Exam 3 Flashcards
Genetic engineering
Using in vitro techniques to alter genetic material in the laboratory
Basic techniques include: restriction enzymes, gel electrophoresis, nucleic acid hybridization, nucleic acid probes, molecular cloning, cloning vectors
Nucleic acid hybridization
Base pairing of single strands of DNA or RNA from two different sources to give a hybrid double helix
What is a nucleic acid probe?
A segment of single-stranded DNA that is used in hybridization and has a predetermined identity
Southern blot
a hybridization procedure where DNA is in the gel and probe is RNA or DNA
Northern blot
a hybridization procedure where RNA is in the gel, probe is RNA or DNA
What are the components of PCR?
DNA sample, primers, nucleotides, taq polymerase, mix buffer, PCR tube
What is the process of PCR?
- Denaturing (95oC - strands separate)
- Annealing (55oC - primers bind template)
- Extension (72oC - synthesize new strand)
- Repeat, repeat, repeat
What are applications of PCR?
Phylogenetic studies
Surveying different groups of environmental organisms
Amplifying small amounts of DNA
Identifying a specific bacteria
Looking for a specific gene
RTPCR
reverse transcriptase, RNA snapshot
laboratory technique combining reverse transcription of RNA into DNA (in this context called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase chain reaction (PCR)
It is primarily used to measure the amount of a specific RNA
qPCR
double strand dye
Molecular cloning
isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated
Three main steps of gene cloning
- Isolation and fragmentation of source DNA
- Insertion of DNA fragment into cloning vector
- Introduction of cloned DNA into host organism
Steps for screening a library by colony hybridization (7 steps)
Start with a master plate with colonies of bacteria containing cloned segments of foreign genes
- Make replica of master plate on nitrocellulose fiber
- Treat filter with detergent (SDS) to lyse bacteria
- Treat filter with sodium hydroxide (NaOH) to separate DNA into single strands
- Add radioactively labeled probes
- Probe will hybridize with desired gene from bacterial cells
- Wash filter to remove unbound probe and expose filter to X-ray film
- Developed film is compared with replica of master plate to identify colonies containing gene of interest
Gene probes
small DNA sequences (oligonucleotide) complementary to only the gene of interest that is “labeled” for detection (such as radioactivity)
Electroporation
a technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane
This may allow chemicals, drugs, electrode arrays or DNA to be introduced into the cell
Microbiota
microorganisms present at a more defined region of our body, can be disturbed by things such as antibiotics
Human microbiome
assemblage of microorganisms on and in the diverse regions/habitats of our body
There are _____ bacteria than cells in the body
more
There are _____ bacteria than people on the planet
more
Metabolic activity is a _____ organ
virtual
The human GI tract contains ____ to _____ microbial cells
10^13, 10^14
Microbiota is influenced by ____ and the _______ in the area
diet, physical conditions
The _____ of the stomach and duodenum prevent many organisms form colonizing here
acidity, they have a pH of ~2
However there is a rich microbiome in the healthy stomach
What types of microbiota are found in the large intestine?
10^12 cells/g - live “fermentation vessel”
Facultative aerobes (E. coli) in low numbers, use up rest oxygen
Mainly obligate anaerobes such as clostridium and bacteroides
Very few microbial eukaryotes (yeast Candida albicans)
no protists
What microbiota are found in the stomach?
10^4 cells/g
Firmicutes, Bacteroidetes, and
Actinobacteria, in acidic lumen
Proteobacteria in gastric mucosa where pH is already close to 7
Helicobacter pylori is present in 50% of people, risk of chronic inflammation
What microbiota are found in the small intestine?
