Antibodies are immune system-related proteins called immunoglobulins. Each antibody consists of four polypeptides– two heavy chains and two light chains joined to form a "Y" shaped molecule. ... This variable region, composed of 110-130 amino acids, give the antibody its specificity for binding antigen.
CLONAL SELECTION THEORY IS AN SCIENTIFIC THEORY IN IMMUNOLOGY THAT EXPALINS THE FUNCTION OF CELLS OF THE IMMUNE SYSTEM IN RESPONSE TO SPECIFIC ANTIGEN INVADING THE BODY.
Antibodies are immune system-related proteins called immunoglobulins. Each antibody consists of four polypeptides– two heavy chains and two light chains joined to form a "Y" shaped molecule. ... This variable region, composed of 110-130 amino acids, give the antibody its specificity for binding antigen.
CLONAL SELECTION THEORY IS AN SCIENTIFIC THEORY IN IMMUNOLOGY THAT EXPALINS THE FUNCTION OF CELLS OF THE IMMUNE SYSTEM IN RESPONSE TO SPECIFIC ANTIGEN INVADING THE BODY.
Immunodiffusion -Different Types,Principle,procedureand application. it is a diagnostic technique for the detection or measurements of antibodies and antigens by their precipitation which involves diffusion through a substances such as agar or gel agarose .common types -oudin procedure,oakley fulthorpe procedure ,mancini technique ,ouchterlony double immuno diffusion
Hybridoma technology is a method for producing large number of identical antibodies called monoclonal antibodies.
It was discovered by G.kohler and C.milstein in 1975. they were awarded nobel prize for physiology and medicine in 1975.
The hybrid cells are produced by fusing B- lumphocyte with myeloma cells or tumour cells.
The B-lymphocyte have the ability to produce large number of antibodies and tumour cells have indefinite growth.
This is why two cells are used for the production of hybrid cell
It includes general introduction to antibodies; Monoclonal antibodies; comparison between Polyclonal & Monoclonal antibodies; Hybridoma Technology & Hyridoma Selection; advantages & disadvantages of mABs; Applications of mABs; Recombinant Monoclonal antibodies production through Antibody Engineering.
Immunodiffusion -Different Types,Principle,procedureand application. it is a diagnostic technique for the detection or measurements of antibodies and antigens by their precipitation which involves diffusion through a substances such as agar or gel agarose .common types -oudin procedure,oakley fulthorpe procedure ,mancini technique ,ouchterlony double immuno diffusion
Hybridoma technology is a method for producing large number of identical antibodies called monoclonal antibodies.
It was discovered by G.kohler and C.milstein in 1975. they were awarded nobel prize for physiology and medicine in 1975.
The hybrid cells are produced by fusing B- lumphocyte with myeloma cells or tumour cells.
The B-lymphocyte have the ability to produce large number of antibodies and tumour cells have indefinite growth.
This is why two cells are used for the production of hybrid cell
It includes general introduction to antibodies; Monoclonal antibodies; comparison between Polyclonal & Monoclonal antibodies; Hybridoma Technology & Hyridoma Selection; advantages & disadvantages of mABs; Applications of mABs; Recombinant Monoclonal antibodies production through Antibody Engineering.
What are Antibody
Monoclonal Antibody (mAb)
Structure of mAb
Types of Monoclonal Antibody (mAb)
Preparation of Monoclonal Antibody
Hybridoma Technique, Phage display Technique
Application of Monoclonal Antibody
Advantage and Disadvantage of Monoclonal Antibody
What are antibodies?
An antibody is a protein used by immune system to identify and neutralize foreign agents like bacteria and viruses.
Each antibody recognizes a specific antigen unique to its target.
Our bodies are constantly under attack by an army of microorganisms, toxins, allergens and other substances that are recognized as foreign (non-self).
The ways in which the body protects itself from pathogens can be thought of as an army consisting of three lines of defense.
Immune system consists of two organs primary and secondary lymphoid organs.
