Antigens-Types-Hapten, Complete antigen, Epitope (antigenic determinants)

An antigen is defined as any substance which, when introduced parenterally into the body,stimulates the production of an antibody with which it reacts specifically and in an observable manner. Some antigens may not induce antibodies but may sensitize specific lymphocytes leading to cell mediated immunity or may cause immunological tolerance.

The word “parenteral” means “outside the intestinal tract”. When antigens are orally given, they are denatured by the intestinal enzymes and their antigenicity is destroyed, so no antibody formation takes place. When given parentarally, they are not denatured, hence induce antibody production.  However, some antigens given as oral vaccines are exceptions.  The word “specifically” is important as specificity is the hallmark of all immunological reactions.  An antigen introduced into the body reacts only with those particular immunocytes (B or T lymphocytes) which carry the specific marker for that antigen and which produce an antibody or cells complementary to that antigen only.  The antibody so produced will react only with that particular antigen and with no other, however cross reactions may occur between closely related antigens.

The two attributes of antigens are:

1. Induction of an immune response (immunogenicity)

2. Specific reaction with antibodies or sensitized cells (Antigenicity/immunological reactivity)

Immunogenicity and antigenicity are related, but distinct. Immunogenicity is the ability to induce a humoral or cell mediated immune response.

B cells + antigen→ effector B cells + memory B cells

                          (plasma cells)

T cells + antigen→effector T cells + memory T cells

Although a substance that induces a specific immune response is usually called an antigen, it is more appropriately called an immunogen.

Antigenicity is the ability to combine specifically with the final products of the above responses (ie., antibodies or cell-surface receptors).  All molecules that have the property of immunogenicity also have the property of antigenicity, but the reverse is not true. Based on the ability to carry out the above mentioned attributes, antigens may be classified into different types.  A complete antigen is able to induce antibody formation and produce a specific and observable reaction with the so produced antibody.  Haptens are substances that are incapable of inducing antibody formation by themselves but can react specifically with antibodies.  In other words, haptens are antigenic, but lack immunogenicity.  The term hapten is derived from the Greek haptein which means “to fasten”.  Haptens become immunogenic on combining with a larger molecule carrier. Or they are low molecular weight molecules that can be made immunogenic by conjugation to a suitable carrier.  Haptens may be complex or simple.  Complex haptens can precipitate with specific antibodies, simple haptens are nonprecipitating. Complex hapten is polyvalent and simple hapten is univalent, since it is assumed that the precipitation requires the antigen to have two or more antibody combining sites.

The smallest unit of antigenicity is known as the antigenic determinant or epitope.  Epitope is the portion of an antigen that is recognized and bound by an Ab or TCR/MHC complex.  The epitope is that small area on the antigen, usually consisting of four or five aminoacid or monosaccharide residues, possessing a specific chemical structure, electric charge and spatial configuration, capable of sensitizing an immunocyte and of reacting with its complementary site on the specific antibody or T cell receptor.  Epitopes when present as a single linear segment of the primary sequence is called sequential or linear epitope.  When formed by bringing together on the surface residues from different sites of the peptide chain during its folding into tertiary structure, it is called conformational epitope.  T cells recognize sequential epitopes where as B cells recognize conformational epitopes.  The combining area on the antibody molecule, corresponding to the epitope, is called the paratope.  Epitopes and paratopes determine the specificity of immunological reactions.

Adjuvants

Adjuvants (from Latin adjuvare-to help) are substances that, when mixed with an antigen and injected with it, enhance the immunogenicity of that antigen.  Adjuvants are often used to boost the immune response when an antigen has low immunogenicity or when only small amounts of an antigen are available.   They are known to exert one or more of the following effects:

·         Prolong antigen persistence

·         Enhance co-stimulatory signals

·         Induce granuloma formation

·         Stimulate lymphocyte proliferation nonspecifically

Examples- Freund’s complete adjuvant, Aluminium potassium sulfate (alum), Mycobacterium tuberculosis, Bacterial lipopolysaccharide (LPS) etc.

Eukaryotic Chromosome Organization

1. Eukaryotic and prokaryotic cells differ in a number of aspects. In Greek, Eu means true, Pro means before and karyon means nucleus. Main difference is in their nucleus. Presence of a well-defined nucleus is the main feature that distinguishes the eukaryotic cell from a prokaryotic one. Nucleus serves as the cell’s control center and genetic information is packed up there. DNA replication, transcription, RNA processing etc take place in the nucleus, and only final stage of translation occurs in cytoplasm.      

