Microbio FINAL: Elisa, Intro to viruses & Influenza

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Radhika316
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Microbio FINAL: Elisa, Intro to viruses & Influenza
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2013-05-28 20:07:31
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ELISA LAB --(✔)-- 05.07-05.09:Intro to Virus & Phages (#13) --(✔)-- 05.09:Influenza Lecture & Other Viruses (#13) --(✔)--
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  1. ELISA:
    -purpose, Detection method, Limitation, Avoiding source of error
    enzyme-linked immunosorbent assay  

    • --> Purpose:determine whether a particular antibody is present in a patient's blood sample; can also detect antigens
    • --> uses specificity of antibodies to captures and stick to antigen

    --> detection method: using enzyme

    • -->Limitations of test: people can be poor produces of antibodies and won't be detected, false positives are possible: unrelated antibody reacts w/ antigent nospecifically
    • -->AVOID SOURCES of error: use replicates, wash properly, use positive & neg controls and follow instructions carefully.
  2. ELISA background
    • Centrifuge for serum
    • second antibody is added and antigen antibody complex signsals enzyme attached to second antibody that converts a added chemical to another color....
    • "Detection becomes possible when a second antibody is added. This antibody is prepared from the serum of an animal injected previously with human antibody; the human antibody in this case serves as an antigen and the animal thus produces an antibody against the human antibody. Once isolated, the second antibody can be chemically linked to a system that can produce a detectable signal."
    • Finally, because the first antibody binds to antigen, the more antigen that is accessible, the more first antibody will be retained. The measure of color, therefore, reflects the amount of antigen initially present.
  3. Why Do Elisa Washes?
    To Prevent False positive...

    Washing helps remove any antibody that did not react with the SLE antigen in the well. When the fluid is removed from the well, antibody that has reacted with antigen remains attached to the well surface. Unreacted (unbound) antibody may also remain in the well in the small amount of fluid that is left behind. This unbound antibody must be removed, because the anti-human antibody added in the next step will recognize and react with any antibody remaining in the well, regardless of whether that antibody is specific for the SLE antigen. A reaction with non-SLE antibody will produce a false-positive result.
  4. ELISA Method:
    • 1. Centrifuge blood and transfer serum sample and dilute.
    • 2. Add Phosphate-Saline buffer to regulate pH of serial dilutions
    • 3. Transfer dilutions to ELISA plate (pretreated with)
    • 4. Add positive controls: anti-DNA primary antibody and Negative control: PBS
    • 5. Incubate so that primary antibodies stick to antigens
    • 6. Wash: to remove antibodies that didn't react and avoid a false positive
    • 7. ADD Horse Radish Peroxidase (secondary antibody)  
    • 8. Incubate again:so that secondary antibody will bind to primary/human antibodies.
    • 9. Wash step.
    • 10. Add the clear HRP substrate to each well
    • 11. Look for color change...yellow=positive
  5. How the ELISA Method works in detecting ANTIGEN:
    A first antibody is adhered to the well, then the solution that may contain antigen (pentagons) is added, and finally an enzyme-labeled antibody against different parts of the antigen. Substrate is again converted to colored product.

    • 1. Antibody is captured with specificity for the protein that is used (pretreated)

    2. Human blood Sample then added to well and incubated, washed off.

    3. Detection of antibody: secondary antibodies which is attached to an enzyme ( ex: HRP) is added that will specifically bind the antigen in sample if antigen is present

    4. Substrate for HRP added and color change is observed if HRP is prsent in wells.
  6. How ELISA method works in detecting ANTIBODY (LAB)
    • Antigen (pentagons) is bound to the bottom of a well, and patient's serum that may contain antibodies to that antigen is added. Next, enzyme-labeled second antibody (made in a diff species)to the first antibody is added. A colorless enzyme substrate (S) is converted into a visible
    • product (P)

    • 1: antigen is coated onto plasatic wells.
    • 2. Serum from patient is added to wells; plate is washed
    • 3. detection antibody w/ enzyme attached is added. Wash.
    • 4. Enzyme substrate is added: color change indicates patient has antibodies
  7. Bronchiolitis:
    Inflammation of the bronchioles (bronchi branches)
  8. Antigens & Antibodies & Immunoglobulins
    antigen: any substance that provokes an immune response from the host; often viral/bacterial proteins.

    antibodies: prtoeins which consist of 4 parts which are produce by B cells (wbc); antibodies specifically bind antigens and help the immune system get rid of infectious organisms.