1-Primary (Central) lymphoid organs.
2-Secondary (Peripheral) lymphoid organs.
1-Thymus Gland.
2-Bone Marrow.
They consist of ….
Spleen.
Lymph nodes.
Mucosa- associated lymphoid tissue.
The sites of immune cell birth and the locations where they mature are considered primary lymphatic organs.
Locations in the body where immune cells become activated, reside, or carry out their functions are called secondary lymphatic organs
The cells of the immune system arise from a pluripotent Hematopoietic Stem Cells (HSCs) through a process known as haematopoiesis.
Hematopoiesis involves the production, development, differentiation, and maturation of the blood cells (erythrocytes, megakaryocytes and leukocytes) from HSCs.
Differentiation of the HSC will occur along one of two pathways, giving rise to either a common myeloid progenitor or a common lymphoid progenitor cells in the presence of specific cytokines or soluble mediates (growth factor).
Title: Sense of Taste
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the structure and function of taste buds.
Describe the relationship between the taste threshold and taste index of common substances.
Explain the chemical basis and signal transduction of taste perception for each type of primary taste sensation.
Recognize different abnormalities of taste perception and their causes.
Key Topics:
Significance of Taste Sensation:
Differentiation between pleasant and harmful food
Influence on behavior
Selection of food based on metabolic needs
Receptors of Taste:
Taste buds on the tongue
Influence of sense of smell, texture of food, and pain stimulation (e.g., by pepper)
Primary and Secondary Taste Sensations:
Primary taste sensations: Sweet, Sour, Salty, Bitter, Umami
Chemical basis and signal transduction mechanisms for each taste
Taste Threshold and Index:
Taste threshold values for Sweet (sucrose), Salty (NaCl), Sour (HCl), and Bitter (Quinine)
Taste index relationship: Inversely proportional to taste threshold
Taste Blindness:
Inability to taste certain substances, particularly thiourea compounds
Example: Phenylthiocarbamide
Structure and Function of Taste Buds:
Composition: Epithelial cells, Sustentacular/Supporting cells, Taste cells, Basal cells
Features: Taste pores, Taste hairs/microvilli, and Taste nerve fibers
Location of Taste Buds:
Found in papillae of the tongue (Fungiform, Circumvallate, Foliate)
Also present on the palate, tonsillar pillars, epiglottis, and proximal esophagus
Mechanism of Taste Stimulation:
Interaction of taste substances with receptors on microvilli
Signal transduction pathways for Umami, Sweet, Bitter, Sour, and Salty tastes
Taste Sensitivity and Adaptation:
Decrease in sensitivity with age
Rapid adaptation of taste sensation
Role of Saliva in Taste:
Dissolution of tastants to reach receptors
Washing away the stimulus
Taste Preferences and Aversions:
Mechanisms behind taste preference and aversion
Influence of receptors and neural pathways
Impact of Sensory Nerve Damage:
Degeneration of taste buds if the sensory nerve fiber is cut
Abnormalities of Taste Detection:
Conditions: Ageusia, Hypogeusia, Dysgeusia (parageusia)
Causes: Nerve damage, neurological disorders, infections, poor oral hygiene, adverse drug effects, deficiencies, aging, tobacco use, altered neurotransmitter levels
Neurotransmitters and Taste Threshold:
Effects of serotonin (5-HT) and norepinephrine (NE) on taste sensitivity
Supertasters:
25% of the population with heightened sensitivity to taste, especially bitterness
Increased number of fungiform papillae
Lung Cancer: Artificial Intelligence, Synergetics, Complex System Analysis, S...Oleg Kshivets
