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Immunology > Lab > Flashcards

Lab Flashcards

1
Q

What’s prozoning?

A

RBC coated w/ too much Ab that Ab con’t crosslink w/ adjacent cells ≠ (proper) haemagglutination = button looks like a star

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2
Q

In a haemagglutination assay how do you find the titre of serum?

A

the last pos. rxn: reciprocal of highest dilution (lowest [ ] that shows a pos. rxn)

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3
Q

What does a pos. & neg rxn look like in a haemagglutination assay

A

pos: large button - completely covers bottom of cell
neg: small button

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4
Q

how do you find titre of neat (OG) solution?

A

reciprocal of final dilution x dilution factor

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5
Q

What information does titre give?

A

[Ab]

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6
Q

In haemagglutination assay, why did we use 2 negative controls (NRS, PBS)?

A

NRS: check for contamination
PBS: double check it’s not reacting w/ RBC

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7
Q

Apart from RBC, what other particles can be used in agglutination assay?

A

latex particles coated w/ antigens

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8
Q

Describe appearance of WBC & their function

A
  • Lympho.: large purple staining nucleus > involved in innate & adaptive IR (B cells, T cells, NK cells)
  • Mono.: kidney-shape cytoplasm, irreg. cytoplasm > APC, defend against virus, intracell. bact. parasites
  • Baso.: large basophilic granules covering nuclei > release chemotactic agents to attract WBC to site; defend against parasites; release cytotoxic granules
  • Eosino.: bilobed nucei, red cytopl. granules > digest worms; allergies & asthma
  • Neutro.: 2-5 lobed nuclei > bact. infecitions; inflamm. resp.
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9
Q

Ab isotypes & functions of each

A
  • IgG: 2º IR; activation, opsinisation, neutralisation
  • IgA: neutralisation
  • IgM: 1º IR; agglutination
  • IgE: allergies & parasites
  • IgD: receptor on B cells
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10
Q

human serum contains

A

Ig & Alb

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11
Q

How do you separate Ig & Alb in human serum?

A
  1. add NH4SO4 (salt) dropwise => Ig precipitate
  2. Separate Ig precipitate & supernatant (Alb)
  3. Re-dissolve Ig precipitate in PBS
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12
Q

Describe SDS-PAGE (& western blotting technique)

A
  1. SDS (anionic detergent) denatures 2º & 3º structures & binds to protein = uniform neg. charge = separation based on MW
  2. B-mercapethanol (reducing agent) breaks S-S bonds
  3. proteins Electrophorhesed in polyacrylamide gel => separated due to MW
  4. transfer protein onto nitrocellulose by electric current
  5. proteins reacted to specific Ab => form Ag-Ab complexes
  6. detect protein of interest
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13
Q

method of sandwich ELISA

A
  1. Ab coats plastic tube & is blocked w/ protein (bovine serum albumin) > prevent non-specific binding = Fpos.
  2. wash & add sample & incubate
  3. wash & add conjugate Ab & incubate
  4. wash & add substrate & incubate
  5. colour change = detactable
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14
Q

interpreting IgM & IgG results about RRV

A
  • IgM = recent exposure
  • IgM + IgG = recent “
  • IgG = past “
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15
Q

Describe the 3 types of ELISA

A
  1. Sandwich/Ag capture: Quantitative = determine [Ag]
  2. Indirect: Quantitative = determine titre
  3. Direct: Qualitative = detect presence/absense of Ag
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16
Q

Apart from ELISA what other assays could be used to detect IgM & IgG Ab to RRV

A
  • haemaglutination assays
  • latex particle agglutination assays
  • Western blotting?
  • immunofluorescence
17
Q

Why is a cut-off value used?

A
  • results above a threshold is relevant
  • detect if the protein of interest is there = if it’s above the threshold. & accounts for minuet values that could be Fpos.
  • if results are below this may be bc of errors
18
Q

What type of assay is the ANA assay? What purpose is the assay used? What does a +ve result mean?

A

a) Immunohistochemistry
b) Screen for autoimmune disease like SLE, scleroderma
c) follow up by ENA or anti-dsDNA assay

19
Q

Difference b/w concentration, yield, purity

A

yield: mg (amount recovered)
purity: % (ideally Ig has a high %)

20
Q

Describe the Homogenous staining pattern of ANA & list the diseases associated

A
  • Not: Fine even staining in nuclueus.
  • Undergoing metaphase: Densely stained mitotic body & surrounding nucleus is unstained
    e. g. SLE & other CT diseases
21
Q

Describe the Speckled staining pattern of ANA & list the diseases associated

A
  • Not: Uneven granular/speckled stain in nucleus.
  • Undergoing mitosis: Unstained mitotic body cell
    e. g. SLE, scleroderma, mixed CT disease, Sjoren’s syndrome
22
Q

Describe the Nucleolar staining pattern of ANA & list the diseases associated

A
  • Not: 3-6 dense granules in nucleus
  • Undergoing metaphase: unstained mitotic body & stained granules around mitotic body
    e. g. scleroderma & Sjoren’s syndrome
23
Q

Describe the Centromere staining pattern of ANA & list the diseases associated

A
  • Not: fine granular/speckled staining in nucleus
  • Undergoing metaphase: linear pattern in the centre of the mitotic body w/ unstained surrounding nuclei
    e. g. progressive systemic sclerosis

Immunology (20 decks)

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