elisa2

Question 1

elisa

Answer 1

Enzyme linked immunosorbent assay (ELISA) tests were originally developed
for antibody measurement. These immunoassays have also been
adapted to successfully detect samples that contain antigens.

Question 2

This HIV

ELISA simulation experiment has been designed to

Answer 2

detect a hypothetical

patient’s circulating IgG directed towards the viral (HIV) antigen.

Question 3

ELISAs are done in

Answer 3

microtiter plates which are generally made of polystyrene
or polyvinyl chloride. The plates are somewhat transparent and
contain many small wells, in which liquid samples are deposited.

Question 4

step 1

Answer 4

Antigens are added to the wells where some
remain adsorbed to the wells by hydrophobic
bonds.
- There is no specificity involved with
the adsorption process to the wells, although
some substances may exhibit differential
binding. In certain cases, the antigens can be
covalently cross-linked to the plastic using UV
light.

Question 5

antiens can b

Answer 5

Antigens can be from whole HIV
lysates, specific HIV proteins, or a mixture of
the two.

Question 6

step 2

Answer 6

Wells are washed to remove unadsorbed

antigens.

Question 7

step 3

Answer 7

Block the unoccupied sites on the walls of the
plastic wells with proteins, typically gelatin or
bovine serum albumin.

Question 8

step 4

Answer 8

2ndary antibody

Question 9

Infection by HIV-1 causes the individual to

Answer 9

mount an antibody response which eventually
results in plasma IgG molecules that bind to
different HIV proteins (or different areas or the
same protein).

Question 10

2ndary antbodies

Answer 10

Secondary antibodies are usually raised in rabbits and goats immunized
with human IgG tractions. Secondary antibodies (anti - HIV - IgG) are
purified and covalently cross linked to horseradish peroxidase. This
modification does not usually affect the binding specificity and affinity of
the antibody or the enzymatic activity of the peroxidase.

Question 11

if antibodies present

Answer 11

If these antibodies are present, as in the plasma sample of an HIV postive
patient, they will bind to the adsorbed antigens in the well and remain
there after washing.

Question 12

if antibodies not present

Answer 12

If the patient is negative for HIV, there will be no
primary andtibody to bind to the antigen and in turn, no secondary
antibody binding.

Question 13

step 5

Answer 13

Wells are washed to remove unbound secondary antibody.

Question 14

step 6

Answer 14

After washing, a solution containing hydrogen peroxide and

aminosalicylate is added to each well.

Question 15

peroxidase

Answer 15

• possesses a high
  catalytic activity and can exceed turnover rates of 106
  per second.
  Consequently, amplification of an HIV positive sample can occur over
  several orders of magnitude.
• Many hydrogen donor co-substrates can be
  used by peroxidase. these co-substrates develop color upon oxdation
• H2O2 (H peroxide) to H2O + O2

Question 16

aminosalicylate

Answer 16

Peroxidase converts the peroxide to H2
O + O2
using the salicylate as the
hydrogen donor. The oxidized salicylate is brown and can be easily
observed in wells containing anti - HIV - 1 IgG (positive plasma).

Question 17

polyclonal antibody preps

Answer 17

It should be noted that polyclonal antibody preparations to a given
antigen can have variable binding affinities due to differences in the
immunological responses between animals. Different immunizations with
the same antigen in the same animal can also produce variable binding
affinities. The use of monoclonal antibodies directed against a single
epitope eliminates this variability.

Question 18

HIV infects

macrophages by

Answer 18

binding to the cells’ CD4 receptors

Question 19

macrophages

Answer 19

Macrophages are circulating monocytes and are involved in the nonspecific
engulfment of foreign material and normal cellular debris. These
materials are degraded in the lysosomes of the cells. Peptides from
foreign degraded proteins are transported to the macrophage surface
where they remain bound by specialized receptors.
- Immunologically
inactive TH cells interact with these surface bound antigen-receptor
complexes which enables them to become fully activated.
(the rest of the CD4 receptors are internalized and gp 120
appears on the T cell surface.)

Question 20

Fully activated TH

cells secrete

Answer 20

several types of protein factors collectively known as lymphokines.

Question 21

interleukins

Answer 21

several lymphokines are the interleukins which stimulate
antibody secretions from B cells enable macrophage activation, stimulate
general T cell growth and activate cytotoxic T cells.

Question 22

cytotoxic T cells

Answer 22

Cytotoxic T cells
are involved in the actual destruction of foreign cells and body cells
infected with different viruses.

Question 23

When a TH cell containing provirus undergoes antigenic activation

Answer 23

the
integrated copy DNA becomes open to the transcription of viral RNA.Viral replication causes the destruction of the TH cells.

Question 24

syncytia

Answer 24

Infected TH cells
also form syncytia, i.e. fused cells. Syncytia occur since the gp 120 on the
infected T cell surface binds to CD4 receptors on other TH cells. Cell to
cell transmission of virus can occur in syncytia without the need for a free
viral intermediate.