Virus quantification involves counting the number of
viruses
A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea.
Since Dmitri Ivanovsky's 1 ...
in a specific volume to determine the virus concentration. It is used in both research and development (R&D) in commercial and academic laboratories as well as production situations where the quantity of virus at various steps is an important variable. For example, the production of viral
vaccines
A vaccine is a biological preparation that provides active acquired immunity
The adaptive immune system, also known as the acquired immune system, is a subsystem of the immune system that is composed of specialized, systemic cells and pro ...
,
recombinant proteins
Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be foun ...
using viral vectors and viral
antigens
In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. ...
all require virus quantification to continually adapt and monitor the process in order to optimize production yields and respond to ever changing demands and applications. Examples of specific instances where known viruses need to be quantified include
clone screening,
multiplicity of infection
In microbiology, the multiplicity of infection or MOI is the ratio of agents (e.g. phage or more generally virus, bacteria) to infection targets (e.g. cell). For example, when referring to a group of cells inoculated with virus particles, the MOI ...
(MOI) optimization and adaptation of methods to cell culture. This page discusses various techniques currently used to quantify viruses in liquid samples. These methods are separated into two categories, traditional vs. modern methods. Traditional methods are industry-standard methods that have been used for decades but are generally slow and labor-intensive. Modern methods are relatively new commercially available products and kits that greatly reduce quantification time. This is not meant to be an exhaustive review of all potential methods, but rather a representative cross-section of traditional methods and new, commercially available methods. While other published methods may exist for virus quantification, non-commercial methods are not discussed here.
Traditional methods that allow quantification of infective particles
Plaque assay
![Plaque assay macro](https://upload.wikimedia.org/wikipedia/commons/7/76/Plaque_assay_macro.jpg)
Plaque-based assays are the standard method used to determine virus concentration in terms of
infectious dose.
Viral plaque
A viral plaque is a visible structure formed after introducing a viral sample to a cell culture grown on some nutrient medium. The virus will replicate and spread, generating regions of cell destruction known as plaques. For example, Vero cell or ...
assays determine the number of
plaque forming units
A plaque-forming unit (PFU) is a measure used in virology to describe the number of virus particles capable of forming plaques per unit volume. It is a proxy measurement rather than a measurement of the absolute quantity of particles: viral part ...
(pfu) in a virus sample, which is one measure of virus quantity. This assay is based on a microbiological method conducted in
petri dishes
A Petri dish (alternatively known as a Petri plate or cell-culture dish) is a shallow transparent lidded dish that biologists use to hold growth medium in which cells can be cultured,R. C. Dubey (2014): ''A Textbook Of Biotechnology For Class- ...
or multi-well plates. Specifically, a confluent monolayer of
host
A host is a person responsible for guests at an event or for providing hospitality during it.
Host may also refer to:
Places
* Host, Pennsylvania, a village in Berks County
People
*Jim Host (born 1937), American businessman
* Michel Host ...
cells is infected with the virus at varying dilutions and covered with a semi-solid
medium
Medium may refer to:
Science and technology
Aviation
*Medium bomber, a class of war plane
*Tecma Medium, a French hang glider design
Communication
* Media (communication), tools used to store and deliver information or data
* Medium of ...
, such as
agar
Agar ( or ), or agar-agar, is a jelly-like substance consisting of polysaccharides obtained from the cell walls of some species of red algae, primarily from ogonori (''Gracilaria'') and "tengusa" (''Gelidiaceae''). As found in nature, agar is ...
or
carboxymethyl cellulose
Carboxymethyl cellulose (CMC) or cellulose gum is a cellulose derivative with carboxymethyl groups (-CH2-COOH) bound to some of the hydroxyl groups of the glucopyranose monomers that make up the cellulose backbone. It is often used as its sodi ...