Eukaryotic nucleus has nuclear membrane, chromatin and nucleolus which is the most prominent substructure in nucleus. Nucleolus are cell’s ribosome factories.  Chromatin, RNA and nuclear proteins move freely in aqueous solution in nucleus.  

·     In prokaryotes, usually a single chromosome is present per cell in the form of covalently closed circular DNA. They also contain extra chromosomal DNA called plasmids. But in eukaryotes, several chromosomes are present per cell (2 to many). Here also extra chromosomal DNA is found in mitochondria and chloroplast. Amount of chromosome in eukaryotic cells is much more than a prokaryotic cell. A human cell contains DNA more than a 1000 times than E coli. In E coli 4x10⁶ base pairs (bp) are seen in single cell whereas in human cell 5.5x10⁹ bp of DNA is present in its diploid nucleus. Each eukaryotic species has a characteristic number of chromosomes.

·     3. C Value- For eg: In humans 46 chromosomes (diploid set) are seen, ie., 23 pairs of chromosomes. Half or haploid set is derived from the egg and half from sperm cell. Eukaryotic chromosomes exist as a single linear unbroken double stranded DNA (DS DNA) molecule running throughout the length of chromosome, coupled with double amount of protein. Chromosomes are not always distinct in a cell. They are well defined structures at the time of cell division. At other times, chromosomes are not well organized. Total amount of DNA in a haploid genome of a species is called the “Species C value”. In other words, C value is DNA contained within a haploid nucleus (e.g. a gamete) or half the amount of DNA in a diploid somatic cell of a eukaryotic organism. C value of human beings is 3.4 x 10⁶ kb. 

·                        ·     5. Chromosome organization in Eukaryotic cells- In eukaryotes, cell cycle has 4 phases viz. G₁, S, G₂ and M phase. G₁ phase is also called the first gap phase. In this phase cells grow in size, copies organelles and makes the molecular building blocks needed in later steps. During S phase, the cell synthesizes a complete copy of the DNA in its nucleus. During the second gap phase G₂, the cell grows more, makes proteins and organelles, and begins to reorganize its contents in preparation for mitosis. G₁, S and G₂ phases together are known as interphase.  In other words, interphase is the time between two mitotic divisions. As mitosis begins, G₂ phase stops. Mitosis has 4 phases called prophase, metaphase, anaphase and telophase. Mitosis is followed by Cytokinesis, the process of the parent cell division into 2 daughter cells. In G₁ phase chromosome is single. During S phase, chromosome duplicate to produce two sister chromatids held together by centromere. Chromosome continues in the same structure in G₂ phase. Centromeres separate and sister chromatids become daughter chromosomes in M phase. 

·                   6. In G₁ phase chromosome exist as one linear DS DNA molecule. This DNA is usually complexed with double amount of proteins. In S phase, sister chromatids have one linear DS DNA running the length of each chromatid. DNA is least organized in G₁ phase and most organized just before cell division. DNA is usually coupled with proteins. DNA and proteins together form the structure called chromatins.  Chromatins are stainable material in cell nucleus and amount of protein is twice the amount of DNA. Basic structure of chromatin in all eukaryotes is identical.

·     7. Chromatin structure- In chromatin, two types of proteins are associated with the DNA called histones and non-histones. Both proteins play a crucial role in the physical structure of the chromosome. Histones are the most abundant proteins. They are small basic proteins with overall positive charge. Large amount of arginine and lysine residues are responsible for the net positive charge and this property helps histones to bind with the negatively charged DNA.  5 types of histones are found in eukaryotes namely H1, H2A, H2B, H3 and H4. In chromatin amount of histone is equal to amount of DNA. Remaining protein part of chromatin is made up of non-histones. Amount and proportion of histone relative to DNA is constant from cell to cell in eukaryotes.

·     8. Among these 5 types of histones, amino acid sequences of H2A, H2B, H3 & H4 are very similar & highly conserved between even distantly related species. H3 & H4 are among the most conserved proteins.  H2A and H2B show slight species specific variation in sequence. This evolutionary conservation shows that histones play some basic role in eukaryotes in the organization of chromosomes. DNA of chromosomes in a single human cell measures >2m (6.5 feet). Human DNA is 700 times greater than E coli. Several levels of packing is necessary to reduce it into cm or even mm to nm and to pack into the nucleus of the cell. Histones play a very crucial role in this chromatin packing.

