May 20, 2020 | Adamas University

Immunodiagnostics- An Emerging Opportunity in Biotechnology

Student contributors:

Ananya Chakraborty, Rahul Dutta & Saloni Ghosh (all B. Tech Biotechnology, SEM-VI)

Immunodiagnostics is a technique of detection by using the ability of antibodies to bind to an antigen specifically. This is a very powerful, sensitive, and specific technique to detect a wide range of targets and has applications in the field of infectious diseases and autoimmune disease detection, cancer diagnostics, food safety, environment, etc.
Enzyme-linked Immunosorbent Assay (ELISA) is the most common term used in the field of immunodiagnostics and has contributed remarkably in the market grown to tens of billions of dollars (Fig.1.*).

Antibodies are molecules produced by our immune cells against antigens (foreign bodies) and have the property of binding to the antigens in a specific manner. These specific antibodies are easy to identify and we can get indirect proof of the presence of antigens.

A correct diagnosis is a key factor towards a better treatment of any ailment and recent advances in the field of immunodiagnostics have accelerated the treatment procedure. Scientists around the world are working constantly in developing more specific, more sensitive, and more cost-effective procedures of diagnosis.

Types of Immunodiagnostics

Radioimmunoassays (RIA), developed in the 1950s, were the earliest immunoassays and are one of the most sensitive methods that rely on radiolabeling. Nonetheless, their use has been precluded owing to the health risk due to radioactive exposure.

ELISAs replaced RIAs and are one of the most common and reliable diagnostic tools until now. The antigen-binding antibody is linked with an enzyme that gives a color reaction in the presence of a substrate. The market is constantly changing and researchers are developing new methods of immunodiagnostics that are furthermore accurate and cost-effective as discussed below:

Chemiluminescent immunoassays (CLIAs) couples immune reaction with chemical reactions that emit a photon (light) that can be easily detected.

Fluorescent immunoassays (FIAs): here antibody-binding antigen is tagged with a fluorescent chemical compound that can re-emit light upon excitation with a particular wavelength of light. Its utility may be further enhanced by the use of flow cytometers.

Lateral flow assays (LFAs) or Lateral Flow Test is a simple diagnostic device to confirm the presence or absence of a pathogen or some other entities that may be present in water, foodstuff, urine, or blood sample. It typically contains a line to confirm the validity of the kit and other line/s to confirm the presence/absence of the analyte. The most common example is the pregnancy test kit. They can be used at home i.e point of care testing and require the least training. They have wide applications in health, food and agriculture, and the environment sector.

Application of Immunodiagnostics

Immunodiagnostics has applications (but not limited to) in the following fields

Immunodiagnostics in oncology

Tumor-associated antigen or antibodies are the primary targets for cancer detection. This is also important in finding tumor relapse. Many cancer markers have been identified which can help in the accurate and early diagnosis of breast cancer, renal neoplasm, oral cancer, pancreatic cancer, and many others. For example, recently OPKO Health Inc. has got the US FDA approval to use microfluidics approaches, for prostate-specific antigen (PSA) testing to increase the accuracy of prostate cancer diagnosis and reduce unnecessary biopsies.

Immunodiagnostics in infectious diseases, Allergy, and autoimmune diseases

Polymerase chain reaction (PCR) is the best choice for the detection of an active infection. However, in many cases such as chronic infection (when a parasite goes in the dormant phase), immunoassays are more reliable. One limitation of ELISA is that it has low sensitivity in the case of the low analyte. This has been overcome by the use of IPCR or immuno-PCR which combines the amplification power of PCR and the sensitivity of ELISA. Here the primary immobilized antibody binds with a secondary antibody tagged with a nucleic acid capable of amplification (Fig.2.). Antibody-based approaches are now very helpful in the diagnosis of autoimmune diseases and allergic reactions. These methods also reduce the pain of invasive biopsies.

