Laboratory Diagnosis of Human Immunodeficiency Virus (HIV) by Elisa and Chemiluminescence Presented by Dr. Abdulrahman Amer

1. Presentedby Dr. Abdulrahman Amer
Laboratory Diagnosis of Human
Immunodeficiency Virus (HIV) by Elisa and
Chemiluminescence

3. HIV-Structure
(Viral Antigens)
1- Gag = Group Antigen: ( (Capsid), P17 (Matrix), & P7
(Nucleocapsid).
2- Pol= polymerase and associated enzymes: P66 (reverse
transcriptase -RT), P32 (integrase) , P9 (protease).
2- Env= Envelope glycoproteins.

6. Why AIDS is more important today
Apart from Men, there is a increasing burden of
 Infections in Women.
 Implication on Mother to Child Transmission
 Female Adolescents are infected 3 – 6 times higher than male
counterparts.
 AIDS has turned out to be Social Problem.

7. Implication of AIDS
 Being diagnosed as having HIV/AIDS has
 life time repercussions.
 Every body understands AIDS as a life threatening
condition.
 A casual way of dealing as being HIV + will
 have Moral, Legal, and Social consequences.

8.  Importance of precise Diagnosis of AIDS
 All patients should be informed about consequences of testing
for HIV infection,
 Legality of respective National Laws should be followed.
 A pretest and posttest counseling is Growing importance, even
in Developing countries.
 Today’s debate on testing for HIV status as matter of Health
protocol ?

9. Purpose of Testing for HIV Infection
 A matter of great importance as in Blood donors to
 prevent infected blood being transfused.
 To diagnose the patients infected with HIV virus.
 For surveillance purpose.
 Persons with high risk behavior.
 In Pregnant women to prevent Mother to Child
 transmission.
 Patient's presenting with opportunistic infections.

10. Diagnosis of HIV/ AIDS
Basic Tests.
1. We can detect Antigen or Antibody
2. We can detect Antigen and Antibody.
3. Majority of Laboratories depend on
Commercial Kits.
4. Devloped Nations going for Molecular and more precise
Methods

11. Screening Tests for HIV/AIDS Detection
 A screening test posses high Sensitivity.
 We rarely miss the Diagnosis in Infected patients.
 Done as mass screening procedure.
 Economical.
 Developing countries depend on these tests even for
Diagnosis.

12. Confirmatory Tests in AIDS
It is important to confirm all screening tests with
Confirmatory tests, or we brand some one without
infection as infected.
Confirmatory tests differentiates false
reactive tests. and identifies truly infected or not.

13. Screening test
 High degree of
Sensitivity
 Few false negatives
Confirmatory test
 High degree of
Specificity
 Few / No false
positive results.
Differences of Screening and
Confirmatory Tests.

14. Why patients should we tested with
Screening and Confirmatory tests.
 Before you declare a person infected with
HIV / AIDS you should perform both methods.
 Faulty testing methods can lead to catastrophic
consequences, and legal litigations.

15. Spectrum of HIV Tests
HIV diagnosis (Antibody/Antigen testing)
Enzyme Immunoassays (EIAs)
Rapid tests
Western blot (WB)
Early diagnosis in infants
p24
Initiation and monitoring of ART
CD4
Viral Load

16. Challenges of HIV Testing
 Early detection of seroconversion
 Early detection in infants born to HIV
positive mothers
 Effect of HIV subtypes on test
performance
 Impact of other health conditions on
test performance
 Product specific equipment
 Technical skill

17. Diagnostic lab test for HIV infection
A- Tests to diagnose HIV infections:
1- Screening 2- Confirmatory
B- Tests for follow up the disease:
1- Viral load by Quantitative PCR 2- Th-cells count
(CD4+)
C- Tests for the complication of the disease:
1- TB infection 2- HBV
3- HCV 4- Toxoplasma
5- Liver function tests 6- UTIs
7- Others.

18. A- Tests to diagnose HIV infections
1- Screening tests
These test are rapid in giving results however, they are not
diagnostic and need confirmation by the confirmatory tests.
The Screening tests include:
 - ELISA tests (combined Ag-Ab Immunoassays) for
detecting HIV-1 & HIV-2 antibodies and P24 Ag of HIV-1.
 chemiluminescent (immunoassay testing).
 - Rapid tests

19. ELISA or chemiluminescent (immunoassay testing)
Were previously for detection of HIV Antibodies
Now detection of P24 Antigen of HIV is included (Combo
ELISA).
Even though, need confirmation
Done upon serum sample
If initially reactive, repeat in triplicate, if positive send for WB or
PCR.

