Section 2: Agglutination and Precipitation Reactions (cont.)

Question 1

What are two optical methods to enhance the detection of lattice formation?

Answer 1

1. nephelometry (reading at an angle, the refractive light)
2. turbidometry (reading the transmittance through a solution or sample)

Question 2

Lattice formation

Answer 2

-the formation of a lattice structure will scatter light in a reaction well, these optical methods ar emore sensitive than the naked eye
-used to measure IgG, IgM, kappa, and lambda light chains, complement proteins, and acute phase proteins

Question 3

agglutination

Answer 3

drawing together of particles that contain antigen to form a visible lattice or clump

Question 4

How to test for the presence of antibody in a sample

Answer 4

1. direct aggluntination
2. passive aggluntination

Question 5

direct agglutination

Answer 5

-particles have naturally occuring antigens on their surface (RBC)

Question 6

passive agglunitation

Answer 6

-particles that have been covalently coated with antigens
-latex beads

Question 7

What do we use to test for the presence of antigens in a sample?

Answer 7

1. inhibition aggluntination assay
2. reverse passive aggluntination

Question 8

inhibition aggluntination assay

Answer 8

-kit contains both antigen coated latex beads and control Abs
-first patient sample is mixed with control Ab
-then latex beads are added, if antigen is present in patient sample it will bind to Ab and INHIBIT aggluntination of the beads

Question 9

Reverse passive aggluntination

Answer 9

-particles have been coated with Ab with Fab regions available to bind antigen. Binding of two or more beads to the antigen creates an aggluntination

Question 10

Rheumatoid Latex aggluntination test (reverse passive aggluntination reaction)

Answer 10

-the reagent in this case is a human IgG Ab bound to the latex bead with the Fc portion facing out
-detecting patient IgM specific to the Fc portion of IgG (Rheumatoid factor)

Question 11

Hemagglutination (agglutination with RBC)

Answer 11

-involves IgG and anti-human Immunoglobulin-mediated aggluntination

Question 12

direct antigloblin test (DAT)

Answer 12

-test if Ab is present on a patient’s red blood cells
-anti-human IgG is added directly to RBCs
- Agglutination read (Infant’s red blood cells coated with mother’s anti-Rh, transfusion rxn, hemolytic anemia)
-this test to see if antibodies are already coated

Question 13

Indirect antiglobulin Test (IAT)

Answer 13

-test the patient for Abs against RBC antigens
-patient serum incubated with test RBCs
-anti-human IgG is added
-agglutination read
-testing to see if the patient has the antibodies in general

Question 14

Particle-enhanced turbidimetric inhibition (Petina)

Answer 14

-used to determin serum levels of a therapeutic drug (Coumadin)
-kit contains drug linked to particles and Ab directed towards that drug
-drug in patient serum inhibits the aggluntination reaction

Question 15

Particle-counting immunoassay (Pacia)

Answer 15

-a detector is used to count non-aggluntination particles, exluding aggregated particles
-sensitive to the 10-10 molar level

Question 16

Quasi-elastic light scattering method (Quels)

Answer 16

-measures changes related to diffusion coefficients (particle size, shape, flexibility, and particle-particle interactions)
-laser light is shined on a sample
-the amplitude of scattered light is proportional to particle size

Question 17

Rapid plasma reagin (RPR)

Answer 17

-card test for screening of syphillis

Question 18

Aggluntination test

Answer 18

-is when Ag is bound to a particle which then forms a lattice structure in the presence of Ab

Question 19

Flocculation

Answer 19

-the process by which particles come out of suspension to form flake-like structures that can be visualized macro- or microscopically
-ex: charcoal

Question 20

causative agent of syphilis

Answer 20

-treponema pallidum
subspecies: pallidum, endemicum, carateum, and pertenue
-each cause a different disease

Question 21

T.pallidum

Answer 21

-spirochete
-motile bacteria with a characteristic screw shape allowing organisms to penetrate mucous and mucosal tissue, gaining access to the bloodstream
-lack many surface proteins commonly present on other microorganisms (coats itself with host serum proteins also like schistosome and parasitic helminths)
-shape mostly anaerobic and weekly staining gram negative
-obligate parasite (cannot live without host)
-these characteristics help pathogens evade the host immune response and prevented the development of an effective vaccine due to low immunogenicity (do not generate antibodies that well)

Question 22

Other major diseases caused by spirochetes

Answer 22

1. lyme disease (borrelia burgdorferi)
2. leptospirosis (leptospira species)

Question 23

What was syphilis named after?

