The document discusses various serological assays used to detect antibodies and antigens. It describes the basic principles of agglutination tests, precipitation tests, immunofluorescence, radioimmunoassay, ELISA, complement fixation tests, and serum neutralization tests. It provides examples of the application of these assays for diagnosing diseases like brucellosis, influenza, and foot-and-mouth disease. The document emphasizes the importance of assay sensitivity, specificity, reproducibility, and using appropriate antigen and antibody concentrations.
Direct
Passive
Reverse Passive
Agglutination Inhibition
Coagglutination
Agglutination tests can be done :
On slides
In tubes
In microtritation plates
-Difference between precipitation and agglutination reaction.
The lecture was presented to the students of Saudi board of Community Medicine to help them know about the various serological methods applicable in the diagnosis of infectious diseases in general with attention upon the specificity and sensitivity of various diagnostic modalities. The lecture covers the basic principles of each test and the clinical applications with the advantages and disadvantages of each.
Direct
Passive
Reverse Passive
Agglutination Inhibition
Coagglutination
Agglutination tests can be done :
On slides
In tubes
In microtritation plates
-Difference between precipitation and agglutination reaction.
The lecture was presented to the students of Saudi board of Community Medicine to help them know about the various serological methods applicable in the diagnosis of infectious diseases in general with attention upon the specificity and sensitivity of various diagnostic modalities. The lecture covers the basic principles of each test and the clinical applications with the advantages and disadvantages of each.
Antigen antibody interactions play important role in immunological assays which help in detection of disease.Such interaction are of various types e.g.Precipitation,Flocculation, Agglutination, Complement fixation, ELISA,RIA, Immunoflourescence,Immunoprecipitation.
Antigen antibody interactions play important role in immunological assays which help in detection of disease.Such interaction are of various types e.g.Precipitation,Flocculation, Agglutination, Complement fixation, ELISA,RIA, Immunoflourescence,Immunoprecipitation.
Antigen ,Antibody and Ag-Ab reactions ppt by DR.C.P.PRINCEDR.PRINCE C P
An immunogen refers to a molecule that is capable of eliciting an immune response, whereas an antigen refers to a molecule that is capable of binding to the product of that immune response (Ab).
So, an immunogen is necessarily an antigen, but an antigen may not necessarily be an immunogen
The terms immunogen and antigen are often used interchangeably but the later is more common.
Antibodies are Globulin Protein (Immunoglobulin) that are synthesized in the Serum and Tissue fluids.
It reacts specifically with the antigen that stimulated their production.
There are two types serum proteins: albumin and globulin
There are Three types of globulins .
1. Alpha globulin
2. Beta globulin
3. Gamma globulin (Antibodies)
Gamma globulins are responsible for immunity. So they are called as Immunoglobulin (Ig)
The binding of an antibody with an antigen of the type that stimulated the formation of antibody that results in the following reaction
Agglutination
Precipitation
Complement fixation
Phagocytosis
Neutralization of an exotoxin
Opsonization
Tissue fixation
Chemotaxis
Activation of mast cells and basophils
PPT prepared by:
DR.PRINCE C P
Associate Professor , Department of Microbiology,
Mother Theresa Post Graduate & Research Institute of Health Sciences (Government of Puducherry Institution)
serological techniques for detection of plant virus.pptxReddykumarAv
Serological tests involve diagnostic procedures for identifying antibodies and antigens in a patient's blood sample. Serology definition tells that Serological tests could be used to diagnose infections and autoimmune disorders, as well as to see whether a person is resistant to these kinds of diseases and for a variety of other purposes, including assessing a person's blood type.
ELISA, principle and method by kk sahuKAUSHAL SAHU
What is ELISA.
Principle.
History.
Types of ELISA method.
1.Direct ELISA.
2.Indirect ELISA.
3.Sandwhich ELISA.
Conclusion.
References.
Antibodies (also known as immunoglobulins abbreviated Ig) are gamma globulin proteins that are found in blood and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.
An approache for different kinds of sampling for diagnosis of animal diseases
( Scientific activity done at Al Ain , UAE . Under the supervesion of Department of Agriculture and livestock
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
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
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Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
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
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.
Title: Sense of Smell
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 primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
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Knee anatomy and clinical tests 2024.pdfvimalpl1234
This includes all relevant anatomy and clinical tests compiled from standard textbooks, Campbell,netter etc..It is comprehensive and best suited for orthopaedicians and orthopaedic residents.