Immunodiagnostics in environmental pollution

The growing concern over environmental pollution and its link to human diseases have warranted the authorities to implement stringent policies. Immunodiagnostic tools have served greatly in this regard by detecting environmental contaminants like Dichlorodiphenyltrichloroethane(DDT), Pentachlorophenol (PCP), polychlorinated biphenyls (PCB), polycyclic aromatic hydrocarbons (PAH), organophosphorus pesticides, explosives, biological warfare materials, etc.

Immunodiagnostics in Food Technology

Immunodiagnostics has been widely used to monitor contaminants in different foodstuffs such as pathogenic microorganisms in food, staphylococcal enterotoxin B, melamine, and antibiotics in milk, wheat proteins in milk powder and pesticides in fruits, vegetables, and grains.

Immunodiagnostics in proteomics study

Proteomics deals with the protein profile produced by our cells. Mass spectrometry (MS) has been a useful tool in proteomics research. MS coupled with immunoassay provides extraordinary ability to perform targeted proteome screening. This is called mass spectrometric immunoassays (MSIAs) which is very sensitive and fast and can screen several proteins at a time.

Next-generation immunodiagnostics

Rapid advances in the Interdisciplinary field of biomedical instrumentation and immunodiagnostics is enabling us to step towards the next-generation immunodiagnostics. The major revolutionizing forces have been the microfluidics and magnetic beads based immunoassays.

Microfluidic assays

The precise control and manipulation of mass transport of minute quantity of fluids confined in a sub-millimeter space through capillary movement governs the principle to microfluidics and has wide application in physics, chemistry, and biotechnology. It has applications in paper-based immunodiagnostics like portable glucose detection and environmental testing.

Magnetic immunoassays

Magnetic beads are nanosized magnetic particles glued together with polymers that show the properties of superparamagnetism in the presence of an external magnetic field. These beads can be conjugated to antibodies for immunobased assays. These are superiority over ELISA or IFA. Magnetic beads based immunoassays are more sensitive, give no false-positive results, are easy in separation and automation. This makes it the next choice of researchers and clinicians.

Lab-On-a-Chip (LOC)

As the name suggests, this is a compact device of the dimension of a few square millimeters to a few square centimeters that can perform several laboratory functions in a single integrated circuit or chip. They are a part of the microelectromechanical system and involve microfluidics. The ability to handle picolitres of the sample, accurate automation, and high throughput make LOCs the future of diagnosis.

According to the latest research report[1] , the global immunodiagnostics market size was valued at USD 15,777.5 Million in 2017. This is projected to reach USD 22,732.7 Million by the end of 2025 with a CAGR of 4.7%. Increased applications of immunodiagnostics in diverse areas like disease burden, environment pollution, and food safety is attracting huge market and will provide an opportunity to biotechnologists skilled in this field in both R&D and production industries that manufacture diagnostic kits.

[1]https://www.fortunebusinessinsights.com/industry-reports/immunodiagnostics-market-100444. accessed on 19^th July, 2020.

*All the diagrams are self drawn by the author, Dr. Manoj Kumar Singh using Microsoft Paint 3D software.

MICRONEEDLE BASED VACCINES DELIVERY IN THE ERA OF NANOBIOSCIENCE

Student coordinator: -Rahul Dutta, Srijani Basak (3^rd-year B.tech biotechnology)

Nano-biotechnology is one of the fascinating and latest areas of science dealing with the conceptualization and designing of biological machines at the nanoscale level. The application of these biomachines also known as nanobots may vary from targeted drug delivery to real-time diagnostics. The nanoscale size of nanobots provides them easy access to targeted areas through the blood circulatory system and its biological origin reduces side effects largely post-application. In Adamas University, Nano-biotechnology is a part of the curriculum for the students of B.Tech Biotechnology.

The novel coronavirus SARS-CoV 2 (Severe acute respiratory syndrome coronavirus 2) isolated from Wuhan, China is posing a challenge to the scientific community and mankind throughout the world. It’s a mutated version of earlier identified similar viruses like SARS-CoV in 2003 and MERS-CoV in 2014 all belong to Coronaviridaefamily. The World Health Organization (WHO) coined this disease as COVID-19 and also declared it a pandemic in March 2020. COVID 19 common symptoms are – Fever, dry cough, shortness of breath which leads to severe acute respiratory syndrome that can turn fatal in a comorbid condition of the patient.

