Section 4: Labeled Immunoassays (ELISA, ELISPOT, and Western Blot)

Question 1

Enzyme-Linked Immunosorbent Assay (ELISA)

Answer 1

-is a heterogenous Assay separating portions of the sample Ag/Ab and immobilizing it on a solid phase
-originally designed for the detection of antibodies but has been adapted to detect antigen
-different forms: direct, sandwich (capture), or competitive
-used in clinical lab for the detection of various analytes: toxins, antibodies, certain drugs, specific pathogen antigens (viral proteins)
-simple and low-cost procedure, test is designed around 96-well plates for high throughput processing

Question 2

What is the ELISA test commonly used to scan ?

Answer 2

HIV-1 antibodies

Question 3

HIV-1 ELISA

Answer 3

-can come in various forms, the simplest has HIV-1 gp120/160 or gp41 immobilized in the wells
-can detect IgM and or IgG depending on assay design
-but this screening test depends on the production of host Ab which can take 6-12 weeks post-infection
- gp= glycoproteins

Question 4

seroconversion

Answer 4

the point at which a person begins producing Ab to an infection

Question 5

What are p24 (HIV-1 capsid) and HIV-1 RNA/proviral DNA

Answer 5

-these are test that can detect HIV-1 infection earlier

Question 6

gag protein

Answer 6

-cleaved into multiple structural components
-p17 (matrix protein)
-p24 (capsid/nucleocapsid)

Question 7

Steps for ELISA screen for HIV-1

Answer 7

1. HIV-1 antigens are immobilized in the well
   * can be HIV whole lysate or specific Ag
2. Wells washed to remove unabsorbed Ag
3. Wells are blocked to cover unoccupied sites in the well (albumin)
4. patient serum is added and incubated to allow patient Ab to bind to HIV-1 Ag
5. Wash
6. Add secondary Ab labeled with enzyme
7. Wash
8. Add substrate read color change

Question 8

Enzyme reaction of ELISA

Answer 8

-the most common enzyme/substrate reaction:
Horseradish peroxidase-enzyme
Hydrogen peroxidase + aminosalicylate- substrate
-horseradish peroxidase has a high catalytic activity which amplifies a positive sample by several orders of magnitude
-substrate is nearly colorless when added, peroxidase converts H202 to H20 +02 creating oxidized salicylate which has a brown color
-read color change visually or using optical density

Question 9

Enzyme-linked immunosorbent spot (ELISPOT)

Answer 9

is an assay similar to an ELISA sandwich capture that is used for assessing immune cell function (cytokine secretion)

Question 10

Steps of ELISPOT

Answer 10

1. primary Ab specific to a cytokine is coated on the bottom of the well
2. Cells are placed in the well in varying concentrations and incubated for a period of time
   * Cells secrete cytokines that are captured by the Ab on the bottom of the well
3. Then cells are removed by washing
4. Secondary Ab labeled with biotin (biotinylated) is added
5. Streptavadin conjugate (streptavidin +peroxidase) is added which binds to the biotin
6. Substrate is added resulting in a color change proportional to the amount of cytokine secreted

Question 11

Western blot

Answer 11

-is an adaptation of an enzyme immunoassay
-proteins are separated using sodium dodecyl sulfate-polyacrylamide Gel electrophoresis (SDS-PAGE)
-protein bands are transferred to nitrocellulose
-nitrocellulose acts as a solid phase for the final step of direct or indirect enzyme immunoassay

Question 12

What are the types of immunoblotting

Answer 12

Western Blot- Protein
Northern Blot- RNA
Southern Blot- DNA

Question 13

Western blot and HIV-1

Answer 13

-A western blot assay is used in the clinical diagnosis of HIV-1 infection
-cells are infected with HIV, the proteins are electrophoretically separated by SDS-PAGE
-Proteins are transferred to nitrocellulose, which is then blocked using non-fat milk (block nonspecific binding sites)
-nitrocellulose cut into strips and used to test patient serum for the presence of AB’s to two or more of the following HIV proteins :
p24 (GAG), p31 (INTERGRASE), gp41 (ENV), gp120/160 (ENV)

Question 14

Recombinant Imminoblot assay (RIBA)

Answer 14

-a variation of western blot that is used to diagnose Hepatitis C
-nitrocellulose strips are manufactured with recombinant purified viral antigens placed on them (not electrophoresed)
-nomenclature: RIBA, strip immunoblot, line immunoassay

Question 15

Steps of a western blot

Answer 15

Take a patient sample and mix it with SDS which unravels proteins and the polyacrylamide gel allows proteins to be separated by their linear size and vertical
1. Preparative polyacrylamide gel electrophoresis. One uniform electrophoresis of the relevant pathogen. Protein bands are shown in blue. but they would be clear
2. Transfer separated proteins to nitrocellulose
3. Add patient antibody (primary antibody)
4. Add an enzyme-labeled anti-human immunoglobulin (detecting antibody, also called secondary antibody)
5. Add substrate which precipitates to form a colored line, and compare strip to control to determine molecular weight of proteins that reacted with patient’s antibody