Ethanol (CH3CH2OH), or beverage alcohol, is a two-carbon alcohol
that is rapidly distributed in the body and brain. Ethanol alters many
neurochemical systems and has rewarding and addictive properties. It
is the oldest recreational drug and likely contributes to more morbidity,
mortality, and public health costs than all illicit drugs combined. The
5th edition of the Diagnostic and Statistical Manual of Mental Disorders
(DSM-5) integrates alcohol abuse and alcohol dependence into a single
disorder called alcohol use disorder (AUD), with mild, moderate,
and severe subclassifications (American Psychiatric Association, 2013).
In the DSM-5, all types of substance abuse and dependence have been
combined into a single substance use disorder (SUD) on a continuum
from mild to severe. A diagnosis of AUD requires that at least two of
the 11 DSM-5 behaviors be present within a 12-month period (mild
AUD: 2–3 criteria; moderate AUD: 4–5 criteria; severe AUD: 6–11 criteria).
The four main behavioral effects of AUD are impaired control over
drinking, negative social consequences, risky use, and altered physiological
effects (tolerance, withdrawal). This chapter presents an overview
of the prevalence and harmful consequences of AUD in the U.S.,
the systemic nature of the disease, neurocircuitry and stages of AUD,
comorbidities, fetal alcohol spectrum disorders, genetic risk factors, and
pharmacotherapies for AUD.
1. Up dating on different
Serological Assays
Dr.Tariq Mustafa Mohamed Ali
Al Ain Veterinary Laboratory
Animal Health section,Agriculture sector
Department of Municipalities and Agriculture
3. Antigen (Ag):
A molecule which elicits a specific immune
response when introduced into an animal.
1. Generally large molecules (>10,000 daltons
in molecular weight).
2. Structurally complex (proteins are usually
very antigenic).
3. Accessible to the immune system .
4. Foreign (not recognizable as "self").
6. Antigenic structure of Brucella
A- Surface antigen (OM)
Lipopolysaccharide (LPS) It is either S-LPS or R-LPS
Constitute the major Ag interact in SAT, CFT, RB , MRT
and represents the A &M epitops
OMP consists of 2 Ag
1. NH 25-27 K ( Not existed in the Rough strain)
2. Poly B 36 – 37 K ( Not existed in the Rough strain)
3. NA 31 K ( Existed in B. melitensis )
B- Internal Antigens free from LPS
More than 20 protenious Ag
(NH and Poly B and A2 ( one of internal Ags) used for
differentiation between Vaccinated and infected animals ).
8. Examples for virus antigens
Foot and mouth
disease viral antigens
146 S (VP1,2,3 & 4).
75 S ( VP1, 2 &VP0)
12 S ( Vp 1, 2 &3)
9. Antibody (Ab):
A glycoprotein produced in response to an
antigen that is specific for the antigen and
binds to it via non-covalent interactions.
The term “Immunoglobulin" is often used
interchangeably with "antibody".
Immunoglobulins (Ig) come in different forms
(IgA, IgD, IgE, IgG, IgM) that reflect their
structure.
13. Monoclonal antibodies
Typically made by fusing myeloma cells with
the spleen cells from a mouse that has been
immunized with the desired antigen to
produce what is called Hybridoma cell
15. Antigen / Antibody Reactions
(A) The hinge region of an
antibody molecule opens
and closes to allow better
binding between the
antibody and antigenic
determinants on the surface
of an antigen. (B) Hinge
flexibility also facilitates the
cross-linking of antigens into
large antigen-antibody
complexes.
16. Requirements of valid diagnostic
assays :
Selection of optimal reagent concentration
and protocols parameters
Use the test of optimal Sensitivity and
Specificity to the target disease.
Use the test of good Repeatability and
Reproducibility
Applied in the Reference animal population
Use the test of high prediction or efficiency
17. Sensitivity of Diagnostic methods
It is the proportion of True positive (TP) that
are detected by this method
Increase sensitivity accompanied with a
proportion of false positive (FP) .
A test with 90% sensitivity implies 10% will
be false negative.
Sensitivity = TP/ (TP+FN) X 100
18. Specificity of Diagnostic methods
It is the proportion of true negative (TN) that
are detected by this method
Increase specificity accompanied with a
proportion of false negative ( FN ).
A test with 90% specificity implies 10% will be
false positive .