·     9. Non- histones- These are less abundant than histones. All other proteins associated with DNA apart from histones are called non- histone proteins. These include the proteins that bind during DNA replication, repair, transcription, gene regulation, recombination etc. They are mainly negatively charged. So will bind usually with the positively charged histones. Many different non-histones are present in each eukaryotic cell. Amount and types of histones vary from organism to organism. They also differ markedly in number & type from cell to cell in same organism. Even they differ at times in the same cell.

·     10. Levels of packing in Chromatin - Chromatin structure differs in different stages of the life cycle. It is least organised in the G1 phase and highly condensed at the metaphase, the phase just before cell division. The least compact structure is called 10 nm chromatin fibre. It is morphologically similar to beads on a string. Diameter of bead region is 10 nm and it forms the core region of nucleosomes which are the basic structural units of chromatin. 

·     11. Bead like region is wound around by the string of DNA. This structure is called nucleosome and its diameter is 11nm. Bead like core region consists of 8 histone proteins – 2 each of H2A, H2B, H3 & H4. Also called core histones. This core region is surrounded by DNA of 147bp length wound around by 1.65 times. This arrangement makes the linear DNA compact by a factor of about 6. Thus nucleosome formation is considered as the 1st level of chromatin packing. In other words, 10nm chromatin fibre formation is the first level of chromatin packing. Strands of DNA between nucleosomes are called “linker DNA”. Means two nucleosomes are connected by linker DNA strands. Amount of linker DNA not constant within & among organisms. In humans, linker DNA range between 38-53 bp, thus total amount of DNA per nucleosome is between 185-200 bp. 

·     12. Second level of chromatin condensation is by the formation of “30 nm chromatin fibre” or “Solenoid fibre” which is brought about by H1 Histones.  H1 are larger than other histones. H1 binds to linker DNA at one end of nucleosome and to middle of DNA segment around histone core, making the 10 nm chromatin fibre more compact.   A nucleosome core plus H1 is called Chromatosome. This condensed DNA spiral helically & becomes regular pattern with 6 nucleosome per turn. Now the diameter of DNA is  30 nm and is called “30 nm chromatin fibre” or “Solenoid fibre”.  Solenoid fibre is seen during interphase of cell cycle. This structure brings the packing ratio of DNA to ~40.

 · 13. Next level of chromosome condensation is by the formation of Chromosome Scaffold. However, the exact mechanism behind it is not clearly understood. Non-histone proteins are mainly involved in Scaffold formation. Non histone proteins bind to 30 nm chromatin fibre to form chromatin loops of 30-90 kb long DNA.  180-300 nucleosome will be usually present per loop. In humans around 2000 such looped domains are present. Stretches of DNA that bind to non-histone proteins to form the loop are called Scaffold associated Regions (SAR).  These chromatin loops attach spirally around the Chromosome Scaffold which is again made up of non-histone proteins and form “Rosettes of chromatin loops”. Around 15 loops are present per turn. This makes the chromosomes now 10,000 times shorter & 400 times thicker than naked DNA. 

·     14. Naked DS DNA is 2nm in its diameter but around 2m long. 10-20 µm is the size of a typical eukaryotic cell and its nucleus is even smaller, having a size ranging between 2-10 µm. So, in order to accommodate the very long DNA into this very small nucleus of eukaryotic cell, it has to undergo different levels of packing.  When it undergoes first level of folding into nucleosome or Bead-on-string pattern, the diameter is 11 nm. Means DNA is getting thicker but even shorter. When chromatin undergoes second level of folding into solenoid fibre, its diameter becomes 30 nm. Further folding in to scaffold loop, makes it to a dimension of   300nm in diameter. Further spiralling of loop around chromosome scaffold makes DNA 700nm in diameter. Entire condensed chromosome is 1400 nm in diameter as consists of two sister chromatids. Such 46 chromosomes are packed in the nucleus of each eukaryotic cell (somatic cells).

