ANTIBODIES B2L1 PART 2

What are the two methods to fuse hybridomas?

Electrical and chemical

Electrical fusion

Hit cells with different currents causing them to fuse together

Which is the more common method to fuse hydridomas?

Chemical

What chemical is used for fusion?

PEG

What is cloning by limited dilution?

Lowering cell source to lower and lower volume until reaching 1 cell per well, grow for 10 days, the result is a monoclonal population

What method is used to screen hybridomas?

ELISA

96 well plate is coated with protein of interest and antibodies produced by cloning

Why are rabbits and goats used to make polyclonal antibodies vs mice?

These animals are large enough to get a good sample of antibodies vs mice

What are nanobodies good for?

These are good for deeper tissues, higher binding affinity, less domains interacting with other cell types making them more stable

Do nanobodies have light chains?

No, only heavy chains

Which species produce nanobodies?

Cameloids, alpacas, llamas

Unique advantages of nanobodies?

• Higher degree of specificity

• Higher stability

• Higher solubility

• Higher penetration (easier to get into deep tissues) 

Steps of Nanobody production

1. Immunized Alpaca or Llama

2. Collect lymphocytes at the right time

3. Amplify VHH genes

4. Create and screen VHH library

5. Express and purify VHH hits

6. Validate VHH activity

Steps of Polyclonal antibody production

1. Inject antigen into rabbit

2. Antigen activates B cells

3. Plasma B cells produce polyclonal antibodies

4. Obtain antiserum from rabbit containing polyclonal antibodies

Disadvantages of using polyclonal antibodies

• lower purity

• lower concentration levels

Steps of hybridoma production

1. A mouse is immunized against the antigen, produces B cells

2. B cells are isolated from the spleen and fused with myeloma cells

3. Fused cells are screened to isolate fused cells from unfused cells using HAT

4. Hybridoma cells are cloned by limiting dilution to generate a population of cells

5. Clonal population is screened by ELISA

Western Blot

Technique used in the lab to detect and quantify specific proteins in a sample 

2 types of protein folding?

Protein can be unfolded (denatured) or folded (native) 

Western Blot Workflow

1. Sample Prep - denature proteins with SDS

2. Gel Electrophoresis (SDS-PAGE)

3. Transfer to Membrane (electroblotting)

4. Blocking - Incubate in blocking solution, provides nonspecific binding to the membrane

5. Primary Antibody Incubation

6. Secondary Antibody Incubation

7. Detection - Xray or imaging

8. Analysis

Direct Labeling

The primary antibody itself is conjugated with a detection agent (enzyme or fluorophore) 

Indirect Labeling

A secondary antibody that binds the primary antibody is labeled with a detection agent. This generates a stronger detection signal 

Signal Amplification

Techniques used to increase sensitivity by multiplying the signal generated at the antigen-antibody binding site 

Types of Enzyme based amplifications

TSA and ABC

Tyramide Signal Amplification (TSA)

Horesradish peroxidase (HRP) conjugated antibodies catalyze the deposition of many fluorophore labeled tyramide molcules at the target site. This results in a strong fluorescent signal 

Avidin-biotin complex (ABC)

Target proteins are labeled with avidin linked antibodies which will interact with biotin conjugated fluorophores. Much like secondary antibody amplification, multiple biotin molecules can bind a single avidin. 

Oligonucleotide-based amplification 

Antigens are labeled with antibodies conjugated to oligonucleotides. Fluorophore conjugated complementary oligos can then hybridize to the bound oligos, amplifying the signal. 

Macrofluorophore labeling 

Multiple fluorophores are linked onto a common scaffold, which is then attached to an antibody against an antigen of interest

What antibodies are good for western blots?

Ones that are highly specific and do not recognize or bind to multiple antigens. 

CAR-T- therapy

• T cells extracted from a patient are modified to carry a chimeric antigen receptor (CAR) on the cell surface.

• The CAR acts as an antibody to bind a specific antigen, like an antigen present on the surface of cancer cells.

• The modified CAR-T cells are expanded in the lab then transfused into the patient.

• The CAR-T cells will target and destroy cancer cells 

Antibody Applications

• to identify specific proteins and determine what tissues and cells they reside in

• determine an increase or decrease in specific proteins during a challenge, pathology or disease state

• used with flow cytometry for cell sorting or isolating cells of a specific time from a mixed population

• therapeautics - neutralizing antibodies

• cancer therapies - antibodies targeting cancer cells

Alternatives to Antibodies

1. Aptamers

2. DARPins

3. Affibodies

Aptamers

• Synthetic, short, single stranded oligonucleotides that form/fold into a 3-dimensional shape that bind tightly to a target molecule

• chemical antibodies

• Generally, more stable then traditional antibodies, easier to produce, and have less immunogenicity (ability to trigger an immune response) 

DARPin

• Designed Ankyrin Repeat Proteins

• Engineered proteins designed to mimic an antibody's ability to bind a target.

• Composed of repeating ankyrin domains, leading to a groove-like structure that interacts with a specific target

• High affinity and specificity to target, and are stable, resistant to heat and denaturation. Smaller than antibodies making them easier to produce and purify 

Affibodies

• Engineered proteins derived from the Z domain of staphylococcal protein A, and act as protein recognition tools

• Small in size, highly stable, and easy to produce compared to antibodies