Specificity = TN/ (TN+FP) X 100
19. Factors yielded false results
Cross reaction with similar epitops
Presence of non specific inhibitors in the tested
serum.
Some animals show natural or induced tolerance
(BVD).
Non specific inhibitors (Anti complementary serum).
Kind of AB ( Excess IgG 1 block IgG 2 in brucella
testing.
Using unsuitable test (Incomplete antibody).
Improper timing for testing the animals (cows for
brucellosis by CFT before abortion might give FN)
20. Diagnostic objectives of serological
tests .
Detection of Target Antigen.
Study the relation between the field
isolate and reference isolates.
Detection of rising antibody titers
between acute and convalescent stage
of infection.
22. Serological assays
It is either
Qualitative
Quantitative test
( Dilution takes place to the unknown
object )
23. Agglutination tests
Card or plate Agglutination
Rose Bengal test (RB).
Serum Agglutination test (SAT).
Micro-agglutination (MAT)
Latex Agglutination (LA)
24. Principals of Agglutination tests
The antigen is reacted with the antibody
inducing (clumping) of the antigen.
25. Application of agglutination tests
Typing of different isolates as in case
of E.coli ,salmonella, Pasteurella
spp.etc..
Diagnosis of Brucellosis :
Rose Bengal test .
Milk ring test.
SAT
Micro agglutination test
26. Principals of Latex Agglutination
(LA)
LA tests are similar in principle to bacterial
agglutination .
Latex particles could be coated with antibody
or antigen and will agglutinate when mixed
with the corresponding antigen or antibody.
Used for Identification of many
(Streptococcus ,salmonella typing and
toxoplasmosis)
27. Disadvantages of quantitative
agglutination tests
Prozone Phenomena :
Lack of agglutination at high concentrations of
antibodies due to excess antibody yielded a very
small complexes that do not clump to form visible
agglutination.
28. Agglutination but use erythrocytes
as targets or carrier
Indirect Hemagglutination (IHA)( Coomb
test).
Hemagglutination inhibition tests (HI).
Passive hemagglutination (PHA)
29. Principals of Hemagglutination
Detect antibody in the mare colostrum in
Equine infectious anemia by Coomb’s test
(Antiglobulin test)
34. Criteria of HI
Diagnosis of haemagglutining viruses such
as Influenza ,Newcastle disease etc .
Evaluating the immune response of
vaccinated animals or birds.
It is serotype specific
35. Principals of Passive Hemagglutination
(PHA)
Passive hemagglutination is a classical
immunological test in which antigen is linked
chemically to preserved red blood cells (RBC)
using tannic acid, glutraldehyde or CRCl3 .
The labeled cells are then used to detect the
appropriate antibody in a simple
hemagglutination test.
36. End point
The results are reported as the reciprocal of the maximal dilution
that gives 50% visible agglutination
Serum
37. Precipitation tests
Agar gel immunodiffusion tests “Double
immunodiffusion” (AGID).
Single radial immunodiffusion (SRID)
Immunoelectrophoresis (IE)
Countercurrent immunoelectrophoresis
(CCIE)
Immunochromatography
39. Principals of Precipitation tests
The serum antibody and the antigen
preparations are placed in holes
punched into into agar or agarose .
The antigen as well as Antibodies
diffuses into the gel, and at the
equivalence zone they form a
precipitation line.
40. Principals of Radial Immunodiffusion(Mancini)
(SRID)
In SRID assay the antibody is incorporated
into the agar gel as it is poured and different
dilutions of the antigen are placed in holes
punched into the agar to generate a standard
curve.
As the antigen diffuses into the gel, it reacts
with the antibody and at the equivalence point
a ring of precipitation is formed.
42. Principals of
Immunoelectrophoresis
In immunoelectrophoresis, both antigen
preparation and antibody are placed in a
well punched out of an agar gel and
exposed to weak electric current for a
period of time .
As the Ag and Ab diffuse into the agar a
precipitin lines are produced at the
equivalence zone when an antigen/antibody
reaction occurs
43. Countercurrent electrophoresis CIE
The antigen preparation into the wells
situated on the cathode side of each pair
Antigens flow towards the anode (+).
46. Principals of RADIOIMMUNOASSAY
(RIA) test
Radioactively labeled antibody competes with the
animal’s unlabeled antibody for binding sites on a
known amount of antigen.
A reduction in radioactivity of the antigen-animal
antibody complex compared with the radioactive
counts measured in the control test with no anti- body
is used to quantitate the amount of serum antibody
bound to the antigen.
