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BIOL 135 > 1+2 - Investigations into immune system function > Flashcards

1+2 - Investigations into immune system function Flashcards

1
Q

Important white blood cells

A

lymphocyte, monocyte, eosinophils, basophils, neutrophils

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2
Q

Lymphocytes

A

B cells, Tc cells, Th cells

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3
Q

B cells

A

Cells manufactured in the bone marrow that create antibodies for isolating and destroying invading bacteria and viruses.

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4
Q

Tc cell (CD8)

A

Effector form of a cytotoxic T cell; it induces apoptosis in infected or cancerous “self” cells

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5
Q

TH cells (CD4)

A

Helper T cells
MHC II complex
- recognizes APC and able to release cytokines and attract other cells to the area

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6
Q

B cell receptor

A

The antigen receptor on B cells: a Y-Shaped, membrane-bound molecule consisting of two identical heavy chains and two identical light chains linked by disulfide bridges and containing two antigen-binding sites.

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7
Q

T cell receptor

A

Unlike antibodies, T cell receptors are never produced in a secreted form.

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8
Q

immunoglobulins

A

bind with specific antigens in the antigen-antibody response

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9
Q

Antibody elimination of pathogen

A
  1. The pathogen is opsonised = faster phagocytosis
  2. Antibodies recruit complement proteins
  3. causes formation of membrane attack complex
  4. A hole forms in the pathogen’s membrane.
  5. pathogen destroyed
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10
Q

Acquired immune response

A
  1. activated T helper cells release cytokines, activating Tc and B cells.
  2. Tc cells trigger infected self cells to undergo apoptosis
  3. B cells stimulate antibody production
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11
Q

HIV Virion

A

Consists of RNA surrounded by core proteins and capsid
RNA serves as template for DNA synthesis
Outer envelope contains glycoproteins that are needed to attach to CD4 cells

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12
Q

how does HIV infect T helper cells

A
  • GP120 binds to CD4
  • GP41 binds CXCR4 on the surface of TH HIV destroys T helper cells, B and T cells.
  • mediated immunity lost
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13
Q

AIDS (acquired immune deficiency syndrome)

A

increased susceptibility to opportunistic infections

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14
Q

example infections

A
  • toxoplasmosis
  • tuberculosis (mycobacterium tuberculosis)
  • pnuemonia (pneumocystis jiroveci)
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15
Q

methods for detection of HIV

A

what might be detected?

  1. antibodies to HIV
  2. Viral RNA
  3. Fall in T helper cell numbers
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16
Q
  1. Antibodies to HIV (‘seroconversion’)
A
  • develop 2-8 weeks after infection (prior to this = ‘window period’)
  • Often bind to p24 or gp41
  • Detection via ELISA, immunochromatography, latex particle agglutination,
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17
Q
  1. Viral RNA
A

Useful in the ‘window period’ before antibodies to HIV are detectable in serum.

The genome of HIV is RNA

RT-PCR (Reverse Transcription Polymerase Chain Reaction) detection

18
Q
  1. FALL IN T HELPER CELL NUMBERS
A

Flow cytometry and Fluorescence-Activated Cell Sorting can be used to identify T Helper cells on basis of CD4

19
Q

ELISA

A
  1. A HIV antigen bound to well
  2. Wash off anything that hasn’t bound
  3. Patient serum added. Any antibodies bind to the antigen.
  4. Wash
  5. Anti-human antibody added. This binds human antibodies. An antibody also has an enzyme attached.
  6. Wash
  7. E Substrate added. Enzyme converts substrate to a coloured product which is detected by absorbance of a certain wavelength of light
20
Q

chromogenic substrate

A

Antibody conjugated (linked) to: ALKALINE PHOSPHATASE (AP) ENZYME Substrate may be BCIP (5-bromo-4-chloro-3-indolyl phosphate)

  • Can use different enzymes conjugated to antibody
  • purple product detected by light abosrbance
21
Q

Immunochromatography

A
  • Detection of antibodies to HIV
  • Serum, plasma or whole blood added to absorbent pad
  • Any anti-HIV antibodies bind HIV antigen in test
  • HIV test line = antibody which binds antigen/antibody complex
  • Control line binds HIV antigen
22
Q

Latex particle agglutination

A
  1. Latex particles covered with HIV antigen e.g. gp41
  2. Add blood which may contain Anti HIV antibodies
  3. Visual clumping if HIV present
23
Q

PCR

A
  1. REACTION Double stranded DNA is DENATURED (separated) when heated to 95oC
  2. Primers added specific to sequence on either side of target sequence ANNEALING (54oC) (A-T; C-G)
    DNA polymerase and DNA building blocks (base + sugar + phosphate) joined to make copy of target sequence. EXTENSION
24
Q

benefit of pcr

A

amplifies a target DNA sequence

25
Q

window period detection of HIV using RT-PCR

A
  1. DNA copy (cDNA) made of viral RNA using Reverse Transcriptase enzyme
  2. DNA copy of cDNA made by DNA polymerase
  3. PCR reaction to amplify DNA
26
Q

FLOW CYTOMETRY AND FLUORESCENCE-ACTIVATED CELL SORTING (FACS)

A

Can be used to distinguish TH cells from other blood cells by virtue of the CD4 on the TH cell surface. Therefore, identify number of T helper cells in patient’s blood.