, to prevent the virus infection from spreading indiscriminately. A viral plaque is formed when a virus infects a cell within the fixed cell monolayer. The virus-infected cell will lyse and spread the infection to adjacent cells where the infection-to-lysis cycle is repeated. The infected cell area will create a plaque (an area of infection surrounded by uninfected cells) which can be seen with an optical microscope or visually (pouring off the overlay medium and adding a
crystal violet
Crystal violet or gentian violet, also known as methyl violet 10B or hexamethyl pararosaniline chloride, is a triarylmethane dye used as a histological stain and in Gram's method of classifying bacteria. Crystal violet has antibacterial, antif ...
solution for 15 minutes until it has colored the cytoplasm, gently removing the excess with water will show uncolored the location of dead cells). Plaque formation can take 3–14 days, depending on the virus being analyzed. Plaques are generally counted manually and the results, in combination with the dilution factor used to prepare the plate, are used to calculate the number of plaque forming units per sample unit volume (pfu/mL). The pfu/mL result represents the number of infectious particles within the sample and is based on the assumption that each plaque formed is representative of one infectious virus particle.
Focus forming assay (FFA)
![Virus Infected Cells](https://upload.wikimedia.org/wikipedia/commons/0/07/Virus_Infected_Cells.jpg)
The focus forming assay (FFA) is a variation of the plaque assay, but instead of relying on cell lysis in order to detect plaque formation, the FFA employs
immunostaining
In biochemistry, immunostaining is any use of an antibody-based method to detect a specific protein in a sample. The term "immunostaining" was originally used to refer to the immunohistochemical staining of tissue sections, as first described by A ...
techniques using fluorescently labeled
antibodies
An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the ...
specific for a viral
antigen
In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. ...
to detect infected host cells and infectious virus particles before an actual plaque is formed. The FFA is particularly useful for quantifying classes of viruses that do not lyse the cell membranes, as these viruses would not be amenable to the plaque assay. Like the plaque assay, host cell monolayers are infected with various dilutions of the virus sample and allowed to incubate for a relatively brief incubation period (e.g., 24–72 hours) under a semisolid overlay medium that restricts the spread of infectious virus, creating localized clusters (foci) of infected cells. Plates are subsequently probed with fluorescently labeled antibodies against a viral antigen, and fluorescence microscopy is used to count and quantify the number of foci. The FFA method typically yields results in less time than plaque or fifty-percent-tissue-culture-infective-dose (TCID
50) assays, but it can be more expensive in terms of required reagents and equipment. Assay completion time is also dependent on the size of area that the user is counting. A larger area will require more time but can provide a more accurate representation of the sample. Results of the FFA are expressed as focus forming units per milliliter, or FFU/mL.
Endpoint dilution assay
The TCID
50 (50% Tissue Culture Infectious Dose) assay is the measure of infectious virus
titer
Titer (American English) or titre (British English) is a way of expressing concentration. Titer testing employs serial dilution to obtain approximate quantitative information from an analytical procedure that inherently only evaluates as positiv ...
. This endpoint dilution assay quantifies the amount of virus required to kill 50% of infected hosts or to produce a
cytopathic effect
Cytopathic effect or cytopathogenic effect (abbreviated CPE) refers to structural changes in host cells that are caused by viral invasion. The infecting virus causes lysis of the host cell or when the cell dies without lysis due to an inability to ...
in 50% of inoculated tissue culture cells. This assay may be more common in clinical research applications where the lethal dose of virus must be determined or if the virus does not form plaques. When used in the context of tissue culture, host cells are plated and serial dilutions of the virus are added. After incubation, the percentage of cell death (i.e. infected cells) is manually observed and recorded for each virus dilution, and results are used to mathematically calculate a TCID
50 result.
Due to distinct differences in assay methods and principles, TCID
50 and pfu/mL or other infectivity assay results are not equivalent. This method can take up to a week due to cell infectivity time.
Two methods commonly used to calculate TCID
50 (can also be used to calculate other types of 50% endpoint such
EC50
]
Half maximal effective concentration (EC50) is a measure of the concentration of a drug, antibody or toxicant which induces a Stimulus%E2%80%93response_model, response halfway between the baseline and maximum after a specified exposure time. Mo ...
,
IC50
The half maximal inhibitory concentration (IC50) is a measure of the potency of a substance in inhibiting a specific biological or biochemical function. IC50 is a quantitative measure that indicates how much of a particular inhibitory substance ...
, and
LD50
In toxicology, the median lethal dose, LD50 (abbreviation for "lethal dose, 50%"), LC50 (lethal concentration, 50%) or LCt50 is a toxic unit that measures the lethal dose of a toxin, radiation, or pathogen. The value of LD50 for a substance is the ...