47. Application of RADIOIMMUNOASSAY
(RIA)
Radioimmunoassay (RIA) is primarily used to
measure antigens (notably certain hormones
or proteins) in serum samples
This automated method of detecting antigens
is usually performed in a chemistry
laboratory.
Of limited application in Veterinary diagnostic
laboratory
48. Principals of Immunofluorescence (IF) test
Immunofluorescence (IF) depends on the
coupling of intracellular viral or bacterial
antigens with fluorescein isothiocyanate
(FITC) conjugated specific antibodies .
The reaction is detected by expose the
reaction site to ultraviolet (UV) or blue light.
Yielding apple green fluorescence
52. 2 - Principals of Enzyme-Linked
Immuno Sorbent Assay (ELISA).
In a simple terms, antigen is fixed to the
solid plate surface, and then the serum in
question is added .
The mixture is washed away followed by
addition of anti-species Ab conjugated with
an enzyme .
In the final step a substance is added that
the enzyme can convert to some detectable
signal.
55. ELISA for Antigen or antibody
Microplate ELISA for HIV antibody: coloured wells indicate reactivity
56. Indirect ELISA (i ELISA) :
Plate is coated with captured antibody
Sample is added, and any antigen present binds to the
captured antibody then washed
GP HIS is added, and binds to antigen if exists .
Conjugated Rabbit anti GP Ig is followed and binds to
detecting antibody
The substrate is added and is converted by enzyme to
detectable form.
58. Competitive ELISA (cELISA) for
detection of RP antibodies.
ELISA plates coated with RP Ag.
Add tested serum samples and RP Mab in blocking
buffer then Incubate plates with continuous shaking
for 1 H at 37oC
Add HRPO conjugated Anti mouse Ab followed by
incubation for 1 H at 37oC.
Add the substrate/chromogen and allow colour to
develop for ten minutes.
Stop colour development
Read plates on ELISA reader at an absorbance of
492 nm.
59.
60. Standard tests
Complement Fixation test (CFT).
Serum neutralization tests (SNT).
Protective tests (PD50)
61. Principals of Complement Fixation
Test (CFT)
The test consists of 2 Ag/AB reactions. One of them
is the target Ag or serum against the corresponding
refrence Serum or Ag .
The 2 nd Ag/Ab called hemolytic system (SRBCs +
Rabbit antisheep RbCs “hemolysine”) .
The complement is activated by Ag-Ab complex.
If C’ consumed in the first reaction . The HS is not
affected and the HS not been lysed. And vise is
versa.
63. Criteria of CFT
Tested serum should be inactivated at 56°C for 30
minutes.
Complement and haemolysin should be titrated first.
All Reagents used in the test should be tested for
QC(C’C, RBCs C, Haemolysin C, Serum C)
100 % and 0% lysis) should be included in the test.
Complement and haemolysin titration should precede
the test proper.
CFT could be done slow or rapid test
ICFT could be applied.
64. Complement (C’) titration
Each C’ dil 0% 100%
VB 0.1 0.2 0.1
Tested Ag or
0.1 0.1 0.1
Serum
C’ dilution 0.1 0 0.1 (1/10)
HS 0.2 0.2 0.2
65. Each
Ag C SC C’ C HS C
Ag dil
VB 0 0.1 0.2 0.2 0.3
Tested
Ag 0.1
HIS
(1/10) 0.1 0.1
C’ (2-
4Unit)
0.1 0.1 0.1 0.1(1/10)
Incubate at 37 oC for 30 m
HS
0.2 0.2 0.2 0.2 0.2
Incubate at 37 oC for 15 m
66. What does a CFT look like?
Complement Fixation Test in Microtiter Plate. Rows 1 and 2 exhibit complement
fixation obtained with acute and convalescent phase serum specimens,
respectively. (2-fold serum dilutions were used) The observed 4-fold increase is
significant and indicates recent infection.
68. Principle of Serum Neutralization
(SNT)
It demonstrate the amount of antibodies
necessary to inhibit the activity of known
amount of virus particles(10 2) .
If it done in tissue culture (TC) tube , the
cytopathic effect (CPE) was recorded.
If it done in embryonated eggs death is
recorded .
