Chapter 18= Practical Applications

Antibodies

Serum proteins, highly specific

* 4 protein chains= heavy and light
* 2 regions= constant and variable
* Monomer has 2 antigen binding sites, 2 identical epitopes

Therapies

Treatments for different disorders or diseases, and Diagnostics

Vaccination

\-Type of therapy, Relies on idea of primary and secondary responses.

\-To produce artificially induced active immunity:


1. Inoculate person with antigen, inject antigen into persons body. Hoping body will produce primary response.
2. End of primary response, generates memory B-cells.
3. Person comes in contact with antigen naturally, will immediately produce secondary response= strong, fast, won’t get actual disease.

Jenner

Physician in late 1700s, created 1st vaccine for smallpox, would inoculate individuals with cowpox. Individuals seemed resistant to smallpox.

* Cow= Vaca= where word vaccine came from.

Types of Vaccines

\-What is being injected into the person, what form the vaccine will have different effects on level of immunity and possibility of getting the disease.

Live or Attenuated Vaccines

\-Whole cell vaccine. Injecting whole microbe into individual.

* Organisms are alive or active, are weakened state, Virulence has been reduced. Able to replicate but weakened, mild disease.
* Will give best immunity because injecting all possible antigens and every antigen has different epitopes.
* Microbe able to replicate at low levels, give immune system good experience of fighting live microbe.
* Live= for bacteria cells
* Attenuated= for viruses
* Negative effect= Are alive so its possible for small percentage of individuals to actually get the disease, mild form.

Killed or Inactivated Vaccines

\-Whole cell vaccine, Injecting whole organism but has been treated with heat or chemicals to kill it. Organism not capable of producing disease, either dead or completely inactivated

* Killed= for bacteria
* Inactivated= for viruses
* Positive effect= No way to acquire/get disease from vaccine.
* Negative effect= Treating with heat or chemicals will affect antigens on outside of microbe. Antigens are proteins, may alter their shape.
* When have immune response to antigen, immune response may not recognize actual normal antigens, overall immunity not as strong.

Subunit Vaccine

\-Only injecting the Antigen from the microbe, not whole microbe.

\-Viruses= could be protein fibers, or spikes being injected.

\-Bacteria= could be flagella, fimbrea, glycoproteins for capsule

* Positive effect= No way of getting disease from vaccine.
* Negative effect= Lowest immunity, only injecting few antigens, few epitopes.

Recombinant Vaccines

Subunit vaccine, use Recombinate DNA technology.


1. Find the gene for surface antigens, put gene into plasmid and transform microbe
2. The microbe produces the antigen, and use antigen in the vaccine.

DNA Vaccines

New vaccine, Find gene for antigen= put into plasmid, inject plasmid directly into person. Human cells take plasmid and make antigen. Present antigen outside the cells. Should stimulate the immune system.

\-Hypothesis= Lifelong immunity, need another booster.

Herd Immunity

Protecting a population from infectious disease.

* 1st= Came from Agriculture= farmers were worried about protecting herds from spread of infectious disease.
* Humans are basically herds. Meant to those susceptible will not get sick.
* Everyone else immune means they are not reservoirs, cannot transmit microbe to susceptible individuals.
* Different diseases have different immunity thresholds

Heard Threshold

Percentage an individual in population that must be immune to prevent an outbreak of disease.

\-Vaccines have a failure rate of between 2-10%

Primary Vaccine Failure

Failure to develop primary response.

* 2-10% of people’s primary response does not occur.
* If don’t get primary response then not getting memory cells. When exposed to antigen its not secondary response, but primary response.
* Reason primary vaccine failure is B-cells. B-cells in lymph nodes is random(genetic shuffling), what antigens they respond to.
* If vaccinated with specific antigen, it’s possible that at that time you don’t have B-cells that recognize it. No colonel expansion, no memory cells, no plasma cells, not produce any antibodies

Vaccine Schedule

For children takes primary vaccine failure into account.

* When giving vaccine to child they are; physically smaller, fewer lymph nodes, and have fewer B-cells. Children need several boosters, several months apart.
* 2-4 months= A lot B-cells where not activated, will start die off. New B-cells going to lymph nodes, new B-cells respond to antigen.
* 4-6 months= Have complete different set of B-cells and increasing your chances there is B-cell responding to particular antigen. B-cells not activated in 3 months will die off.

Antitoxin

Using antibodies to Neutralize toxins.

\-Normal= toxin molecules target cell and cause damage to cell by toxin.

