ode Microsoft PowerPont (Product Actvation

Failed
Sign Transitions Animations Slide Show Review View

HUMAN IMMUNODEFICIENCY VIRUS

Eiological agent of AIDS-

Has emerged as the biggest threat in the past 3 decades

Belongs to the family Retrovinidae and gemus Lentivius

o add notes

U.S) E 48% -0
 Paint (Product Actaation Failed)

Transitions Animations Slide Show Review View

Epidemiology
2013/2015
Preralence 0.28%/0.26%
PLHIV: 2.26 milkon/2.17 milhon
Newlr infected with HIV: 0.116 millkon/0.086
1989 frst cinical descnption of I V sesistance to
ARV drugs pubished about patients taking AZT
monotherapr accumulation of mutations within RT
gene

es, 0.22 in females

milliuan alohall
88 48%
 Transitions Animations Slide Show Review View

Structure

HIV, is a spherical enveloped viras about 80 -110 nm in

$1ze.

Earelope- ipid part- derived from host cell

Protein part- gp 120, gp 41
The mucleocapsid- Capsid- icosahedral- made of core
proteins and an inner cone shaped cone, enclosing

Two identical single stranded positire sense RNA

Viral enzvmes- reverse transciptase, integrase and
proteases

id notes
 ct Activation Failed)
Transitions Animations SlideShow Review View

gp41 RNA
gp120 Envelopee
Envelope Proteln
Protein

p17
Matrix
Protelns

Lipid
Membrane

p24
Capsule
Protelns

Reverse
Transcriptase
Anatomy of the AIDS VIirus

notes
 or
Transitions Animations Slide Show Review View

Genes and antigens

3 structural genes-
Figure 1.2. HIV genome
Gag
Pol
LTR
Env
p12

6non structural or p4

regulatorr genes
tranecripease

(Souroe HIv Medune 2007)

 INCUBATION PERIOD:

Few months to 10 vs or even more- from HIV NFECTION to

development of AIDS

The viras can be silent for many years (uwindow period)

tes
 Replication

Begins when the virus enters the blood or tissues of a

person and comes into contact with a suitable host cel,
principally the CD 4 LYPHOCYTES

The receptor for the virus is CD4 antigen and therefore

the virus may infect any cell bearing CD 4 antigen on
surface.

Glal cells and microgla in CNS are also susceptble.

ld notes
 ***********************

*****
***********************

ww
ww

notes
04

ELISA(ENZYME LINKED IMMUNOSORBENT

ASSAY
Named because the technique involves use of an
immunosobent, an absorbing material specific for Ag
or Ab

106
Itmay be particulate (cellulose or agarose) or solid
phase (polystyrene, polyvinyl or polycarbonate tubes
or microwells or membranes or discs of
DOlyacylamide, papper or plastic)
182
Usually done using microtitre plate, suitable for
automation.8 cmx 12 cm plastic plate which
an 8 x 12
containsS
matrix of 96 wells, each of which are about
1 cm high and 0.7 cm in diameter.

Click to add notes

Slide 104 of 126 Trek English (U.S)
 Review View
Transitions Animations SlideShow

ENZYME LABELLED ANTIBODIES WITH

RESPECTIVE SUBSTRATE COMPLEXES

AntDudiesCuyatad to SIILSUate

Horse radsh peroidase Hydrogen peroxide+orthoph enylene

diamine

Alkaiine phosphate nitrophend phosphate

B-galactosictase o-nitro pheny-8-d galadoovranoside

notes
 TYPES OF ELISA

Qualitative ELISA
- Postive or Negative results

Quantitative ELISA
is
optical density or fluorescent units of the sample
interpolated into a standard curve, which is
typically a serial dilution of the target.

d notes

S) 88 48%
 x Noncompetative ELISA:

xAntigen measuring system

+Ttre wells coated with suitable antibody
+Add patient sample containing the antigen
+Incubate: till antigen antibody reaction is complete.
Wash remove unbound antigen
+Add Antibody labelled with Enzyme
Incubate till antigen binds labelled antibody
Wash remove unbound labelled antibody
Addsubstrate(paranitrophenyl phosphate)
incubate in room temp
Enzyme+Substrate> Product> measure colour
Colour proportional to antigen in patient sample

d notes

8 7 489%
 (A) Indirect EUSA

Wash
Wash

Specfic antibody Enzyme-linked Substrate is added and

coated wel binds to antgen antbody binds to comerted by enzyme nto
specific antibody colored product; the rate
of color formation is
proportional to the amount
f specfic antbody
B) Sandwich ELISA

Wash Wash Wash

YY
Antigen binds Substrate is added and
Monodonal A second monodonal
antbody to antibody antibody, Iinked to comverted by enzyme into
coated well entyme, bnds to colored product; the rate
immoblized antigen of color formation is
proportional to the
amount o antgen

add notes
Design Transitions Animations Slide Show Review View

ample Diu

dd notes
Design Transitions Animations SlideShow Review View

For competitive ELISA, the higher the sample antigen

concentration, the weaker the eventual signal.