RESULTS: Overall life span (LS) was 2252.1±1742.5 days and cumulative 5-year survival (5YS) reached 73.2%, 10 years – 64.8%, 20 years – 42.5%. 513 LCP lived more than 5 years (LS=3124.6±1525.6 days), 148 LCP – more than 10 years (LS=5054.4±1504.1 days).199 LCP died because of LC (LS=562.7±374.5 days). 5YS of LCP after bi/lobectomies was significantly superior in comparison with LCP after pneumonectomies (78.1% vs.63.7%, P=0.00001 by log-rank test). AT significantly improved 5YS (66.3% vs. 34.8%) (P=0.00000 by log-rank test) only for LCP with N1-2. Cox modeling displayed that 5YS of LCP significantly depended on: phase transition (PT) early-invasive LC in terms of synergetics, PT N0—N12, cell ratio factors (ratio between cancer cells- CC and blood cells subpopulations), G1-3, histology, glucose, AT, blood cell circuit, prothrombin index, heparin tolerance, recalcification time (P=0.000-0.038). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and PT early-invasive LC (rank=1), PT N0—N12 (rank=2), thrombocytes/CC (3), erythrocytes/CC (4), eosinophils/CC (5), healthy cells/CC (6), lymphocytes/CC (7), segmented neutrophils/CC (8), stick neutrophils/CC (9), monocytes/CC (10); leucocytes/CC (11). Correct prediction of 5YS was 100% by neural networks computing (area under ROC curve=1.0; error=0.0).
CONCLUSIONS: 5YS of LCP after radical procedures significantly depended on: 1) PT early-invasive cancer; 2) PT N0--N12; 3) cell ratio factors; 4) blood cell circuit; 5) biochemical factors; 6) hemostasis system; 7) AT; 8) LC characteristics; 9) LC cell dynamics; 10) surgery type: lobectomy/pneumonectomy; 11) anthropometric data. Optimal diagnosis and treatment strategies for LC are: 1) screening and early detection of LC; 2) availability of experienced thoracic surgeons because of complexity of radical procedures; 3) aggressive en block surgery and adequate lymph node dissection for completeness; 4) precise prediction; 5) adjuvant chemoimmunoradiotherapy for LCP with unfavorable prognosis.
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
NYSORA Guideline
2 Case Reports of Gastric Ultrasound
Couples presenting to the infertility clinic- Do they really have infertility...Sujoy Dasgupta
Dr Sujoy Dasgupta presented the study on "Couples presenting to the infertility clinic- Do they really have infertility? – The unexplored stories of non-consummation" in the 13th Congress of the Asia Pacific Initiative on Reproduction (ASPIRE 2024) at Manila on 24 May, 2024.
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Ve...kevinkariuki227
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
TEST BANK for Operations Management, 14th Edition by William J. Stevenson, Verified Chapters 1 - 19, Complete Newest Version.pdf
MANAGEMENT OF ATRIOVENTRICULAR CONDUCTION BLOCK.pdfJim Jacob Roy
Cardiac conduction defects can occur due to various causes.
Atrioventricular conduction blocks ( AV blocks ) are classified into 3 types.
This document describes the acute management of AV block.
New Directions in Targeted Therapeutic Approaches for Older Adults With Mantl...i3 Health
i3 Health is pleased to make the speaker slides from this activity available for use as a non-accredited self-study or teaching resource.
This slide deck presented by Dr. Kami Maddocks, Professor-Clinical in the Division of Hematology and
Associate Division Director for Ambulatory Operations
The Ohio State University Comprehensive Cancer Center, will provide insight into new directions in targeted therapeutic approaches for older adults with mantle cell lymphoma.