    Immunoglobulins: antibodies produces in one host and transferred to a new host who is at risk of eveoloping the disease, this is a way to acquire temporary immunity to a disease when there is not enough time to giave a vaccine or when it's not available..
  9. Hypoxia:
    deficiency of oxygen reaching the tissues; not enough oxygen in the blood.
  10. Orthomyxovirus:
    Enveloped, ssRNA; segmented type A genome pandemics (Influenza)
  11. Herpes Virus:
    enveloped, dsRNA; latency; large genome; ex: VZV, HSV1&2, EBV,CMV,HHV8
  12. Retro Virus:
    enveloped,2copies +ssRNA; latency, proviral integration, reverse transctipion; ex: lentivirus ( HIV &SIV) and oncoviruses
  13. Picornovirus:
    icoshadedral; +ssRNA; smallest-22 to 20 nm; ex: Rhinoviruses/cold and Polio
  14. Paramyxovirus:
    enveloped, -ssRNA; cause of several childhood diseases;ex: RSV, measles-rubeola, and mumbs
  15. Pox virus
    complex shaped; dsDNA; VERY LARGE-250 nm; ex: smallpox-size: 20-250 Nm)
  16. Intro to Virues:
    -Structure
    -Nucleic Acid Genomes: +ssRNA vs -RNA mean?
    -Size of genome
    -Viral shapes
    • STRUCTURE:
    • 1."Nucleocapsid": capsid: protein coat with nucleic acid (DNA/RNA) on the inside; seen in all viruses
    • 2. Envelope: lipid layer + proteins (only in some viruses); stolen from cell membrane as it leaves the cells-unlike cells: no cytoplasm, no organelles, ribosomes, mitochondria

    • Nucleic Acid Genome: either DNA or RNA; has different varieties;
    • 1. dsDNA: doublestranded; like in living things
    • 2. ssDNA: singlestranded; cyclic
    • 3.  "+ssRNA": plus tells you if it's the same for messenger RNA sequence
    • 4. "-ssRNA": the minus means it's complementary to viral messenger RNA
    • 5. dsRNA

    --> size of genome: smaller than bacteria; virus has 7 genes vs. E.coli has 4,000 genes

    • Viral Shapes (what's seen under microscope)
    • 1. Helical-Capsid shape
    • 2. Icosahedral capsid-20 triangular sides o:
    • 3. enveloped shape (can't see capsid)-Influenza
    • 4. complex capsid: umbrella term
  17. Generic Viral Life Cycle:
    • 1. Attatchment:attach to host cells, sticks to protein/carb on host membrane.
    • 2. Entry: has to go inside the cell
    • 3. Biosynthesis: making copies of it's genome, transcriptions for mRNA, translation for viral protein and maybe some enzymes along with capsid.
    • 4. Assembly/Packaging: making virus pieces to make many complete viruses; virions mature.
    • 5. Release of virions: "virions" = one virus particle; leave the host cell and usually the cell dies.
  18. Common Causes of viral respiratory infections:
    1. Rhinoviruses
    2. coronaviruses
    3. adenoviruses
    4. RSV (on another card)
    • 1. rhinoviruses: cause colds
    • 2. coronaviruses: cause colds & Severe Acute Respiratory Syndrome (SARS)
    • 3. adenoviruses: causes colds, gastrointestinal infections, and pneumonia.
    • 4. Respiratory Syncytial virus (RSV) 
  19. Bacteriophages:
    viruses that infect bacteria
  20. Respiratory Syncytial virus (RSV)
    -Transmitted & characteristics
    -Initial symptoms & complications
    -Diagnosis
    -Prevetnion & treatment
    -"syncytium"
    • -Common Causes of viral respiratory infections
    • -Trasmitted: Respiratory droplets
    • -Characteristics: enveloped negative ssRNA (complementary mRNA) virus, easily killed by soap/water/disinfectants
    • -Intials symptoms: wheezing, cough, runny nose, croup in babies  (inflammation of larynx/trachea which lead to difficulty in breathing, a barking cough and noisy breathing)
    • -complications: the most common cause of bronchiolitis especially in babies (Inflammation of the bronchioles) and pneumonia in children <12 months

