W7.3

Question 1

Turbidimetry and Nephelometry sensitivity comparison

Answer 1

“Since the amount of scattered light is far greater than the transmitted light in a turbid suspension, nephelometry offers higher sensitivity than turbidimetry
“

Question 2

steps to maintain good accuracy (Turbidimetry and Nephelometry)

Answer 2

”- Because some precipitation and settlement of particles may occur with time, in order to obtain good accuracy it is important to:
* mix the sample well prior to placing the cuvette in the instrument, and,
* keep the same time for measurement of every sample throughout the measurement
“

Question 3

Clinical applications for turbidimetry

Answer 3

”- Quantification of protein activity

- Quantification of bacterial suspension “

Question 4

Clinical applications for nephelometry

Answer 4

”- Quantification of immunoglobulins (total, IgG, IgE, IgM, IgA) in serum and other biological fluids
- Quantification of individual serum proteins; complement or c-reactive protein”

Question 5

Detection of precipitation in a solid matrix (passive immunodiffusion)

Answer 5

“(classical method)
-Radial immunodiffusion * Diffusion of only antigen
- Ouchterlony double diffusion
* Diffusion of both antigen and antibody
* Used to detect the specificity of a polyclonal antiserum
“

Question 6

Detection of precipitation in a solid matrix (electrophoresis)

Answer 6

” - Immunoelectrophoresis
- Protein electrophoresis
- Immunofixation electrophoresis
“

Question 7

Double immunodiffusion

Answer 7

”- Antibody and antigen are added to wells; incubate 12 to 48 hrs; view precipitin lines (antibody and antigen meet); density of line reflects the amount of immune complex formation
-Technique can be used to test the similarity/differences between antigens
“

Question 8

Agglutinins

Answer 8

Visible aggregation of particles caused by combination with specific antibody

Question 9

Agglutination main characteristics

Answer 9

”- Reaction takes place on the surface of the particle, antigen must be exposed and able to bind with antibody
- Types of particles used in agglutination reactions include:
* Erythrocytes, bacterial cells, and inert carriers like latex particles
“

Question 10

Steps in agglutination

Answer 10

sensitiation and lattice formation

Question 11

Sensitization (Step of agglutination)

Answer 11

“(antibody binds to cells and “sensitizes” it)
- Antigen-antibody combination through single antigenic determinants (epitopes) on the particle surface
- Affected by the nature of the antibody: affinity and avidity
- Affected by the class of antibody: IgM is more efficient than IgG
- Affected by the nature of the antigen-bearing surface: sparse epitopes or obscured epitopes are less likely to interact with antibody
“

Question 12

Lattice formation (Step of agglutination)

Answer 12

”- The Fab portion of the antibody attaches to antigens on 2 adjacent
cells
- Formation is governed by: ionic strength, pH (best between 6.5 and 7.5), and temperature
“

Question 13

Seroconversion

Answer 13

“(rise in titer)

Time period during which an antibody develops (and can be detectable in blood) “

Question 14

Titer

Answer 14

” is the reciprocal of the highest dilution where agglutination occurs
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Question 15

Serial dilutions

Answer 15

”- To allow for a visible end-point in a serological reaction, the relative proportions of antigen and antibody must be adjusted
* Too much antibody present = may not reach end-point
“

Question 16

Direct agglutination

Answer 16

“Includes antiglobulin-mediated agglutination reactions

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Question 17

Agglutination categories

Answer 17

Direct agglutination, Passive (indirect) and reverse passive agglutination, Agglutination inhibition

Question 18

Hemagglutination

Answer 18

agglutination reaction involves red blood cells

Question 19

Coomb’s test

Answer 19

”- detects nonagglutinating antibody by means of coupling with a second antibody
* Antibody to human globulin that is made in animals will react with the Fc portion of the human antibody attached to red blood cells
* Agglutination takes place as the antihuman globulin is able to bridge the distance between cells that IgG alone cannot do
* Strength of the reaction is proportional to the amount of antibody coating the red blood cells
* Direct or indirect types of reaction
“

Question 20

Passive and reverse passive agglutination (passive)

Answer 20

”- Utilizes particles that are coated with antigens, not normally found on their surfaces
* Examples of particles include: erythrocytes, latex beads
** Use of latex bead provides stability and consistency
** Use of erythrocytes could lead to cross-reactivity
- Used to detect rheumatoid factor, antibodies (IgM) to group A streptococci, antibodies to CMV, rubella, Varicella zoster, HIV
“

Question 21

Passive and reverse passive agglutination (reverse passive)

Answer 21

”- Utilizes particles that are coated with antibodies
- Used to detect microbial antigens
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Question 22

Agglutination inhibition

Answer 22

”- Inhibition reactions are based on competition between particulate and soluble antigens for limited antibody- combining sites
- Lack of agglutination is an indicator of a positive reaction
- Involves haptens that are complexed to proteins; hapten- protein conjugate is attached to a carrier particle
- Examples: pregnancy test (human chorionic gonadotrophin or HCG)
“

Question 23

Labeled Immunoassays

Answer 23

”- Uses antibodies and antigen complexes as a means of generating measurable results
- Presence of antigen or antibody can be measured “