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ASSAY OF VIRAL PARTICLES.

          PRESENTED BY
       IDOWU TEMITAYO .O.
    MATRIC NUMBER: 0903010024
   DEPARTMENT OF BIOLOGICAL
            SCIENCES,
 COLLEGE OF NATURAL SCIENCES,
JOSEPH AYO BABALOLA UNIVERSITY,
         IKEJI-ARAKEJI,
           OSUN STATE.
OUTLINE
DEFINITION OF TERM.
VIRAL GROWTH RECOGNITION.
COMMONLY USED LABORATORY METHODS
 FOR THE DETECTION OF VIRUSES.
QUANTIFICATION OF VIRUSES.
TRADITIONAL METHODS.
MODERN METHODS.
CONCLUSION.
DEFINITION OF TERMS

WHAT ARE VIRUSES?
“A PIECE OF BAD NEWS WRAPPED UP IN A PROTEIN”
WHAT IS AN ASSAY?
ASSAY IS AN INVESTIGATIVE (ANALYTIC) PROCEDURE
  IN LABORATORY MEDICINE, MICROBIOLOGY,
  ENVIRONMENTAL BIOLOGY, AND MOLECULAR
  BIOLOGY FOR QUANTITATIVE ASSESSING OR
  QUALITATIVELY MEASURING THE PRESENCE OR
  AMOUNT OR THE FUNCTIONAL ACTIVITY OF A
  TARGET ENTITY (Mckean et al., 2005).
VIRAL GROWTH RECOGNITION.

Recognition of a diseased state
Systemic infection.
The production of a local lesion such as
  plaque.
In tissue culture the culture will undergo
change with the production of cytopathic
effect (CPE), namely the destruction of the
cell, syncytia formation, or cellular
transformation.
Plate 1: CYTOPATHIC EFFECT (CPE)
Source: Wikipedia
COMMONLY USED LABORATORY
    METHODS FOR THE DETECTION OF
             VIRUSES.

Measure of virus infectivity.
Examination of viral serology (that is,
 host antibody responses to the virus).
Molecular methods.
QUANTIFICATION OF VIRUSES

• TRADITIONAL METHOD-: These are industry
  standard methods that have been used for
  decades but are generally slow and labour
  intensive.
• MODERN METHOD-: These are relatively new
  commercially available product and kits that
  greatly reduce quantification time.
TRADITIONAL METHOD
PLAQUE ASSAY




  Plate 2: Viral Plaques of Herpes Simplex Virus.
                  Source: Wikipedia
FLUORESCENT FOCUS ASSAY (FFA):




      Plate 3: Fluorescent Focus Assay
                Source: C.D.C
50% TISSUE CULTURE INFECTIVE DOSE
  (TCID50)

This endpoint dilution assay quantifies the
 amount of virus required to kill 50% of
 infected hosts or to produce a cytopathic
 effect in 50% of inoculated tissue culture cells.
PROTEIN ASSAY:
There are several variations of protein-
 based virus quantification assays.
In general, these methods quantify either
 the amount of all protein or the amount of a
 specific virus protein in the sample rather
 than the number of infected cells or virus
 particles.
HEMAGGLUTINATION ASSAY:
The hemagglutination assay (HA) is a common
 non-fluorescence protein quantification assay
 specific for influenza.
    SINGLE RADIAL IMMUNODIFFUSION
     ASSAY:
Single Radial Immunodiffusion Assay, also
 known as the Mancini method, is a protein
 assay that detects the amount of specific viral
 antigen by immunodiffusion in a semi-solid
 medium (e.g. agar).
MODERN METHODS
Flow cytometry:
          MODERN METHODS:
 A virus counter quantifies the number of intact
  virus particles in a sample using fluorescence to
  detect proteins and nucleic acids.
Quantitative Polymerase Chain Reaction
 (qPCR):
Quantitative PCR utilizes polymerase chain reaction
 chemistry to amplify viral DNA or RNA to produce
 high enough concentrations for detection and
 quantification by fluorescence.
Enzyme-Linked Immunosorbent Assay
   (ELISA):
 ELISA is a more modern variation of a protein
 assay that utilizes a specific antibody linked to
 an enzyme to detect the presence of an
 unknown amount of antigen (i.e. virus) in a
 sample.
CONCLUSION

A basic principal of science is
measurement or quantitation,
therefore being able to accurately
quantify virus numbers became a
necessity.