\-Antitoxin= toxin molecules immediately inject antitoxin antibodies against toxin, Antibodies bind to toxin, neutralize toxin, no effect on cell.

* TIG= Tetanus Immunoglobulin= antibodies from humans from donated plasma, give injection, neutralizes tetanus toxin before interacts with neuromuscular junction and prevents contraction.
* Rabies= interacting with wild animal, give injection of human rabies immunoglobulin= HRIC- antibodies neutralize rabies virus prevent virus/infection.

Antivenom

If bit by rattlesnake may inject Cro Fab, generating Cro Fab, inject rattlesnake into sheep. Cause primary response in sheep, expose sheep again to venom, generate secondary response, purify antibodies from plasma of sheep.

\-If bit get injection of Cro Fab, sheep antibodies, which neutralize the neurotoxin before it has negative effect on tissue.

IVIgG

Gamma Globulin= By IV giving immunoglobulin gamma antibodies. Antibodies are from donated plasma, usually used to treat individuals that are immunocompromised.

* Undergo chemotherapy, other conditions where immune system is depressed, giving infusions of Iv IgG is formed Artificial passive immunity.
* Good at protecting them from a lot of infections, since its passive immunity, there is no memory, Not long term.

Diagnostic

Used to determine if individual has particular disease.

Diagnostic- Indicate

\-Infection= If they have been invaded by microbe if expressing signs or symptoms.

\-Disease= Determine what sort of infections disease a person has.

\-Immunity= Determine if person is immune, if stimulated primary response and if they have antibodies against it.

\-Pathogens= indicating whether person has actual pathogen

\-Antibodies= evidence of antibodies which indicates person came in contact with particular pathogen.

\-Other Molecules= Have some sort of antigen, have large protein

\-Sensitivity= Needs high level of sensitivity, ability test to determine identity positive sample

\-Specificity= Test need high level of specificity ability to test to not react with negative sample.

Designing Diagnostic Test

Need to figure out which one to identify; antigen or antibody.

* To identify Antigen= Test must include antibody, used to identify antigen.
* To identify Antibody= Need antigen in test
* Antigen= indicates the actual presence of microbe
* Antibody= indicates individual was exposed to microbe and generated an immune response
* Specificity is important if you want to know exactly what strain of microbe has infected individual

Stimulate Primary response

Primary response has mature B-cells in lymph nodes, waiting to be stimulated by antigen. Injecting an antigen has B-cells goes through clonal expansion, generating memory cells, plasma cells= secrete antibodies.

Polyclonal Antibodies

* Many B-cells clones are responding the antigen will have several epitopes. B-cells could be responding epitope 1, another epitope 2 and epitope 3
* Have 3 clones producing antibodies to particular antigen
* All antibodies are not identical, some recognize epitope 1 other epitope 3
* If polyclonal antibodies in diagnostic test, you may get cross reactivity, positive reactions/false positives reactions, don’t have high specificity.

Monoclonal Antibodies

Prevent cross reactivity that can happen with polyclonal antibodies, developed method to generate antibodies from 1 B-cell clone.

\-Very effective, cheap way to generate highly specific uniform diagnostic tests

* Uniform= come from 1 B-cell clone, identify exactly same epitope on particular antigen.
* Highly Specific= identify particular antigen because bind to 1 particular epitope.
* Large quantities= can produce antibodies in large quantities

Steps of Monoclonal Antibodies

1. Take antigen and inject into individual
2. Individual will go through primary response. Remove B-cells from spleen or lymph nodes from individual.
3. Mix B-cells from individual with cancerous cells go through mitosis.


1. Mix cells, some fuse together to form Hybrid cells, produce antibodies, maintain in culture.
4. Separate cells into individual wells so every cell has own well
5. Identify Hybridomas= B-cells that fuse with cancer cells, can live forever.
6. Identify 1 that is producing antibody you want. Hybrid cell that can be maintained in culture forever. Is producing antibodies that recognize antigen, injecting into individual.

Precipitation Reactions

Use monoclonal antibodies.

\-Involve IgG or IgM antibodies to identify soluble antigen-floating antigen in a solution.

\-Using the result of Agglutination

\-Is concentration dependent need particular concentration= Zone of Equivalence.

Zone of equivalence

where the concentration of antibody and antigen is in balance, get clumps forming, actually see reaction, mix 2 clear fluids, mix together, and form solid particles.

\-Too much of antigen or antibody won’t get clumping

Agglutination Tests

Takes advantage of result of antibody binding to antigen.

\-Not looking for soluble antigen, looking for antigens on large particles. Involves IgG or IgM antigens are large objects not soluble.