·     15. To Summarise, in first level of chromatin packing, DS DNA is wound around histones to form nucleosomes and 10 nm chromatin fibre is produced. H1 histones cause second level of condensation of chromatin into 30 nm chromatin fibre or Solenoid fibre. Third level of packing is brought about by non-histone proteins and Scaffold loops are formed which helically get arranged around the chromosome scaffold.  Thus DNA in chromosome becomes 10,000 times shorter and 400 times thicker and accommodated in the nucleus of eukaryotic cell.

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Culture Media PART 1- Solid media, Liquid Media & Use of Agar

    

1. Culture Media

·     In nature as well as in clinical samples, microorganisms exist in mixed populations. In order to study particular organisms or to know their clinical importance, their pure cultures have to be isolated from source by cultivating in suitable culture media. A suitable culture media makes the cultivation of microorganisms possible in laboratories. A culture media can be either solid or liquid which is used to grow, transport and store microorganisms. A culture media should contain all the necessary nutrients required for the growth and multiplication of the organisms. In general, all microorganisms require source of energy, micro and macro nutrients for growth.  However, the precise composition of the media will depend upon the types of organisms being cultivated as microbes vary considerably in their nutritional requirements. Knowledge of normal habitat of a particular organism is useful in the selection of media as their natural habitat reflects their nutritional requirements. In order to support the growth of microorganisms in culture media, physical factors like optimum temperature, pH etc. also have to be maintained properly.

·      Culture media can be classified on the basis of several parameters (Table 1.1). Based on physical nature or consistency media can be classified into Liquid, Solid and Semi-solid media.  Based on chemical constituents, media are classified into Simple, Complex, Defined or Synthetic media, Semi defined media or Semi Synthetic media. Based on their application or function media can be divided into Supportive media (General purpose media) and special media.  Special media includes a number of preparations like Enriched media, Enrichment media, Selective media, Differential media, Indicator media, Sugar media and Transport media. However, sometimes a single media may fulfil more than one functions. For example; Blood agar can act as both Enriched and Differential media.           

           Eosin Methylene Blue (EMB) agar can act as both Selective and Differential media

           MaConkey (MAC) agar can act as Selective, Indicator and Differential media

           Mannitol salt agar can act as both Selective and Differential media.

Based on oxygen requirements, media are classified as Aerobic media and Anaerobic media.

Table-1.1. Types of media
	Basis for Classification	Types
1	Physical nature or consistency media	Liquid media Solid    media                                                Semisolid media
2	Chemical constituents	Simple media Complex media Defined or Synthetic media  Semi defined media.
3	Application or function	Supportive media (General purpose media) Special media ·       Enriched media ·       Enrichment media ·       Selective media ·       Differential media ·       Indicator media ·       Sugar media ·       Transport media
4	Oxygen requirements	Aerobic media Anaerobic media

1.1.       Media based on physical nature

1.1.1.                  Liquid Media

Liquid media is liquid in consistency. Louis Pasteur used liquid media for first time to cultivate microbes. He used original urine and meat broth as liquid media. When inoculated into suitable media, microbes will produce clone (cells originating from single parental cell) of cells. On solid media they can be seen as discrete colonies with specific colony morphology which will be helpful in the identification of microorganisms. But in liquid media, microbes grow diffusely. Hence microbes cannot be easily identified from liquid culture. Also it is difficult to isolate pure culture from mixed populations of microbes when inoculated in liquid media. Inoculated liquid media are first incubated at 37⁰ C for 24 hrs, and then sub cultured to solid media to get pure, isolated colonies. However, liquid media are useful when large volume samples like blood and water are to be tested. They are also widely used for the bulk culture preparation of microorganisms for antigen and vaccine production.  Most widely used liquid media now a days is Nutrient broth. In liquid media microbes show growth patterns like uniform turbidity, pellicle formation at the top region, sedimentation at the bottom or floccules throughout the medium (Figure-1.1). 

1.1.1.                  Solid Media

Solid media is solid in consistency. Solid media contains the solidifying agent agar in it. Earliest solid media was cooked cut potatoes used by Robert Koch. Later he used gelatine to solidify the liquid media. But it was not satisfactory as gelatine liquefied at 24⁰ C and also digested by proteolytic bacteria. Use of agar as solidifying agent was suggested to him by Frau Hesse, wife of one of the investigators in Koch’s lab, who had seen her mother using agar as solidifying agent during preparation of jellies.

