Proteins.pptx SYNTHESIS.pptx SYNTHESIS.pptx

Of course. Here are detailed, lecture-style notes on ELISA designed for medical students, organized for clarity and understanding, moving from basic principles to clinical applications.

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Lecture Notes: Enzyme-Linked Immunosorbent Assay (ELISA)

Target Audience: Medical Students
Objective:To understand the principle, types, procedure, applications, and interpretation of ELISA in clinical diagnostics and research.

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1. Introduction: What is ELISA?

· Definition: ELISA is a quantitative, plate-based immunoassay used to detect and measure the presence of a specific substance, usually an antigen (Ag) or an antibody (Ab), in a liquid sample.
· Core Principle: It uses antibodies conjugated to a detectable enzyme. The enzyme catalyzes a reaction with a substrate to produce a measurable signal (usually a color change). The intensity of this signal is proportional to the concentration of the target molecule in the sample.
· Key Feature: Highly sensitive and specific due to the use of immunological reagents (antibodies). It is a cornerstone of modern clinical serology.

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2. The "Workhorses" of ELISA: Key Components

1. Solid Phase: Typically a 96-well polystyrene microtiter plate. Proteins (antigens or antibodies) passively adsorb to the well surfaces.
2. Antigen (Ag): The molecule of interest that you want to detect (e.g., a viral protein, hormone, cytokine).
3. Antibody (Ab): The detection molecule.
· Capture Antibody: Binds to the antigen and immobilizes it on the plate.
· Detection Antibody: Binds to a different epitope on the immobilized antigen and is linked to an enzyme.
4. Enzyme: Conjugated (linked) to the detection antibody. Common examples:
· Horseradish Peroxidase (HRP): Uses substrates like TMB (3,3',5,5'-Tetramethylbenzidine), which turns blue, then yellow when stopped with acid.
· Alkaline Phosphatase (AP): Uses substrates like pNPP (p-Nitrophenyl Phosphate), which turns yellow.
5. Substrate: The chemical the enzyme acts upon to produce a detectable signal.
6. Stop Solution: An acid (e.g., Sulfuric Acid) that halts the enzyme-substrate reaction, stabilizing the final color.
7. Wash Buffer: Used to remove unbound reagents between steps, minimizing non-specific background signal.

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3. The Four Main Types of ELISA (with Detailed Protocols)

A. Direct ELISA

· Principle: A single enzyme-labeled antibody is used to detect the antigen.
· Procedure:
1. Coat: Antigen is directly adsorbed to the well.
2. Block: Add a blocking protein (e.g., BSA, Casein) to cover any empty plastic surface.
3. Detect: Add the enzyme-conjugated primary antibody. It binds directly to the antigen.
4. Wash to remove unbound antibody.
5. Add Substrate: Measure the color development.
· Advantages: Fast, only one antibody needed, fewer steps.
· Disadvantages: Lower sensitivity, no signal amplification, every primary antibody must be labeled.

B. Indirect ELISA

· Principle: Uses two antibodies: an unlabeled primary antibody and an enzyme-

1. Proteins Njihia Martha

2. Introduction There are approximately 300 amino acids present in various animals, plants, and microbial systems, but only 20 amino acids are coded by DNA to appear in proteins. Cells produce proteins with strikingly different properties and activities by joining the same 20 amino acids in many different combinations and sequences. This indicates that the properties of proteins are determined by the physical and chemical properties of their monomer units, the amino acids

3. Definition Amino acids are the basic structural units of proteins consisting of an amino group, (-NH2) a carboxyl (-COOH) group a hydrogen (H) atom and a (variable) distinctive (R) group. All of the substituents in amino acid are attached (bonded) to a central α carbon atom. This carbon atom is called α because it is bonded to the carboxyl (acidic) group

4. Amino acid structure

5. Amino acid structure In neutral solution (PH = 7), both the α- amino and α carboxyl group are ionized resulting the charged form of an amino acids called zwitterion (dipolar) In dipolar (zwitterion) form the amino group is protonated (-NH3 +) and the carboxyl group is dissociated (deprotonated) (-COO-) leading to a net charge zero

6. Stereochemistry (Optical activity) Stereochemistry mainly emphasizes the configuration of amino acids at the α carbon atom, having either D or L- isomers Out of the 20 amino acids, proline is not an α amino acid rather an α - imino acid. Except for glycine , all amino acids contain at least one asymmetric carbon atom (the α - carbon atom).

