1+2 - Investigations into immune system function Flashcards
Important white blood cells
lymphocyte, monocyte, eosinophils, basophils, neutrophils
Lymphocytes
B cells, Tc cells, Th cells
B cells
Cells manufactured in the bone marrow that create antibodies for isolating and destroying invading bacteria and viruses.
Tc cell (CD8)
Effector form of a cytotoxic T cell; it induces apoptosis in infected or cancerous “self” cells
TH cells (CD4)
Helper T cells
MHC II complex
- recognizes APC and able to release cytokines and attract other cells to the area
B cell receptor
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.
T cell receptor
Unlike antibodies, T cell receptors are never produced in a secreted form.
immunoglobulins
bind with specific antigens in the antigen-antibody response
Antibody elimination of pathogen
- The pathogen is opsonised = faster phagocytosis
- Antibodies recruit complement proteins
- causes formation of membrane attack complex
- A hole forms in the pathogen’s membrane.
- pathogen destroyed
Acquired immune response
- activated T helper cells release cytokines, activating Tc and B cells.
- Tc cells trigger infected self cells to undergo apoptosis
- B cells stimulate antibody production
HIV Virion
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
how does HIV infect T helper cells
- GP120 binds to CD4
- GP41 binds CXCR4 on the surface of TH HIV destroys T helper cells, B and T cells.
- mediated immunity lost
AIDS (acquired immune deficiency syndrome)
increased susceptibility to opportunistic infections
example infections
- toxoplasmosis
- tuberculosis (mycobacterium tuberculosis)
- pnuemonia (pneumocystis jiroveci)
methods for detection of HIV
what might be detected?
- antibodies to HIV
- Viral RNA
- Fall in T helper cell numbers
- Antibodies to HIV (‘seroconversion’)
- develop 2-8 weeks after infection (prior to this = ‘window period’)
- Often bind to p24 or gp41
- Detection via ELISA, immunochromatography, latex particle agglutination,
- Viral RNA
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
- FALL IN T HELPER CELL NUMBERS
Flow cytometry and Fluorescence-Activated Cell Sorting can be used to identify T Helper cells on basis of CD4
ELISA
- A HIV antigen bound to well
- Wash off anything that hasn’t bound
- Patient serum added. Any antibodies bind to the antigen.
- Wash
- Anti-human antibody added. This binds human antibodies. An antibody also has an enzyme attached.
- Wash
- E Substrate added. Enzyme converts substrate to a coloured product which is detected by absorbance of a certain wavelength of light
chromogenic substrate
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
Immunochromatography
- 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
Latex particle agglutination
- Latex particles covered with HIV antigen e.g. gp41
- Add blood which may contain Anti HIV antibodies
- Visual clumping if HIV present
PCR
- REACTION Double stranded DNA is DENATURED (separated) when heated to 95oC
- 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
benefit of pcr
amplifies a target DNA sequence
window period detection of HIV using RT-PCR
- DNA copy (cDNA) made of viral RNA using Reverse Transcriptase enzyme
- DNA copy of cDNA made by DNA polymerase
- PCR reaction to amplify DNA
FLOW CYTOMETRY AND FLUORESCENCE-ACTIVATED CELL SORTING (FACS)
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
Flow Cytometry/FACS
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.
FLOW CYTOMETRY/FACS USING MORE THAN ONE FLUOROCHROME
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
FLOW CYTOMETRY/FACS EXAMPLE – PATIENT WITH HIV
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.
Multiple Myeloma (MM)
cancerous proliferation of plasma b cells
Osteoclasts dissolve bone.
Activity up-regulated in MM.
Fractures of long bones, ribs and vertebrae.
presenting features of MM
Anaemia
weakness, tachycardia Recurrent infection
Bone pain/fractures
DIAGNOSIS OF MULTIPLE MYELOMA (MM)
- 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
DETECTION OF ‘PARAPROTEIN’ IN BLOOD SERUM OF PERSON WITH MULTIPLE MYELOMA
Cellulose acetate electrophoresis 1. normal SERUM 2. multiple myeloma SERUM - PARAPROTEIN 3. normal PLASMA (Free light chains excreted in urine = BENCE-JONES PROTEIN
difference between serum and plasma
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
cellulose acetate electrophoresis
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.
CELLULOSE ACETATE ELECTROPHORESIS PH 8.6
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-
VISUALISATION OF PROTEIN IN CELLULOSE ACETATE ELECTROPHORESIS
- 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
PERNICIOUS ANAEMIA
- 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
INDIRECT IMMUNOFLUORESCENCE
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
TRANSMITTED LIGHT FLUORESCENCE MICROSCOPY
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