Useful in monitoring HIV infection

27
Q

Flow Cytometry/FACS

A

Blood cells mixed with antibody specific for CD4, which is engineered to contain a fluorescent label. Cells are streamed through a flow cell

Measures physical and chemical characteristics of cells as they flow one cell at a time through a laser beam. The light scatter is a characteristic of the cells and their components.

28
Q

FLOW CYTOMETRY/FACS USING MORE THAN ONE FLUOROCHROME

A

Mix lymphocytes with range of antibodies (e.g. to CD4, CD8), each with different fluorochrome.
• Antibodies engineered to have different fluorescent labels (fluorochromes).
• Identification of more than one antigen in the same sample e.g. CD4, CD8.
• Gives you number of Tc and TH etc

29
Q

FLOW CYTOMETRY/FACS EXAMPLE – PATIENT WITH HIV

A

Normal TH 30-50% of lymphocytes (Expect 500-1200 cells/ul
200-500 CD4+/ul= abnormal
<200 CD4+/ul= AIDS
CD4 cells labelled with FLUORESCEIN
CD8 cells labelled with PHYCOERYTHRIN
provides number so are able to quantify how far the disease has mobilised.

30
Q

Multiple Myeloma (MM)

A

cancerous proliferation of plasma b cells
Osteoclasts dissolve bone.
Activity up-regulated in MM.
Fractures of long bones, ribs and vertebrae.

31
Q

presenting features of MM

A

Anaemia

weakness, tachycardia Recurrent infection

Bone pain/fractures

32
Q

DIAGNOSIS OF MULTIPLE MYELOMA (MM)

A
  • Large amount of one IgG produced (PARAPROTEIN).
  • Detect via CELLULOSE ACETATE ELECTROPHORESIS.
  • Malignant plasma cells stay in bone marrow and activate OSTEOCLASTS
  • Bone broken down
33
Q

DETECTION OF ‘PARAPROTEIN’ IN BLOOD SERUM OF PERSON WITH MULTIPLE MYELOMA

A 
Cellulose acetate electrophoresis 
1. normal SERUM 
2. multiple myeloma SERUM - PARAPROTEIN 
3. normal PLASMA 
(Free light chains excreted in urine = BENCE-JONES PROTEIN
34
Q

difference between serum and plasma

A

serum is when you have had a blood sample and allow it to clot and spin it out, clot is pellet, serum is top layer

in plasma you prevent clotting; spin serum, clot is pellet, plasma is top layer

35
Q

cellulose acetate electrophoresis

A

Blocking OH groups in cellulose with acetyl group (to form cellulose acetate) inhibits protein and water binding.

Cellulose acetate made into paper.

Proteins migrate through holes.

36
Q

CELLULOSE ACETATE ELECTROPHORESIS PH 8.6

A

Proteins have overall negative charge at pH 8.6 and migrate to ANODE.

  • NH2 not protonated at pH 8.6 to -NH3 +
  • COOH deprotonated at pH 8.6 to -COO-
37
Q

VISUALISATION OF PROTEIN IN CELLULOSE ACETATE ELECTROPHORESIS

A
  • Immerse cellulose acetate in trichloroacetic acid
  • Protein reprotonated (-COOH and –NH3 + )
  • Ponceau S stain added
  • Negative charge binds to + protein
  • Hydrophobic rings bind any hydrophobic groups in protein
38
Q

PERNICIOUS ANAEMIA

A
  • Absorption of vitamin B12 requires a combination with INTRINSIC FACTOR (IF) made by gastric parietal cells
  • Pernicious anaemia = autoantibodies against gastric parietal cells so less B12 absorbed
  • Vitamin B12 needed to make DNA
  • Red blood cell production requires rapid DNA synthesis.
  • fewer RBC made, less O2 transport = FATIGUE
39
Q

INDIRECT IMMUNOFLUORESCENCE

A

FIXED GASTRIC PARIETAL CELLS ON GLASS MICROSCOPE SLIDE
TEST BLOOD SERUM (Autoimmune antibody?)
wash
Add fluorescently-labelled Anti-human antibody
EXAMINE WITH MICROSCOPE AND UV-LIGHT SOURCE
Autoimmune antibody binds antigen in test sample
Anti human antibody
binds autoimmune antibody
Fluorescent label on
anti-human antibody detected

40
Q

TRANSMITTED LIGHT FLUORESCENCE MICROSCOPY

A

Enables detection of location of Anti-human immunoglobulin which
has been labelled with fluorescein.
Barrier filter – enables visible fluorescent light to pass to eye
Exciter filter – maximum amount of appropriate wavelength light reaches specimen

BIOL 135 (10 decks)

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