First Aid, Chapter 4 Laboratory Tests, Immunoglobulin Measurement Flashcards Preview

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Flashcards in First Aid, Chapter 4 Laboratory Tests, Immunoglobulin Measurement Deck (32)
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1
Q

What are the three zones of antibody-antigen interactions? What is the ratio in each zone and how many antigens bound per antibody?

A
  • Zone of Antibody Excess: Complexes exist as single antibody to single antigen. -Zone of Equivalence: Single antibody bind to two antigens, forming large insoluble lattices (i.e., can increase turbidity of solution, appear as precipitin lines).
  • Zone of Antigen Excess: Binding sites on the antibodies are saturated, and antibodies exist as single antibody to two antigens.
2
Q

What is used to measure antibody excess?

A

Immunoblotting Enzyme-Linked Immunosorbent Assay (ELISA)

3
Q

What is used to measure antigen-antibody equivalence?

A

Radial Immunodiffusion (RID) Double Immunodiffusion (Ouchterlony) Nephelometry

4
Q

What is used to measure antigen excess?

A

Radioimmunoassay (RIA) ELISA

5
Q

How are serum immunoglobulins measured?

A

Nephelometry

6
Q

What is nephelometry? What quantities of protein are measured?

A

Nephelometry is a method by which the turbidity of a solution is determined by a pattern of scattered light (Figure 4-2). In the determination of Ig levels, Ig are injected into a reaction chamber with antigen-specific antibody. As antibody-antigen complexes form, light is applied and the extent of light scatter is measured by a nephelometer. Photons are reflected from the immune complexes at an angle and measured with a photomultiplier tube.The amount of light detected is quantified as a function of time and the concentrations of antibody and antigen. This automated technique allows laboratories to process a high volume of samples. Nephelometry is considered to be more sensitive than RID and can measure quantities of protein from 1–10 µg/mL.

7
Q

Describe how radial immunodiffusion (RID) works. Is it qualitative or quantitative? What quantities can be measured.

A

Antiserum is mixed into a warm gel matrix while it is still in liquid form, then poured into a flat plate and allowed to cool. Antigen (IgG) is then placed into wells that have been cut into the gel and diffuses into the gel matrix. At the zone of equivalence, a precipitin line is seen (Figure 43). The amount of antigen is determined by the diameter of the precipitin ring, which is compared with a standard curve, created by known serially diluted concentrations of the antigen (see Figure 4-3). This is a quantitative method. Limitations include the inability to accurately measure serum protein concentrations less than 10 µg/mL.

8
Q

What are advantages of Laurell Rocket over RID? What methods does it incorporate? How much time difference is there between this method and RID?

A

The Laurell rocket method is considered to be less timeconsuming and more accurate than RID. This method incorporates features of immunoelectrophoresis and the immunodiffusion technique. The addition of immunoelectrophoresis decreases the time involved considerably, from up to 48 hours in RID to several minutes for the Laurell rocket technique.

9
Q

Describe how the Laurell Rocket method works. Is it qualitative or quantitative?

A

Samples are added to gel plates containing antiserum in holes made throughout the gel layer. A voltage is applied for a predetermined length of time. A precipitin line is seen at the zone of equivalence, which adopts the shape of a “rocket” (Figure 4-4). The height of the precipitin line is used to determine the amount of antigen concentration, compared against a standard curve. This represents both a qualitative and quantitative method of the antigen placed in the gel.

10
Q

What is the method of choice to determine immunoglobulin levels?

A

Nephelometry.

11
Q

Which methods of determining immunoglobulin levels are insensitive?

A

RID and Laurell Rocket.

12
Q

What are the methods of characterizing immunoglobulins?

A
  • Immunodiffusion (Ouchterlony)
  • Enzyme-Linked Immunosorbent Assay (ELISA)
  • Western Blot (Protein Immunoblot)
  • Immunofixation Electrophoresis (IFE)
13
Q

Describe immunodeiffusion (Ouchterlony). What is the Wadsworth method and how long does it take?

A

The Ouchterlony method utilizes a doubleimmunodiffusion (DID) technique. Holes are punched into an agarose gel. Antigens are placed in wells, and serum is placed in adjacent wells. Antigen and antibodies diffuse into the gel, and complexes form at the zone of equivalence, which is seen as a white precipitin line (Figure 4-5). The Wadsworth method simplifies the Ouchterlony method and utilizes agar on glass slides. Diffusion occurs within hours and requires fewer reagents.

14
Q

Which test can be used to detect both excess antibody and excess antigen?

A

ELISA

15
Q

How does ELISA work? What types of labels are used? How is the signal detected?

A

ELISA refers to assays performed on a microtiter plate in either a zone of antigen excess or zone of antibody excess. Antigens and reagents are immobilized by adsorption onto microtiter plates. ELISAs can be performed in many different ways by modifying the basic principle of identifying the bound protein by an antibody, which is conjugated to a label (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, streptavidin, and fluorescent tags). Figure 4-6 illustrates common ELISA formats. The appropriate substrate is added, and the signal is detected by use of a spectrophotometer, fluorometer, or luminometer.

16
Q

What are three different ELISA formats?

A

Direct Assay
Indirect Assay
Capture assay sandwich

17
Q

What is Western blot? What antigen-antibody zone does it measure? What types of samples is it used in? Describe the process.

