What is Serology & Serology Tests?

The morphological characteristics of plants and animals are used by descriptive natural sciences as classification criteria.

However, the development of serology in the form of an independent science has made it possible to clarify the differences between living organisms on the basis of chemical verbs by determining their structures.

These thoughts come indirectly and not as a result of simplicity. The concept of uniqueness arises in this knowledge that after recovery, an infectious disease is a certain resistance to that disease (immunity).

Edward Jenner used this fact to develop the first practical vaccine.

In search of an explanation of this incident, a strange group was discovered in the blood called antibody.

Some antibodies protect organisms against infectious agents like bacteria and Viruses or neutralize toxins etc.

These substances, which have now been identified as modified globulin, are not only produced as a result of a transition, but also as the result of the administration of some hazardous substances, high molecular weight Bacteria, animals, or toxins of plants or dead bacteria.

In addition to the initial interest in immunology, other findings in immunology found that due to immunity caused by the bacteria, toxins are only a part of a more general principle:

when animals were added to substances obtained from foreign species or cells containing proteins. So, the same mechanism is controlled.

In this case, antibodies are caused by the erosion of the blood, cell destruction or the rainfall of those proteins.

All immuno-antibodies are specific. This means, literally, that they react to an antigen (used for immunization).

Antigens, a separate species, cells with bacteria or virus etc. Various biological sciences have developed efficient and accurate biochemical and immunological techniques to diagnose various traditional diseases, thus replacing more conventional methods.

History of Serology

Edward Jenner (1749-1823) pioneered the use of vaccines for the prevention of infectious diseases.

Louis Pasteur (1822-1895) studied various methods for the Attenuepettingone virus, which developed a vaccine.

Bence-Jones (1847) discovered an abnormal protein in the urine of multiple myeloma patients. This protein, produced in large amounts, is the retardant of light (L) series.

Paul Erlich (1854-1915) found that antibody is synthesized as antigen reaction.

George Neutoll (1888) discovered the bactericidal property of blood.

Gruber and Durham (1896) generated the first synergistic agglutination reaction of the Vibrio cholerae, and this antiserum.

Albert Connes (1942) developed histochemical methods to detect pathogens using fluorescent antibodies.

Ouchterlony, J. Ondin, and S. Elek (1946-1948) described prison proliferation trials for the study and detection of viruses. Grabber and C. Williams (1953) developed immuno-electrophoresis technology.

Alic Isaac (1957) produced Interferon.

Solomon Burson and Rosalin Yalv (1959-1960) developed radioimmunoassay.

In 1962, Rodney Porter proposed a unique but basic four-chain model for the structure of immunoglobulin molecules.

Edelman et al. (1968) established a complete amino acid sequence of an IgG antibody with a multiple myeloma patient’s blood.

Voller et al. (1976) used the Enzyme-Linked Immunosorbent Test (ELISA) for the first time to detect the three plant viruses.

George Koller and Caesar Milstein (1981) developed the technique of producing monoclonal antibodies.

Immunochemistry

Leukocytes are blood cells that protect organisms. There are various kinds of leukocytes present in our body but the most important of these includes:

Monocytes –

15 to 18 μm long; Up to 8% of all leukocytes. They are macrophages and are responsible for the consumption of large molecules with phagocytosis.

Lymphocytes –

8-9 microns long; constitute 30-35% of all leukocytes and take part in antigen-antibody reactions.

Eosinophils –

12-13 microns long; There are blood components up to 8%. They take part in antigen-antibody reactions, especially in allergies.

The immune system responds by cell immune or comedic immune system in two ways –

Lymphocytes are the primary cells involved in these reactions.

Two different lymphocyte populations have been identified: T cells (T lymphocytes) and B cells (B lymphocytes).

T lymphocytes are responsible for cell immunity, including cutaneous reaction, graft rejection, anti-tumor immunity and cellular defense against fungal and intracellular pathogens.

B lymphocytes develop in the Fabrician sack of birds, but it is believed that it emits from the bone marrow of mammals. They are in charge of comic immunity expressed in the production of specialized circulating plasma proteins called antibody or immunoglobulin.

What is an Antigen?

Substances capable of inducing an immune response (comedic, cellular, or mixed T and B cells), when presented in animals, are called antigens or immunogens.

Most antigens are macro-molecular proteins, but can also be like immunogenic, polysaccharides, synthetic polypeptides, and other synthetic polymers.

Immunogenic molecules have the following characteristics:

The molecule should be foreign to the host.

Molecules with more than 10,000 molecular weight are weak immunogens; Proteins with more than 100,000 proteins from a molecular weight are strong immunogens.