The primary virulence factor of SARS-CoV 2 is the surface Spike(S) protein. The S-protein is susceptible to mutations which bind strongly to the angiotensin-converting enzyme (ACE2) receptor present on the cell surface of the respiratory tract epithelium. ACE2 binding mediates the entry of virus particles into the cell cytoplasm where it undergoes replication. The coat protein then synthesized is a product of the translation of the RNA. Once fully assembled the virus particles are released from the vesicles. In the lungs, the virus infects trachea, bronchi which triggers inflammation. Subsequently, it damages the alveolar lining which causes fluid leakage in the alveoli and hinders oxygen exchange, leading to a reduction in oxygen saturation.

Among the increasing number of COVID-19 related casualties worldwide, a glimpse of hope and success can be seen from the news of vaccines that entered clinical trials recently. US-based biotechnology company Moderna, Inc, Inovio Pharmaceuticals, and China-based CanSino Biologics are main competitors in the race of vaccine development. Moderna, Inc leads by entering Phase-II clinical trials while casino recently received approval for initiating clinical trials.

As vaccine development in its way, its effective delivery in our physiological system is also very important. Nanotechnology-based designing of microneedle array is an upcoming area of drug delivery with a several-fold increase in potency. Microneedles consist of several projections of about 25 to 2000 um in length arranged in the form of an array on base support in the form of a patch. Microfabrication technology has the potential to revolutionize drug delivery and has successfully been demonstrated in clinical procedure includes in vitro and in vivo delivery of biomolecules. The first microneedle (MN) devices were developed using silicon but other materials like dextrin, glass, maltose, and other polymers have also been tested as per requirement. This procedure is called micromachining or micro-electromechanical systems(MEMS). Fabrication is done on thin polymeric films of the size of a contact lens, using suitable etching techniques.

Keeping in view, the upsurge of the novel SARS-CoV-2 pandemic and the subsequent demand for a lasting cure, researchers of the Pittsburgh School of Medicine, USA, and Carnegie Mellon University have jointly developed a Microneedle array that successfully delivered a COVID 19 vaccine to mice, eliciting an immune response. The vaccine PittCoVacc is delivered through microneedles on a fingertip-sized patch.

Unlike the mRNA vaccine, PittCoVacc uses synthetic fragments of the viral spike (S) protein, which is injected through the patch with about 400 dissolvable microneedles. The microneedle-based vaccine is highly scalable which an important factor in a pandemic situation is. The vaccine does not need refrigeration and maintains its potency at room temperature for a long time. Unlike traditional hypodermal vaccines, it creates transient micropores in the skin without stimulating the dermal nerves and causing pain. Microneedle-based vaccines penetrate the skin which has an abundance of cells triggering an immune response. As a result, the vaccine can be effective at much lower doses than traditional vaccines.

COVID-19 affecting people with a prevailing ailment and a window of opportunities for future biotechnologists

Student Contributors:

Nirsanka Roy (UG-VI B.Sc. Biotechnology) & Soumili Sen (UG-VI B.Sc. Biotechnology)

Coronavirus also is known as COVID-19(Corona Virus Disease 2019) originating in the city of Wuhan in 2019 has created a pandemic all over the world, shutting down all kinds of business around the world bringing it to a standstill. As of 29th April 2020, COVID-19 has affected over 213 countries affecting 2,959,929 people and the confirmed number of deaths recorded is 202,733. COVID-19 is not sparing anyone and affecting whoever is coming in contact with it. Now the question arises is that whether people who are suffering from chronic diseases or any kind of disease are at greater risk of getting affected by this virus or the threat is equal for all?

People with a chronic metabolic condition such as diabetes may be under a higher level of risk of severe illness from the COVID-19 virus. Cases of coronavirus in diabetic people are more serious than other people. A good immune system is required to fight the virus but a diabetic patient has a weak immune system which makes it more difficult to fight the virus if one is affected. The novel coronavirus “may thrive in an environment of elevated blood glucose.” Diabetes also slows the rate of healing response to any disease as it keeps the body in a low-level state of inflammation. A high level of glucose and constant state of inflammation makes it way more difficult to recover from COVID-19 infection.

