunology, it is really useful to identify the different types of leukocytes within a sample. But distinguishing leukocytes according to their morphological or functional characteristics is not an easy job.
However, scientists discovered that leukocytes express on their surface a large number of molecules which can be used as markers for their specific identification. The concept of cell immunophenotypic identification was born.
This identification is based on the detection of these membrane markers using monoclonal antibodies followed by analysis in flow cytometry. Since 1982, these markers have been designated according to the cluster of differentiation nomenclature or CD, followed by a number. And the combination of present (CD+) and absent (CD-) markers allows the identification of the state of maturation of the cells.
CD25: a difficult youth
As early as February 1988, a Diaclone monoclonal antibody anti-CD25, the B-B10 clone (now known as Inolimomab or Leukotac) was used in vivo to treat a severe case of acute Graft versus Host disease.
CD25, also known as the interleukin-2 high-affinity receptor alpha chain (IL-2Rα), has been observed on activated B, T lymphocytes and macrophages. Alternative names for the antigen are often used: IDDM10, IL2R, TCGFR, p55 or Tac.
Inhibiting the binding of IL-2 to CD25, anti-CD25 monoclonal antibodies were first developed as therapeutic agents to treat organ transplant rejection (Basilixumab, a Novartis chimeric mouse-human CD25 antibody, 1998 approval) and multiple sclerosis (Daclizumab, a Biogen humanized CD25 antibody, 1997 approval then withdrawn due to safety issues).
But since - as CD25 is constitutively and highly expressed on Treg cells - this marker was rapidly investigated as a potential target for inflammatory, auto-immune and cancer immunotherapies.
In 2013, ADC Therapeutics and Genmab partnered to develop a new antibody format, an Antibody Drug Conjugate (ADC), offering anti-CD25 a second life, this time in the oncology field. Camidanlumab was in the pipeline.
But tackling cancer with a Treg cell depletion tactic - like companies such as Hifibio Therapeutics have promoted since the beginning - was quite a contested therapy approach. So far, clinical trials have proven these strategies wrong. Until recently…
CD25: an oncology boat with the wind in its sails
- Anne Goubier and her team at Tusk Therapeutics made the headlines when they showed that a blockade of IL-2 signalling limits Teff responses. They consequently decided to develop a new version of anti-CD25 antibody that does not block the IL-2 signalling in order to deplete Treg and stimulate Teff at the same time. This promising antibody is going to be tested in cancer. But not by Tusk itself. At the end of 2018, Roche paid €70 million ($81 million) upfront to buy Tusk Therapeutics and in doing so getting complete control on this promising anti-CD25 antibody.
- In 2019, NIH conjugated a near infrared silica-phthalocyanine dye to a monoclonal antibody. This process led to a new cell-specific cancer therapy that locally kills specific cells in the tumour. In this particular context, comparing full length anti-CD25 antibody with its fragment version, it appears that the absence of the Fc portion leads to faster clearance and therefore promotes a superior activated T cell response in tumours.
- January 2020, Alderaan Biotechnology, a preclinical company focused on the development of anti-CD25 monoclonal antibodies for the treatment of cancer, announced that it raised €18.5 million in a Series A financing.
And any recent updates on dysregulated inflammatory response?
In 2019, a Japanese team from Kyoto University and Asahi Kasei Corporation paved the way to a new approach. Apheresis is a technique that makes it possible to take, via a machine, one or more blood components depending on the needs. The team demonstrated that the removal of Tregs from septic patients by apheresis was a good relief solution. However, the removal of cells other than Tregs caused the adverse effect of lowering the immune response against microbes. So, they decided to immobilise an anti-CD25 antibody on a polyethylene fibre to specifically remove Tregs without impacting the other components of PBMCs.
We might agree that it is not a definitive therapeutic solution like those described above for cancer, but this nice creativity - combining immunoaffinity purification with the apheresis technique - must be applauded and encouraged!
Do you have a current project on CD25? Do you want to specifically target Treg?
Diaclone has developed several anti-CD25 monoclonal antibodies for your research:
Clone B-B10: anti-IL2Ra, a biologically active mAb
Clone B-F2: anti-IL2Ra
Clone B-G3: anti-IL2Ra
CD25 ELISA Pair:
Clone B-G3: Capture antibody
The most common and potentially severe toxicity seen across trials using immunotherapies such as monoclonal antibodies, bispecific antibodies (Bi-specific T-cell engagers or BiTEs) and adoptive T-cell therapies (e.g. CAR-modified T-cell) is CRS.
