Laboratory Use of Antibodies

Question 1

Why does agglutination occur?

Answer 1

Extensive cross-linking between antigen-antibody to form complexes results in agglutination

Question 2

Describe a precipitation test

Answer 2

Increasing concentrations of antigen solution are plated in individual wells. A fixed amount of ab is added to each well and the aggregation of ab-ag complexes are observed by visualization of precipitate

Question 3

What is the zone of equivalence in the antigen-precipitatation technique?

Answer 3

The zone of equivalence is the solution of ag concentration where the amount of ag is approximately equivalent to ab added. This equal proportion results in the largest amount of precipitate

Question 4

Explain how a sandwich enzyme linked immunoabsorbent assay (ELISA) is performed?

Answer 4

1. Abs of known ag specificity are coated on sample plate
2. Ag containing sample of interest is added
3. Excess Ag not bound to Ab is washed off
4. Enzyme linked abs, also of known Ag specificity are added and the excess washed off
5. Concentration of bound enzyme linked ab is determined by spectrometry when a color-changing substrate is added and acted upon by the ab-linked enzyme

Question 5

How can the presence of an ab be tested via ELISA?

Answer 5

Process is identical to identifying an ag, but instead the ag is coated to the sample plate and the sample containing the ab in question is added to ag

Question 6

Is ELISA considered a direct or indirect ab test?

Answer 6

ELISA can be direct or indirect. A direct ELISA uses an enzyme linked ab specific for the ag for detection. An indirect ELISA relies on identifying bound ag-specific ab with a second enzyme linked ab specific for the species of the primary ab bound, which increases sensitivity of the assay

Question 7

What is the conventional confirmatory test to determine the validity of screening ELISAs, such as those for HIV and Lyme disease?

Answer 7

Western blot

Question 8

What are the key steps involved in a Western blot? Describe each step

Answer 8

Gel electrophoresis: denatured/solubilized ags are separated by molecular weight on gel by electrical current polarized positive to negative
Transfer: ags are transferred from gel to a membrane by an electrical current polarized positive to negative (photocopy of gel)
Ab label: next a radioiodinated ab specific for ag of interest is applied and binds ag
Autoradiography: radio labeled membrane is exposed to x-ray film, resulting in identification of an ag as a darkened band on film
(currently most western blots are performed with enzyme linked abs resulting in fluorescence which is exposed against x-ray film)

Question 9

Southern and Northern blot techniques are analogous to Western blots. What do Souther and northern blots identify? What is used instead of radiolabeled ab?

Answer 9

Southern blot identifies specific DNA sequences; Northern blot identifies specific RNA sequences. Both use DNA probes with radioactive phosphate that is complementary to the sequence of interest to be detected

Question 10

In a radioimmunoassay (RIA) an Ag specific Ab competes for a known Ag concentration of radiolabeled ag and an unknown concentration of nonradiolabeled ag. Next, the amount of radioactivity is measured, but what is needed to determine the unknown concentration?

Answer 10

A pregenerated standard curve of concentrations that correlates the amount of radioactivity measured to a final concentration of ag

Question 11

What is measured in the radioallergosorbent test (RAST)?

Answer 11

RAST is a specialized RIA in which the amount of serum immunoglobulin E (IgE) that reacts with a known allergen is quantified

Question 12

What is the primary purpose of using affinity chromatography on a serum sample?

Answer 12

Allows a desired ag to be separated from a mixture; the sample is run through a gel column with bound abs specific for ag; the sample is washed while the ag remains bound to the fixed ags, separating the desired molecule from the mixture

Question 13

In affinity chromatography how is the desired ag extracted from the bound abs?

Answer 13

A change in pH in the column buffer changes the carge and therefore binding affinities of the ab and ag, allowing the ag to be eluted from the column

Question 14

In the complement fixation technique an ag of interest and patient’s serum is mixed with complement. If addition of the sensitized red blood cells (RBCs) (RBC with the ag of interest) results in hemolysis, what can be said about the result?

Answer 14

If sensitized RBCs hemolyze, then it is a negative reaction. This implies that the patient’s serum lacks the ab to bind the ag of interest. In a positive reaction, the sensitized RBCs do not hemolyze. This implies that the patient’s serum have the specific ab to bind the ag of interest. The ab-ag complex then activates the complement so that when sensitized RBCs are added to the mixture, there is no complement left to hemolyze the RBCs

Question 15

Limus amoebocyte lysate (LAL) is a purified extract from the horseshoe crabs that is used to test the sterility of surfical equipment. Upon exposure to endotoxin from gram-negative bacteria, LAL will rapidly clump together, indicating contaminated equipment. This is best characterized as what type of test?

Answer 15

Active hemagglutination

Question 16

What is the difference bw passive and active hemagglutination?

Answer 16

Active hemagglutination results from the clumping of native blood cells in response to ag impurities such as viruses. By contrast, passive hemagglutination requires the blood cells to first passively absorb ags (eg, viral ags) from solution, and then clump upon administration of abs specific for the ag

Question 17

What is the difference in the target of detection bw the direct and indirect versions of the Coombs test?

Answer 17

The Coombs test is an agglutination test that detects anti-RBC abs. The direct test measures those foreign abs attached to the host’s RBCs; the indirect test measures the host’s abs, which must first attach to foreign RBCs

Question 18

How are cells identified in flow cytometry?

Answer 18

Cells are tagged with fluorescently labeled abs to various surface markers (eg, CD4, CD8). A laser then detects the wavelength of light emitted from each flurescent ab (red vs. green) and records the number of times that wavelength was encountered. Cells may be not labeled, labeled by only one fluorescent ab, or by multiple fluorescent abs

Question 19

How are cells sorted in flow cytometry?

Answer 19

A fluorescence activated cell sorter (FACS) separates cells by the electromagnetic charges applied to the fluorescent signals. Cells with no fluorescence, only one fluorescent ab or multiple fluorescent abs are deflected (and thus sorted) differently

Question 20

Describe agglutination test to determine ABO blood type:

Answer 20

First, the sample blood is mixed separately with antiserum against both types A and B. Agglutination or clumping with anti-serum suggests that the sample is of that blood group (no agglutination for type O)