7. Classification of Amino Acids I. Structural Classification This classification is based on the side chain radicals. Each amino acid is designated by three letter abbreviation eg . Aspartate as Asp L-Amino acids are the building blocks of proteins. They are frequently grouped according to the chemical nature of their side chains. Common groupings of amino acids are aliphatic, hydroxyl/sulfur , cyclic, aromatic, basic, acidic and acid amides

8. Structural Classification

9. Structural Classification

10. II. Electrochemical classification Amino acids could also be classified based on their acid – base properties Acid amino acids (Negatively charged at pH = 6.0 ) eg aspartic acid( CH2 – COO-), glutamic acid(CH2 – CH2 – COO) Basic amino acids(positively charged at PH = 6.0) e.g. Lysine ( CH2 – CH2 - CH2 - CH2 – NH3+) Neutral amino acid. Example : ♦ Serine - CH2- OH ♦ Asparagine - CH2- CO-NH2 ♦ Glutamine - CH2- CH2 - CO-NH2

11. III. Biological or Physiological Classification This classification is based on the functional property of amino acids 1. Essential Amino Acids Amino acids which are not synthesized in the body and must be provided in the diet to meet an animal’s metabolic needs are called essential amino acids. About ten of the amino acids are grouped under this category indicating that mammals require about half of the amino acids in their diet for growth and maintenance of normal nitrogen balance. 2. Non- Essential Amino Acids These amino acids are need not be provided through diet, because they can be biosynthesized in adequate amounts within the organism 3. Semi-essential amino acids Two amino acids are grouped under semi-essential amino acids since they can be synthesized within the organism but their synthesis is not in sufficient amounts. In that they should also be provided in the diet Eg . Arginine and Histidine

12. III. Biological or Physiological Classification

13. IV. Classification Based on the Fate of Each Amino acid Amino acids can be classified as: Glucogenic (potentially be converted to glucose ) eg . Alanine, cysteine, glycine, Arginine, glutamine, Isoleucine, tyrosine ketogenic (potentially be converted to ketone bodies) eg . Phenylalanine, tyrosine, tryptophan, isoleucine, leucine , and lysine both glucogenic and ketogenic - in severe starvation and untreated diabetes mellitus Alanine, Cysteine and serine, can also potentially form acetoacetate via acetyl CoA

14. V. Classification Based on Participation in Protein Synthesis I. Non-Standard Amino Acids In addition to the 20 standard amino acids, proteins may contain non- standard ( proteogenic ) amino acids, which are normally components of proteins but created by modification of the standard amino acids . Eg . Among the non – standard amino acids 4 – hydroxyproline a derivative of proline , 5- hydroxylysine derivative of lysine where both are found in collagen a fibrous protein of connective tissues. 6 N – methyllysine a constituent of myosin, a contractile protein of muscle γ- carboxy glutamate a derivative of glutamate, which is found in the blood clotting protein prothrombin .

15. V. Classification Based on Participation in Protein Synthesis II. Non – Proteogenic Amino Acids These amino acids occur in free or combined state, unlike in proteins , and play important roles in metabolism in plasma Eg : Citrulline used as a signalling molecule

16. ACIDIC AND BASIC PROPERTIES Amino acids in aqueous solution contain weakly acidic α-carboxyl groups and weakly basic α-amino groups. In addition, each of the acidic and basic amino acids contains an ionizable group in its side chain. Thus , both free amino acids and some amino acids combined in peptide linkages can act as buffers. Acids may be defined as proton donors and bases as proton acceptors. Acids (or bases) described as weak ionize to only a limited extent.

17. Peptides Proteins are macromolecules with a backbone formed by polymerization of amino acids in a polyamide structure. P eptide bonds are formed by linkage of α - carboxyl group of one amino acid with α- amino groups of the next amino acid by amide bonds. A peptide chain consisting of two amino acid residues is called a dipeptide, three amino acids tripeptide (e. g Glutathione) etc

18. Protein structure The 20 amino acids commonly found in proteins are joined together by peptide bonds . The linear sequence of the linked amino acids contains the information necessary to generate a protein molecule with a unique three-dimensional shape that determines function. The complexity of protein structure includes: primary, secondary , tertiary, and quaternary

19. Primary structure The sequence of amino acids in a protein is called the primary structure of the protein In proteins, amino acids are joined covalently by peptide bonds For example, valine and alanine can form the dipeptide valylalanine through the formation of a peptide bond Linkage of ≥50 amino acids through peptide bonds results in an unbranched chain called a polypeptide , or protein

20. Primary structure

21. Characteristics of peptide bonds has a partial double-bond character The bond is rigid and planar The peptide bond is almost always in the trans configuration instead of the cis configuration The peptide group is uncharged and polar-neither accept nor release protons over the pH range of 2–12

22. b) Secondary Structure The secondary structure of a protein refers to the local structure of a polypeptide chain , which is determined by Hydrogen bond. The Interactions are between the carbonyl oxygen group of one peptide bond and the amide hydrogen of another near by peptide bond. There are two types of secondary structure, the ∝ - helix and the β- pleated sheet.

