Immunology And Molecular Diagnostics

Immunology is the science which deals with the study of immune system and immune response to invading of foreign organisms. The study of the molecular and cellular components that comprise the immune system, including their function and interaction, is the central science of immunology. Immunity is defined as a specific reaction by which the foreign organisms are inactivated, inhibited and destroyed. The whole process is carried out by immune system of our body. Immunology (in general) deals with the physiological functioning of the immune system in states of both health and disease; malfunctions of the immune system in immunological disorders, the physical, chemical and physiological characteristics of the components of the immune system. The immune system has been divided into innate immune system, and acquired or adaptive immune system of vertebrates, the latter of which is further divided into humoral and cellular components. An immunologist is a research scientist who investigates the immune system of vertebrates.

The immune system in our body is a defensive system in host which consists of widely distributed cells, tissues and organs which recognize foreign substances and many micro-organisms and then neutralizes or destroys them. The cell which is responsible for both non-specific and specific immunity mainly consists of the leukocytes or white blood cells. Leukocytes generally originate from pluripotent stem cell in the fetal liver and in the bone marrow of the animal. Pluripotent stem cells which are present in the bone marrow get divided into two blood cell forms. First is the lymphoid stem cell which gives rise to B cells, T cells and natural killer cells (NK cells). The common myeloid progenitor cell gives rise to the granulocytes (neutrophils, eosinophils, basophils), and monocytes gives rise to macrophages and dendritic cells. There is unknown procedure that give rise to mast cells, megakaryocytes produces platelets, and the erythroblast produces RBCs.

3.1 ANTIGENS Adaptive immune responses generally arise as a result of the foreign compounds. The compound that generates the response is referred to as antigen. An antigen is an agent which is capable of binding specifically to components of the immune response, such as the B cell receptor (BCR) on B lymphocytes and soluble antibodies. The ability of an antigen to bind with antibodies and with cells of immune system is referred to as antigenicity. 3.1.1 Functional Distinction Between the Antigen and Immunogens The compound that generates the immune response is generally referred to as antigen; it is more appropriately called an immunogen and is therefore known as immunogenic. The difference between the two (antigen and immunogen) terms is necessary because there are certain compounds which are incapable of inducing an immune response, but then also they are capable of binding with components of the immune system that have been induced specifically against them. Thus all immunogens are antigens, but all antigens are not immunogens. Some small molecules (haptens) are antigenic but incapable of inducing a specific immune response by them. Therefore, they lack immunogenicity. The study of antigen-antibody reactions in vitro is called serology. Serological reactions are the basis for all diagnostic immunology tests.

Monoclonal antibodies (MAb) are produced by one type of immune cell (that are all clones of a single parent cell) and are mono-specific antibodies. To detect or purify a particular molecule or substance (polypeptide hormones, tumor markers, cytokines), we can generate specific monoclonal antibodies against that particular molecule/substance. Thus, monoclonal antibodies (MAb) are important reagents used in biomedical research, in diagnosis of diseases, and in treatment of such diseases as infections and cancer

Various applications of monoclonal antibodies can be broadly classified into three categories: 1. Diagnostic 2. Purification 3. Therapeutic In diagnostic applications monoclonal Abs are used to detect specific antigen or antibody. In purification applications, property of specific interaction of an antibody to the antigen is exploited to purify antigen present in small quantities as a mixture with other molecules. The purification and diagnostic applications come in “in vitro” uses. Therapeutic applications include treatment and protection from diseases and come under “in vivo” uses of monoclonal antibodies. Monoclonal antibodies (mAb) are important reagents used in biomedical research, microbio- logical research, in diagnosis of Hepatitis, AIDS, influenza, herpes simplex (various bacterial and viral diseases), and in treatment of such diseases as infections and cancer. onoclonal antibodies form the basis of a number of diagnostic tests under in vitro conditions,. For example, monoclonal antibodies against the hormone (human chorionic gonadotrophin, hCG) can detect pregnancy only 10 days after conception. Rapid diagnosis of hepatitis, influenza, herpes simplex, and Chlamydia infections is done with the aid of specific monoclonal antibodies. “Monoclonal antibodies are playing a valuable role in diagnostic medicine in tests to determine the concentration of specific proteins in the blood or urine. For example, an unusually high blood level of a prostate-specific antigen, which is measured by its interaction with a monoclonal antibody, provides an early warning that a man may have developed prostate cancer. Antibodies can also be used in protein purification. When a purified antibody is added to a crude mixture of proteins, the specific protein being sought selectively combines with the antibody and precipitates from solution. Monoclonal antibody diagnostic kits are being used routinely to identify communicable dis- eases including transfusion transmissible infections. More than 300 different monoclonal antibody diagnostic products are currently available. Such monoclonal antibodies are produced by in vitro and in vivo method