But what does CRS mean?
CRS stands for Cytokine Release Syndrome. Cytokine Release Syndrome is a clinical syndrome resulting from generalized immune activation correlating with marked elevations of serum inflammatory markers and cytokines. The first clinical signs of CRS are fevers, myalgias, and fatigue. But starting from fever with or without constitutional symptoms (CRS grade #1), symptoms can rapidly evolve to hypoxia requiring O2 (grade #3) and finally death (grade #5).
But is the probability of a CRS event really alarming?
The risk of CRS is influenced by factors related to the type of therapy, underlying disease, and characteristics of the patient. Treatment with most conventional monoclonal antibodies carries a relatively low risk of CRS, whereas CAR-T therapies carry a significantly higher risk of CRS incidence.
With regard to recently approved CAR-T therapies, Yescarta was studied in 107 adults with large B cell lymphoma and ninety-four percent of treated patients developed CRS and 12% developed grade ≥3 CRS. Kymriah was tested in 68 paediatric and young adult patients with relapsed/refractory ALL and seventy-nine percent developed CRS with 49% developing grade ≥3 CRS!
But what can we do when CRS occurs?
Corticosteroids may be necessary in some cases, but in high doses have proven to have a detrimental effect on CARTs.
Extracorporeal blood purification techniques, such as high-volume hemofiltration, cascade hemofiltration, plasma exchange, and coupled plasma filtration adsorption, have also been reported as a potential alternative to treat CRS syndrome. But they remain time consuming and labour extensive solutions.
Since the initial observation that tocilizumab, an antibody against the IL-6 receptor, rapidly reversed severe CRS, the drug remains at the forefront of CRS treatment.
For the future, many CARTs in development incorporate suicide targets as methods to mitigate toxicity. Another approach is manufacturing CARTs that can be regulated without actually killing them, for example requiring infusion of an additional agent for activation.
But diagnosis is better than treatment.
Early prediction is necessary as the side effects appear rapidly - within 1 to 2 weeks - following CAR-T infusion. The elevation of the signature set of 24 different cytokines is then an interesting and crucial measure to monitor in patients.
The main cytokines implicated in the pathogenesis of CRS include effector cytokines released from activated T cells, but also cytokines mainly secreted by monocytes and macrophages. IFN-γ and sgp130 levels, for example, rise early and their elevation is different for severe versus non-severe CRS.
But we can also mention sIL6R, interleukin-6 (IL-6), interleukin-10 (IL-10), interferon INF-α, chemokines that are chemotactic for monocytes/macrophages (MCP1, MIP1α, MIP1β), granulocyte-macrophage colony-stimulating factor (GM-CSF), but also TNF-α, IL-1b, IL1RA, IL-2, sIL2Rα, C-reactive protein (CRP) and IL-8. Other biomarkers of endothelial cell activation, such as Angiopoietin-2 and von Willebrand factor, have also been recently described to predict CRS severity.
In order to effectively monitor the risk linked to immunotherapy, Diaclone has developed a large panel of standard recombinant cytokines and monoclonal antibodies targeting specific cytokines engaged in Cytokine Release Syndrome, as well as ELISA and DIAplex kits. As a first evaluation test, and for 7 of the targeted set of cytokines, Diaclone DIAplex is a particularly useful tool for providing a rapid response and with a reduced sample volume requirement starting from only a few µl.
Question: What am I? Antagonist, inductive, repressive, blocking or neutralizing.... Answer: Biologically active antibodies!
They are sometimes qualified as antagonist, sometimes as agonist. Sometimes named inductive, sometimes repressive, sometimes blocking, or sometimes neutralizing. But they are always called antibodies. Have you ever guessed what it was about? Yes, that’s them, the biologically active antibodies!
Depending on the nature of the antigen, a specific antibody can demonstrate different biological functions:
- if the target is a cell surface marker for instance, the desired effect of the mAb may involve proliferation (induction/activation effect through a signaling cascade ), inhibition, cell maturation or even the killing of the target cell. Cell depletion will then occur through the recruitment of immune mediators with the Fc portion of the mAb to trigger antibody-dependent cellular cytotoxicity (ADCC), antibody-mediated phagocytosis cytotoxicity (ADCP) or complement-dependent cytotoxicity (CDC).