STATEMENT OF NEED
Mantle cell lymphoma (MCL) is a rare, aggressive B-cell non-Hodgkin lymphoma (NHL) accounting for 5% to 7% of all lymphomas. Its prognosis ranges from indolent disease that does not require treatment for years to very aggressive disease, which is associated with poor survival (Silkenstedt et al, 2021). Typically, MCL is diagnosed at advanced stage and in older patients who cannot tolerate intensive therapy (NCCN, 2022). Although recent advances have slightly increased remission rates, recurrence and relapse remain very common, leading to a median overall survival between 3 and 6 years (LLS, 2021). Though there are several effective options, progress is still needed towards establishing an accepted frontline approach for MCL (Castellino et al, 2022). Treatment selection and management of MCL are complicated by the heterogeneity of prognosis, advanced age and comorbidities of patients, and lack of an established standard approach for treatment, making it vital that clinicians be familiar with the latest research and advances in this area. In this activity chaired by Michael Wang, MD, Professor in the Department of Lymphoma & Myeloma at MD Anderson Cancer Center, expert faculty will discuss prognostic factors informing treatment, the promising results of recent trials in new therapeutic approaches, and the implications of treatment resistance in therapeutic selection for MCL.
Target Audience
Hematology/oncology fellows, attending faculty, and other health care professionals involved in the treatment of patients with mantle cell lymphoma (MCL).
Learning Objectives
1.) Identify clinical and biological prognostic factors that can guide treatment decision making for older adults with MCL
2.) Evaluate emerging data on targeted therapeutic approaches for treatment-naive and relapsed/refractory MCL and their applicability to older adults
3.) Assess mechanisms of resistance to targeted therapies for MCL and their implications for treatment selection
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
The prostate is an exocrine gland of the male mammalian reproductive system
It is a walnut-sized gland that forms part of the male reproductive system and is located in front of the rectum and just below the urinary bladder
Function is to store and secrete a clear, slightly alkaline fluid that constitutes 10-30% of the volume of the seminal fluid that along with the spermatozoa, constitutes semen
A healthy human prostate measures (4cm-vertical, by 3cm-horizontal, 2cm ant-post ).
It surrounds the urethra just below the urinary bladder. It has anterior, median, posterior and two lateral lobes
It’s work is regulated by androgens which are responsible for male sex characteristics
Generalised disease of the prostate due to hormonal derangement which leads to non malignant enlargement of the gland (increase in the number of epithelial cells and stromal tissue)to cause compression of the urethra leading to symptoms (LUTS
These lecture slides, by Dr Sidra Arshad, offer a quick overview of physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar leads (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
4. ANTIBODY
Antibodies (Abs) or Immunoglobulins
(Igs) are glycoprotein having combing
sites for antigen & are produce by plasma
cells in response to stimulation of B cell
by foreign antigen.
6. BONDS OF ANTIGEN-ANTIBODY INTERACTION
1. Hydrogen bonds,
2. Electrostatic bonds,
3. hydrophobic interactions
4. van der Waals forces,
Antibody and antigen reactions are reversible
7. Affinity refers to how a specific combining site on the
antibody is attracted to a specific epitope on the antigen
(remember that the antigen has multiple epitopes).
Avidity refers to the overall interaction between the
antigen-antibody complex. In other words, avidity is the
sum of all affinities.
8. The most important function of antibodies is to confer
protection against microbial pathogens, by different ways.
1- Neutralization.
2- Opsonization.
3- Killing by Oxidation.
4- Agglutination and precipitation.
5- Antibody-dependent cellular cytotoxicity (ADCC).
6- Antibodies activate the complement system.
FUNCTIONS OF IMMUNOGLOBULINS:
9. IMMUNOGLOBULIN CLASSES (ISOTYPE)
The immunoglobulins can be divided into five different
classes, based on differences in the amino acid sequences
in the constant region of the heavy chains. So, there are 5
classes of Igs are IgM, IgA, IgG, IgE and IgD.
Mnemonic rule = (MAGED)
10. EPITOPES (ANTIGENIC DETERMINANTS)
Epitope is immunologically active regions of an
immunogen that binds to antigen-specific membrane
receptors on B lymphocyte cell.
The whole antigen dose not trigger immune response and
only a small part of it induces B and T cell response.
Each antigen carries many & different epitopes.
11. *Antigens (Ag) are molecules (substances) recognised by
the immune system which induce an immune response.
*Although all antigens are recognized by specific
lymphocytes or by antibodies, only some antigens are
capable of activating lymphocytes.