10^8 cells/g
becomes gradually less acidic and more anoxic
mainly anaerobic Fusobacterium (long slender rods), attached with one end to intestinal wall
Where are microbiota found in the large intestine?
bacteria are in lumen and outer mucus layer
not in inner, partially aerobic mucus layer, contains also antimicrobial peptides to protect gut mucosa
Bacteria make vitamins ____, _____, and ____ out of 20 amino acids for us that we can’t make
K, B12, 9
Cell _____ within intestine replaces lost microbes
doubling (1-2 divisions/day)
Material leaves human body after ~24 hours, with ~10^13 bacteria/day
The vast majority (~98%) of all human gut phylotypes fall into one of three major bacterial phyla:
firmicutes, bacteroidetes, and proteobacteria
Individual people may have >90% firmicutes, >90% bacteroidetes, or a mix of the two
Individuals are one of three enterotypes:
- Bacteroides-dominated (bacteroidetes)
- Prevotella-dominated (bacteroidetes)
- Ruminococcus-dominated (firmicutes)
Not correlated with ethnicity or diet, but species of individual’s gut microbiome is often shared among family members
Gut microbiome influences our ______, ________, ________, etc.
heath, propensity for leanness/obesity, response to drug therapy
The immune system does not properly develop in the absence of ___________________
microbial stimulation
Early life exposure to a variety of microorganisms is essential to develop tolerance for beneficial microorganism, and to recognize harmful ones as foreign
Excessive hygiene with infants and during early childhood results in:
poorly trained immune system and frequent inflammatory response to harmless bacteria, promotes autoimmune conditions (allergies)
Germ-free mouse study
Studies in germ-free (GF) animals show the crucial role of gut microbiota in the development and maturation of the immune system
Immaturity of gut-associated lymphoid tissue (Galt) in GF mice
Decreased serum immunoglobulin levels (“antibodies”)
Underdeveloped, smaller thymus and spleen
Gnotobiotic mice
refers to mice in which every microorganism present is defined
Germ-free mice are one class of gnotobiotic animals, but mice associated with defined bacterial communities (e.g. Altered Schaedler’s Flora) are considered gnotobiotic
Gut colonization
Not yet completely understood
Significant difference in naturally born vs. caesarian section in infants
Also, breastfeeding vs. formula influences gut microbiota and immune system development
Gut of naturally-born, breast-fed infants contain mainly bifidobacteria (actinobacteria) - anaerobic fermenters, make vitamin K, B6, and B7
Microbiota changes several times until it establishes adult-like at 3 years
Protective functions of gut microbiota
Pathogen displacement
Nutrient competition
Receptor competition
Production of anti-microbial factors (bacteriocins, lactic acids)
Structural functions of gut microbiota
Barrier fortification
Induction of IgA
Apical tightening of tight junctions
Immune system development
Metabolic functions of gut microbiota
Control IEC differentiation and proliferation
Metabolize dietary carcinogens
Synthesize vitamins (biotin, folate)
Ferment non-digestible dietary residue and endogenous epithelial-derived mucus
Ion absorption
Salvage of energy
Gut-associated lymphoid tissue (GALT)
the key antigen sampling and adaptive immune inductive sites within the intestinal wall
Human GALT includes the multi-follicular Peyer’s patches of the ileum, the vermiform appendix, and the numerous isolated lymphoid follicles (ILF) which are distributed along the length of the intestine
A wide range of circumstances can influence bacterial balance:
Age and diet
Susceptibility to infections
Use of antibiotics or other drugs
Excess alcohol
Immunologic status of the host and stressors
Exposure to toxic substances
The GIT PH
Transit time
Probiotics
A usually dairy food or a dietary supplement containing live bacteria that replace or add to the beneficial bacteria normally present in the gastrointestinal tract
Examples: lactobacillus, bifidobacteria, enterococcus, lactococcus, streptococcus, saccharomyces/yeast
Probiotic mechanism of action
Metabolic end-products, such as SCFAS
Competitive effects from occupation of normal colonization sites
Direct antagonism through natural antimicrobial compounds (bacteriocins)
Competition for nutrients
Enhancement of immune system
Increase absorption of vitamins and minerals
Prebiotics
A non-digestive food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria
Prebiotics block pathogens early on by:
- Non-digestibility (gut bacteria convert SCFA)
- Selective fermentation by one or a limited number of potentially beneficial bacteria in the colon
- Alteration of the composition of the colonic microbiota towards a healthier composition
Short chain fatty acids (SCFA or VFAs)
Derived from prebiotics undigestable oligosaccharides
Main source of energy for colonic epithelial cells
Maternal immune activation (MIA)
Changes mouse pup’s gut microbiota induced by poly(I:C), caging!