- if the target is a soluble molecule such as plasma protein (TNF, VEGF…) or a drug, the binding may trigger a blocking effect. When bound to the mAb, these drugs are not able to interact with their normal targets anymore. Of course, blocking effect can also occur via the cell surface receptor like the well-known immune checkpoint inhibitors (targeting CTLA-4, LAG3, PD1 and PDL1).
- If the target is an infectious organism, the desired function of antibodies may be neutralization of the foreign host, so disabling the virus, bacteria or other. In fact, most licensed vaccines teach the body how to make neutralizing antibodies.
Cell proliferation induced by IL-6 and Neutralization by anti-IL-6 antibody
(a) Recombinant human IL-6 stimulates proliferation in XG1 cell line in a dose-dependent manner. The ED50 is typically 30-40 pg/mL.
(b) Proliferation elicited by rhIL-6 (1ng/ml) is neutralized by increasing concentrations of human IL-6 monoclonal antibody. The ND50 is 0.07nM .
Unfortunately, many difficulties can appear during the process of raising biologically functional antibodies. We can name:
- epitope masking by variable loops or multimerization event,
- the presence of cryptic binding domains,
- the very high binding affinity of the target for its receptor, which makes the generation of inhibitory antibodies extremely challenging,
- the very narrow window for neutralizing antibodies to act before the establishment of a virus infection and its genome integration into the cell host.
Either from a classical hybridoma approach or by phage display technique, DIACLONE has over the years learned to tackle all these obstacles to offer you the best possible antibody references. Thanks to its bioassay platform, all our references are highly validated via proliferation, cytotoxicity, neutralization or various cell functionality tests.
KD is the equilibrium dissociation constant, a calculated ratio of koff/kon, between the antibody and its antigen. The association constant (kon) is used to characterize how quickly the antibody binds to its target. The dissociation constant (koff) is used to measure how quickly an antibody dissociates from its target.
KD and affinity are inversely related. A high affinity interaction is characterized by a low KD, a fast recognizing (high Kon) and a strong stability of formed complexes (low Koff).
10-4 to 10-6 Micromolar (μM)
10-7 to 10-9 Nanomolar (nM)
10-10 to 10-12 Picomolar (pM)
10-13 to 10-15 Femtomolar (fM)
At Diaclone, we measure using the SPR (Surface Plasmon Resonance) technology and the Octet instrument. This technique can help you to rank your antibodies but also to better understand the performance of your antibodies.
For example, this ELISA development showed that the pair worked only when mAb1 is coated (Fig.1)
Fig. 1 : mAb1-mAb2 pairing evaluation
The determination of KD and association/dissociation profiles of antibodies by Octet permitted us to understand why the pair works only in one way.
Fig. 2 : Profiles of association dissociation of mAb1 (top) and mAb2 (bottom)
The mAb2 has a quick dissociation time (Fig. 2), so, when it’s coated and after all the washing steps, the antigen doesn’t stay on mAb2. Using this antibody as revelation avoids this dissociation which is linked to accumulated washing steps.
Octet analysis can also help to validate the accessibility of tags (HIS, GST…), to study the interaction receptor-ligand and the potential inhibition of interaction with an antibody, and to perform epitope binning (also known as epitope mapping or pairing).
The advantage of Octet compared to the highly popular Biacore technique is the lower price, the rapid execution of experiments and the small amount of proteins required.
Whatever the interaction to study, Octet is a valuable tool.
Use Diaclone’s expertise to further characterize your antibody
The Octet technology is fully integrated into our custom monoclonal antibody development so that it can be implemented to further study monoclonal antibody candidates.
A large range of custom services, dedicated to your needs:
· Biological activity: agonist or antagonist, secretion enhancer, blocking signal transduction, cellular growth activation or inhibition,….or any new model to design
· Effector activity: ADCC, ADCP or CDC
· Antibody applications: ELISA, Western Blotting, Flow cytometry…
· Antibody labeling
Contact us to discover how Diaclone can support your activity - firstname.lastname@example.org
A little bit of history ...
