Glossary
We've compiled all of the key terms we use throughout Molecular Probes School of Fluorescence in one location. As we expand the School of Fluorescence, we will continue to add key terms to this glossary. Browse alphabetically to find the definition for the term you're interested in.
A
Affinity
The binding strength between a single antigen-binding site on an antibody and a corresponding epitope on an antigen. Affinity is determined by the rate of antibody-antigen association and dissociation at an individual binding site and is quantified by the dissociation constant (Kd).
Agglutination
The visible clumping or aggregation of particulate antigens (such as cells or latex beads) when crosslinked by antibodies. This antigen-antibody reaction is used diagnostically to detect antibodies or antigens in samples and occurs optimally when reactants are in equivalent proportions.
Antibody
A protein (immunoglobulin) produced by B cells of the immune system that specifically recognizes and binds to a foreign antigen. Antibodies consist of two identical heavy chains and two identical light chains and are fundamental components of adaptive immunity.
Antigen
Any foreign substance that triggers an immune response and can be recognized by antibodies or T cells. Antigens are typically proteins or carbohydrates on the surface of pathogens, vaccines, or other foreign materials.
Antibody Isotype
The classification of antibodies based on their heavy-chain constant region determines their structural and functional properties. In humans, there are five isotypes: IgM, IgD, IgG, IgA, and IgE, each with distinct roles in immune responses and tissue distribution.
Antibody Titer
A measurement of the concentration of specific antibodies in serum or other body fluids, expressed as the highest dilution level that still produces a positive result. Titer is determined through serial dilution and is commonly used to assess immune response following infection or vaccination.
Avidity
The total binding strength between a multivalent antibody and a multivalent antigen represents the cumulative effect of all simultaneous binding interactions. Avidity is typically stronger than individual affinities because if one binding site unbinds, others keep the complex intact.
Adjuvant
A substance that increases or modulates the immune response to a vaccine or immunogen. Adjuvants enhance antibody production and cell-mediated immunity by stimulating the immune system through mechanisms that mimic pathogen-associated molecular patterns. Common adjuvants include aluminum-based compounds and oil-in-water emulsions.
B
Blocking Buffer
A solution containing inert proteins, typically bovine serum albumin (BSA) or non-fat milk, is used to prevent non-specific antibody binding to solid surfaces in immunoassays. Blocking buffers saturate unoccupied surface sites, improving assay specificity and reducing background noise.
BSA (Bovine Serum Albumin)
A major protein fraction derived from bovine blood plasma, widely used as a blocking reagent in immunoassays, as a protein standard in quantification assays, and as a carrier protein. BSA is valued for its stability, non-reactivity, and ability to occupy non-specific binding sites.
Bioconjugation
The process of covalently attaching one molecule (often a label, drug, or protein) to another molecule through chemical bonds. Bioconjugation is used to create antibody-enzyme conjugates, antibody-fluorophore conjugates, and other molecular tools for detection and therapy.
Biomarker
A measurable biological indicator of disease state, normal biological process, or response to treatment. Biomarkers can be cellular, biochemical, molecular, or genetic alterations detected in blood, tissues, or other body fluids and are essential for diagnosis and monitoring.
Binding Site
The specific region on an antibody (paratope) or receptor that physically binds to its complementary structure on an antigen (epitope). The three-dimensional complementarity between the binding site and target determines specificity and affinity.
Baculovirus Expression System
A viral-based protein expression platform employing baculovirus-infected insect cells (typically Sf9) to produce recombinant proteins. It excels in generating eukaryotic proteins with proper folding and posttranslational modifications, though it may involve higher costs and longer timelines compared to prokaryotic systems.
C
Capture Antibody
The immobilized primary antibody in sandwich immunoassays binds and captures the target antigen from a sample. Capture antibodies are typically coated onto the solid phase of assay plates and must have high affinity for the target antigen.
Cell Lysis
The controlled breaking or rupture of cell membranes to release intracellular contents. Cell lysis can be achieved through mechanical disruption (sonication, freeze-thaw), osmotic pressure, chemical treatment, or enzymatic digestion and is essential for obtaining cellular proteins.
Chromogen
A substrate that produces a visible colored product when acted upon by an enzyme, commonly used in immunohistochemistry and ELISA for visualization of antibody-antigen complexes. Common chromogens include TMB and DAB.