20. ELISA METHOD
Universally accepted test ,most popular even in the
developing nations.
Useful in large scale screening.
A common method used in Blood banks in mass
screening of Human blood.

21. ELISA plateHIV EIAs
•Based on color change/fluorescence
•Change compared with standardized cut-off
•Result positive or negative
•No specific antibody reaction information
•Multiple samples run with traditional EIA
96-Well Microtiter Plate EIA Interpretation of EIAs

22. Sequence of Events in ELISA test

24. ELISA (Enzyme-linked immuno-sorbent assay) is
one of immunoassay method used to detection of
1-Antibodies
2-Proteins
3-Peptides
4-Biomolecules
ELISA

25. Immunoassay
The term “immunoassay” is a combined term of
“immuno”(= immunological, practically immunochemical
antigen-antibody-reaction) and “assay” means the
determination of the purity of a substance or the amount
of any constituent of a mixture.

26. 1. Antigen/antibody of interest is absorbed on to plastic surface
(sorbent).
2. Antigen is recognised by specific antibody (immuno).
3. This antibody is recognised by second antibody (immuno) which has
enzyme attached (enzyme-linked).
4. Substrate reacts with enzyme to produce product, usually coloured.
Enzyme Linked Immunosorbent Assay

27. Radioimmunoassay was first described in a scientific paper by Rosalyn
Sussman Yalow and Solomon Berson published in 1960.
In 1971, Peter Perlman and Eva Ingval at the University of Stockholm
in Sweden, Anton Schuurs and Bauke van Weemen in the Netherlands
published research papers that synthesized this knowledge into
methods to perform EIA/ELISA.

28. 1-Antibody (Antiserum)
2-Antigen
3-Labeling materials
Important components in immunoassay

29. 1-Antibody (antiserum)
Antibody: proteins produced by the immune system which help defend
against antigens
SYMBOL FOR
ANTIBODY
The variable regions are though to be the
place for recognition and binding with the
antigen.

30. Any molecule that induces production of antibodies when
introduced in the body is called antigen.
e.g. bacteria, viruses, (or their parts), pollen, etc.
SYMBOL FOR ANTIGEN
2-Antigen

31. 3-Labeling materials
In immunoassay, it is necessary to use any marker to know the
antigen-antibody binding. For such purpose, we label either antigen
or antibody with some materials that do not interefere with the
binding.

32. ELISA
READER

33. ELISA KIT
Lisawash

34. Components of Kit
 Pre-Coated, Stabilized 96-well Microtiter Plate.
 Sample Diluent
 Standards and controls
 Conjugated Detection Antibody
 10X Wash Solution
 Substrate
 Stop Solution

37. Advantages of ELISA
• Reagents are relatively cheap & have a long shelf life
• ELISA is highly specific and sensitive
• No radiation hazards occur during labelling or disposal of waste.
• Easy to perform and quick procedures
• Equipment can be inexpensive and widely available.
• ELISA can be used to a variety of infections.

38. Disadvantages of ELISA
Measurement of enzyme activity can be more complex than
measurement of activity of some type of radioisotopes.
Enzyme activity may be affected by plasma constituents.
Kits are commercially available, but not cheap
Very specific to a particular antigen. Won’t recognize any other antigen
False positives/negatives possible, especially with mutated/altered
antigen

40. 1-Direct ELISA
2-Indirect ELISA
3-Sandwich ELISA
4-Competitive ELISA
5-Ogives ELISA
Types Of Elisa

41. The direct detection method uses a labeled primary antibody
that reacts directly with the antigen. Direct detection can be
performed with antigen that is directly immobilized on the
assay plate . Direct detection is not widely used in ELISA but
is quite common for immunohistochemical staining of tissues
and cells.