Pups exhibit autism-like disorder
Adding back B. fragilis increases 4-EPS ameliorates ALD
Inflammatory bowel disease (IBD)
chronic inflammation of GI tract
diarrhea, blood in the stool, weight loss, and abdominal pain
not caused by a specific pathogenic microorganism
an imbalance between immune system and normal gut flora
microbial community exhibits significantly less functional capacity
IBD cause and transmission not well understood
possible: early antibiotic treatment, or, protein-rich diet
Obesity
excessive amount of body fat
indirectly increases your risk of other diseases and health problems, such as heart disease, diabetes, high blood pressure and certain cancers
not caused by a specific pathogenic microorganism
in mice: energetic imbalance of microbial gut flora
more Firmicutes and methanogenic Archaea
stronger H2-consumption leads to more turnover in fermentation
increased production of acetate, propionate, butyrate, … taken up by host
Transfer of obese conditions
A microbiota transplant can transfer the obese gene to organisms
Mice are coprophagic, and mice in a cage can transfer the obese gene to each other that way
The microbiota mean something and can be transferred
Research with humans is challenging, less controllable diet, behavior, genotype
Account for side symptoms, e.g., hypertension, glucose intolerance, diabetes
Contradicting hypotheses, e.g., increased VAFs would bind to fatty acid receptors in gut, signaling feeling of “full”
Healthy gut is maintained by ___________
akkermansia muciniphila, without it you have a leaky gut
Innate immunity
Built-in capacity of the immune system of multicellular organisms to target pathogens that are seeking to colonize the host
Does not require previous exposure to pathogen or its products
targets common pathogens and/or their products
Responds within hours, non-inducible
Does not require pre-exposure
Does not generate memory
Driven by phagocytes (white blood cells able to ingest, kill, and digest pathogens)
Three types of innate immunity
Physical (skin, nasal hair, eyelashes and eyelids, mucous membranes, mucocilliary clearance, urination)
Chemical (low pH, antimicrobial molecules)
Biological (microbiome)
Opportunistic pathogens
Many elements of the normal flora may act as opportunistic pathogens, especially in hosts rendered susceptible by rheumatic heart disease, immunosuppression, radiation therapy, chemotherapy, MRSA
Obligate pathogens
Never part of normal flora, always cause disease, usually require host for multiplication
HIV, Hansen’s disease (leprosy)
Successful infection is:
influenced by virulence of the pathogen and health condition of the host
What are the steps of infection?