Clone
A group of genetically identical cells or organisms derived from a single parent cell. In antibody production, clones refer to hybridoma cell lines that secrete identical monoclonal antibodies.
Coating Buffer
A solution used to dilute and apply capture antibodies or antigens to solid surfaces (such as microplate wells) during immunoassay setup. Coating buffers typically have a neutral pH and may contain buffering salts to optimize antibody adsorption.
Cross-Reactivity
The ability of an antibody to bind to antigens other than its original target antigen, particularly those sharing similar epitopes. Cross-reactivity can lead to false-positive results and is minimized through antibody selection and cross-adsorption techniques.
Cytokine
A small regulatory protein secreted by cells that acts as a signaling molecule between cells. Cytokines regulate inflammation, immune responses, cell growth, and tissue repair and include interferons, interleukins, and growth factors.
D
Dot Blot
A simplified protein detection technique where samples are spotted directly onto a membrane rather than first being separated by gel electrophoresis. Dot blots are rapid and cost-effective but provide no information about protein molecular weight.
Dissociation Constant (Kd)
A quantitative measure of binding affinity between two molecules at equilibrium. A lower Kd indicates higher affinity (tighter binding), while a higher Kd indicates weaker binding. Kd is expressed in molar concentration units.
Dilution Factor
The ratio by which a sample is diluted from its original concentration. Dilution factors are used in serial dilutions to determine antibody titers, LOD calculations, and assay sensitivity.
Denaturation
The loss of the native three-dimensional structure of proteins or nucleic acids due to heat, pH extremes, chemical treatment, or other factors. Denatured proteins lose biological activity and are often used intentionally in techniques like SDS-PAGE to separate proteins by molecular weight.
Direct ELISA
An ELISA format where the primary antibody is directly conjugated to an enzyme, eliminating the need for a secondary antibody and reducing assay steps. This format is faster but typically less sensitive than indirect ELISA.
E
E. coli Expression System
A bacterial protein expression platform utilizing Escherichia coli cells to produce recombinant proteins rapidly and cost-effectively. While ideal for simple proteins due to high yields and ease of genetic manipulation, it lacks the ability to perform complex posttranslational modifications like glycosylation.
ELISA
Enzyme-Linked Immunosorbent Assay - A widely used immunoassay technique that detects and quantifies antigens or antibodies using enzyme-conjugated detection antibodies. ELISA formats include direct, indirect, sandwich, and competitive, each suited for different applications.
Epitope
The specific amino acid sequence or three-dimensional region on an antigen recognized by an antibody or T cell receptor. Each antigen typically contains multiple epitopes that can be recognized by different antibodies.
Epitope Mapping
The process of identifying the specific region or sequence of an antigen that is recognized by an antibody. Epitope mapping is important for understanding antibody specificity and for designing diagnostic assays and vaccines.
Enzyme Conjugate
An antibody or protein covalently linked to an enzyme (such as horseradish peroxidase or alkaline phosphatase) to enable enzymatic detection in immunoassays. Enzyme conjugates generate measurable signals through substrate conversion.
Excitation Wavelength
The wavelength of light that is absorbed by a fluorophore to promote it to an excited electronic state. Each fluorophore has a characteristic excitation spectrum with a peak wavelength for maximum absorption.
F
Fc Region
The crystallizable fragment of an antibody that comprises the constant domains of two heavy chains. The Fc region is responsible for binding to Fc receptors on immune cells and mediating effector functions like complement activation.
Fluorophore
A chemical compound that absorbs light energy at specific wavelengths and re-emits light at longer wavelengths (fluorescence). Fluorophores are used as labels in immunofluorescence, flow cytometry, and other optical detection methods.
Flow Cytometry
An analytical technique that measures properties of individual cells suspended in fluid as they pass through a laser beam. Flow cytometry can analyze cell size, fluorescence intensity, and other properties to identify and sort cell populations.
Fluorescence Intensity
The measured brightness of emitted light from a fluorophore is proportional to the number of fluorophores and their quantum yield. Fluorescence intensity is used quantitatively in immunoassays and flow cytometry to determine analyte concentration.
G
Growth Factor
A secreted signaling protein that promotes cell growth, proliferation, differentiation, or survival. Growth factors bind to specific receptors and activate intracellular signaling pathways controlling gene expression and cellular behavior.