) are:
* Spearman-Karber
*
Reed-Muench method
The ''theoretical'' relationship between TCID
50 and PFU is approximately 0.69 PFU = 1 TCID
50 based on the
Poisson distribution
In probability theory and statistics, the Poisson distribution is a discrete probability distribution that expresses the probability of a given number of events occurring in a fixed interval of time or space if these events occur with a known co ...
, a
probability distribution
In probability theory and statistics, a probability distribution is the mathematical function that gives the probabilities of occurrence of different possible outcomes for an experiment. It is a mathematical description of a random phenomenon i ...
which describes how many random events (virus particles) occurring at a known average rate (virus titer) are likely to occur in a fixed space (the amount of virus medium in a well). However, it must be emphasized that in practice, this relationship may not hold even for the same virus + cell combination, as the two types of assay are set up differently and virus infectivity is very sensitive to various factors such as cell age, overlay media, etc.
But the following reference defines the relationship differently:
Assuming that the same cell system is used, that the virus forms plaques on those cells, and that no procedures are added which would inhibit plaque formation, 1 mL of virus stock would be expected to have about half of the number of plaque forming units (PFUs) as TCID
50. This is only an estimate but is based on the rationale that the limiting dilution which would infect 50% of the cell layers challenged would often be expected to initially produce a single plaque in the cell layers which become infected. In some instances, two or more plaques might by chance form, and thus the actual number of PFUs should be determined experimentally.
Mathematically, the expected PFUs would be somewhat greater than one-half the TCID
50, since the negative tubes in the TCID
50 represent zero plaque forming units and the positive tubes each represent one or more plaque forming units. A more precise estimate is obtained by applying the Poisson distribution. Where is the proportion of negative tubes and m is the mean number of infectious units per volume (PFU/ml), . For any titer expressed as a TCID
50, . Thus and which is ~ 0.7.
Therefore, one could multiply the TCID
50 titer (per ml) by 0.7 to predict the mean number of PFU/ml. When actually applying such calculations, remember the calculated mean will only be valid if the changes in protocol required to visualize plaques do not alter the expression of infectious virus as compared with expression under conditions employed for TCID
50.
Thus as a working estimate, one can assume material with a TCID
50 of 1 × 10
5 TCID
50/mL will produce 0.7 × 10
5 PFUs/mL.
Protein assays
There are several variations of protein-based virus quantification assays. In general, these methods quantify either the amount of all protein or the amount of a specific virus protein in the sample rather than the number of infected cells or virus particles. Quantification most commonly relies on
fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...
detection. Some assay variations quantify protein directly in a sample while other variations require host cell infection and incubation to allow virus growth prior to protein quantification. The variation used depends primarily on the amount of protein (i.e. virus) in the initial sample and the sensitivity of the assay itself. If incubation and virus growth are required, cell and/or virus lysis/digestion are often conducted prior to analysis. Most protein-based methods are relatively fast and sensitive but require quality standards for accurate calibration, and quantify protein, not actual virus particle concentrations. Below are specific examples of widely used protein-based assays.
Hemagglutination assay
The hemagglutination assay (HA) is a common non-fluorescence protein quantification assay specific for
influenza
Influenza, commonly known as "the flu", is an infectious disease caused by influenza viruses. Symptoms range from mild to severe and often include fever, runny nose, sore throat, muscle pain, headache, coughing, and fatigue. These symptoms ...
. It relies on the fact that
hemagglutinin
In molecular biology, hemagglutinins (or ''haemagglutinin'' in British English) (from the Greek , 'blood' + Latin , 'glue') are receptor-binding membrane fusion glycoproteins produced by viruses in the ''Paramyxoviridae'' family. Hemagglutinins ar ...
, a surface protein of influenza viruses, agglutinates red blood cells (i.e. causes red blood cells to clump together). In this assay, dilutions of an influenza sample are incubated with a 1%
erythrocyte
Red blood cells (RBCs), also referred to as red cells, red blood corpuscles (in humans or other animals not having nucleus in red blood cells), haematids, erythroid cells or erythrocytes (from Greek ''erythros'' for "red" and ''kytos'' for "holl ...
solution for one hour and the virus dilution at which agglutination first occurs is visually determined. The assay produces a result of hemagglutination units (HAU), with typical pfu to HAU ratios in the 10
6 range. This assay takes ~1–2 hours to complete and results can differ widely based on the technical expertise of the operator.