- Exposure to pathogens
- Adherence to skin or mucosa
- Invasion through epithelium
- Multiplication/colonization - growth and production of virulence factors and toxins
- Enter bloodstream (go on to cause disease)
Steps of bacteria causing disease
1a. Toxicity - toxin effects are local or systemic
OR
1b. Invasiveness - further growth at original and distant sites
- Tissue or systemic damage
Adherence
ability of bacteria to stick to host cell
3D shape that determines host range and success of an infection
Chromosomal island
big hunk of DNA that can be passed through conjugation
Several genes that direct invasion are clustered together as pathogenicity islands
Presence of __________ differentiates virulent from non-virulent strains in streptococcus pneumoniae
slime capsule
Evade phagocytosis of white blood cells and severely infect lung tissue
Presence of __________ differentiates virulent from non-virulent strains in Escherichia coli
fibriae
attach to mucus membrane of urinary tract and implement infection
Presence of __________ differentiates virulent from non-virulent strains in Neisseria gonorrhoeae
cell surface protein/adhesin, binds only to host cell surface proteins found in genitourinary tract, eye, rectum, and throat
All gram negative bacteria share a particular virulence factor:
Lipopolysachharide (LPS)
A structural part of the outer membrane, replaces most of phospholipids in outer half of outer membrane
Is released when:
1. Secreted as part of the normal physiological activity during (outer) membrane vesicle trafficking while also excreting other virulence factors and proteins
- Set free passively when cells die (attack from immune system) and cell structures get disintegrated
Lipid A of the LPS layer
is important for host receptor recognition
Lipid A chemistry differs among distinct bacterial species
The exact LPS structure, and especially lipid A structure, determines immunogenicity
Can cause strong or weak inflammatory reactions
LPS layer causes dysregulation of:
immune system (induces inflammation)
clotting cascade
blood pressure regulation
Results in fever, altered white blood cell count, hemorrhaging/clotting, lowered blood pressure, shock, death
Exotoxins
Inhibit host cell function or kill host cells
Proteins released from the pathogen cell as it grows
Three categories: cytolytic toxins, AB-toxins (active (A) domain and a binding (B) domain), superantigen toxins
Cytolytic toxins
Work by degrading cytoplasmic membrane integrity, causing cell lysis and death
Hemolysin, leukocidin, phospholipase
Hemolysin
Toxin that lyses red blood cells
E.g. staphylococcal alpha-toxin
Leukocidin
Toxin that lyses white blood cells
Phospholipase
Hydrolyze phospholipids (membranes) into fatty acids
Diphtheria AB-exotoxin: blockage of protein synthesis
Corynebacterium diphtheriae
Causes thick covering in the back of the throat
Can lead to difficulty breathing, heart failure, paralysis, and even death
Neurological exotoxins: botulinum and tetanus toxins
Clostridium tetani and clostridium botulinum produce potent AB-exotoxins that affect nervous tissue
Botulinum toxin consists of several related AB-toxins that are the most potent biological toxins known
Enterotoxin
AB-exotoxins whose activity affects the small intestine
Generally cause massive secretion of fluid into the intestinal lumen, resulting in vomiting and diarrhea
Cholera toxin in vibrio cholerae (lysogen)
Superantigens
Cause an overstimulation of the immune system
Can lead to shock and death
Generally due to localized infection, but with systemic effects (staphylococcus aureus toxins shock syndrome)
In severe cases cause bacteremia or septicemia (streptococcus pyrogenies toxic shock syndrome)
Specifically, it causes non-specific activation of T-cells resulting in polyclonal T-cell activation and massive cytokine release
Fever, functional disruption of kidney, liver, etc
Invasion
Pathogens produce enzymes that:
enhance virulence by breaking down or altering host tissue to provide access to nutrients (hyaluronidase)
protect the pathogen by interfering with normal host defense mechanisms (coagulase, staph aureus fibrin coat)
Virulence can be estimated from experimental studies of the ________
LD50 (lethal dose50)
The amount of an agent that kills 50% of the animals in a test group
Highly virulent pathogens show little difference in the number of cells required to kill 100% of the population
Compromised host
One or more resistance mechanisms are inactive
Genetic predisposition, or a permanent primary infection (e.g. HIV)