Genetic Expression
The process by which information encoded in DNA is transcribed into RNA and translated into proteins. Genetic expression can be measured at the RNA level (transcription) or protein level (translation) using various molecular techniques.
Gel Electrophoresis
A laboratory technique that separates DNA, RNA, or proteins through a gel matrix based on size and charge using an electric field. SDS-PAGE is commonly used to separate proteins by molecular weight.
H
Host Species
The animal species from which an antibody was derived, such as mouse, rabbit, goat, or sheep. The host species determines the compatibility of secondary antibodies and potential cross-reactivity in multiplex applications.
Hybridoma
A hybrid cell line created by fusing an antibody-producing B cell with an immortal myeloma cell line. Hybridomas produce monoclonal antibodies continuously and are fundamental to hybridoma technology for antibody generation.
Hemagglutination
The agglutination or clumping of red blood cells (erythrocytes) is caused by viruses or antibodies binding to antigens on their surface. Hemagglutination assays are used for blood typing and viral antigen detection.
I
Immunohistochemistry (IHC)
A technique that uses antibodies to detect and localize specific antigens in tissue sections, typically visualized with a light microscope. IHC combines antibody specificity with chromogenic detection to show antigen distribution in tissues.
Immunofluorescence (IF)
A microscopy technique using fluorophore-labeled antibodies to visualize specific antigens within cells or tissues. Immunofluorescence allows simultaneous detection of multiple targets using different fluorophore wavelengths.
Immunoprecipitation (IP)
A technique for isolating a specific antigen from a complex mixture using a specific antibody. The antibody-antigen complex is immobilized on beads or solid supports, separated from other components, and analyzed.
Immunogen
Any substance capable of eliciting an immune response and antibody production. Immunogens are used in animal immunization protocols to generate antibodies for research or therapeutic purposes.
Incubation Time
The duration for which reactants are allowed to interact at a specific temperature in an assay. Incubation time affects reaction kinetics and is a critical parameter for optimizing immunoassay performance.
J
J-Chain (Immunoglobulin J chain)
A polypeptide that links two immunoglobulin molecules in IgM pentamers and IgA dimers, facilitating their secretion. The J-chain is essential for the formation and stability of these multimeric antibody forms.
K
Kinetic Assay
An assay that measures the rate of an enzyme-catalyzed reaction over time rather than at a single endpoint. Kinetic assays provide information about enzyme activity and substrate concentration through continuous monitoring of product formation.
L
Ligand
A small molecule, such as a hormone, cytokine, or drug, that binds to a larger molecule like a protein or receptor. In immunoassays, antigens can be considered ligands that bind to antibody binding sites.
Lysate
The mixture of cellular components released after cell lysis contains proteins, nucleic acids, and other intracellular molecules. Cell lysates are used in downstream applications like Western blotting, immunoprecipitation, and protein quantification.
Limit of Detection (LOD)
The lowest concentration of an analyte that can be reliably detected above background noise, typically defined at a signal-to-noise ratio of 2:1 or 3:1. LOD is a critical performance parameter for assay validation.
M
Mammalian Cell Expression System
A protein expression platform using mammalian cells (such as CHO or HEK293) to produce recombinant proteins that closely mimic human biology. This system is preferred for complex proteins requiring authentic glycosylation and folding, but it is limited by higher production costs, slower growth rates, and scalability challenges.
Monoclonal Antibody
An antibody derived from a single B cell clone that recognizes a single epitope on an antigen. Monoclonal antibodies are highly specific and uniform, produced through hybridoma technology or recombinant methods.
Multiplex Assay
An immunoassay capable of simultaneously detecting multiple target analytes in a single sample using different detection labels or spatial separation. Multiplex assays increase efficiency and reduce sample volume requirements.
Molecular Weight
The sum of the atomic masses of all atoms in a molecule, typically expressed in Daltons (Da). Molecular weight is used to characterize proteins and is determined by techniques like gel electrophoresis and mass spectrometry.
Marker Protein
A protein of known molecular weight used as a reference standard in gel electrophoresis and protein analysis. Marker proteins help identify the sizes of unknown proteins in samples.