23. A. α- Helix It is a rigid, right-handed spiral structure, consisting of a tightly packed, coiled polypeptide backbone core, with the side chains of the component L-amino acids extending outward from the central axis to avoid interfering sterically with each other A very diverse group of proteins contains α-helices. For example, the keratins are a family of closely related, rigid, fibrous proteins whose structure is nearly entirely α- helical . They are a major component of tissues such as hair and skin. In contrast to keratin, myoglobin, whose structure is also highly α-helical, is a globular , flexible molecule found in muscles all but the first and last peptide bond components are linked to each other through intrachain hydrogen bonds. Hydrogen bonds are individually weak, but they collectively serve to stabilize the helix.

24. A. α- Helix

25. Each turn of an α-helix contains 3.6 amino acids. Thus , amino acids spaced three or four residues apart in the primary sequence are spatially close together when folded in the α-helix . Proline and Glycine are “helix breaker” because the R group (a hydrogen) confers high flexibility .

26. B. β- Sheet A β-sheet is formed by two or more peptide chains ( β- strands ) aligned laterally and stabilized by hydrogen bonds between the carboxyl and amino groups of amino acids that either are far apart in a single polypeptide ( intrachain bonds) or are in different polypeptide chains ( interchain bonds). The adjacent β-strands are arranged either antiparallel to each other (with the N-termini alternating or parallel to each other

27. c) Tertiary Structure The three dimensional, folded and biologically active conformation of a protein is referred to as tertiary structure The structure reflects the overall shape of the molecule. The three - dimensional tertiary structure of a protein is stabilized by interactions between side chain functional group, covalent, disulfide bonds, hydrogen bonds, ionic bonds, and hydrophobic interactions .

28. Bonds in the tertiary structure Disulfide bonds: A disulfide bond (–S–S–) is a covalent linkage formed from the sulfhydryl group (−SH) of each of two cysteine residues to produce a cystine residue Hydrophobic interactions: Amino acids with nonpolar side chains tend to be located in the interior of the polypeptide molecule, where they associate with other hydrophobic amino acids Hydrogen bonds: Amino acid side chains containing oxygen- or nitrogen-bound hydrogen, such as in the alcohol groups of serine and threonine , can form hydrogen bonds with electron-rich atoms, such as the oxygen of a carboxyl group or carbonyl group of a peptide bond Ionic interactions: Negatively charged groups, such as the carboxylate group (−COO−) in the side chain of aspartate or glutamate, can interact with positively charged groups such as the amino group (− NH3 +) in the side chain of lysine

29. Bonds in the tertiary structure

30. Bonds in the tertiary structure

31. V. QUATERNARY STRUCTURE Many proteins consist of a single polypeptide chain and are defined as monomeric proteins. However, others may consist of two or more polypeptide chains that may be structurally identical or totally unrelated. The arrangement of these polypeptide subunits is called the quaternary structure of the protein. Subunits are held together primarily by noncovalent interactions (for example, hydrogen bonds, ionic bonds, and hydrophobic interactions). Subunits either may function independently of each other or may work cooperatively, as in hemoglobin , in which the binding of oxygen to one subunit of the tetramer increases the affinity of the other subunits for oxygen

32. Chaperones in protein folding Chaperones, then, facilitate correct protein folding by binding to and stabilizing exposed, aggregation-prone hydrophobic regions in polypeptides , preventing premature folding The chaperones, also known as heat shock proteins (HSP), interact with a polypeptide at various stages during the folding process. Some chaperones bind hydrophobic regions of an extended polypeptide and are important in keeping the protein unfolded until its synthesis is completed

33. Protein denaturation Denaturation results in the unfolding and disorganization of a protein’s secondary and tertiary structures without the hydrolysis of peptide bonds. Denaturing agents include heat, urea, organic solvents, strong acids or bases , detergents, and ions of heavy metals such as lead. Denaturation may, under ideal conditions, be reversible, such that the protein refolds into its original native structure when the denaturing agent is removed. However, most proteins remain permanently disordered once denatured. Denatured proteins are often insoluble and precipitate from solution.