MHC complex is a large genomic region or group of genes found in most vertebrates on a single chromosome that codes the MHC molecules which plays a vital role in immune system. Major histocompatibility antigens (also called transplantation antigens) mediate rejection of grafts between two genetically different individuals. HLA (human leukocyte antigens) were first detected on leukocytes and so they are called MHC antigens of humans. H-2 antigens are their equivalent MHC antigens of mouse. A set of MHC alleles present on each chromosome is called an MHC haplo- type. Monozygotic human twins have the same histocompatibility molecules on their cells, and they can accept transplants of tissue from each other. Histocompatibility molecules of one indi- vidual act as antigens when introduced into a different individual. George Snell, Jean Dausset and Baruj Benacerraf received the Nobel Prize in 1980 for their contributions to the discovery and understanding of the MHC in mice and humans MHC gene products were identified as responsible for graft rejection. MHC gene products that control immune responses are called the immune response genes. Immune response genes influence responses to infections. The essential role of the HLA antigens lies in the induction and regulation of the immune response and defence against microorganisms. The physiologic function of MHC molecules is the presentation of peptide antigen to T lymphocytes. There are two general classes of MHC molecules: Class I and Class II. Class I MHC molecules are found on all nucleated cells and present peptides to cytotoxic T cells. Class II MHC molecules are found on certain immune cells themselves, chiefly macrophages, B cells and dendritic cells, collectively known as professional antigen-presenting cells (APCs). These APCs specialize in the uptake of pathogens and subsequent processing into peptide fragments within phagosomes. The Class II MHC molecules on APCs present these fragments to helper T cells, which stimulate an immune reaction from other cells.

Cytokines (Greek cyto - cell; and, kinos - movement) are small cell-signaling protein molecules. “Cytokine” refers to the immuno-modulating agents, such as interleukins and interferons. Cytokines are secreted by the cells of the immune system and glial cells of the nervous system and are used extensively in intercellular communication. Cytokines can either be proteins, peptides or glycoproteins. All nucleated cells and especially endothelial cells, epithelial cells and macrophages are potent producers of IL-1, IL-6, and TNF-. On the basis of function, cell of secretion, or target of action, cytokines can be classified as lymphokines, interleukins and chemokines. The term interleukin was earlier used for cytokines whose targets were principally leukocytes. Majority of the interleukins are produced by T-helper cells. The term chemokine refers to cytokines that mediates chemoattraction (chemotaxis) between cells.

The complement system is an enzyme cascade that helps to defend against an infection. Many complement proteins are present in serum as zymogens (inactive enzyme) and others reside on cell surfaces. The interaction of antibodies and antigens is sometimes useful by itself. For example, coating of a virus/bacterium, prevents it from binding and invading a host cell. But most of the time, this binding performs no useful function until and unless it can activate an effector mecha- nism. The complement system serves several effector roles. Therefore, the complement system provides the actual protection from the response and the antibodies and antigen interaction provides the specificity of the response. We can also say that antibodies “finger” the target and comple- ment destroys it. The complement system acts as a bridge between innate and acquired immunity

“Immuno” refers to an immune response that causes body to generate antibodies and “assay” refers to a test. Thus an immunoassay is a test that utilize antibody-antigen complex to generate a measurable signal. These assays utilize one or more selected antibodies to detect analyte of interest. The analyte being measured can be naturally present in the body like thyroid hormone, or not typically present but produced by the body like a cancer antigen or not naturally present in the body like an abused drug. These assays are used for identifying as well as quantifying the organic and inorganic compounds. They are widely used in the hospital labs, special area of forensic science and field analysis in the environment. The first immunoassay RIA (Radioimmunoassay) was invented by Rosalyn Yalow and Soloman in 1959 which applied the use of radioisotopes for the measurement of insulin.