*Molecules that stimulate immune responses are called
Immunogens.
*All immunogens must be antigens, but not all antigen
must be immunogens.
14. PARATOPE
*Each Y-shaped antibody molecule has at least two binding sites
(paratope) that can attach to a specific epitope on an antigen.
*The site in the variable (V) region of an antibody which is
complementary to the epitope and interacts with the epitope.
*The combination is very much like the lock and key analogy
(identity).
15. MONOCLONAL ANTIBODIES
Mono=single=one
These singular types of antibody are produced by
harvesting purified cell lines derived from a single
B cell within the immunity, allowing them to bind to
one unique epitope region of an antigen.
(mAB,moAB)
16. PRODUCING MONOCLONAL ANTIBODIES
mAbs are produced by immunizing (injection an
immunogen) an animal, often a mouse, multiple times with
a specific antigen.
After the immunogen has caused an immune response, the
B-cells from the spleen are removed.
Since normal B cells are unable to proliferate forever, they
are fused with immortal, cancerous B cells called myeloma
cells, to yield hybridoma cells.
17. PRODUCING MONOCLONAL ANTIBODIES
All of the cells are then placed in a selective medium
(Hypoxanthine Aminopterin Thymidine -HAT) that allows
only the hybridomas to grow; unfused myeloma cells
cannot grow, and any unfused B cells die off.
Mechanism of grow hybridoma cell in this selective media:
B cell is contain a gene hypoxanthine-guanine
phosphoribosyltransferase (HGPRT) but myeloma don’t
have it this gene.
18. PRODUCING MONOCLONAL ANTIBODIES
This gene is necessary for replication of the cell.
This HAT media only cell that have HGPRT
positive will survive ,while cells HGPRT negative
will die.
So, HAT media only kill HGPRT negative cells only.
19. PRODUCING MONOCLONAL ANTIBODIES
The hybridomas, which are capable of growing
continuously in culture while producing antibodies, are then
screened for the desired mAb. Those producing the desired
mAbs are grown in tissue culture; the culture medium is
harvested periodically and mAbs are purified from the
medium.
20. PRODUCING MONOCLONAL ANTIBODIES
Once a monoclonal antibody hybridoma clone is created, it
is a stable renewable source of antibodies. This ensures
that each batch is identical to the previous one.
Hybridomas can also be injected in the peritoneal cavity of
a mouse in order to create a tumor that secrets a fluid rich
in antibodies called ascites fluid.
22. MONOCLONAL ANTIBODIES: ADVANTAGES
1. Can produce large quantities of identical
antibodies.
2. Batch to batch homogeneity.
3. Have high specificity to a single epitope which
reduces the risks for cross reactivity.
4. Better results in assays requiring quantification of
the protein levels
5. Very efficient in affinity purification.
23. MONOCLONAL ANTIBODIES: DISADVANTAGES:
1. more expensive.
2. Requires significantly more time 6 months.
3. More demanding storage conditions for the clone.
4. Less strong for detecting the protein in a denatured state
or altered conformation. Why?
5. Less ideal for application requiring quick capture of the
target protein
6. More sensitive to pH and buffer conditions.
7. More susceptible to binding changes when labelled.
24. Poly=many
Describe a collection of antibodies which are
grown from different B cells. This makes them
capable of recognizing multiple epitopes on the
same antigen.
POLYCLONAL ANTIBODIES
25. Antibodies used for research and diagnostic purposes are
often obtained by injecting a lab animal such as a rabbit
with a specific antigen.
Within a few weeks, the animal’s immune system will
produce high levels of antibodies specific for the antigen.
These antibodies can be harvested in an antiserum, which
is whole serum collected from an animal following
exposure to an antigen.
PRODUCING POLYCLONAL ANTIBODIES
26. Because most antigens are complex structures with
multiple epitopes, they result in the production of multiple
antibodies in the lab animal.