34. Denaturing agents 1 . physical factors Temperature, pressure, mechanical shear force, ultrasonic vibration and ionizing radiation causes the protein to lose its biological activity. 2. chemical factors Acids and alkalis, organic solvents (acetone, ethanol), detergents (cleaning agents), certain amides urea, guanidine hydrochloride, alkaloids, and heavy metal salts

35. Properties of a Denatured Protein An increase in number of reactive and functional group in the composition of the native protein molecule ( side chain group of amino acids, COOH, NH2, SH, OH … etc ) Reduced solubility Configurational alteration of the protein molecule. Loss of biological activity evoked by the disarrangement of the native structural molecular organization. Access of proteolytic enzymes in comparasion with the native protein

36. Clinical Application of Denaturation The amounts of proteins found in the urine, serum, CSF are utilized to asses various pathological conditions. The appearance of proteins like Albumin and Globulin in the urine can be detected by precipitating them using ammonium sulphate . This could be used to asses the degree of kidney impairment and glomerular permeability

37. Globular hemeproteins Hemeproteins are a group of specialized proteins that contain heme as a tightly bound prosthetic group Hemoglobin and myoglobin are the two most abundant hemeproteins in humans, the heme group serves to reversibly bind oxygen (O2). Hemoglobin ( Hb ) is restricted to the Erythrocytes which is responsible for the movement of O2 between lungs and other tissues

38. Heme structure Heme is the O2 – binding molecule common to Mb and Hb protophorphyrin IX is the backbone of heme when iron is complexed with protophorphyrin IX it is called Heme . So heme is the prosthetic group in Hemoglobin, Myoglobin and Cytochrome b, c, and c1 . Heme become an integral part of the globin proteins during poly peptide synthesis. It is the heme molecule that give globin proteins their characteristic red brown colour . Once the Fe2 + ( Ferrious ) is incorporated, the protein is called hemoglobin. Such structural coordination creates an environment essential for Globin to bind and release O2.

39. Heme structure

40. Myoglobin and hemoglobin Myoglobin contains a single polypeptide chain folded about a prosthetic group, the heme , which contains the oxygen binding site. Hemoglobin is a tetrameric protein. Each polypeptide subunit closely resembles myoglobin Myoglobin can bind only one molecule of O2, because it contains only one heme group. In contrast, hemoglobin can bind four molecules of O2, one at each of its four heme groups

41. Hemoglobin Adult Hb ( HbA ) Hemoglobin A, the major hemoglobin in adults, is composed of four polypeptide chains (two α chains and two β chains) held together by noncovalent interactions Fetal Hb ( HbF ) contain 2 alpha and 2 γ subunits Sickle Cell Hemoglobin ( HbS ) HbS , the variant most commonly associated with sickle cell disease, cannot tolerate high protein concentration when deoxygenated. At low oxygen concentrations, deoxy HbS polymerizes, forms fibers, and distorts erythrocytes in to sickle shapes. The mutation is Glu6 β - val a surface localized charged amino acids is replaced by a hydrophobic residue

42. HbS

43. Digestion and Absorption of Proteins Entry of a protein in to stomach stimulates the gastric mucosa to secrete a hormone gastrin which in turn stimulates the secretion of Hcl by the parietal cells of the gastric glands and pepsinogen by the chief cells The HCL thus produced lower the pH of stomach to (pH1.5 – 2.5) and acts as an antiseptic and kills most of the bacteria and other foreign cells ingested along with. The acid denatures the protein and the whole protein susceptible to hydrolysis by the action other proteolytic enzymes Pepsinogen is converted into pepsin This active pepsin cleaves the ingested protein at their amino terminus of aromatic amino acids ( Phe , Tyr, and Trp .) The major products of pepsin action are large peptide fragments and some free amino acids

44. Digestion and Absorption of Proteins The entry of large peptide fragments and some free amino acids in the upper part of the small intestine (Duodenum), excites the release of a hormone cholecytokinin (CCK ) which: 1) stimulates gall bladder contraction. 2) stimulate secretion of several pancreatic enzymes whose activity is between pH 7and 8 in proenzyme forms A low pH as gastric content enter the duodeumtriggers the secretion of a hormone Secretin in the blood. Secretin stimulates the pancreas to secrete HCO-3 (bicarbonate), which in the small intestine neutralizes the gastric HCL and abruptly change the pH to 7.0.

45. Proenzyme conversion

46. Digestion and Absorption of Proteins By the sequential action of these proteolytic enzymes and peptides ingested proteins are hydrolyzed to yield a mixture of free amino acids which can be transported across the epithelial lining of the small intestine Since pancreatic juice does not contain appreciable aminopeptidase activity final digestion of di and Oligopeptides depends on the small intestinal enzymes. The lumenal surface of epithelial cells is rich in endopeptidase , and dipeptidase aminopeptidase activity The end products of the cell surface digestion are free amino acids and di and tripeptides

47. Digestion and Absorption of Proteins These are passed in to the interior of the epithelial cell where other specific peptidases convert almost all of them to a single amino acids they are transported to the blood stream and carried to liver (primarily) and other tissues for oxidative degradation . This process complete the absorption of 99% of digested proteins