The hemagglutination inhibition assay is a common variation of the HA assay used to measure flu-specific antibody levels in blood serum. In this variation, serum antibodies to the influenza virus will interfere with the virus attachment to red blood cells. Therefore, hemagglutination is inhibited when antibodies are present at a sufficient concentration.
Bicinchoninic acid assay
The
bicinchoninic acid
Bicinchoninic acid is a weak acid composed of two carboxylated quinoline rings.
Bicinchoninic is an organic compound with the formula (C9H5NCO2H)2. The molecule consists of a pair of quinoline rings, each bearing a carboxylic acid group. Its s ...
assay (BCA) is based on a simple
colorimetric
Colorimetry is "the science and technology used to quantify and describe physically the human color perception".
It is similar to spectrophotometry, but is distinguished by its interest in reducing spectra to the physical correlates of color ...
measurement and is the most common protein quantification assay. BCA is similar to the
Lowry or
Bradford
Bradford is a city and the administrative centre of the City of Bradford district in West Yorkshire, England. The city is in the Pennines' eastern foothills on the banks of the Bradford Beck. Bradford had a population of 349,561 at the 2011 ...
protein assays and was first made commercially available by Pierce, which is now owned by
Thermo Fisher Scientific
Thermo Fisher Scientific Inc. is an American supplier of scientific instrumentation, reagents and consumables, and software services. Based in Waltham, Massachusetts, Thermo Fisher was formed through the merger of Thermo Electron and Fisher S ...
. In the BCA assay, a protein's peptide bonds quantitatively reduce Cu
2+ to Cu
1+, which produces a light blue color. BCA
chelates
Chelation is a type of bonding of ions and molecules to metal ions. It involves the formation or presence of two or more separate coordinate bonds between a polydentate (multiple bonded) ligand and a single central metal atom. These ligands are ...
Cu
1+ at a 2:1 ratio resulting in a more intensely colored species that absorbs at 562 nm.
Absorbance
Absorbance is defined as "the logarithm of the ratio of incident to transmitted radiant power through a sample (excluding the effects on cell walls)". Alternatively, for samples which scatter light, absorbance may be defined as "the negative lo ...
of a sample at 562 nm is used to determine the bulk protein concentration in the sample. Assay results are compared with known standard curves after analysis with a
spectrophotometer or plate reader. Total assay time is 30 minutes to one hour. While this assay is ubiquitous and fast, it lacks specificity since it counts all protein, the virus preparation to be quantified must contain very low levels host cell proteins.
Single radial immunodiffusion assay
Single
radial immunodiffusion
Radial immunodiffusion (RID) or Mancini method, Mancini immunodiffusion or single radial immunodiffusion assay, is an immunodiffusion technique used in immunology to determine the quantity or concentration of an antigen in a sample.
Description ...
assay (SRID), also known as the Mancini method, is a protein assay that detects the amount of specific viral antigen by immunodiffusion in a semi-solid medium (e.g. agar). The medium contains
antiserum specific to the antigen of interest and the antigen is placed in the center of the disc. As the antigen diffuses into the medium it creates a precipitate ring that grows until equilibrium is reached. Assay time can range from 10 hours to days depending on equilibration time of the antigen and antibody. The zone diameter from the ring is linearly related to the log of protein concentration and is compared to zone diameters for known protein standards for quantification. There are kits and serums commercially available for this assay (e.g. The Binding Site Inc.).
Transmission electron microscopy (TEM)
TEM Tem or TEM may refer to:
Acronyms
* Threat and error management, an aviation safety management model.
* Telecom Expense Management
* Telecom Equipment Manufacturer
* TEM (currency), local to Volos, Greece
* TEM (nuclear propulsion), a Russian ...
is a specialized type of microscopy that utilizes a beam of electrons focused with a magnetic field to image a sample. TEM provides imaging with 1000x greater spatial resolution than a light microscope (resolution down to 0.2 nm). An ultrathin,
negatively stained sample is required. Sample preparations involve depositing specimens onto a coated TEM grid and negative staining with an electron-opaque liquid. Tissue embedded samples can also be examined if thinly sectioned. Sample preparations vary depending on protocol and user but generally require hours to complete. TEM images can show individual virus particles and quantitative
image analysis
Image analysis or imagery analysis is the extraction of meaningful information from images; mainly from digital images by means of digital image processing techniques. Image analysis tasks can be as simple as reading bar coded tags or as sophi ...
