Principles of Biochemistry Midterm 2/2

Contains AI generated questions from ELISA and Bradford Assay labs. Do not reccomend using fill in the blank. Studied at UNT Fall 2025. Another set by the same name except 1/2 contains micropipetting and SEC.

What is the role of the immune system in protecting the body from disease?

by physically blocking pathogens and by producing molecules and cells that recognize and attack specific pathogens

How do vaccines help protect against disease?

by exposing the immune system to a weakened or inactive pathogen, which allows the body to develop immunity without causing illness

What are common ways that diseases can spread?

ingestion of contaminated food or water, exchange of body fluids, inhalation of airborne pathogens, transfer via vectors such as insects

Which diseases specifically attack the human immune system?

HIV (virus) and Lupus (autoimmune disorder)

What are the factors that can prevent the immune system from functioning properly? :::

overreaction to antigens, infection by pathogens that attack the immune system, use of immunosuppressive drugs, autoimmune disorders like multiple sclerosis

Why are immunosuppressive drugs necessary after organ transplants?

to prevent the immune system from attacking transplanted tissue

Why is rapid detection of disease exposure important? 

to minimize or prevent transmission to others and to identify individuals who need treatment quickly

What does ELISA stand for?

Enzyme-Linked Immunosorbent Assay

Why are enzymes used in ELISA?

to convert a substrate into a colored product that can be detected

Why are positive and negative controls included in an ELISA?

positive controls confirm the assay works when antigen is present; negative controls confirm the assay shows no signal when antigen is absent

What is innate immunity?

the natural, non-specific immune defenses present from birth, such as circulating macrophages and natural killer cells

What is passive immunity?

acquiring antibodies from an external source, such as maternal antibodies or certain post-exposure vaccines; temporary immunity

What is acquired/adaptive immunity?

a specific immune response activated by initial exposure to an antigen, allowing for a stronger response upon subsequent exposures

What are the two main types of adaptive immunity?

humoral immunity (antibody production by B cells) and cell-mediated immunity (T cells destroy infected cells)

What is the function of B lymphocytes (B cells)?

to produce antibodies that recognize specific antigen epitopes and help coordinate the immune response

What is the function of T lymphocytes (T cells)?

to kill infected cells directly and to stimulate B cells and other immune cells

What is an antigen?

any foreign substance that triggers an immune response, including microbes, microbial products, or foreign molecules

What is an epitope?

the specific part of an antigen recognized by a single antibody

What are the five classes of antibodies (immunoglobulins) and their roles?

IgG: most abundant, circulates in body fluids, binds two antigens; IgM: responsible for primary immune response; IgA: found in secretions, protects mucosal surfaces, transferred from mother to infant; IgE: mediates allergic responses; IgD: may regulate immune responses

What are the main functions of macrophages?

1) removing foreign cells/molecules from blood; 2) processing antigens and presenting them to T cells to initiate immune response

What is hypersensitivity?

an exaggerated immune response to an antigen that can cause illness or death; includes anaphylactic, cytotoxic, immune complex, and delayed-type reactions

What is immunodeficiency?

inability to mount an effective immune response, which can be primary (genetic) or secondary (acquired)

What is an autoimmune disease?

a disorder in which the immune system attacks the body’s own cells, such as lupus, rheumatoid arthritis, MS, IDDM, or celiac disease

How are infectious diseases diagnosed in the lab?

by detecting the microorganism itself, microbial products, or the body’s immune response to the pathogen

What are examples of traditional diagnostic tests for microbes?

culture methods, immunoassays (like ELISA), agar diffusion assays, immunofluorescence assays, agglutination, immunochromatography, microplate tests

What are molecular methods for detecting infectious agents?

detection of microbial RNA or DNA, PCR, and tests for antimicrobial resistance

How do immunofluorescence assays work?

microorganisms are detected using fluorescently labeled antibodies under a microscope

How do agglutination tests work?

visible precipitate forms when antibodies bind to their specific antigens

What are immunochromatography tests?

card- or dipstick-based tests using labeled antibodies to produce a rapid visible result

What are microplate tests like ELISA or RIA used for?

to detect microbial antigens, microbial products, or antibodies against microorganisms; RIA uses radioactive labels instead of enzymes

What are microscopy-based diagnostic methods?

visual identification of pathogens using staining or physical characteristics; includes electron and light microscopy

What are the types of vaccines and their characteristics?

Live attenuated: weakened microbes, strong response; Killed/inactivated: microbes killed by heat/chemicals, safer, weaker response; Subunit: pieces of microbes, engineered or extracted; DNA: cloned microbial genes injected to elicit immune response; mRNA: chemically stabilized mRNA encoding antigen; Antibody vaccines: recombinant monoclonal antibodies; Postexposure vaccines: immunotherapy after infection (e.g., rabies)

How do polyclonal antibodies differ from monoclonal antibodies?

Polyclonal: produced by multiple B cell clones in animals, recognize multiple epitopes; Monoclonal: from a single B cell clone, identical antibodies, high specificity

How can recombinant DNA technology improve antibodies?

produces humanized antibodies for therapy, reduces animal use, allows large-scale production, maintains specificity

What is hybridoma technology?

fusion of a single B cell with an immortalized cell to produce monoclonal antibodies indefinitely

What is phage display in antibody production? 

displaying antibody genes on bacteriophages to screen for specific binding to antigens

How are antibodies labeled for detection?

by attaching chemical, fluorescent, or enzymatic labels that produce a detectable signal

What is indirect detection of antigens using antibodies?

primary antibody binds antigen, secondary enzyme-linked antibody binds primary, amplifying signal

What are some applications of antibodies in research and diagnostics?

immunostaining, immunoblotting/western blotting, dot blotting, dipstick tests, FACS, ELISA

What are the main steps of an ELISA procedure?