This so-called polyclonal antibody response is also
typical of the response to infection by the human immune
system.
Antiserum drawn from an animal will thus contain
antibodies from multiple clones of B cells, with each B cell
responding to a specific epitope on the antigen.
PRODUCING POLYCLONAL ANTIBODIES
27. Antiserum obtained from animals will not only contain
antibodies against the antigen artificially introduced in the
laboratory, but it will also contain antibodies to any other
antigens to which the animal has been exposed during its
lifetime.
For this reason, antisera must first be “purified” to remove
other antibodies before using the antibodies for research or
diagnostic assays.
PRODUCING POLYCLONAL ANTIBODIES
28. Figure: This diagram illustrates the process for harvesting polyclonal antibodies produced in
response to an antigen.
specific
specific epitope
multiple
Varying paratope
29. POLYCLONAL ANTIBODIES: ADVANTAGES
1. Inexpensive.
2. Easy to store.
3. Quick to produce.
4. Ready to use in under four months.
5. Highly stable and tolerant of pH or buffer changes.
6. High affinity as the antibodies bind to more than one
epitope, they can help amplify the signal from target
protein even with low expression level.
30. POLYCLONAL ANTIBODIES: DISADVANTAGES
1. Higher potential for cross reactivity.
2. High variability between different batches produced in
different animals at different times.
3. The affinity purification of the serum will typically be
required to minimize cross reactivity.
31. POLYCLONAL VS. MONOCLONAL ANTIBODIES.
Monoclonal antibodies Polyclonal antibodies
Expensive production Inexpensive production
Long production time Rapid production
More Specificity less Specificity
Recognize a single epitope on an
antigen
Recognize multiple epitopes on an
antigen
Production is continuous and uniform
once the hybridoma is made
Different batches vary in composition
Few host species options Many host species options
A homogenous antibody population. A heterogeneous antibody population.
32. ANTIBODY PURIFICATION
Antibody purification is a multistep process by which
contaminants of source is removed and antibody with
high purity is obtained.
Antibodies are widely used as injectables and parenteral
products for human use.
33. POSSIBLE CONTAMINANTS
Antibodies are produced in mice, rabbits, etc. Nowadays
antibodies are expressed in cell culture with good yield.
When antibodies are taken from animal source possible
contaminants could be serum proteins such as albumin,
transferrins and cell degradation products like DNA and
cellular proteins.
Currently serum free media for cell culture are developed
which can ease the antibody purification process. With a
combination of chromatographic steps and precipitation
methods one can purify the antibody with good yield and
resolution.
34. The choice of a purification method is based on these
factors:
Nature of antibody,
Nature of feedstock,
Scale of production,
Economics – cost and other factors,
Process Timings, and
Desired purity.
PURIFICATION METHODS
35. ANTIBODY PURIFICATION PROCESS
Antibody purification is multistep process, which mainly
includes:
1. Sample Preparation
2. Capture
3. Initial Purification
4. Secondary Purification
5. Polishing / Formulation
36. ANTIBODY PURIFICATION: STEP 1: SAMPLE
PREPARATION
sample preparation is the initial step in which crude protein
sample is conditioned or making it ready for the initial
capture step.
Generally this step involves changing pH or Ionic strength,
dilution of the crude sample or addition of salts for the
ionic strength.
use buffer exchange by size exclusion chromatography or
to use ultrafiltration or diafiltration.
37. These are the various chromatographic techniques which
are widely used for antibody purification.
Immunoaffinity (most used).
Immobilized Metal Affinity Chromatograhy (IMAC)
Ion – Exchange Chromatograhy (IEC)
Hydrophobic Interaction Chromatography (HIC)
Hydroxyapatite
Size – Exclusion chromatography (SEC)
ANTIBODY PURIFICATION: STEP 2: CAPTURE
38. ANTIBODY PURIFICATION: STEP 3:
SECONDARY PURIFICATION
The secondary purification step is selected based on the
nature and the optimization requirement of the crude
antibody source.