Membrane based rapid immunoassays are also known as immuno-blotting assays. Immuno- blotting is a solid phase immunoassay because the membrane matrix involved is insoluble, particulate and non diffusible reaction ingredient. These techniques are basically used to charac- terize proteins after electrophoretic separation. Two major categories of membrane based immunoassays are: 1. Protein Transfer—This method refers to immunoblot assay which involve the transfer of electrophoretically separated proteins from gel to an immobilized matrix. 2. Dot Blot Assay—This assay was developed in 1982 in order to simplify the screening of hybridoma clones for specific antibody production. In this assay, small amount of protein sample (i.e., 0.2—10l) is directly applied to the surface of membrane and allowed to penetrate in the membrane by diffusion. The membrane is then allowed to dry and blotted substance is detected by using immunochemical method. In case to accumulate the protein from diluted solution, large volumes can be tested by repeated application onto the same spot. This results in greater accumulation and adsorption of proteins on the membrane surface until maximum capacity of membrane has been reached. The actual amount of protein binds to the membrane is variable and depends on: a) Concentration of protein solution b) The chemistry of protein as well as membrane c) Conditions of the binding reaction.

Antigen (Ag) antibody (Ab) reactions occur when an antigen combines with a corresponding antibody to produce an immune complex. Therefore, an antigen-antibody reaction is thus a bimolecular association which is similar to an enzyme-substrate interaction but the only difference is that antigen-antibody reaction does not lead to an irreversible chemical interaction. The basis for antigen-antibody reactions are the non-covalent interactions like hydrogen bonds, ionic bonds, van der Waal interactions, hydrophobic interactions, etc. These interactions are individually weak, therefore, a large number of such interactions work together in an antigen-antibody reaction. The in vitro study of antigen antibody reactions is known as serology. The principle for all diagnostic immunological tests is serological reactions. The binding of an antibody with an antigen of the type that stimulated the formation of the antibody, results in agglutination, precipitation, complement fixation, greater susceptibility to ingestion and destruction by phagocytes, or neutralization of an exotoxin. The main use of antigen-antibody reactions is in the determination of blood groups for transfusion, serological ascertainment of exposure to infectious agents, and development of immunoassays for the quantification of various substances. Schematically

ELISA or EIA is a method widely-used for measuring the concentration of a particular molecule (e.g., a hormone or drug) in a fluid such as serum or urine. The molecule is detected by antibodies that have been made against it. In simple words, in ELISA an antigen (unknown amount) is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a “sandwich” ELISA). Then a specific antibody is added over the surface so that it can bind to the antigen. This antibody is linked (conjugated) to an enzyme. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. Then a substrate is added that the enzyme can convert to some detectable signal which indicates the quantity of antigen in the sample. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates enabling much higher sensitivity.

Biomarkers in physiological specimens serve as useful sensors for clinical diagnosis. Accurate detection of specific markers is crucial for the diagnosis of diseases, monitoring drug therapy and patient screening. In vitro immunoassays are probably the most common, simple and relatively inexpensive serological tools used in clinical laboratories for the diagnosis and management of disease. Despite continued efforts to improve the performance of immunoassays in the past three decades, there is a need for highly sensitive assays that can detect the lowest levels of disease markers with greater accuracy. Efforts are made towards increasing the sensitivity of immunoassays by amplifying detection signals, with implications for the development of highly sensitive diag- nostic systems. In a conventional enzyme immunoassay, the enzyme labeled on the antigen or antibody converts the substrate into product. The product is then detected depending upon the type of substrate used. If the substrate used is a fluorescent molecule (fluorophore), then fluorescence is observed and if the substrate used is chromogenic or chemiluminescent, then change in color is measured spectrophotometrically and light emission is measured by luminometer. But in an enzyme immunoassay, if the enzyme concentration is low, a weak signal will be produced and it is difficult to observe a weak signal because of background noise. Sensitivity of an assay is determined mainly by the detectability of the molecules. Assay sensitivity can be enhanced by amplification. Enzymes (such as alkaline phosphatase and horseradish peroxidase) are widely used as non-radioactive labels because they provide signal amplification through the high turnover of substrate to detect- able products. Further signal amplification can be introduced either by attaching multiple enzyme molecules on the target molecule, through the branched chain DNA system, or by using enzyme- coding DNA fragments as labels which, upon expression, generate several enzyme molecules in solution.