42. The indirect ELISA utilizes an unlabeled primary antibody
in conjunction with a labeled secondary antibody.
The secondary antibody has specificity for the primary
antibody

43. Direct and Indirect ELISA

44. The sandwich measures the amount of antigen between two layers of
antibodies.
Sandwich are especially useful if the concentration of antigens is low or
they are contained in a mix of high concentrations of contaminating
protein
To utilize this assay, one antibody (capture) is bound to a microtiter plate
well. Antigen is then added and bound to the antibody. Unbound products
are then removed, and 2ry antibody is added (detection), then add the 3rd
labeled antibody to complete the sandwich
Major advantages of this technique are that the antigen does not need to
be purified prior to use, due to its high specificity.
Sandwich ELISA

46. In this Unlabeled antibody is incubated in the presence of its antigen. These
bound antibody/antigen complexes are then added to an antigen coated well.
The plate is washed unbound antibody is removed. The secondary antibody,
specific to the primary antibody is added. This second antibody is coupled
to the enzyme. A substrate is added, and remaining enzymes elicit a
chromogenic or fluorescent signal. For competitive ELISA, the higher the
original antigen concentration, the weaker the eventual signal.
Competitive ELISA

48. A newer technique uses an solid phase made up of an immuno-sorbent
polystyrene rod with 8-12 protruding ogives.
The entire device is immersed in a test tube containing the collected
sample and the following steps (washing, incubation in conjugate and
incubation in chromogenous ) are carried out by dipping the ogives in
microwells of standard microplates pre-filled with reagents
Multiple and portable ELISA

51. 1. Coating of Wells with Antibody
Add 25µL of the sample diluent IG4 to all the wells
Add 100 μl of the anti-HIV Negative control in well A1,B1 and C1
Add 100 μl of the anti-HIV Positive control in well D1.
Add 100 μl of the HIV P24 Antigen Positive control in well E1.
2- Add 100 μl of the first sample in the well E1, second sample in the well G1 and
so on.
3- Incubation with Test Samples
Cover the wells with strip sealers &block cover, incubate for 60 minuts
at 37 °C. Remove the sealers

52. 5. Incubation with enzyme- Conjugated Antibody.
. Add 50 μL Assay buffer IG4 and 50μL conjugate G4 to all the wells
Cover the wells with strip sealers and black cover, 30 minutes at 37 °C.
The enzyme-conjugated antibody should be directed against the antigen to be
determined
The conjugatec antibody must be specific for the antigen of interest
4. Washing
6 cycles
350 μl/well and 30 seconds soak time with diluted wash buffer(1:20).
For 500mL wash buffer, mix 25mL concentrare in 475 mL distilled
water

53. 6. Wash as described
6 cycles (350 μL/well and 30 seconds soak time with diluted wash buffer(1:20)).
7. Colour Development
Add 50μL color reagent to each well(chromogenic substrate ).
Incubution 30 minutes at room temperature (20-30 °C) in dark.
The plate should preferably be protected against light during this incubation.
8. Stopping the Colour Development
Add 100μL stop solution to each well to stop the reaction .
9. Reading of Results
Read results directly through the bottom of the micro well plate using an automated
(ELISA-reader). The subtraction of the absorbance at a reference wavelength
(between 620 and 650 nm) is recommended.

55. Add mach. photo

56. HOW TO READ

57. Important points in performing ELISA and
improvement of assay performance
1-Sample treatment.
3-Stability of assay samples.
2-Infleunce of humidity and air stream.

58. Important points in performing ELISA and improvement of assay
performance
Sampling and treatments of samples
Serum or plasma

59. In general, we recommend using
Use of fluoride must be avoided because fluoride ion is a potent
inhibitor of peroxidase.
sampling
When getting
heparin is most often used as an anti-coagulant

60. An important phenomenon with frozen plasma is that an insoluble
substance (fibrin) will be formed when thawed. In this case, the sample
must be mixed and centrifuged, then the insoluble cluster flowing in the
plasma should be taken out by a thin wire needle sharply bent at an end.
If such fibrin remains in the sample, it may clog the tip of a pipette and
influences assay variability

61. Hemolysis and Lipemia

62. Serum or plasma, when fresh, shows pH near neutral, however, it very
quickly goes to alkaline more than pH 8 by losing CO2.
In alkaline pH, the antigen-antibody reaction is interfered. resulting in
cancellation of the assay or giving inaccurate assay values. So, in such case,
before hand dilution of serum or heparinized plasma with assay buffer will
be helpful.
pH Of the sample

63. Sample storage temperature is better to be lower than -35 C. Ultra-
low temperature such as -80 C is recommended for a long-term
storage.
Repeated freezing and thawing is also harmful to the protein, and
may cause inactivation.
Storage temperature and freezing-thawing.

