Serological Assays

Question 1

Serological tests

Answer 1

detect interactions between antigens and antibodies. These are widely used for diagnostic purposes, i.e.,to detect antibodies to specific infectious agents, to identify microorganisms in clinical specimens, or to detect autoantibodies in patients with autoimmune diseases

Question 2

Point-of-care tests

Answer 2

are serological assays performed in the healthcare professional’s office or at the patient’s bedside. These tests are simple to perform and results are usually available within minutes

Question 3

Cellular assays

Answer 3

measure the ability of B cells to make antibodies, T cells to make cytokines, Tc and NK cells to kill target cells, and lymphocytes to proliferate in response to mitogens(polyclonal activators).Mitogens are natural products, usually of plant origin, that trigger proliferation and differentiation of many lymphocyte clones. T cell mitogens include concanavalin A (ConA) and phytohemagglutinin (PHA). Pokeweed mitogen (PWM) is mitogenic for both T and B cells.

Question 4

Titer

Answer 4

the reciprocal of the highest dilution of an antiserum to yield a positive reaction

Example: a 1:32 dilution of a patient’s serum yields a positive result but a 1:64 dilution does notthe titer of the patient’s serum is 32

Question 5

Serum

Answer 5

liquid portion of blood that remains after clotting factors have been removed

Question 6

Plasma:

Answer 6

liquid portion of blood that includes clotting factors; clotting factors may be inactivated by heparin, sodium citrate, etc. but they’re still present in plasma

Question 7

epitope

Answer 7

antigenic determinant) is a specific site on an antigen where an antibody or TCR binds. Most antigens have several epitopes.

Question 8

paratope

Answer 8

site on an antibody or TCR that binds to an epitope on an antigen

Question 9

Heterophile antibody test

Answer 9

(Monospot test)

Epstein Barr virus (EBV) produce heterophile (“different loving”) antibodies early during infection. These heterophile antibodies will cross-link antigens on the surface of red blood cells of certain animal speciesto cause their agglutination. Thus, apositive heterophile antibody test is confirmatory for infectious mononucleosis caused by EBV.

Question 10

ABO and Rh(D) blood typing

Answer 10

agglutination rxn

If patient’s blood agglutinates on a particular antigen factor, it indicates their blood type.

Options include A/B/O as well as +/-

Question 11

The direct Coombs test

Answer 11

Often used to test the red blood cells of a newborn suspected of having hemolytic disease of the newborn (erythroblastosis fetalis)for the presence of maternal IgG antibodies bound to the Rh antigen

Question 12

Indirect Coombs test

Answer 12

detects circulating IgG antibodies that are specific for surface antigens on red blood cells, platelets, or other cells

Often used to detect anti-Rh IgG antibodies in the circulation of Rh-women who have given birth to Rh+ children

Question 13

prozone effect

Answer 13

antibody concentration is high relative to antigen concentration, resulting in very small complexes that do not clump to form visible agglutination

Question 14

equivalence zone

Answer 14

s the serum is diluted further, agglutination becomes visible

Question 15

Solid phase immunoassays

Answer 15

use solid supports (plastic, nitrocellulose, polyvinylidene fluoride membranes, spherical beads, etc.) to immobilize the antigen and antibody reactants. Solid phase assays are the most sensitive and widely used of all the serological assays and are a popular choice for point-of-care tests

Question 16

Lateral flow immunoassays (immunochromatographic assays)

Answer 16

use colored particles (e.g. beads or colloidal gold) as solid supports for antigen-antibody reactions. However, the assays do not involve agglutination of the colored particles. The home pregnancy test is an excellent example of a solid phase immunoassay that uses lateral flow technology.

Question 17

Enzyme immunoassay (EIA)

Answer 17

In the EIA, antigen is recognized by a detection antibody tagged with an enzyme that cleaves a colorless substrate to yield a colored product, indicating a positive reaction. The sandwich assay is also known as the double-antibody or two-site capture assay. It uses a capture antibody to pull a specific antigen out of a solution containing a complex antigen mixture.

Eg- HIV-1/2 Ag/Ab combo

Question 18

ELISA

Answer 18

enzyme immunoassay performed in a microtiter plate so that color formation in the fluid phase can be measured in a spectrophotometer.

Question 19

radioimmunoassay (RIA)

Answer 19

immunoassay in which the detection antibody is linked to a radioisotope instead of an enzyme. The reaction between antigen and antibody is measured by scintillation counting

Question 20

Solid phase Direct assay

Answer 20

the antigen, which is directly adsorbed onto the solid phase, is detected by an enzyme-labeled antibody specific for the antigen

Question 21

solid phase Indirect assay

Answer 21

the antigen, which is directly adsorbed onto the solid phase, is first incubated with patient serum, and then with an enzyme-labeled secondary antibody specific for human IgG, IgM, IgA, or IgE

Question 22

Immunofluorescence assays

Answer 22

localizing an antigen in cells or tissues by the use of fluorescently labeled antibodies that emit colored light when exposed to UV light. For example, cold sore tissue might be examined for herpes simplex viral antigens with a fluorescently tagged anti-viral antibody. Common fluorescent tags include fluorescein isothiocyanate (green) and tetramethyl rhodamine isothiocyanate (red).

direct or indirect

Question 23

Flow cytometry and fluorescence-activated cell sorting (FACS)

Answer 23

The tissue lineage, maturation stage, or activation status of a cell can be determined by analyzing the cell surface or intracellular expression of different molecules

label fluorescently, use flow cytometer, cells are separated by fluorescent and non-fluorescent

Question 24

Standard HIV Algorithim

Answer 24

Screening with Combo Immunoassay

HIV-1/2 Ab differentiation immunoassay

HIV-1 NAT (if step 2 is negative)

Question 25

COVID Testing recommendations

Answer 25

NAAT- best test to detect current infection (RT-PCR, IAT, NEAR, TMA, LAMP, HDA, CRISPR, SDA)

Antigen Testing- asymptomatic exposures, vaccinated, previous recent dx of covid

Serology- only for evidence of past infection NOT for vaccination status