Solid media are widely used than liquid media. On solid media, microbes produce discrete colonies with particular colony morphology. Also organisms exhibit other characteristics like pigmentation or haemolysis.  For example, Serratia sp. and Pseudomonas sp. colonies   produce red and green pigmentation respectively on solid media.  Streptococci sp. produce α or β hemolysis on blood agar. Hence isolation of pure culture and identification of organisms are easier when cultivated on solid media compared to liquid media. Most widely used solid media is Nutrient agar. Its composition is same as nutrient broth except the use of additional ingredient, agar.  

Composition of Nutrient agar and Nutrient broth

Nutrient agar	Nutrient broth	Amount
Agar	--	20 g
Peptone	Peptone	5 g
Yeast extract / Beef extract	Yeast extract	3 g
NaCl (Sodium chloride)	NaCl (Sodium chloride)	3 g
Distilled water	Distilled water	1 Litre
pH	pH	7±0.2

1.1.1.1.              Agar

Agar, also called agar-agar is the universally used solidifying agent in the preparation of solid media. Agar is extracted from seaweeds or marine algae of species Gelidium, mainly Gelidium corneum. Agar is long chain polysaccharides with varying amount of inorganic salts and small quantities of protein like substances.   It is a sulphated polymer composed mainly of D-galactose, 3,6 anhydro- L-galactose and D-glucuronic acid. Commercially available agar is sulphuric acid esters of linear galactan, insoluble in cold water and soluble in hot water. Agar is well suited as a solidifying agent for many reasons. Agar is an excellent solidifying agent with no nutritive value and most microbes do not degrade it. Means, agar is not effected by the growth of the microorganisms. Most unique property of agar is that it melts at 90⁰ C and sets at 45⁰ C. Hence after being melted, it can be cooled to a temperature that is tolerable to human hands as well as to microbes. Also solid media with agar can be incubated at a wide range of temperatures depending on the type of organisms cultivated. Usually 2% agar is used for the preparation of solid media. Based on concentration of agar jellifying capacity also varies. Agar is commercially available as dehydrated powder or long shreds.

1.1.1.1.              Peptone

Peptone is another universal agent used in solid media. It is a complex mixture of partially digested proteins resulting from digestion of protenaceous materials like meat, casein and gelatine. Digestion of these materials can be done either by acid or with enzymes. As hydrolysis proceeds, large colloidal protein molecules are broken up into a series of smaller fragments like proteoses, peptones, polypeptides and amino acids respectively. Constituents of peptone usually include proteoses, polypeptides, amino acids, variety of inorganic salts like Phosphate, Potassium, Magnesium and accessory growth factors like riboflavin. It has nutritive value and main source of organic nitrogen and contain vitamins and carbohydrate based on the material digested. Commercially different brands of peptone are now available. Different brands differ in composition and growth promoting properties. Other common ingredients of solid media are meat/beef extract and yeast extract. Peptone also act as buffering agent.

1.1.1.1.              Beef Extract

Aqueous extract of lean beef tissue concentrated to paste like form is called beef extract. It has nutritive value and consists of water soluble substances of animal tissue which include carbohydrate, organic nitrogen compounds, water soluble vitamins and salts.

1.1.1.2.         Yeast Extract

It is aqueous extract of yeast cells available commercially in powder form. It is nutritious with rich source of B Vitamins, also contains organic nitrogen and carbon compounds.

1.1.1.3.         Sodium chloride (NaCl)

It is not necessary for bacterial growth and also does not act as buffer. The presence of sodium chloride in nutrient agar maintains a salt concentration in the medium that is similar to the cytoplasm of the microorganisms. If the salt concentration is not similar, osmosis takes place transporting excess water into or out from the cell. Sometimes, either to cultivate some microorganisms or to recognize properties like haemolysis, blood agar which is an enriched media is usually used.  In this media blood is added. Red Blood Cells get haemolysed when added to water or to media having low osmotic pressure. This may be prevented by addition of NaCl.

1.1.1.4.         Distilled water

Water is necessary for the existence of living organisms. Usually distilled water is used for preparation of culture media since it is of definite composition. Calcium and Magnesium present in tap water react with phosphates present in peptones, beef extract and other ingredients of culture media to give insoluble phosphates. Insoluble phosphates may not form in cold, but during sterilisation such media throw down considerable precipitate.

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