In this modern age, one of the most common health issues is obesity. Obese people usually have other related medical problems. Abdominal obesity compresses the diaphragm, lungs, and capacity of the chest. Obesity is also known to cause chronic, inflammation in a low-grade scale and an increase in the circulation of pro-inflammatory cytokines, this may play a pivotal role in outcomes of COVID-19. Although the link between obesity and COVID-19 is still a hypothesis, however, if it turns out to be true then a great deal of world population is in danger. One of the severe chronic diseases is kidney failure or malfunction. Patients suffering from kidney failure have to undergo dialysis which can, in turn, weaken the patient’s immune system thus making it harder to fight against any infections. People who need kidney transplants usually need to take anti-rejection medicines which are basic immunosuppressive medicines. These medicines suppress the immune response towards any infection. Hence people taking this type of medicine are at more risk than normal healthy people. So, it is advised to stock up on supplies and maintain social distancing and limit going outside.

In this current pandemic situation, it is a unique opportunity for biotechnologists to flex their muscles and get to work. As biotechnologists, we can develop solutions that will help the human race to get back on their feet. In developing testing kits a biotechnologist plays a major role and in developing the vaccine many biotechnologists are in constant working mode. Advancements in genetic technology have helped in fighting the coronavirus, which is now easier, faster, and cheaper. As soon as the outbreak of the virus was reported to the World Health Organisation (WHO) it took only two weeks to isolate the virus and understand the entire sequence of its genome. Once the genome was mapped and made public biotechnologists began creating synthetic copies of the virus that is being used in research and creating a vaccine. According to Wetsman “this all happened much faster than it ever has before”, during the outbreak of the SARS virus (2002) creating a synthetic copy of the virus took months and hence delayed the process of vaccine making. “Speed is important because the outbreak is unlike anything public health experts have seen before”, she writes.

Fight against the invisible enemy is getting intense day by day affecting our daily lives. COVID-19 has shattered our social life and trapped us within the four walls of our house so that we can stay safe and healthy. So, in this pandemic, we as future biotechnologists need to think about what we can do to help the fight against the invisible enemy. We need to think of innovative technologies, new types of drugs and vaccines, better and cheap testing kits, or at least designing some new protective gear for the workers who are working on the field.COVID-19 has affected the world economy the most, many countries have faced their worst economic quarter in years. This economic turmoil has led to many people losing their job but there is a silver lining to this dark cloud for biotechnologists. COVID-19 has opened a window of opportunities for people pursuing a career in the field of biotechnology, microbiology, virology, and related subjects. Job opportunities have opened up in different sectors of industry such as:

Research Lab:As soon as the virus was reported by WHO research labs all around the lab started working to understand the working of the virus and how to find a cure and prevent the spread of the virus. For this reason, research labs would require more employees to have a biotechnology background so that they can help in the prevention of the virus.
Biotech Industry: Biomolecular and cellular processes are used by medical biotechnologists to develop new technologies and products that are used in the field of medicine and other related fields. The biotech industry recruits people temporarily from the medical biotechnological background and relative subject specialists to design medical devices, diagnostic kits, etc.
Drug Industry: A person working in the drug industry needs to know subjects like microbiology, molecular biology, genetics, recombinant DNA technology, and bioinformatics. A biotechnologist, having a blend of all these subjects can more efficiently design new drugs and vaccines. Now in this COVID-19 situation once a drug or vaccine for this virus is created, the drug industry will have to produce the drug on a large scale to meet the demand of the world. Huge demand for quality control and production management will open new opportunities for skilled biotechnologists.
Hospitals and Diagnostic Centres: Two types of tests are done for detection of COVID-19 first type is testing for infection and the second type is testing of antibodies. These both types of tests require various steps of using high tech equipment for detection of the virus for example in case of testing for infection first a nasal or throat swab is collected then the sample is developed using an RTPCR so that the amount of viral genetic material is increased and it can be detected. One must have proper knowledge and training to use such sophisticated equipment, so in this sector also there is a great scope for biotechnologists.