* Ex of direct agglutination test= involves antibodies, antigens on surface of bacterial cell. Identifying antigen still bound to bacterial cell

Direct Agglutination

Blood typing

\-Blood types involve antigen on red blood cells

\-Determining person’s blood type, use 3 types of antibodies= Anti-A, Anti-B, and Anti-D.

* Anti-A= identifies A antigen
* Anti-B= identifies B antigen
* Anti-D= identifies Rh factor
* Rh factor= determines if person has a positive or negative type of blood.

Indirect Agglutination Reaction

Looking for antigen that is not soluble bound to larger structure.

\-Antigens are found on Latex beads, generate Latex beads in the test, put antigen on beads, use latex beads to identify presence of antibody in patients sample.

* Can be used to identify, if individual has produced antibodies to particular antigen.
* Can identify presence of microbe in a sample
* Latex beads= very small plastic beads/balls.

Conjugated Antibodies

Antibodies that have another molecule attached to Constant region of heavy chain.

\-Molecule can be- radioactive molecule, fluorescent molecule, or enzyme. Labeled antibody= Allows you to identify presence of antibody.

Fluorescent-Antibodies

Are labeled with a molecule that will fluoresce or glow.

\-Ex: directly staining step throat

\-Steps:


1. Streptococci from patients throat.
2. Add Fluorescent dye
3. If positive will grow.

FACS

Florescent Activated Cell Sorting= cell sorter

* Cell sorting, take a suspension of many different cells and separate them out by type.
* Help monitor health of HIV patients, bone marrow transplant


1. Mixture of cells, add antibodies, antibodies bind to cells labeled cells. Antigens with fluorescent-antibody markers
2. Mixture put in FACS machine
3. Laser beam strikes each droplet.
4. Fluorescence detector identifies fluorescent cells, electrode gives positive charge to identified cells.
5. As cells drop between electrically charged plates, cells with positive charge move on side, Negative to other.
6. Separates cells, fall into different collection tubes.

Western Blot

Use Fluorescent Antibodies

\-**Process to identify presence of a Protein**

\-Steps=


1. Protein extract- the antigen samples, run on electrophoresis and separated by size, transfer pattern to blot.
2. Transferred to blot, ass your probe= antibody that is conjugated to Fluorescent molecule.
3. If antigen is present the antibody will bind and use X-ray film
4. Look for fluorescent, glowing band- means protein is present in sample.

Enzyme-Linked Antibodies

Conjugated antibody is enzyme linked antibody

* The molecule that is attached to constant region of heavy chain is an enzyme. Enzyme catalyze a reaction and gives positive results in different types of tests.
* Often the reaction enzyme catalyzes is color change
* Ex; pregnancy test

ELISA

Enzyme-Linked Immunosorbent Assay

* Used to identify the presence of an antigen or antibody, very sensitive, can determine levels of antigen or antibody at extremely low concentrations, and are Quantifiable= can determine concentration of antigen or antibody

`Direct` **ELISA**

* Where enzyme-linked antibody binds directly to antigen
* Once enzyme-linked antibody binding to antigen, then substrate, and its antibody is present will cause reaction and change color.
* **Primary Antigen conjugate bind to antibody**

Indirect ELISA

* Enzyme-linked antibody binds to antibody, which recognizes antigen.
* Then add substrate, if enzyme is present, substrate will be converted into product.
* ***Antigen bound to Antibody***

Sandwich ELISA

* 1st start with capture antibody, then add the antigen, add 2nd antibody to recognize the antigen.
* Then add enzyme-linked antibody that recognizes 2nd antibody, add substrate. If enzyme is present the convert substrate to color substrate.
* ***Antigen between 2 capture antibody***

Direct ELISA- steps

Used to detect Antigen(protein) in a sample, used to detect microbe antigen, hormones, cytokines, drugs, any type of protein in sample.

\-Steps=


1. Antibody is absorbed to well
2. Patients sample is added; complementary antigen binds to antibody.
3. Enzyme-linked antibody specific for test antigen is added, binds to antigen, forming sandwich.
4. Enzyme’s substrate is added, reaction produces a product that causes a visible change.

Indirect ELISA- steps

Determine presence of Antibody in a sample, used when checking a person titer- determine amount of antibodies person has against a microbe.

\-Steps:


1. Antigen is absorbed to well
2. Patients serum is added; complementary antibodies bind to antigen
3. Enzyme-Linked anti-HISG is added and binds to antibody
4. Enzymes substrate is added, reaction produces product that causes a visible color change.