In 1983, two laboratories on opposite sides of the world described almost simultaneously what would later be called the ELISpot: Cecil Cervinsky in Gothenburg, Sweden and Jonathon D. Sedgwick in Perth, Australia. This “invention” was first dedicated to enumerate frequency of B hybridroma cells secreting an antigen specific immunoglobulin and then, a few years later, to measure the frequency of T lymphocytes secreting a specific lymphokine.
At that time, Monoclonal Antibodies were rare, the ELISA was still a very new technique and the specificity of most polyclonal antibodies was highly questionable, but with the proximity of the team of Dr Outcherlony at Gothenburg University, Cecil Cervinsky had the idea to imagine an “Antibody forming cell ELISA assay” in a gel matrix.
Since the first description, key advances have included the first use of membrane bottomed plate (nitrocellulose and then PVDF) that enables increased sensitivity, improvement in substrates and application of computer-assisted spot counting technologies (1993) and the commercialization of these technologies by numerous instrumentation companies.
The assay was called by a range of names including spot-ELISA, ELISA-spot, ELISA-immunospot and, of course, ELISpot ultimately chosen by the scientific community.
Diaclone’s ELISpot ...
ELISpot is now the method of choice in clinic for monitoring Interferon-gamma secretion in cancer immunotherapy and viral vaccine trials.
The Diaclone IFNg ELISpot assay is used in several clinical studies and published in Clinical Cancer Research by Dosset et al.,Godet et al. and Teixeira, L. et al.*
Diaclone ELISpot Kits and ELISpot Sets
Diaclone ELISpots (Enzyme-Linked Immunospot Assays) are highly specific immunoassays for the analysis of cytokine and other soluble molecule production and secretion from T-cells at a single cell level in conditions closely comparable to the in-vivo environment with minimal cell manipulation.
Utilising sandwich immuno-enzyme technology, Diaclone ELISpot and Dual ELISpot assays can detect both secreted cytokines and measure the frequency of single cells that simultaneously produce multiple cytokines or other effector molecules.
- ELISpot Kits: Pre-coated PVDF plate(s), Detection antibody, Alkaline Phosphatase conjugate, BSA and BCIP/NBT ready- to-use substrate buffer.
- ELISpot Sets: Capture and Detection antibody, Alkaline Phosphatase conjugate, BSA and BCIP/NBT ready-to-use substrate buffer.
- ELISpot Pair: Capture and Detection Antibodies.
- Dual ELISpot Sets: Capture and Detection antibodies for two analytes, Alkaline phosphatase conjugate, blocking reagent, Ready-to-use BCIP/NBT substrate buffer, Peroxydase conjugate, BSA, Ready-to-use AEC substrate buffer.
- Dual Fluorospot Sets: Capture antibody for two analytes, FITC-conjugated Detection antibody for cytokine 1, anti-FITC antibody green fluorescence conjugate, biotinylated detection antibody for cytokine 2, streptavidin-phycoerythrin conjugate, BSA.
Valuable Investigative Tool
ELISpot techniques are amongst the most-sensitive methods available (up to 400x more sensitive than conventional ELISA) for cytokine research and benefit from a technically easy performance, rapid detection time and no requirement for expensive equipment or analysis software.
- Highly Sensitive assay can allow the detection of a single cell out of 100,000
- Fast procedure following cell stimulation
- Accuracy and reliability are guaranteed as all our reagents have been validated according the ISO 9001:2000 quality systems
- Reagents generate well focused, defined and easy to analyse ‘spots’
- No cross reactivity with other human cytokines tested
- Extensive kit range across different species with high specificity and affinity for a number of different cytokines and soluble molecules
- Mono and Dual cytokine analysis available using both enzymatic and fluorescent detection systems
- ELISpot reagents available in a number of formats from whole pre-coated kits for easy analysis and increased throughput to matched Antibody Pairs a great tool for developing research
A NEW target for monitoring or treating cancers?
The cell adhesion molecules (CAMs) family includes more than 50 proteins with four main groups: immunoglobulin (Ig)-like CAMs, cadherins, selectins, and integrins.
Many cellular functions are directly linked to cell adhesion such as signal transduction, cellular communication and recognition, embryogenesis, inflammatory and immune responses, apoptosis and some of them also act as viral receptors (Cohen MB, Am J Clin Pathol. 1997, 107(1):56-63).