In addition to protein contaminants, other impurities such
as DNA, endotoxins, viruses, and aggregates need to be
removed. In such cases, a multistep procedure is almost
inevitable.
39. ANTIBODY PURIFICATION: STEP 4:
POLISHING / FORMULATION
Final polishing / formulation step can be considered as a
part of purification in which it removes conditions that would
impair the stability or utility of the antibody in its intended
use.
Ultarfilteration
Diafiltration
Lyophilization
40. POLYCLONAL ANTIBODIES VS. MONOCLONAL
ANTIBODIES: DIAGNOSTIC STUDIES
Which is better, a monoclonal or a polyclonal
antibody?
It depends on the different characteristics of
monoclonal and polyclonal antibodies.
41. POLYCLONAL ANTIBODIES VS. MONOCLONAL
ANTIBODIES: DIAGNOSTIC STUDIES
The best use of polyclonal antibodies is to detect unknown antigens.
Polyclonal antibodies are used as a secondary antibody in
immunoassays (e.g. ELISA, western blotting, microarray
assays, immunohistochemistry, flow cytometry).
Their role is to bind to different epitopes and amplify the signal, leading
to better detection. (sensitization and stronger)
Polyclonal Antibodies: Polyclonal antibodies are used in general
research applications.
42. POLYCLONAL ANTIBODIES VS. MONOCLONAL
ANTIBODIES: DIAGNOSTIC STUDIES
Monoclonal antibodies are often used as primary
antibodies in immunoassays due to their ability of
specifically binding to a single epitope of an antigen.
Monoclonal antibodies, provide an unlimited source of
antibody that is homogeneous.
Monoclonal Antibodies: Monoclonal antibodies are used as
therapeutic drugs.
43. WHAT ARE THE USES FOR MONOCLONAL ANTIBODIES?
The use of monoclonal antibodies to treat diseases is called
immunotherapy because each type of monoclonal antibody
will target a specific targeted antigen in the body.
Uses for monoclonal antibodies includes: cancer,
MS ,Crohn's disease, ulcerative colitis, rheumatoid
arthritis, systemic lupus erythematosus …
44. In these conditions the monoclonal antibody targets and
interferes with the action of a chemical or receptor that
is involved in the development of the condition that is being
treated.
For example, a monoclonal antibody used for treating
cancer may block a receptor that cancer cells use for
preventing the immune system from the destroying the
cancer cell. Blocking this receptor allows the immune
system to recognize cancer cells and destroy them.
45. SUMMARY
Monoclonal antibodies provide higher specificity than
polyclonal antisera because they bind to a single epitope
and usually have high affinity.
Polyclonal antisera are useful for some types of
laboratory assays, but other assays require more
specificity. Diagnostic tests that use polyclonal antisera are
typically only used for screening because of the possibility
of false-positive and false-negative results.
Antigens which are present on the body’s own cells are called the auto-antigens or self antigens. The antigens on the non-self cells are known as foreign antigens or non-self antigens.
Also called Ligand
Elicit = triiger = stimulate= evoke =
one hundred thousand
Analogy=identity.
Monoclonal antibodies consist of one (mono) type of antibody that binds to a specific epitope on the target antigen
hypoxanthine-aminopterin-thymidine medium
hypoxanthine-aminopterin-thymidine medium
Batch دفعه
hypoxanthine-aminopterin-thymidine
higher specificity
This immunogen causes an immune response in the animal and the activation of multiple B-cells which all target a specific epitope on the immunogen.
lack of specificity
high degree of cross reactivity
Crude antibody sample need to be concentrated which is done either by centrifugation or Filtration, sometimes both the methods are combined to get faster results.Antibody precipitation can be done to precipitate out, salts used for this purpose include ammonium sulphate, Poly ethylene glycol, etc. if the antibody is expressed in cell line media contaminants (dye – phenol red) need to be removed which in-turn can bind to the column and reduce the efficiency of the purification process