The major advantage of a competitive ELISA is the

ability to use crude or impure samples and still
selectively bind any antigen that may be present.

add notes

(U.S) 48% 0
dooo ndi. vITcrosonEFOweOnt.roauct Actvauon rdileo)
n Transitions Animations Slide Show Review View

Sandwich ELISA is used when we are looking

for antigens, in this case a primary, unlabelled
antibody is bound to the wells, if there are
antigens they will bind to these antibodies, a
second set of labelled antibodies is then
introduced. These antibodies bind to the antigen
bound to the primary antibody. Again a substrate
for the enzyme is added and this produces an
optically measurable signal.

H notas
antiboby reaction nam. c
Animations Slide Show Review View
Transitions
n

Sandwich ELISA protocol

1. Coet primary antibody

onto micropiete

1a. Allow aribody atsorption

and blook
unocrupied site
with neutrai protein BSA

2. Add entigen to be detectes

in bioiogic (dinicel) meterel
into each wel

3. Asd second primery artibooy

g e s t e n t e n intoesdh wel

4. Acs HR or AP conjegetes
secondsry ntiboay mto e
well ana develop colorimetic
reaction with apprapriste substrete

5. Rea absorbence in EUSA Spectrophotometer

with eppropriete filter ad qusntitate reistive
antigen evet

notes
E 9 Eg 48% (-
antiboby reaction n a m . MicrosoE HOwerrolnt o 0 u c t A c t v a l o n i r d e

n Transitions Animations Slide Show Review View

APPLICATIONS OF ELISA
Serum Antibody Concentrations
Detecting potential food allergenss
(milk, peanuts, walnuts, almonds and eggs)
Disease outbreaks- tracking the spread
of disease
e.g. HIV. bird flu, common, colds, cholera.
STD etc
Detections of antigens
e.g. pregnancy hormones, drug allergen
GMO. mad cow disease
Detection of antibodies in blood sample for
past exposure to dissease
eg Lyme Disease tichinosis HIV bird flu

notes
L8 48% O-
 Transitions Animations Slide Show Review View

(a) Add SDS-reated protein »)Eectrophorese in

mixture to well ot g e SOS polyacrylamide oo

Direcuon
migration
Protein antioSAS
Oenatured n sbs

Western blot- (c) Ronmove

Dertorm asel and
elecirotransfe

finding 1
protein out of
many in serum Electric
curren
or cytosol Porou
membrane
eet

td) Bind a n t i g e n of interest

with enzyme-inked
antubodies

(e) Add substrate to

actvate color resctlon

notes
CE 48% 0
 Transitions Animations Slide Show Review View
n

IMMUNOELECTROBLOT TECNIQUE:
to individual antigenic
Used to detect antibodies targetted mixture.
determinants in a crude whole cell antigen
Initially the antigen mixture is subjected to electrophoretic
separation in a gel ( SDS-PAGE).
separated protein bands are then blotted on
The
nitrocellulose sheets
The sheets are then incubated along with patients serumn,
when antibodies bind to the individual protein antigen.
After washing awayisthe unbound material, an enzyme
to
labelled conjugate added. This material binds
antibodies that have already bound to the antigen
done followed by addition of enzyme
chromogen substrate
Colour bands appear on the strip at the sites of initial
antibody reactivity.

notes

8 48% 0
 PowerPoint (Product Activation Falled)
tigen-aniboby reaction ram. Micosoft
Transitiois Animatlan SllideShow RevieW View
Deslgn

IMMUNOELECTROMICROSCOPY

For visual detection of viral agents that are

not cultivable, such as hepatitis A.
Specific antiviral antibodies are used to
cause aggregates of viral particles, which
are detected by electron microscop
Immune specific aggregation increases
electron microscopy detection by 100-1000
folds

o add notes
C 48% -
sh |U.S

Manasi is presenting
 EntgEn-artiab escian a n eTaseT FOWErEOm IF rodugtEVaton kailed

Deslgm Transitions Arlmatlan SlldeShow Review Mew

IMMUNOCHROMATOGRAPHIC TEST
The test system small cassette containing a
is a
membrane impregnated with an antibody -colloid gold
dye conjugate
The lest serum is dropped into the first window
As the serum travels by capillary action a coloured
band appears at the second
serum contains an antibody
window (test site ) if the
due to formation of
,

antibody -conjugate complex This Is the positive

reaction
Simultans0usly coloured
third window which is an bard
should
appear at the
inbuilt control
Egs for HbsAg , clostridium difficle toxins
H pyorn fecal antigen AandB
test, pregnancy tesi

lick to add notes

Englsh145)
File HOme fnserf Deslgn
valionalled
ransifiOTs Anmaficns Slde Show ReviEw View

131
IMMUNOENZYME TEST
Stable enzymes like peroxidase can be
conjugated with antibodies.
Tissue sections containing corresponding
antigens are treated with peroxidase labelled
antisera.
The peroxidase bound to the antigen can be
124
visualised under electron microscope, by
microhistochemical methods.
Other enzymes glucose oxidase
125
phosphatases, tyrosinases.

126
Click to add notes

Slde124 of 126 Trek Engsh S . 48s =