The main developmental goals of an assay system are its desired accuracy, precision, sensitivity and specificity. Therefore, the foremost consideration in an assay development and optimization should be the intended application of the assay which may further affect the developmental goals. Immunoassays requires a longer time period for assay development in comparison to other bio-analytical techniques. During the immunoassay development, the generation of the reagent antibody (monoclonal or polyclonal antibody) takes the longest time. It can take even months and thereafter also there is no confirmation that a desired reagent antibody will be produced. To save time and efforts, we can opt for commercially available kits or reagent antibody/antiserum which can be used directly and therefore the longest step can be skipped off. Academia can serve as the best source of reagent antibodies for novel biomarkers. There are a number of immunoassays (as discussed earlier) which needs to be developed on some specific formats. A broad range of different aspects that must be considered

Formulation is a mixture or substance prepared as per a formula. Formulations are made for a particular application and normally are hyperactive than its individual components when used alone. The quality of an assay system largely depends on the source and preparation of reagents. In the commercial kits, the reagents are reconstituted before use. Extra care should be taken while reconstituting the freeze dried reagents. Freeze dried reagents are packed at negative pressure and so they should be opened very carefully allowing air to slowly enter the vial. Then such reagents are reconstituted with correct solvent using calibrated pipettes at the recommended tempera- tures. The solvent and reagent is then mixed carefully to avoid foam formation (which may lead to denaturation of protein reagents). We should strictly follow the manufacturers instructions while preserving the reconstituted reagents. Some general guidelines have been given by Blockx and Martin, 1994 for this purpose

Hypersensitivity is an inflammatory reaction within humoral or cell mediated immune response that leads to significant tissue injury, serious disease or even death. Immediate hypersensitivity is anaphylactic reaction within the humoral branch which is initiated by antibody/antigen-antibody complexes. They are called so because the symptoms manifest within minutes/hours after a sensitized recipient encounters the antigen. Delayed-type hypersensitivity (DTH) is so called in recognition of the delay of symptoms until days after exposure.

Hypersensitivity is an inflammatory reaction within humoral or cell mediated immune response that leads to significant tissue injury, serious disease or even death. Immediate hypersensitivity is anaphylactic reaction within the humoral branch which is initiated by antibody/antigen-antibody complexes. They are called so because the symptoms manifest within minutes/hours after a sensitized recipient encounters the antigen. Delayed-type hypersensitivity (DTH) is so called in recognition of the delay of symptoms until days after exposure.

Autoimmunity is a condition in which structural and functional damage is produced by the action of antibodies or immunologically competent cells against the normal components of the body or it is the failure of an organism to recognize its own constituent parts as self, which allows an immune response against its own cells and tissues. For example, Coeliac disease, diabetes mellitus type 1 (IDDM), systemic lupus erythematosus (SLE), Graves disease, etc. Ideally, minimum three requirements should be met before a disorder is categorized as truly due to autoimmunity

Immune system is a defense system that enables us to resist infections. The immune system is composed of two types of immunity: the innate or non-specific immunity and the adaptive or specific immunity. The innate immunity is the first line of defense against invading organisms while the adaptive immunity acts as a second line of defense and also gives protection against re-exposure to the same pathogen. Each type of immunity has both cellular and humoral components by which they carry out their protective function.