64. When samples are taken out from the freezer and thawed,
never forget to mix these samples because the solution after
thawing is not homogeneous, and the bottom area contains
more solute
DON’T FORGET

65. Stability of assay samples.
In assay, the problem of sample stability, i.e. how long the substance to be
measured can keep its immunoreactivity, in serum or plasma, is very important.
Blood samples also contain enzymes to destroy peptides or proteins, and stability
against those enzymes differs from substance to substance.
Freezer of –20C is not trustable for the constancy of temperature but use of a
freezer of –35 C or lower temperature is recommended.

66. Avoid sunlight
Avoid air fans

67. Influence of humidity and air stream
During all the incubation process, the well-plate should be covered
using the attached plate cover. Plate cover is effective only under the
most suitable condition, i.e. room temperature, humidity more than
50%, and air stream of less than 0.2m/sec.
It is recommend to get a small semi-transparent plastic box, and put
moistened paper towel on the bottom .

68. Trouble shooting in ELISA

69. Poor or no coloration after the last step
1) The standard or samples might not be added.
2) Reagents necessary for coloration might not be added.
3) Wrong reagents related to coloration might have been added.
4) Influence of the temperature under which the kits had been
stored.
5) Excessive hard washing of the well plate.

70. 2)The standard curve obtained
was not smooth.
There might be some mistake in the serial dilution of
the original standard solution.
3)Flat standard curve.
Standard solutions are not added.

71. 4-Big variation between two wells in duplicated assay
was observed.
1) Scratching the bottom of the well by aspirator tip during
aspiration of washing buffer.
3)Assay might be started while the well-plate was still cooler than room
temperature.
2) Scratching the bottom of the well by pipette tip during addition of
standards, samples, or reagents.

72. 4) Air stream, warmer or cooler than room temperature
5) Air stream from air conditioner or other instruments
might dry wells.
6) Insufficient removal of washing buffer from the wells
might dilute reagent solution added in the following step of the
procedure.
7)Big variation would be obtained if the sample is not
homogeneous.

73. Chemiluminescence
Chemiluminescence is a chemical reaction that emits energy in the
form of light.
When used in combination with immunoassay technology, the
light produced by the reaction indicates the amount of analyte in a
sample.
Direct chemiluminescent reactions directly measure the light
energy without the use of added steps or amplifying molecules.
The ADVIA Centaur XP assays use acridinium ester (AE) as the
chemiluminescent label, because AE does not require the addition
of a catalyst or substrate.

74. Direct Chemiluminescence
It is easy to automate direct chemiluminescence using AE and
provides many benefits, such as long reagent shelf life, fast
reaction time, and assay sensitivity. The ADVIA Centaur XP
assays use the dimethyl form of AE because its stability allows
long reagent shelf life.

75. Assay Reaction Formats
The system uses a variety of formats to detect
antigens as well as antibodies.
1 2 3
Sandwich
format
Competitive
format
Antibody-
capture
format

88. SAMPLE HANDLING

91. Reagent Handling

92. Reagent
Cooling
• 30-position reagent tray cooled 4°C to 8°C
Reagent
Integrity
Control
• Barcode reagent identification, automatic inventory tracking and
flagging, calibration validity tracked and flagged, reagent on board
stability tracked and flagged, reagent expired/ reagent low flagging
Reagent
Mixing
• Ready Packs automatically rocked onboard

93. Quality Control
Advanced QC package includes Levy-Jennings
plots and Westgard rules; 65,000 control results
can be stored
Results within ± 2SD accepted

94. TYPES OF ASSAYS

97. • AFP
• BR
• CA 15-3
• CA 19-9
• CA 125II
• CEA
• Complexed PSA
• Free PSA
• PSA
• Serum HER-2/neu
Tumor
Markers/Onc
ology

98. ToRCH
and Special
ID
• CMV IgG
• CMV IgM
• Rubella IgG
• Rubella IgM
• Syphilis
• Toxo IgG
• Toxo IgM

99. • BNP
• CKMB II
• D-Dimer
• Myoglobin
• Tnl-Ultra
• C-Peptide (serum, urine)
• Insulin

100. • Anti-HBe
• Anti-HBs
• Anti-HBs-2
• eHIV 1/O/2†
• HAV IgM
• HAV Total
• HBc Total
• HBc IgM
• HBe Ag
• HBs Ag
• HBs Ag Confirmatory
• HBs AgII
• HCV†
• HIV Combo (CHIV)†