N
Negative Control
A sample or condition without the target analyte, processed identically to test samples, is used to confirm assay specificity and detect non-specific binding. Negative controls should yield no signal or minimal background.
Neutralization Assay
An assay that measures an antibody's ability to block or neutralize the biological activity of an antigen, typically a virus or toxin. Neutralization assays assess functional antibody responses and are important for vaccine efficacy evaluation.
Nanobody
A nanobody is a small, single-domain antibody fragment derived from the unique heavy-chain-only antibodies found naturally in camelids (such as llamas and camels). Nanobodies, also known as VHH domains, retain full antigen-binding capacity but are significantly smaller, more stable, and easier to engineer than conventional antibodies, making them valuable tools for research, diagnostics, and therapeutic applications.
O
Optical Density (OD)
A measure of light absorption by a solution, quantifying how much light is attenuated as it passes through the sample. OD is commonly measured at 450 nm in ELISA and is proportional to analyte concentration.
On-Target Binding
The specific and intended binding of an antibody to its target antigen or protein. On-target binding contrasts with off-target or non-specific binding that contributes to assay background.
Opsonization
The process of coating a pathogen or other antigen with antibodies or complement proteins to enhance phagocytosis and immune clearance. Opsonization is an important effector function of antibodies.
P
Primary Antibody
The first antibody used in an immunoassay directly binds to the target antigen. Primary antibodies determine assay specificity and are often detected through secondary antibodies or enzyme conjugates.
Phosphorylation
The addition of a phosphate group (PO₄³⁻) to amino acid residues (typically serine, threonine, or tyrosine) on proteins. Phosphorylation is a key post-translational modification that regulates protein function and is detected by phospho-specific antibodies.
Peptide
A short chain of amino acids linked by peptide bonds. Peptides typically contain 2-50 amino acids; longer chains are called proteins or polypeptides. Peptides are used as antigens in immunization and as assay standards.
Protein Purification
The isolation and concentration of a target protein from a complex mixture of proteins and other molecules using techniques like chromatography and immunoprecipitation. Purification yields protein preparations suitable for research and therapeutic applications.
Protein Expression
The process of synthesizing protein from DNA through transcription and translation. Protein expression levels can be measured using techniques like Western blotting, immunoassays, and qPCR.
Polyclonal Antibody
A heterogeneous mixture of antibodies produced by multiple B cell clones, each recognizing different epitopes on the same antigen. Polyclonal antibodies have broader reactivity but lower specificity than monoclonal antibodies.
Q
Quantification
The determination of the amount or concentration of a specific molecule or analyte in a sample. Quantification is achieved through various techniques, including ELISA, Western blotting, and spectrophotometry.
QC (Quality Control)
The processes and tests used to verify that assay reagents, equipment, and results meet established standards. QC includes positive and negative controls, reference standards, and validation of assay performance parameters.
R
Recombinant Protein
A protein produced from recombinant DNA, typically by expressing a cloned gene in a host cell system (bacterial, yeast, or mammalian). Recombinant proteins are used extensively in research, diagnostics, and therapeutics.
Reference Standard
A purified preparation of known concentration is used as a benchmark to validate assay performance and quantify unknown samples. Reference standards must be well-characterized and stable over time.
RUO (Research Use Only)
A regulatory classification indicating that a product is intended for laboratory research and not for clinical diagnostic or therapeutic use. RUO products may have lower validation requirements than clinical products.
Reporter Enzyme
An enzyme conjugated to an antibody or other detection molecule that catalyzes a reaction to generate a measurable signal. Common reporter enzymes include horseradish peroxidase (HRP) and alkaline phosphatase (AP).
S
Secondary Antibody
An antibody that recognizes and binds to the constant region of a primary antibody, used to detect primary antibody location in immunoassays. Secondary antibodies are often labeled with enzymes or fluorophores for signal generation.
Substrate
A small molecule that serves as the starting material for an enzyme-catalyzed reaction. In ELISA, the substrate (e.g., TMB) is converted by the reporter enzyme to produce a colored product.
Sandwich ELISA
An ELISA format using two different antibodies - a capture antibody to immobilize the antigen and a detection antibody to generate a signal. Sandwich ELISA is highly sensitive and widely used for quantifying proteins in complex samples.