can be used to determine virus concentrations. These high resolution images also provide particle morphology information that most other methods cannot. Quantitative TEM results will often be greater than results from other assays as all particles, regardless of infectivity, are quantified in the reported virus-like particles per mL (vlp/mL) result. Quantitative TEM generally works well for virus concentrations greater than 10
6 particles/mL. Because of high instrument cost and the amount of space and support facilities needed, TEM equipment is available in a limited number of facilities.
Modern methods that allow quantification of viral particles
Tunable resistive pulse sensing (TRPS)
Tunable resistive pulse sensing (TRPS) is a method that allows high-throughput single particle measurements of individual virus particles, as they are driven through a size-tunable
nanopore
A nanopore is a pore of nanometer size. It may, for example, be created by a pore-forming protein or as a hole in synthetic materials such as silicon or graphene.
When a nanopore is present in an electrically insulating membrane, it can be used a ...
, one at a time. The technique has the advantage of simultaneously determining the size and concentration, of virus particles in solution with high resolution. This can be used in assessing sample stability and the contribution of aggregates, as well as total viral particle concentration (vp/mL).
TRPS-based measurement occurs in an ionic buffer, and no pre-staining of samples is required prior to analysis, thus the technique is more rapid than those which require pre-treatment with fluorescent dyes, with a total preparation and measurement time of less than 10 minutes per sample. TRPS-bases virus analysis is commercially available through
qViro-X systems, which have the ability to be decontaminated chemically by autoclaving after measurement has occurred.
Flow cytometry
While most flow cytometers do not have sufficient sensitivity, there are a few commercially available flow cytometers that can be used for virus quantification. A virus counter quantifies the number of intact virus particles in a sample using fluorescence to detect colocalized proteins and nucleic acids. Samples are stained with two dyes, one specific for proteins and one specific for nucleic acids, and analyzed as they flow through a laser beam. The quantity of particles producing simultaneous events on each of the two distinct fluorescence channels is determined, along with the measured sample flow rate, to calculate a concentration of virus particles (vp/mL). The results are generally similar in absolute quantity to a TEM result. The assay has a linear working range of 10
5–10
9 vp/mL and an analysis time of ~10 min with a short sample preparation time.
Single Virus Inductively Coupled Plasma Mass Spectroscopy (SV ICP-MS)
This technique is similar to Single Particle Inductively Coupled Plasma Mass Spectroscopy (SP ICP-MS)discovered by Degueldre and Favarger (2003) and adapted later for other nanoparticles e.g. gold colloids, see Degueldre et al (2006). The SP ICP-MS was adapted for the analysis of Single Virus Inductively Coupled Plasma Mass Spectroscopy (SV ICPMS) in a comprehensive study i.e. Degueldre (2021). This study suggests to adapting this method for single viruses (SV) identification and counting. With high resolution multi-channel sector field (MC SF) ICP-MS records in SV detection mode, the counting of master and key ions can allow analysis and identification of single viruses. The counting of 2-500 virial units can be performed in 20 s. Analyses are proposed to be carried out in Ar torch for master ions: 12C+, 13C+, 14N+, 15N+, and key ions 31P+, 32S+, 33S+ and 34S+. All interferences are discussed in detail. The use of high resolution MC ICP-MS is recommended while options with anaerobic/aerobic atmospheres are explored to upgrade the analysis when using quadrupole ICP-MS. Application for two virus types (SARS-COV2 and bacteriophage T5) is investigated using time scan and fixed mass analysis for the selected virus ions allowing characterisation of the species using the N/C, P/C and S/C molar ratio's and quantification of their number concentration.