The metastatic dissemination of tumor cells is the leading cause of morbidity and mortality in patients with cancer since it designates the transition from a localized, potentially curable to a generalized, usually incurable disease (Makrilia N, Cancer Invest. 2009, 27(10)).
Across the years, it has become evident that the adhesion properties of neoplastic cells play a pivotal role in the development and progression of cancer. (Okegawa T, Acta Biochim Pol. 2004;51(2):445-57)
(Windisch R, Cancers 2019, 11(3), 311)
Changes in the expression or function of CAMs have been associated with alterations in the adhesive or signalling status of tumor cells, allowing them to acquire a more motile and invasive phenotype prognostic biomarkers or as potential therapeutic targets in malignancies.
Additionally, many of CAMs can be cleaved and released by proteolytic cleavage activity, and their soluble forms were found to be increased in serum levels of cancer patients. Even if elevated levels of soluble CAM are also observed in bacterial and viral infections or in acute inflammation, some of them have been identified to be interesting prognostic markers of cancer progression, such as EpCAM, described to be upregulated in colorectal cancer with clinical relevance (Han S, Ebiomedicine 2017; 20:61–69).
Diaclone has been interested for many years in the adhesion molecules and can provide antibodies against all of the selectin and integrin families, most of IgSF CAM family and against EpCAM, H-CAM, M-CAM, and BL-CAM.
Knowing that the soluble form levels could become an innovative tool of cancer monitoring, Diaclone has also developed ELISA kits for measuring serum levels of a wide range of sCAMs.
Diaclone continues its implication in innovative health technology with its participation in the development of IL-1RAP CAR T cells (Warda W Cancer Res February 1 2019 79 (3) 663-675) and in a CD123 CAR T cells project (both in collaboration with the INSERM U1098 research team in Besançon, France).
Diaclone is renowned as a specialist in antibody development - in particular CD markers and membrane expressed proteins. Diaclone’s competence in cell biology and antibody development coupled with its new molecular biology platform and the antibody engineering capabilities of the mAbexperts team (www.mabexperts.com) can provide:
A case study:
Diaclone, with over 30 years of experience and expertise in immunology products, offers a vast catalogue of mAbs and ELISA kits for use in research and diagnostic applications. A large number of Diaclone’s products are cytokine related and specifically Interleukins (IL).
The development of the anti-IL-8 antibody was initiated in 1991, according to Köhler and Milstein’s technique (Nature-1975, 256, p495-7); six fusions were carried out giving only one specific anti-IL-8 mAb, the clone B-K8, which is still used in Diaclone’s IL-8 ELISA kit paired with a rabbit polyclonal. The aim of this project was to replace the polyclonal.
At the beginning of 2019, a new project of anti-IL-8 mAb development was initiated with a new IL-8 mouse immunization. One part of the cells from the immunized animals was unsuccessfully fused using the classical mAb method and the second was processed by the Diaclone Phage Display Platform. The phage display method (Smith, Science-1985, vol. 228, n°4705, p1315-17) is an alternative way of developing mAbs using molecular biology. The platform is capable of obtaining a strong Fab library of IgG sequences from spleen cells coming from immunized mice.
The library obtained was screened in biopanning steps with IL-8 to select very specific clones. One hundred selected Fabs were tested in ELISA to keep the best candidates. Ten of them were selected and evaluated in pairs with B-K8. Finally, we obtained four good substitutes to the polyclonal anti-IL-8. These four Fabs were sequenced and reformatted by molecular biology in full IgG to be further validated in the IL-8 Elisa kit. As a result of this project, phage display technology has demonstrated its interest, notably in allowing mAb development which was particularly difficult to obtain with a classical fusion.
Case Study Poster available
It has been demonstrated that T helper cells can differentiate into IL-17 producing cells independent of Th1 or Th2 pathways establishing Th17 cells as a unique T-cell lineage challenging the classical two pathway theory.
This new T helper cell lineage is defined by its ability to produce IL-17A, IL-17F and IL-17A/F.
Diaclone has developed a large range of ELISA and ELISpot kits for the detection of Human IL-17A, IL-17F and IL-17A/F.
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