A

Western Blot (Protein Immunoblot)—The Western blot represents an antibody excess assay for recognizing specific proteins within soluble tissue or cell samples. The method is summarized in Figure 4-7 and a Western blot is shown in Figure 4-8.

  • Proteins are separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). SDS is a detergent that unfolds a protein’s tertiary structure and provides a negative charge. Immunoglobulins separate by migrating to a positive pole when placed under an electric field. Shorter, denatured immunoglobulins will migrate further in the polyacrylamide gel.
  • Separated immunoglobulins are then transferred to a nitrocellulose sheet or polyvinylidene difluoride (PVDF) membrane while maintaining the same separation pattern created in the polyacrylamide gel.
  • Labeled specific immunoglobulin antibody is added, binding to the separated immunoglobulins. A secondary antibody is added and then conjugated to an enzyme, such as horseradish peroxidase or alkaline phosphatase.
  • Enzyme substrate is added to provide a colorimetric reaction (see Figure 4-8).
18
Q

What are examples of molecules measured by ELISA?

A

allergen-specific IgE (see later discussion), Bnatriuretic peptide, complement, toxins, histamine, leukotriene C4, and tumor markers.

19
Q

What infections has western blot been used to detect? What other proteins is it used to detect?

A
  • Borrelia burgdorferi
  • HHV6 and HHV7
  • HIV (confirmatory test after positive ELISA)
  • Human T-lymphocyte leukemia virus
  • Prion diseases
  • Respiratory syncytial virus
  • Severe acute respiratory syndrome (SARS) coronavirus
  • Varicella-zoster virus
  • Confirmatory test for hepatitis B
  • Lyme disease
  • It can also be used to determine the presence of antiglomerular basement antibodies, antinuclear antibodies, and antiretinal antibodies.
20
Q

What is immunofixation electrophoresis? How does it compare to immunoelectrophoresis? What is it used to detect?

A
Immunofixation Electrophoresis (IFE)—IFE determines levels of serum immunoglobulins by electrophoresis by measuring immunodiffusion against an antiserum. The test replaces the less sensitive and slower immunoelectrophoresis method and is used to confirm monoclonal gammopathy and determine heavy- or light-chain class type.   
Serum proteins are separated by high-resolution agarose gel electrophoresis. Proteins are separated by size and charge. Once separated, immunoglobulins are exposed to specific antisera and the immunoprecipitate is visualized with a protein stain.
21
Q

What method is used to determine specific Ig levels in Bacterial polysaccharides (pneumococcus, meningococcus, Hib) ?

A

ELISA

22
Q

What method is used to determine specific Ig levels in tetanus?

A

ELISA

23
Q

What method is used to determine specific Ig levels in e. coli 0157 LPS/HUS infection? Which Igs?

A

ELISA (IgG, IgM, IgA)

24
Q

What method is used to determine specific Ig levels in H. pylori infection?

A

ELISA (IgG, IgM, IgA)

25
Q

What method is used to determine specific Ig levels in Sm autoantibody?

A

DID (Ouchterlony)

26
Q

What method is used to determine specific Ig levels in Alport’s syndrome (anti GBM antibody)?

A

Western blot

27
Q

How does HyCor Turbo-RAST: Radioallergosorbent Test work?

A

The first-generation method for determining allergen-specific IgE was the radioallergosorbent test. Paper discs (allergosorbent material) coated with allergens were incubated with a patient’s serum. Antigen would bind specific immunoglobulins of all classes (IgG, IgM, IgA, IgE), and unbound immunoglobulins were washed away. Radiolabeled antihuman IgE was used to detect antiallergen IgE. Results were then compared with a reference curve.
Currently, Phadia ImmunoCAP (cellulose sponge as allergosorbent) and DPC Immulite 2000 (allergen conjugated to biotin) are used due to their excellent degree of quantification and automation.

28
Q

How does ImmunoCAP or FEIA (Fully Automated Fluroroenzyme Immunoassay) work?

A

ImmunoCAP or FEIA (Fully Automated Fluroroenzyme Immunoassay)— ImmunoCAP was developed by Phadia and is the technique employed by both Phadia and Quest. It utilizes a basic “sandwich” ELISA technique (Figure 4-11). The anti-IgE conjugate has a fluorescent enzymatic tag. Fluorescence quantifies the amount of sIgE captured in this process.

29
Q

What is DPC Immulite 2000? Describe it.

A

This is a chemiluminescent enzyme-labeled immunoassay. Fig 4-12.

1) Serum and allergens incubate with ligand-coated bead.
2) Liquid allergens bind to serum antibodies and to the ligand-coated bead.
3) Antibody conjugate binds to serum IgE.
4) Substrate initiates chemiluinescent reaction.

30
Q

Can you use different IgE assays to compare to eachother for food allergies?

A

No, they do not correlate.

31
Q

Describe the steps of immunocap.

A

1) Allergens coupled to Immunocap, patient IgE is added
2) Patient IgE ab bound to immunocap allergens, conjugate enzyme anti-IgE is added.
3) Conjugate bound to patient IgE, fluorogenic substance is added.
4) Conjugate enzyme reacts with substrate forming a fluorescent product.

32
Q

What test(s) would you use to diagnose Lyme disease?

A

ELISA as a screen and Western blot to confirm

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