    -Diagnosis: look for viral RNA, viral antigens/proteins,  viral antibodies against RSV, rapid test is also possible.

    -Treatment: For severe infections, oxygen is given or a ventilator may be used.

    -Prevention: No available vaccine; there is a "Palivizumab" monocloncal (all variable regions attack RSV) antibody for children at high risk for severe disease; circulate in baby and give protection by neutralizing RSV

    *syncytium*: when a several viruses infect several host cells and then virus causes infected cells to merge into one giant multinucleated cell.
  21. Influenza Background
    -Family, types
    -Structure of Type A & what's on surface..
    • -->family: orthomyxovirus
    • -->Three types: A (associated with pandemics; mutates quickly), B and C
    • -->structure of Type A:
    • 1. 8 nuclear capsids: ss-RNA helical capsids; haploid genes (only 1 copy of every gene; not repeated anywhere else)
    • 2. Envelope w/ layer under called M1 proteins and embedded M2 protein (ion channel)
    • 3: ON the surface:  
    • - Hemaglutinin(HA): glycoprotiein; "glutinin"(clumping); sticks to cell it attacks, binding sialic acid which is on host cell (used in attachment)
    • -Neuraminidase (NA): enzyme; cuts sialic acid so that the virions can release after infecting the host cell.
  22. Human flu:
    -Primary symptoms & Secondary infection
    -Risk Factors
    -Diagnosis
    -Treatment-
    -Vaccines
    1. Primary Symptoms: body ache, fever, chills, congestion, cough, fatigue, vomitting, diahrrhea

    2. Secondary bacterial infection: bacterial pneumonia ( caused by staph aureus and strep pyogenes, strep pneumonia, pseuodomonas aeruginosa, and haemophilus influenza--see meningitits)

    --> Risk Factors: pregnancy and lung disease

    • --> Diagnosis:
    • 1. Real time PCR (test for RNA); has an extra step where it takes RNA, makes dna copy of RNA and then makes dna copies; quantitative
    • 2. Viral culture

    • --> Treatment:
    • 1. Zanomivir/Relenza: inhaled
    • 2. Osteltomavir (tamiflu) pill
    • * mode of action: antiviral drugs; both inhibit Neuramindase
    • -viruses are trickier to get rid off; mostly helpful for risk factor groups

    • --> Vaccine:  "Trivalent"-refers to the fact that there are three diff viruses in here; 2 types of A viruses (diff subtypes) and one type B virus;
    • 1. attenuated: weakned; nasal
    • 2. inactivated: injected version, doesn't give virus to patient (safe)
  23. Influenza & Gene Reassortment:
    -Two types of Antigenic variation
    -Quadruple Reassortment
    • Antigenic variation: change the outside of virus; can change in two ways.
    • 1. Antigenic drift: slight change in either HA or NA; from a spontaneous mutation, might not get too protected from flu vaccines
    • 2. Antigenic Shift: Big change in protein; completely new and different HA or NA; occurs by genetic reassortment (the virus actually gains a new HA or NA gene; done when 2 viruses infect same cell and same time)ex: H2N2 became H3N2

    Quadruple reassortment: swin flu origin pandemic 2009; new H1V1 (HA & NA subtypes)
  24. Pandemic Risk Factors (For Flu)
    • 1. New Influenza A subtype
    • 2. Can infect humans and cause illness
    • 3. Spreads easily b/w humans.

    *must meet all three requirements to be considered a pandemic*

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