Temporary immune-suppressing conditions such as pregnancy, or undergoing medical procedures (e.g. surgery) predispose individuals to develop disease
Nosocomial infections affect nearly 2 million people each year
Chemotherapy induced neutropenia (neutrophils WBC first responders)
Attenuation
The decrease or loss of virulence
Attenuated strains of various pathogens are valuable to clinical medicine because they are often used as vaccines
May be a result from keeping a strain in laboratory culture over long periods of time
No selective pressure for expressing/keeping pathogenicity genes
Adaptive immunity
The acquired ability to recognize and destroy a specific pathogen or its products
Dependent on previous exposure to the pathogen or its products (specificity)
Directed toward an individual molecular component of the pathogen (antigen)
Responds after days
Induced by innate response
Requires pre-exposure
generates immune memory
Driven by lymphocytes - white blood cells for making antibodies, mark pathogens for destruction
Natural host resistance
Normal microbiota helps host resist pathogens, particularly on the skin and in the gut
Pathogens do not easily infect tissues because the harmless microbes limit available nutrients and sites for infection (competitive exclusion)
The ability of microbes to cause disease varies between species (e.g variable sensitivity to rabies, host range)
The lymphatic system
Separate circulatory system that drains lymph fluid from extravascular tissues
Blood is pumped through arteries and capillaries and returns from the body through veins
In capillary beds, leukocytes and solutes pass from blood into the lymphatic system
Lymph nodes contain high concentrations of immune system cells
Leukocytes (buff coat)
Nucleated white blood cells
About 0.1 percent of the cells in blood are white blood cells
Include lymphocytes, granulocytes, and monocytes
Whole blood is composed of ________ and ________
Plasma and cells
Plasma contains proteins and other solutes
Serum
The portion of blood that is not cells or clotting proteins
Lymphocytes
Specialized leukocytes involved exclusively in adaptive immune response
B cells: originate and mature in bone marrow
T cells: originate in bone marrow, but mature in thymus
Tissue damage and chemokine release
Tissue damage triggers the recruitment of a large number of phagocytes
Resident leukocytes and damaged cells release cytokines, or chemical mediators, that allow communication between white blood cells
Release of cytokines and chemokines draws macrophages and neutrophils to the area as they leave circulation (extravasion)
Pathogen-associated molecular patterns (PAMP)
Pathogens have structures and molecules not found in or on host cells (e.g. peptiglycan, flagellin, DSRNA)
LPS is a pamp
Pattern recognition receptors
Leukocytes have membrane bound or soluble proteins that recognize pamps
Signal transduction in phagocytes
Upon encountering a pathogen-associated molecular pattern, toll-like receptors (TLR) will send a signal to the nucleus
Each TLR on a human phagocyte recognizes and interacts with a specific pamp
At least 9 TLRs in humans interact with cell surface and soluble pamps from viruses, bacteria, and fungi
Inhibiting phagocytes
Some pathogens can survive the phagolysosome
Mycobacteria tuberculosis produces carotenoids to neutralize singlet oxygen and has a waxy cell wall that absorbs free radicals, this pathogen lives and divides within phagocytes
Some pathogens, such as Streptococcus pyogenes produce leukocidins, which kill white blood cells (dead white blood cells are found in pus)
Lastly, some pathogens contain a capsule, which makes it difficult for the phagocyte to engulf them. Host antibodies can counteract this, which is why pneumococcal vaccines are effective in preventing pneumonias caused by Streptococcus pneumoniae
Steps of antibody production in B cells
- BCR (B cell surface antibodies) recognizing its specific epitope on pathogen or toxin; B cell ingests the antigen (part of entire pathogen) via phagocytosis
- Digestion and presentation of (several) antigens of the encountered pathogen or toxin on their surface (MHC II) to T helper cells
- T helper cell produces cytokines that stimulate division of antigen-reactive B cells, which differentiate into thousands of plasma cells
- Secretion of large amounts of antibodies of identical antigenic specificity
Induction of inflammation
non-specific reaction to damage
symptoms at the site of infection: redness, swelling, pain, heat
Aims of inflammation
Isolation of site
Destruction of invaders
Removal of damaged cells
**Can also inadvertently result in considerable damage to healthy tissue, particularly key organs, such as lungs and brain tissue
Fever