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(Tayo)semina presentation

  • 1. ASSAY OF VIRAL PARTICLES. PRESENTED BY IDOWU TEMITAYO .O. MATRIC NUMBER: 0903010024 DEPARTMENT OF BIOLOGICAL SCIENCES, COLLEGE OF NATURAL SCIENCES, JOSEPH AYO BABALOLA UNIVERSITY, IKEJI-ARAKEJI, OSUN STATE.
  • 2. OUTLINE DEFINITION OF TERM. VIRAL GROWTH RECOGNITION. COMMONLY USED LABORATORY METHODS FOR THE DETECTION OF VIRUSES. QUANTIFICATION OF VIRUSES. TRADITIONAL METHODS. MODERN METHODS. CONCLUSION.
  • 3. DEFINITION OF TERMS WHAT ARE VIRUSES? “A PIECE OF BAD NEWS WRAPPED UP IN A PROTEIN” WHAT IS AN ASSAY? ASSAY IS AN INVESTIGATIVE (ANALYTIC) PROCEDURE IN LABORATORY MEDICINE, MICROBIOLOGY, ENVIRONMENTAL BIOLOGY, AND MOLECULAR BIOLOGY FOR QUANTITATIVE ASSESSING OR QUALITATIVELY MEASURING THE PRESENCE OR AMOUNT OR THE FUNCTIONAL ACTIVITY OF A TARGET ENTITY (Mckean et al., 2005).
  • 4. VIRAL GROWTH RECOGNITION. Recognition of a diseased state Systemic infection. The production of a local lesion such as plaque. In tissue culture the culture will undergo change with the production of cytopathic effect (CPE), namely the destruction of the cell, syncytia formation, or cellular transformation.
  • 5. Plate 1: CYTOPATHIC EFFECT (CPE) Source: Wikipedia
  • 6. COMMONLY USED LABORATORY METHODS FOR THE DETECTION OF VIRUSES. Measure of virus infectivity. Examination of viral serology (that is, host antibody responses to the virus). Molecular methods.
  • 7. QUANTIFICATION OF VIRUSES • TRADITIONAL METHOD-: These are industry standard methods that have been used for decades but are generally slow and labour intensive. • MODERN METHOD-: These are relatively new commercially available product and kits that greatly reduce quantification time.
  • 9. PLAQUE ASSAY Plate 2: Viral Plaques of Herpes Simplex Virus. Source: Wikipedia
  • 10. FLUORESCENT FOCUS ASSAY (FFA): Plate 3: Fluorescent Focus Assay Source: C.D.C
  • 11. 50% TISSUE CULTURE INFECTIVE DOSE (TCID50) This endpoint dilution assay quantifies the amount of virus required to kill 50% of infected hosts or to produce a cytopathic effect in 50% of inoculated tissue culture cells.
  • 12. PROTEIN ASSAY: There are several variations of protein- based virus quantification assays. In general, these methods quantify either the amount of all protein or the amount of a specific virus protein in the sample rather than the number of infected cells or virus particles.
  • 13. HEMAGGLUTINATION ASSAY: The hemagglutination assay (HA) is a common non-fluorescence protein quantification assay specific for influenza. SINGLE RADIAL IMMUNODIFFUSION ASSAY: Single Radial Immunodiffusion Assay, also known as the Mancini method, is a protein assay that detects the amount of specific viral antigen by immunodiffusion in a semi-solid medium (e.g. agar).
  • 15. Flow cytometry: MODERN METHODS:  A virus counter quantifies the number of intact virus particles in a sample using fluorescence to detect proteins and nucleic acids. Quantitative Polymerase Chain Reaction (qPCR): Quantitative PCR utilizes polymerase chain reaction chemistry to amplify viral DNA or RNA to produce high enough concentrations for detection and quantification by fluorescence.
  • 16. Enzyme-Linked Immunosorbent Assay (ELISA):  ELISA is a more modern variation of a protein assay that utilizes a specific antibody linked to an enzyme to detect the presence of an unknown amount of antigen (i.e. virus) in a sample.
  • 17. CONCLUSION A basic principal of science is measurement or quantitation, therefore being able to accurately quantify virus numbers became a necessity.