102. • HA (ELF Marker)
• TIMP (ELF Marker)
• PIIINP (ELF Marker)
Liver
Fibrosis
• Cortisol (serum,
urine)
• Homocysteine
Metabolic

103. • AFP
• DHEAS
• Estradiol-6
• Estradiol-6 III
• FSH
• LH
• Progesterone
• Prolactin
• SHBG
• Testosterone
• Total hCG
Reproductive
Endocrinology

104. • Cyclosporine
• Tacrolimus*
Immunosuppresants
• Procalcitonin
Sepsis

105. Thyroid
Function
• Anti-TG Ab
• Anti-TPO Ab
• Free T3
• Free T4
• Intact PTH
• TSH
• Third Generation TSH
• T Uptake
• Total T3
• Total T4
• TSH 3-UL

106. Solid-phase EIA with immobilized viral antigens to
detect antibodies to specific HIV proteins.
Western Blot (Immunoblotting)

107.  AIDS is caused by at least 2 etiological agents HIV-1
& HIV-2
 Inactivated and denatured protein of HIV-1 are
fractioned by polyacrylamide gel electrophoresis
 Protein bands are transferred into nitrocellulose
strips
 HIV-1 sample diluted with buffer are then incubated
with the strip
Principle

108.  Conjugate peroxidase labeled anti human IgG is
added
 It will bind to the antibodies already bound to the strip
 Chromogen is then added forming color reaction
 Reaction is then stopped by aspiration and reaction

109.  Serum sample
 Maximum 8 days
 Stored 2o C – 8oC or frozen at – 25oC
 Lipemic sample must be centrifuged well
 Avoid heating
Sample requirement:

110. Creating Western Blot Strips
Proteins are
transferred
(blotted) onto
the surface of a
membrane
Strips are
incubated with
patient serum
and antihuman
IgG conjugated
with an enzyme
(and chromagen)
HIV lysate
proteins are
separated by
size using gel
electrophoresis
The membrane
is cut into strips

111. HIV Western Blot Banding Pattern
env gp160
gp120
gp 41
gag p55
p18
p24
pol p65
p51
p31

112. Interpretation of Results
(General Consensus)
Negative: No bands present
Positive: 2 ENV band present
(WHO Guidelines)
Indeterminate Any bands present but
do not meet criteria for
positive

113. *
*
*

114. When should WB be used?
Western Blot assay should not be used as a screening test.
WB should be viewed as a supplemental test which can be used to
confirm positive results obtained from EIA.
HOWEVER:
Specificity is less than that of EIA
A significant number of indeterminate blots are seen in low
risk populations

115. Advantages
Specific interaction of antibody and
antigen can be directly visualized.
Disadvantages
Technically demanding
Expensive
Subject to interpretation
Presence or absence of bands
Intensity of those bands

116. HIV-1 Western Blot Antigens
p = protein
gp = glycoprotein
Number = molecular weight

117. Components Used in HIV-1 Western Blot
Human HIV Antibodies
(from patient serum)
Y YY Y
HIV Western blot Strip
YY
HIV Antigens
(on Western blot)
YY Y
Antihuman IgG Antibodies
Enzyme Detector
Color Reagent

118. Sample HIV-1 Western Blot
YY
Y
YY
Y
Y
Y
YY
Y
Y
Antibodies to gp120
Anti-human IgG
Enzyme Detector
HIV gp120 antigen
Color Reagent
Antibodies to p24
Enzyme Detector
HIV p24 antigen
Color Reagent
Anti-human IgG
Test Completed gp120 & p24 bands Visible

119. HIV-1 Gene Products & Western Blot

120. HIV-1 and HIV-2 Gene Products & Western Blot

121. Interpretive Criteria for HIV-1 Western Blot
Source: CDC. MMWR. 1989:38(S-7):1-7.
Positive Control

122. Interpretive Criteria for HIV-1 Western Blot
Source: CDC. MMWR. 1989:38(S-7):1-7.
Negative
No bands:

123. Interpretive Criteria for HIV-1 Western Blot
Source: CDC. MMWR. 1989:38(S-7):1-7.
Positive
At least two of the following bands:
p24
gp41
gp120/160

124. Interpretive Criteria for HIV-1 Western Blot
Source: CDC. MMWR. 1989:38(S-7):1-7.
Indeterminate
One or more bands present
Not meeting positive criteria
Examples
Most common bands seen with
indeterminate Western blot (IWB)
p17, p24, p55

125. THANK YOU