Quantitative polymerase chain reaction (qPCR)
![ELISA diagram](https://upload.wikimedia.org/wikipedia/commons/b/ba/ELISA_diagram.png)
Quantitative PCR utilizes
polymerase chain reaction
The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) t ...
chemistry to amplify viral
DNA or
RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
to produce high enough concentrations for detection and quantification by fluorescence. In general, quantification by qPCR relies on serial dilutions of standards of known concentration being analyzed in parallel with the unknown samples for calibration and reference. Quantitative detection can be achieved using a wide variety of fluorescence detection strategies, including sequence specific probes or non-specific fluorescent dyes such as
SYBR Green
SYBR Green I (SG) is an asymmetrical cyanine dye used as a nucleic acid stain in molecular biology. The SYBR family of dyes is produced by Molecular Probes Inc., now owned by Thermo Fisher Scientific. SYBR Green I binds to DNA. The resulting ...
. Sequence specific probes, such as
TaqMan TaqMan probes are hydrolysis probes that are designed to increase the specificity of quantitative PCR. The method was first reported in 1991 by researcher Kary Mullis at Cetus Corporation, and the technology was subsequently developed by Hoffmann ...
(developed by Applied Biosystems), Molecular Beacons, or Scorpion, bind only to the DNA of the appropriate sequence produced during the reaction. SYBR Green dye binds to all double-stranded DNA produced during the reaction. While SYBR Green is easy to use, its lack of specificity and lower sensitivity lead most labs to use probe-based qPCR detection schemes. There are many variations of qPCR including the comparative threshold method, which allows relative quantification through comparison of Ct values (PCR cycles that show statistically significant increases in the product) from multiple samples that include an internal standard. PCR amplifies all target
nucleic acid
Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...
, including ones originating from intact infectious viral particles, from defective viral particles as well as free nucleic acid in solution. Because of this, qPCR results (expressed in terms of genome copies/mL) are likely to be higher in quantity than TEM results. For viral quantification, the ratio of whole virions to copies of nucleic acid is seldom one to one. This is because during viral replication, the nucleic acid and viral proteins are not always produced in 1:1 ratio and viral assembly process results in complete virions as well as empty capsids and/or excess free viral genomes. In the example of foot-and-mouth disease virus, the ratio of whole virions to RNA copies within an actively replicating host cell is approximately 1:1000. Products for qPCR-based virus titration are available commercially through numerous companies (e.g. Invitrogen, Roche or Qiagen). Advantages of titration by qPCR include quick turnaround time (1–4 hours) and sensitivity (can detect much lower concentration of viruses than other methods).
Enzyme-linked immunosorbent assay (ELISA)
ELISA
The enzyme-linked immunosorbent assay (ELISA) (, ) is a commonly used analytical biochemistry assay, first described by Eva Engvall and Peter Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presence ...
is a more modern variation of a protein assay that utilizes a specific antibody linked to an enzyme to detect the presence of an unknown amount of antigen (i.e. virus) in a sample. The antibody-antigen binding event is detected and/or quantified through the enzyme's ability to convert a reagent to a detectable signal that can be used to calculate the concentration of the antigen in the sample.
Horseradish peroxidase (HRP) is a common enzyme utilized in ELISA schemes due to its ability to amplify signal and increase assay sensitivity. There are many variations, or types of ELISA assays but they can generally be classified as either
indirect,
competitive
Competition is a rivalry where two or more parties strive for a common goal which cannot be shared: where one's gain is the other's loss (an example of which is a zero-sum game). Competition can arise between entities such as organisms, indivi ...
,
sandwich
A sandwich is a food typically consisting of vegetables, sliced cheese or meat, placed on or between slices of bread, or more generally any dish wherein bread serves as a container or wrapper for another food type. The sandwich began as a po ...
or
reverse
Reverse or reversing may refer to:
Arts and media
* ''Reverse'' (Eldritch album), 2001
* ''Reverse'' (2009 film), a Polish comedy-drama film
* ''Reverse'' (2019 film), an Iranian crime-drama film
* ''Reverse'' (Morandi album), 2005
* ''Reverse'' ...
.
ELISA kits are commercially available from numerous companies and quantification generally occurs via
chromogenic
In chemistry, the term chromogen refers to a colourless (or faintly coloured) chemical compound that can be converted by chemical reaction into a compound which can be described as "coloured". There is no universally agreed definition of the term. ...
reporters or fluorescence (e.g. Invitrogen, Santa Cruz Biotechnology Inc.). This technique is much less labor-intensive than the traditional methods and can take anywhere from 4 to 24 hours based on antibody incubation time.
References
{{DEFAULTSORT:Virus Quantification
Laboratory techniques
Immunology
Virology