69. SNT assay
The test is done using test tubes, microtiter plate ,
2 oz bottle, embryonated eggs , Mice etc…
The target serum is exposed to 4-fold dilutions
followed by the addition of an equal volume of
virus suspension diluted to contain approximately
100 ID50 .Following an incubation of serum + virus
1 - 2 hours at room temperature .Inoculate the
mixture of each dilution into odd number of target
host .Incubated at 37°C, and observed daily for
development of viral CPE.
71. Criteria of SNT
The end point is the reciprocal serum dilution
yielded 50% CPE in case of tissue culture.
This test is considered the most reliable of all
serologic procedures, being less prone to variation
and less subjective in its interpretation.
SNT tests are almost always performed using
cell cultures.
72. Principle of Protection Tests
It has the same principal of SNT but in vivo.
The target host is immunized with several
Vaccine dilution followed by challenge with
constant virus concentration at 3 weeks post
vaccination.
Protection tests are used for Vaccine Evaluation
An example of a protection test once used is that
for FMD vaccine evaluation.
73. Calculation of PD50
No. +ve /Total
Vaccine dilution Positive%
No. of animals
Undiluted 1/5 20%
1/4 2/5 40%
1/16 3/5 60%
1/64 4/5 80%
1/256 5/5 100%
m =xk +1/2d- d (-Spi /100)
74. Immune Electron Microscopy
Classical Immune electron microscopy (IEM) - the sample is
treated with specific anti-sera before being put up for EM.
Viral particles present will be agglutinated and thus congregate
together by the antibody.
Solid phase immune electron microscopy (SPIEM) - the grid is
coated with specific anti-sera. Virus particles present in the
sample will be absorbed onto the grid by the antibody.
75. Uses of Immune Electron
Microscopy
At least 106 virus particles per ml required for visualization,
The magnification power should be 50,000 - 60,000 X
Viruses may be detected in the following specimens.
Feces Rotavirus, and corona virus &
Adenovirus, Calicivirus , Parvoviruses
Skin scrapings Papillomavirus, ORF , Pox
76. Problems with Electron Microscopy
Expensive equipment .
Expensive maintenance .
Require experienced observer .
Sensitivity is often low .
77. Fluorescence polarization assay
(FPA)
The assay works on the principle that
molecules in solution randomly rotate at a
rate inversely proportional to their size.
A small molecule labeled with a fluorochrome
will depolarize plane polarized light at a more
rapid rate than a large molecule such as Ag-
Ab complex.
78. Fluorescence polarization assay
(FPA)
Fluorescence polarization provides a direct readout of the
extent of FITC conjugated Ag binding to serum AB.
In positive cases ; the FITC conjugated Ag bind to the Ab
molecules leads to formation of large , slowly rotating
molecules and high fluorescence polarization.
In negative cases ; When no reaction takes place with
FITC conjugated antigen, the FITC conjugated Ag remain
as small, rapidly rotating molecules resulting in low
fluorescence polarization.
80. Western blotting
Western blotting is based on the principles of
immunochromatography where proteins were
separated into poly acrylamide gel according to
the isoelectric point and molecular weight.
Immunoblotting is performed chiefly in diagnostic
laboratories to identify the desirable protein
antigens in complex mixtures.
81. Criteria of Western blotting
The separated protein bands were detected by :
1. Colorimetric detection or
2. Chemiluminescent detection or
3. Radioactive detection or
4. Fluorescent detection
82. Western Blot
Western Blot
Lane1: Positive Control
Lane 2: Negative Control
Sample A: Negative
Sample B: Indeterminate
Sample C: Positive
83. Immunoblotting
Viral antigens are detected with a
polyclonal or a MAb onto nitrocellulose
paper.
After incubation, the protein bands
(immune complexes) are visualised with
peroxidase-conjugated protein and a
colour reagent.
A colour develops in the bands where
antibody binds to the antigen.
84. Immunochromatography
Test principle.
Antibodies were attached to two different zones on a
nitrocellulose membrane .
Purified monoclonal antibody against nucleoprotein (anti-N
Mab) that had been identified as IgG2a subtype by using
Mab isotyping kit was attached to the test zone at the
concentration of 0.75 μg/strip, and purified goat anti-mouse
IgG (was attached to the control zone at the concentration of
0.70 μg/strip.
The 30 nm colloidal gold conjugated antibodies were dried
on the glass fiber
The test strip was assembled in the following order (Sample
pad , gold pad, nitrocellulose paper, and absorption pad
(cellulose paper).