Specificity (Assay)
The ability of an assay to distinguish the target analyte from structurally similar or related molecules. High specificity minimizes false-positive results and is determined by antibody recognition of unique epitopes.
Sensitivity (Assay)
The ability of an assay to detect the target analyte at low concentrations. Sensitivity is related to LOD, and typically expressed as the lowest detectable concentration or as signal fold-change above background.
Signal-to-Noise Ratio
The ratio of the measured signal from the target analyte to the background noise level. A signal-to-noise ratio >5 is generally considered acceptable, while >12 indicates excellent assay quality.
T
Titer
A titer is the concentration or amount of a substance, such as an antibody, virus, or enzyme, in a solution, determined by measuring the highest dilution at which it still produces a specific effect or reaction. It is commonly used in laboratory settings to quantify antibodies, viruses, or cells within a sample.
Target Antigen
The specific antigen or protein of interest that an antibody or assay is designed to detect and measure. The target antigen determines antibody selection and assay design.
TMB (Tetramethylbenzidine)
A widely used chromogenic substrate for horseradish peroxidase that produces a blue-colored product convertible to yellow in acidic conditions. TMB is the most common substrate in ELISA due to its sensitivity, color purity, and stability.
Threshold Cycle (Ct)
In qPCR, the cycle number at which fluorescence exceeds background indicates the initiation of exponential amplification. The Ct value is inversely proportional to the initial template quantity and is used for quantification.
U
UV Absorbance
The measurement of light absorption in the ultraviolet wavelength region (typically 280 nm for proteins). UV absorbance at 280 nm is proportional to protein concentration and is used for rapid protein quantification.
Unbound Fraction
The portion of antibody or antigen that remains unbound after incubation in an immunoassay. Unbound material is removed through washing steps to reduce the background signal.
V
V(D)J Recombination
The genetic recombination process in B and T lymphocytes randomly combines V, D, and J gene segments to generate diversity in antibody and T cell receptor genes. V(D)J recombination creates the enormous repertoire of antibody specificities.
Viral Antigen
A protein or carbohydrate component on the surface or interior of a virus that is recognized as foreign by the immune system. Viral antigens are targets for antibody-based diagnostics and vaccines.
Validation
The process of confirming that an assay, instrument, or method performs as intended and meets established criteria. Validation includes assessment of accuracy, precision, linearity, and other performance parameters.
W
Western Blot
A protein detection technique that separates proteins by size using SDS-PAGE, transfers them to a membrane, and detects them with specific antibodies. Western blotting is used to quantify proteins and analyze posttranslational modifications.
Wash Buffer
A solution used to remove unbound antibodies, antigens, and other non-specifically bound material during immunoassays. Wash buffers typically contain low concentrations of detergent and buffering salts.
Working Concentration
The optimal concentration of a reagent (typically an antibody) used in an assay for maximum sensitivity and specificity. Working concentration is determined through titration experiments and must be validated for each application.
X
Xenograft
A tissue or tumor graft transplanted from one species to another, typically from humans to immunocompromised mice. Xenograft models are used to study human tumor biology and evaluate therapeutic efficacy in preclinical research.
X-Linking (Crosslinking)
The formation of chemical bonds between molecules, typically proteins, to stabilize complexes or prepare samples for analysis. Crosslinking can be reversible or irreversible and is used in immunoprecipitation and protein studies.
Y
Yeast Expression System
A protein expression platform using yeast cells (typically Saccharomyces cerevisiae or Pichia pastoris) to produce recombinant proteins. Yeast systems offer advantages for producing proteins requiring posttranslational modifications.
Yield (Protein Yield)
The total amount of protein obtained from a purification process or expression system, typically expressed as milligrams or grams of pure protein. Yield is an important metric for assessing the efficiency of protein production.
Z
Z-Factor
A dimensionless statistical measure of assay quality that accounts for both signal separation and signal variability. A Z-factor >0.5 indicates an excellent assay, 0-0.5 indicates an acceptable assay, and <0 indicates a poor assay.
Zeta Potential
Zeta potential is the electrical potential at the slipping plane near the surface of a particle in a liquid. It quantifies the magnitude of electrostatic repulsion or attraction between particles, influencing their stability in suspensions or colloidal systems. A high absolute zeta potential value indicates strong repulsion, leading to better particle dispersion, while a low value suggests a higher risk of aggregation.