Answer 23

-named after a shepherd who was given the disease by the god Apollo for his defiance

Question 24

Transmission of syphilis

Answer 24

-predominately through sexual contact but also may be passed from mother to fetus (congenital syphilis)
-congenital syphilis can cause miscarriages, stillbirths, and death of the newborn

Question 25

Symptoms of syphilis

Answer 25

-complex with the diagnosis being difficult before the advent of serological techniques
-there are multiple stages of the disease with distinct clinical manifestations

Question 26

primary stage of syphilis

Answer 26

-1 week to 3 months after exposure
-symptoms: the point of infection develops a characteristics lesion called chancre
-if left untreated 25% of primary cases progress to the secondary stage
-most likely spread during this stage

Question 27

chancre

Answer 27

-a painless ulceration that will heal by itself

Question 28

secondary stage of syphilis

Answer 28

-1 to 6 months after initial exposure
-symptoms: enlarged lymph nodes, fever, loss of weight, sore throat, headache, stiffness of head and neck, and overall feeling of illness
-widespread rashes and lesions involving the trunk, limbs, palms, and soles of feet
-wart-like growths called condyloma latum can be found in the genital areas
-Highly Infectious Stage with all lesions containing large amounts of live organisms
*** in rare cases can lead to meningitis, kidney and liver disease, arthritic disease, and connective tissue inflammation

Question 29

Latent stage of syphilis

Answer 29

-following the secondary phase there is a latency period which lacks any symptoms of the disease
-can be divided into early and late phases based on time of exposure (<2 years early // >2 years late)
-defined by no symptomology but a positive serological test
-infectious but not as much as the secondary phase
-if left untreated about 50% of patients will advance to tertiary syphilis
-the organism is still replicating
-also likely to spread during this stage (hallmark area)

Question 30

Tertiary phase of syphilis

Answer 30

-can occur as early as 1 yr after exposure but usually manifest after decades
symptoms: key clinical feature is the formation of granulomas called Gummas
-granulomas are chronic festering balls or pockets of inflammatory molecules and cells, that try unsuccessfully to clear the infection
-these granulomas can form in almost any anatomical space and tissue, which can result in major health complications and deformities
-other complications: cardiac abnormalities (aneurysms, heart murmurs, left ventricular hypertrophy)
-cor bovinum (cow heart, huge enlargement of the heart)

Question 31

Neurosyphilis

Answer 31

-can occur in earlier stages ( can occur at any stage)
-clinical manifestations are diverse: behavior changes, personality, emotional reactions, and psychotic symptoms
-irregular reflexes and pupil function
-Tabes dorsalis
-commonly seen in patients with HIV but unclear why

Question 32

Tabes dorsalis

Answer 32

gradual degeneration of sensory neurons, deafness, visual problems, and dementia

Question 33

Treatment for neurosyphilis

Answer 33

-standard antibiotic treatment is very effective (penicillin G) during all stages of infection

Question 34

Syphilis Direct examination- Microscopy

Answer 34

-requires access to active lesions
-can be confused with non-pathogenic spirochetes
-conventional light microscopy and gram staining are unreliable so often darkfield microscopy needs to be used
-organisms must be alive to observe motility

Question 35

Syphilis Direct Examination- Fluorescence microscopy

Answer 35

-Fluorescent Treponemal Antibody- Absorption (FTA-ABS)
-the various lab test involves detection of antibodies produced during a T.pallidum infection

Question 36

Treponemal test

Answer 36

-antibodies that are specific to T. pallidum (confirmatory)
-FTA-ABS

Question 37

Nontreponmeal test

Answer 37

-antibodies to components of cells that arise as a consequence of cellular damage (screen)
- venereal disease research lab (VDRL) test
-rapid plasma reagin (RPR) test

Question 38

Rapid plasma reagin (RPR)

Answer 38

-detects anti-nontreponemal antibodies (REAGIN)
-the test is cheap by the specificity is low because other conditions can produce reagin (Abs)
-some viral infections (hepatitis), other treponemal infections (yaws, pinta, bejel), malaria, and autoimmune disease (SLE) can cause false positives
-used as a screening test which is confirmed with a treponemal test
-can be qualitative or semiquantitative (if serial dilution is performed)

Question 39

Reagin

Answer 39

-an antibody targeting cardiolipin, which is a lipid generated from damaged cells

Question 40

What is needed for rapid plasma reagin (RPR)

Answer 40

-is a flocculation test for the detection of reagin
-reagents needed: cardiolipin (antigen to reagin)
lecithin and cholesterol (enhance binding)
charcoal (activated, visual enhancer)
-patient serum and reagents are mixed and rotated for eight minutes
-the reagents form a complex with the reagin which results in macroscopic flocculation of the charcoal