Research studies have shown that SARS-CoV and MERS-CoV both originated from bats. Since the SARS outbreak in 2002 research has started on coronavirus, different kinds of strains their effects, how they can spread, etc. In 2013 research showed that bat feces had a virus which was 96% similar to SARS-CoV 2. Later studies began by altering parts of coronavirus to find out whether that can transmit to humans or not. The answer is YES. This was back in 2015 but still, there is no cure. Now, among this outbreak, various researches should be conducted with coronavirus and identify the possible threats to humans and possible ways to nullify them.

Designing of vaccines and immunostimulating agents against COVID-19: A novel approach for human welfare

Student co-ordinator: Subidita Pal, Saloni Ghosh, (Students of UG VI B.Tech in Biotechnology)

Introduction

Our universe is currently facing a huge crisis due to the outbreak and challenges of COVID-19 globally. This virus which was identified in Wuhan Market, China in December 2019 uses the bat as the natural host. More than 233,000 persons died worldwide as of May 1, 2020. The identification of coronaviridae was done in the middle of 1960, which infected humans and a variety of mammals. Two coronaviruses (can cause infection of higher animals) were evolved since 2002 and showed outbreaks in humans: the first one was discovered at south China in 2003, i.e. SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus), and the later one at Saudi Arabia in 2012, which is MERS-CoV (Middle East Respiratory Syndrome Coronavirus). The recently discovered coronavirus is identified is known as SARS-CoV-2 and the disease associated with it is called or coronavirus disease 19 (COVID-19), a type of respiratory illness. The World Health Organisation(WHO) declared the disease mediated by this virus as pandemic on 11^th March 2020 due to its pattern of spreading throughout the universe.

SARS-CoV-2 carries a positive-sense single-stranded RNA as its genome. Infection by this virus is usually spread between individuals of the human population through close contact and via respiratory droplets produced from coughs or sneezes, which indicates its highly contagious nature. The symptoms of the disease can range from mild to severe. Individuals who are infected with the virus take 1 to 14 days to develop the symptoms. It enters into the human cells by binding to (ACE2) within the cytoplasm of the target cell.

Initiatives taken to discover the vaccine

Scientific researches are also running to fight against this virus are two different approaches: Vaccination and improvision of antibody-dependent cell-mediated cytotoxicity(ADCC).

The vaccine will be the ultimate weapon through which the whole world needs to fight this outbreak. On the other hand, the defense system of the human body is capable of generating immune response through the activation of both cell-mediated and humoral immune system upon recognizing and engulfing the foreign antigenic particles. Researchers throughout the world are studying to initiate a natural physiological mechanism within our body, which will make our body to become capable to eliminate this deadly virus. It nearly takes 12-18 months to invent a vaccine but our researchers are trying to achieve success within a small period although they need to be very efficient to avoid the spoil of work in haste.

Oxford University, Europe had its first human trial of Coronavirus vaccine. BothSARS-COV-1andSARS-COV-2arecloselyrelatedviruses.ButthevaccineforSARS-COV-1wasnotdevelopedproperly.ThecurrentcrisisdemandsoneforSARS-CoV-2.They are highly confident and optimistic about its chances. By the end of 2020, it is believed that the markets would launch vaccines in a small amount across the globe, where the success rate is still questionable for the kids and aged people. Most recently Italy developed a vaccine and passed. They injected the vaccine in mouse and it generates antibody which blocks the viral protein.

Discussion

“Better late than never” i.e. all outstanding and beneficial discoveries in the scientific field take time. Till then we need to boost up our immunity by exercising and treating the human body with natural household remedies to ensure prevention. This is the time where we have to come together as a community, take the initiative to prevent the transmission of disease from the infected person to noninfected person, to help the doctors(are involved in the treatment of COVID-19) and scientists(are involved to design vaccine and immunotherapeutics). In this way, the life-threatening condition that is mediated by the SARS-CoV2 can easily be overcome by us, which will be accompanied by a glorious sunrise of the corona free world.