Immuno Week 10 lab

what does ELISA stand for

Enzyme Linked Immunosorbent Assays (ELISAs)

what are ELISAs

sensitive, cost-effective, plate-based assays that are versatile, and reliable

what is a hybridoma

a hybrid cell line produced by fusing a specific antibody-producing B lymphocyte with a cancerous myeloma cell

what does this fusion do

it combines the antibody-producing capability of B-cells with the longevity and rapid proliferation of a myeloma cell

what are Hybridomas essential for

producing large, uniform quantities of mAbs for research, diagnostics, and therapeutic uses

what do qualtitative ELISAs do

detect the presence or absence of an antibody or antigen in patient samples such as serum, plasma, or stool, producing a simple “yes” or “no” result similar to a dot blot

how are quantitative ELISAs performed

are performed alongside a standard curve generated by serially diluting a protein of known concentration

what does stronger colour development in the wells indicate

more antibody present

what is the optical density (OD) value used for

to calculate the amount of antigen present in the sample based on the standard curve

what are the 4 types of ELISAs

direct

indirect

sandwich

competitive

how does the direct ELISA work

Uses an enzyme-labelled antibody for detection of a specific analyte (antigen, antibody, protein, hormone, peptide, etc.) within a complex biological sample

what does the indirect ELISA do

Detects antibodies in a sample to quantify immune responses

how does the indirect ELISA work

the plate is first coated with a specific capture antigen, which immobilises the target antibody (from a biological sample), and the Ag-Ab complex is then detected using a secondary antibody

how does the sandwich ELISA work

A capture antibody is bound to the plate, which is followed by the antigen and then a secondary antibody. This method improves sensitivity and specificity

how does a competitive ELISA work

Antibodies are first incubated in solution with a sample containing antigen

This is then added to a plate coated with antigen

High amounts of antigens present in a biological sample create competition for antibodies from the same sample to bind to antigens coated to the plate

what is influenza A virus (IAV)

a common human pathogen that cause seasonal outbreaks of respiratory disease and secondary complications such as pneumonia

what are the 2 main surface glycoproteins IAV expresses

hemagglutinin (HA) and Neuraminidase (NA)

what is the HA protein involved with

entry of the virus into the host epithelial cell

what is the NA protein involved with

release of viruses from the infected host cell

how do Abs help the immune system neutralise IAV

opsonisation, where the immune complexes can be cleared by macrophages

how is conformation of IAV infection done

ELISA where virus-specific proteins (e.g. HA or NA) can be coated onto the ELISA plate and patient serum tested for presence of anti-influenza specific Abs

what are some advantages of an indirect ELISA

Multiple secondary antibodies can bind to one primary antibody, increasing the signal

The same secondary antibody can be used for many different primary antibodies

cost-effective

ELISA plates can be coated with either specific protein or Abs which bind to specific sites on the plate. However, the entire surface will not be covered. That means when you add another protein to the well, they will be able to bind to those other “unbound” sites. How do we overcome this problem, to prevent false positive ELISA readings?

Blocking – using a blocking buffer containing an irrelevant protein (e.g., skim milk powder, BSA, or casein) to coat all remaining unbound surfaces on the plate.

What is the purpose of PBS-Tween 20 in an ELISA?

PBS-Tween 20 is used as a wash buffer to remove unbound reagents and reduce non-specific binding