To be antigenic in the molecule, there must be a certain degree of complexity. The Immunogenicity increases with structural complexity.

In addition, aromatic amino acids contribute to a large degree compared to the residues of non-aromatic amino acids in immunogenicity.

The ability to respond to antigens varies with animal species, and even with its genetic constitution.

Antigenic determinants

In the production of immunoglobulin, the association of the lymphocyte’s antioxidant to the surface is required.

Combination sites on the surface of lymphocytes that contain molecules similar to antibodies are called antigen receptors.

Only limited parts of antigenic molecules are related to antibody combination sites.

These areas are called antigenic determinants, and they determine antigen-antibody responses.

An antigenic determinant can contain only 6 to 7 amino acids of the total number of proteins.

In other words, a virus, for example, induces the production of a mixture of antibody, which in particular affects the different antigen determinants present in the particle.

Thus, we can define Antisera with a mixture of antibodies called “polyclonal Antisera.”

The number of antigen determinants in the single molecule varies with the size and complexity of the molecule.

Hapten

It is a small, chemically defined molecule that can not induce the production of specific antibodies against it. However, when it is connected to the large molecule from the covalent converter, it can act as an antigenic determinant and may stimulate the antibody synthesis.

Adjuvants

Adjuvants are chemical substances that are used to increase the immune response.

They not only stimulate the formation of antibodies, but also localize them at the site of injection, from where they are released gradually during the period of antibody synthesis, either through the adsorption of solid particles or by making a complex with oily emulsions.

The most commonly used calcium is calcium phosphate, inorganic gels, aluminum hydroxide, bentonite, Methylated serum albumin, and incomplete freund-adjuvant.

It is the most commonly used adjuvant with viral antigens and is composed of 9 parts mineral oil (Lanolin) and one part detergent; this allows the formation of a stable emulsion. If the mixture contains dead Mycobacterium sp also, is called a full Freud adjuvant.

What are Antibodies?

Antibodies are immunoglobulins that are produced in response to the invasion of foreign compounds such as proteins, glucoside, or nucleic acid polymers produced by organisms.

The antibody molecule binds non-covalently to the foreign substance, which initiates a process to eliminate the foreign particle(s) from the organism.

The antibody molecules are originally formed by the four polypeptide chains, the original unit is formed: two identical heavy (H) series (MW 53,000 to 75,000), and two light (L) series (MW 23,000) united by Di-sulphur Bond.

The antibody molecule consists of two regions: The Normal Fraction (Fc) and Variable Fraction (Fab):

Fc: Forms a part of the heavy chain.

Fab fraction chain (MW 50,000) determines the specificity of the antibody and is formed by the second part of heavy chains (MW 100,000).

Immunoglobulins made of more than one basic monomeric unit are called polymers.

Various studies have been done by using advanced techniques like Electrophoresis and Ultracentrifugation that enabled the identification of 5 groups of immunoglobulin which are as follows:

1.) IgG

It is the major part of the antibody and contains 80% immunoglobulin with a molecular weight between 150,000 and 160,000.

It contains 2-4% of carbohydrate and shows the lowest Electrophoretic mobility between all immunoglobulins.

It is the only immunoglobulin that is able to cross the placenta.

2.) IGA

The molecular weight of this immunoglobulin occurs between 180,000 & 400,000.

It contains more carbohydrate content (5-10%) than IgG.

IgA is found in high concentration in the blood, colostrum, saliva, tears, bronchial and digestive tract secretion.

IgA cannot cross the placenta.

3.) IgM

This immunoglobulin contains the highest protein and its amino acid sequence has not yet been determined.

It contains 576 amino acids and its molecular weight is 950,000.

This is the first antibody synthesized by a newborn animal or human.

Immunoglobulin M can promote phagocytosis of microorganisms by macrophages and polymorphnuclear leukocytes.

IgM does not cross the placenta.

4.) IGD

No antibody activity is responsible for IgD.

Its role in immunoglobulin generals is still unknown.

5.) IgE

It appears in serum in very low concentrations. There is a molecular weight of 190,000.

In Allergic diseases, about half of patients show high IgE Concentrations.

Monoclonal Antibodies

Monoclonal antibodies are specific antibodies only for one antigenic determinant.

Monoclonal antibodies are produced by cloned cells (hybrids), which is the union of B lymphocytes with carcinogenic myeloma cells.

Lymphocyte provides monoclonal antibody with the ability to produce antibodies, and the myeloma cell gives it the ability to reproduce itself indefinitely.