Bacteria are microscopic, single-celled organisms. There are thousands of different kinds of bacteria which live in every conceivable environment all over the world. They live in soil, seawater, and deep within the earth’s crust. Some bacteria have been reported even to live even in radioactive wastes. Some bacteria live in the bodies of people and animals—on the skin and in the airways, mouth, and digestive and genitourinary tracts—often without causing any harm. Bacterial infections occur when harmful bacteria enter our body or the existing bacteria get out of balance. The bacteria that cause disease are called pathogens. Sometimes bacteria that normally reside harmlessly in the body can also cause disease. Bacteria can cause disease by producing harmful substances like toxins or by invading the tissues.

Cancer is an abnormal, uncontrolled cellular growth. Risk factors linked to cancer include: diet and obesity, family history of cancer, sedentary life style, occupational factors, viruses and biological agents, alcohol, environmental pollution and UV rays.

Transplantation: It is an act of transferring cells, tissues or organs from one site to another. Many diseases can be cured by implementation of healthy organ tissue or cells (a graft) from one individual (donor) to another in need of transplant (host). Alux Carrel reported first systemic study of transplantation in 1908, he interchanged both kidneys in series of nine cats. Organs that can be transplanted are the Kidney, heart, liver, pancreas, lungs, thymus and intestine and tissues include heart valves, bones, tendons, cornea, skin, and veins. The kidneys are the most commonly transplanted organs, followed by the liver and then the heart. A variety of immunosuppressive agents can delay or prevent rejection of transplanted organ, e.g. Calcineurin inhibitor, mTOR inhibitor, Anti-proliferative, corticosteroids and antibodies etc. Different types of grafts/transplants.

Nucleic Acid Hybridization

DNA diagnostics means qualitative and quantitative analysis of DNA, RNA and proteins. The common techniques used are, for DNA – PCR, RFLP (restriction fragment length polymorphism), electrophoresis, Southern hybridization, cloning, etc; for RNA – reverse transcription, real-time PCR, Northern-blotting, etc; for proteins–PAGE electrophoresis, Western-blotting, monoclonal antibodies, immuno-precipitation, ELISA, liquid chromatography, (HPLC), mass spectroscopy, etc.

Nucleic acid hybridization can be detected by labeling the probe with a radioactive isotope or a non-radioactive isotope. Therefore, hybridization procedure can be of two types depending upon the type of label used to label the probe

“DNA Fingerprinting – Greatest breakthrough in forensic sciences in this century” Identification and characterization of individuals is carried out at different levels. These can be social, physical or biological. The biological identity means phenotypic and genotypic markers. Most commonly used biological markers for individual identification include blood groups, serum proteins, enzymes, etc. These markers have proved useful but they are limited in number and their degree of variation. So these cannot be used in precise identification of specific individual. Most of the genomes of animals and plants cannot vary greatly between individuals because it has an essential coding function. In non-coding regions this requirement does not exist and the DNA sequence can accommodate changes. One change, which does occur, is the tandem repetition of DNA sequences. The discovery of hypervariable repeats (HVR) in human DNA has created a pow- erful new class of genetic markers, which promise to revolutionize forensic biology, and opened new vistas in animal and plant sciences. The HVR, also referred to as mini-satellites or variable number of tandem repeats (VNTRs), consists of core tandem repeats of a short nucleotide sequence about 15–30 base pairs in length. They are hyper-variable because the number of tandem repeats, and hence the length of DNA in that region, varies considerably in the general population. DNA probes have been isolated which detect families of these HVR located at many different chromo- somal loci. The probability that two unrelated individuals have identical lengths of DNA at a particular HVR is very low. However, the probes that have been developed to detect 30–40 different HVR simultaneously, so the probability that all of these are the same length in both individuals becomes vanishingly small. The complex banding pattern obtained when southern blots of DNA are hybridized with these probes is therefore individual specific, and is referred to as a DNA fingerprint.

One of the most important threats for human’s health is the genetic diseases. Genetic disease is a disorder caused by genetic factors (especially abnormalities) in the human genome. The four main types of genetic diseases

Polymerase Chain Reaction-Oligonucleotide Ligation Assay (PCR-OLA)