Certain cytokines, particularly IL-1, will cause the host’s body temperature to rise, causing a fever
Fever-causing cytokines are called pyrogens because they generate heat
Fever is beneficial because it increases circulation rate, which allows leukocytes to get to the site of infection
Also beneficial because some pathogens cannot tolerate increased temperature
Associated with an increase in transferrins, which sequester iron, keeping it away from pathogens and limiting their growth
Systemic inflammation and septic shock
Widespread (systemic) inflammation can lead to shock as the increased vascular permeability decreases a host’s blood pressure, which can cause damage to multiple organs at the same time
Gram-negative bacteria are particularly dangerous because they contain LPS, which triggers a pro inflammatory response from leukocytes as their toll-like receptors are activated. This leads to a cytokine storm, which can be fatal
Complement system (C’)
Set of circulating, inactive proteins that are sequentially activated in response to a pathogen
Initiated when complement binds to antibodies that are attached to a pathogen
C1 cleaves C2 and C4 to form C2A-C4B, otherwise known as C3 convertase
Convertase breaks C3 into C3B, which binds to the target cell and C3A, which diffuses into the surrounding area and serves as a chemoattractant
Opsonization
C3B of complement system coating a target makes it easier for the phagocytes to engulf it
Membrane attack complex (MAC)
With C3B added, the C3 convertase also binds C5, leading to C5A being released and C5B binding to the cell
Monnose-binding lectin (MBL) and alternative pathways
Rely on innate mechanisms rather than antibodies to activate the complement pathways
While they start at a different place in pathway, all three complement systems end with opsonization, direct attack, and recruitment of phagocytes
Some chemoattractants (C5A and C3A) are considered anaphylatoxins because they can lead to immune over activation and potentially anaphylactic shock
Major histocompatibility complexes (MHC)
All nucleated cells in the body contain these surface proteins (MHC type I)
Purpose is to present small fragments of protein
Many viruses force body cells to decrease or even stop their expression of MHC I (happens also during differentiation into tumor cell)
This change will be noticed by both leukocytes of the innate immune system (natural killer cells) and leukocytes of the adaptive immune system (cytotoxic T cells)
MHC I cells present ________
self-protein (“I belong to the body”)
MHC II cells present _______
antigens (“I found this foreign thing”)
Are only on special immune cells: phagocytes (macrophages and dendritic cells) and B cells (cells of the adaptive immune system)
Natural killer (NK cells)
Cytotoxic lymphocytes
Recognize cells that do not display MHC I
No MHC and a stress protein
NK kills target (likely old, virally infected, cancer cell)
Granzyme
Enzyme that induces programmed cell death (apoptosis)
Perforin
Chemically and functionally similar to C9, pokes holes in the target membrane
Interferons
Small cytokine proteins that are produced by virally infected cells
Interferons serve as a warning system and prevent viral replication by stimulating the production of antiviral proteins in uninfected cells once they receive the interferon signal from infected cells
Antibody functions
Most of all cases: marking pathogen’s surface for destruction (phagocytes have unspecific antibody recognizing receptors, phagocytosis of IG-coated cell or virus)
Interfering with pathogen’s outer structures, inhibiting infection (e.g. antibodies in mucous membranes hinder influenza virus to attach host cells)
Binding toxins, neutralizing their effect (e.g. antibodies circulating in blood and lymph serum block their binding to host cell receptors)
Cytotoxic T cell
kill cell with antigen (release granule contents that kill target cell by apoptosis)
T helper cell binding MHC II
production of cytokines/interleukins
Natural active immunity
getting a disease and recovering
Natural passive immunity
passing antibodies to nursing infants through breast milk
Artificial active immunity
receiving a vaccination shot and developing immunity
Artificial passive immunity
Receiving pre-formed antibodies (antiserum)
Type I (intermediate) allergies
Increase or decrease of blood pressure or heart rate
Mild or life threatening (anaphylaxis)
Pollen, mold, nuts, shellfish, insect venom
Immune system alert through mucous membranes of lungs or gut
Antibody-mediated
Type IV (delayed) allergies