Because all cell clones from a hybridoma come from single B Lymphocytes, they only produce specific antibodies for an antigen determinant.

Antigen-Antibody Reactions

The antigen-antibody association is irreversible under non-covalent and general laboratory conditions and includes hydrogen bonds, Van-Der-Waals forces, and hydrophobic and coulombic interactions.

As a result, the chemical supplement is similar to the one between the key and the lock.

The association area between antigen and antibody is only 1% of the total globulin surface and its specificity depends on the sequence of amino acids present in that area.

The degree of engagement between the antigen and the antibody depends on the characteristics of each molecule and is determined by the combined effects of interaction with them.

If the affinity and avidity are high, the molecules will be united more rapidly and separation will be very slow.

Antisera with strong precipitation reactions show more avidity than those reacting vulnerable to the same degree of dilution.

Antiserum affinity depends on:

The temperature at which the reaction occurs: High temperature causes a strong reaction. However, the temperature above 40 degrees Celsius is the result of denaturation of proteins.

Concentration of salt: Low salt content favors the union of reactive substances.

The pH level can cause changes in the antigen-antibody relationship.

Concentrations of reactive substances for the formation of precipitates.

Preparation of viral antigen

1.) Viruses that infect Animals

This can be propagated in laboratory animals, embryonic eggs or in vitro cultures.

Animal species used for the production of antiserum should be phylogenetically distant from the common species infected with pathogens.

The preparation of viral antigens should have a high viral concentration, and the virus should be free from host contaminants.

For example, in the case of rabies, the passive virus must be used in the first vaccination in a series.

2.) Viruses that infect Plants

These can be propagated to obtain high particulate concentrations in vivo, allowing the transmission of viruses in the tissues of host plants.

The difficulty of purification depends on the virus and the plant host.

For such viruses, whose multiplication is limited to the phloem, the concentration obtained will be low and the purification can be difficult.

Immunization and production of antibodies

i.) Vaccination

Animals used in the production of antisera for research purposes usually includes rabbits, guinea pigs, mice, goats, sheep, monkeys, horses and hens.

Selection of species depends on the availability and quantity of essential antiserum.

In the presence of antibody synthesis and viral particles the reactions that occur in the immune system cells are extremely complex, and some characteristics are still unknown.

Vaccination process can be summarized as follows:

a.) Some viruses entering the organism

b.) Intake is done by macrophages cells.

c.) Some of the many millions of Helper T lymphocytes

d.) Generally, the blood circulation in the system is activated to identify new enemies, in this case, a viral particle.

T cell is activated by its interaction with the macrophage, which catches the particle.

The association occurs when T lymphocytes recognizes one of their own markers in macrophages and another for the antigen.

The active hellper T Lymphocytes multiplies itself and thus stimulates the product of killer T lymphocytes and B lymphocytes.

e.) Once B lymphocytes are multiplied (approximately one million unique lines), they are prompted to start producing a large number of T Lymphocytes antibody which will be involved in the blood stream.

f.) When a virus infects an organism, some particles begin to penetrate and multiply in infected cells.

Killer T lymphocytes cause chemical damage to the membranes of infected cells, which inhibit the virus replication.

Some of the synthesized antibodies neutralize the remaining virus particles.

When the infection stops, the suppressor T Lymphocyte stops all activity of the immune system, thus preventing uncontrolled responses.

g.) T lymphocytes and memory B Lymphocytes store information on viral particles and live in blood and lymphatic systems.

Thus, they are ready to give feedback and react to the same viral particles attacking the organism again.

ii.) Methods of Vaccination

Vaccination can be Intravenous (IV), Intramuscular (IM), Subcutaneous (SC), Intraperitoneal (IP), Intradermal (ID), Intraarticular (IA), or Intranodular (IN). It enhances the stimulating effect on the immune response.

iii.) Antiserum production

During the first days after the first vaccination, the production of antibody increases, so that it can be maintained for a few days.

If a new dose of antigen is injected, then the synthesis of antibodies increases rapidly higher concentration than the first dose of antigen started.

Antibodies are found in the blood serum of the immune animals. This is simply called as serum.

To produce antiserum, allow blood samples to stand out from the other blood components for the serum.

Finally the components are centrifuged to remove the debris and to obtain the Antiserum from the reaction mixtures.

The antiserum can be stored with frozen (-20°C to 70°C) and Lyophilized, or as a liquid with antiseptic solution (usually at 4°C).

If the Antiserum is stored adequately in required conditions, then it will remain active for many years depending on the type and sensitivity of antiserum.

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