All pads are overlapped to enable migration of sample
86. Diagram of the test strip for the detection of anti-canine parvovirus antibody.
Serum is added to the sample pad where serum antibodies can interact with
CPV. Addition of buffer enables the complex to migrate along the test strip
where gold-conjugated antibodies are captured by the immobilized anti-porcine
or anti-canine IgG.
87. Quantitative application of the test
The intensity of the
color developed at the T
line (T) correlated with
the HI assay-
determined titer of the
reference serum
sample shown on each
test strip
88. Diagram of the test strip for the
detection of Rabies virus
Saliva is added to the
sample pad.
sample pad► gold pad
Mab against
nucleoprotein ► go
through nitrocellulose
paper to test line
(Rabies Mab) ► Control
line ( goat anti-mouse)
and absorption pad
(cellulose paper).
All pads overlapped to
enable migration of
sample
91. Polymerase Chain Reaction (PCR)
The amplification of DNA by the PCR is
accomplished via a succession of
incubation steps at different temperatures.
PCR can amplify copies of a small region
of 100-400 or more base pairs into millions
of copies.
92. Basic steps of PCR
Separation of DNA strands (Denaturation) By
heating to 95 oC .
Activate primer’s links (Annealing) By heating
to 55 oC .
Activate polymerase activity in 3’ to 5’
direction (Extension) By heating to 72 oC, to
synthesis new strand .
99. Criteria of PCR
PCR is a highly sensitive procedure for detecting
infectious agents in host tissues and vectors, even when a
small number of host cells are infected
PCR is very useful in the diagnosis of chronic-persistent
infections such as :
Bovine leukemia virus
Caprine arthritis/encephalitis virus, etc.)
Latent infections by IBR
PCR could be used for testing vaccines contamination.
It does not differentiate between viable and nonviable
organisms.
100. Criteria of PCR
Extremely liable to contamination
Require high degree of operator skill
Not easy to set up a quantitative assay.
A positive result may be difficult to interpret,
especially with latent viruses such as CMV, where
any seropositive person will have virus present in
their blood irrespective whether they have disease
or not.
101. Restriction fragment length
polymorphisms (RFLP)
Detect differences in the genomes of closely
related microbial species
DNA is extracted and clipped into fragments of
specific nucleotide sequences with restriction
endonucleases.
The resultant DNA fragments are then separated
in agarose gel by electrophoresis and visualised
with ethidium bromide.
The fragments can then be hybridised with
complementary DNA (cDNA) tagged with 32P to
determine the differences or similarities in the
genomes.
102. Criteria of FRLP
Uses :
Used to detect differences in the genomes of
closely related microbial species or compare
field isolates of a given virus.
A distinct limitation of the method is that the
presence of a mutation cannot be detected
unless that mutation happens to fall within the
recognition sequence of the restriction
endonuclease being used for digestion of the
DNA.
103. Solid-phase of NA hybridization
assays
Principals of the test
The double-stranded nucleic acid of a virus is
denatured with alkali to separate strands.
The single strands of nucleic acid are attached to
a solid support, usually a nylon or nitrocellulose
membrane, to prevent the strands from
reannealing.
The nucleic acid attaches to the membrane by its
sugar-phosphate backbone; the nitrogenous
bases are thus projecting outward.
104. Nucleic Acid probe
A probe (single-stranded DNA or RNA molecule of known
origin - containing the nucleotide sequence specific to that
of the target virus - labeled with a radioactive atom or
enzyme) is added to the membrane.
Formation of hydrogen bonds occurs between the
complementary bases.
Unreacted probe is removed by washing and hybridization
is detected by an assay for the probe.
107. Dot blot hybridization.
The nucleic acid is
placed onto
nitrocellulose in an
apparatus that
focuses the
individual spots into
concentrated areas,
similar to a microtiter
plate
108. Application of Microarray Analysis:
The development of microarrays has been
fueled by the application of robotic technology
to routine molecular biology, rather than by
any fundamental breakthrough.
It used for Identification of specific viruses or
specific viral sequences as many as included
in the test.
109. DNA microarray
(gene chip, DNA chip or biochip) is a collection of
DNA probes attached to a solid surface, such as
glass, plastic or silicon chip forming an array. Sample
DNA or RNA is extracted, RNA is reverse transcribed
to cDNA and the DNA or cDNA is labelled with
fluorescent labels. The labelled DNA is denatured
and hybridized with the probes on the array.
Unbound probes are washed away and the array is
visualized using confocal laser microscope scanner