Often after contact with skin (contact dermatitis)
Itchiness, reddening, edema, tissue damage, blisters
Jewelry, cosmetics, poison ivy, latex
Cell-mediated
Immunodeficiency
Active adaptive immunity is critical for infectious disease resistance
Animals (humans included) with deficiencies in B cells are prone to bacterial infections, while those with T cells are prone to viral infections and cancers
Severe combined immune deficiency (SCID)
Serious, congenital deficiency of both B and T cells
Patients live a restricted life, limiting their exposure to pathogens
X-SCID interlukin receptor missing, expresses on T, B, and NK cells
Omen syndrome RAG gene mutations (recombination activating gene)
Acquired immunodeficiency syndrome (AIDS)
Caused by HIV infection that progresses and kills CD4 + T cells
Patients are prone to opportunistic infections and cancer, since they are deficient in T cell help
If CD4 count stays the same, HIV medication is working
If CD4 count drops, virus has mutated and medication is no longer working
Antigen-positive relapse
One of two main forms of disease relapse after car-T cell infusions
In the early phase and antigen escape relapse in a later phase
The disease relapse of antigen-positive cells is closely related to car-T cell persistance
Factors that affect the persistence of car-T cells require further exploration
CD137, which is significantly different from the CD28 costimulatory domain, has been shown to stimulate fatty-acid oxidation in metabolism and to increase the central memory phenotype, thereby improving car-T cell persistence
Antigen escape
one of two main forms of disease relapse after car-T cell infusions
Including antigen loss or downregulation
Alternative splicing is the primary cause of CD19 antigen loss and induces the generation of CD19 isoforms that gradually develop into dominant clusters under the strong immune pressure
Minimum antigen density threshold is required for car-T cells to achieve therapeutic efficacy. In preclinical models, Sadelain et al demonstrated that car-T cells extract and transfer target antigens into T cells via trogocytosis, which leads to a reversible decrease in antigen density
Researchers have also verified that combinatorial multi-antigen-targeted strategies could effectively prevent the tumor escape caused by low antigen density that results from car-T cell trogocytosis
Cytokine release syndrome/storm
A condition that may occur after treatment with some types of immunotherapy, such as monoclonal antibodies and car-T cells
Caused by a large, rapid release of cytokines into the blood from immune cells affected by the immunotherapy
Monoclonal antibodies
Produced by isolating single clones of B cells that are fused with cancer cells to make immortalized cell lines that produce a single type of antibody
Clinical diagnostic tests that use MABs include immunological typing of bacterial pathogens, identification of cells containing foreign surface antigens and highly specific blood and tissue typing, and are also used to detect and treat human cancers
Direct agglutination
Results when soluble antibody causes clumping due to interaction with an antigen that is an integral part of the surface of a cell or other insoluble particle
Used for the classification of antigens found on the surface of red blood cells
Passive agglutination
The agglutination of soluble antigens or antibodies that have been adsorbed or chemically coupled to cells or insoluble particles (e.g. latex beads, charcoal)
Reactions can be up to five times more sensitive than direct agglutination tests
Enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA)
Very sensitive immunological assay
Widely used in clinical diagnostic and research applications
EIAs employ covalently bonded enzymes attached to antibody molecules
Rapid tests are similar to EIAS
Both allow detection of antigen-antibody complexes
Rapid tests
Similar to EIAs except that results can often be reported within minutes instead of hours
Provide point-of-care diagnostics but are generally not as specific or sensitive
Reagents are absorbed to support material
Body fluid is applied to the support matrix
Matrix contains a soluble antigen conjugated to a colored molecule (chromophore)
Matrix also contains a line of fixed antigen which antibodies bind to, detect color change
Immunoblot (western blot)
Electrophoresis of proteins, followed by transfer to a membrane and detection by addition of specific antibodies
Immunoblot methods detect antibodies to specific antigens or the antigens themselves
Detects anti-HIV antibodies, confirmatory test for HIV rapid