1) Label wells and prepare controls and samples; 2) Bind antigens to wells; 3) Wash unbound proteins; 4) Add primary antibody; 5) Wash; 6) Add enzyme-linked secondary antibody; 7) Wash; 8) Add substrate to produce color and record results

Why must ELISA wells be washed multiple times?

to remove unbound proteins or antibodies, preventing nonspecific signal

How do dipstick immunochromatography assays work?

labeled antibodies bind antigens and migrate up a strip; positive and control lines indicate presence/absence of target

Why is indirect detection more efficient than direct labeling in ELISA?

a single labeled secondary antibody can detect multiple primary antibodies, amplifying signal and reducing cost

What is the role of controls in ELISA experiments?

positive controls confirm the assay can detect the antigen, negative controls confirm no signal occurs when antigen is absent

Biophotonics

The technology that focuses on the interaction of biological materials with light and other forms of radiant energy, whose quantum unit is the photon.

Radiation

Energy that comes from a source and can travel through material or space.

Four ways light can interact with biomolecules

Absorption, light scattering, reflection, transmission.

Absorption in the Bradford assay

As light passes through a material, light energy is absorbed at specific wavelengths; removal of these wavelengths gives the material its color.

Protein-dye complex color at 595 nm

Blue

Color of Coomassie dye alone (470 nm)

Reddish-brown

Spectrophotometer

An instrument with a light source that passes through a cuvette containing the sample; a detector measures the amount of light absorbed by the material.

Peak absorbance of unprotonated Coomassie G-250

595 nm

Colorimetric methods for total protein determination

Bradford, Lowry, Biuret

Sensitivity of colorimetric assays

Bradford is the most sensitive, Lowry is moderately sensitive but affected by reagents, Biuret is least sensitive.

Coomassie G-250 dye states

Cationic reddish-brown form at 470 nm; unprotonated stable blue form at 595 nm when bound to protein.

Protein-binding amino acids in the Bradford assay

Arginine (primary), tryptophan, tyrosine, and histidine (weaker interactions).

Primary interaction between Coomassie G-250 and protein

Electrostatic interactions between arginine residues and the sulfate groups of the dye.

Secondary interactions in Bradford assay

Electron stacking between aromatic rings (tryptophan) and hydrophobic interactions with polar amino acids like tyrosine.

Relationship of blue intensity to protein concentration

The more intense the blue color, the higher the protein concentration.

Bradford assay main steps

1. Prepare dilution series of standards and unknowns. 2. Add Bradford dye and incubate >5 minutes. 3. Dye binds to protein; read absorbance at 595 nm. 4. Compile data into standard curve to determine unknown concentrations.

Standard curve

Plot of absorbance vs. known protein concentrations used to determine unknown protein concentrations.

Correlation coefficient (R²)

Statistical measure of how well the regression line fits the data; close to 1.0 indicates high linearity and accuracy.

Effect of pipetting on R²

Low R² indicates variability in pipetting, reducing accuracy of unknown protein concentration determination.

Incubation time for Bradford assay

At least 5 minutes, not exceeding 60 minutes.

Qualitative comparison of unknowns

Compare color of unknowns against standard series to estimate concentration visually.

Calculation of dilutions using C₁V₁=C₂V₂

V₁ = C₂V₂ / C₁; volume of PBS = V₂ - V₁.

Casein in milk

Most abundant protein; 224 amino acids; 24,967 daltons molecular mass.

Casein amino acids that strongly react with Coomassie dye

4 arginines (R), 1 tryptophan (W), 4 tyrosines (Y), 4 histidines (H).

Molecular weight comparison

Coomassie dye = 854 daltons vs. average amino acid = 110 daltons; few dye molecules can coat a protein.

PBS blank

Accounts for absorbance of solvent; used to zero the spectrophotometer.

Qualitative vs. quantitative assessment

Qualitative = visual comparison to standards; quantitative = spectrophotometer measurement and standard curve.

Importance of accuracy in pipetting

Small pipetting errors lead to inaccurate protein quantitation, affecting R² and experimental outcomes.

Purpose of R² in Bradford assay

Indicates how well measured absorbances of standards fit the regression line; assesses precision and pipetting accuracy.

Electrostatic binding in Bradford assay

Arginine side chains interact with dye sulfate groups, causing dye to turn blue.

Dye-protein color change mechanism

Cationic dye binds to protein → unprotonated blue dye → absorption shifts from 470 nm to 595 nm.

Incubation importance

Ensures complete binding of dye to protein and stable color development

Color shift explanation

Removal of yellow wavelengths by protein-dye complex makes the solution appear blue.

Electrostatic interaction specificity

Arginine residues are highly basic, making them the primary binding site for Coomassie dye.

Weaker dye-protein interactions

Include hydrophobic and aromatic interactions (tyrosine, tryptophan, histidine).

Importance of the incubation time range

Too short = incomplete binding; too long (>60 min) may destabilize color.

Dye binding principle

Coomassie dye “coats” proteins, allowing absorbance-based quantification.