Accelerō® Bioanalytics - Good Laboratory Practice (GLP) Compliant
During the development of new nucleic acid therapies, pharmaceutical companies often outsource critical drug testing procedures to contract research organization (CROs) for quick, efficient results performed under reliably accurate Good Laboratory Practice (GLP) protocols. Quantitative analysis of proteins and peptides are among the most frequently outsourced immunoassay procedures, including immuno PCR (iqPCR) or proximity ligation testing.
Accelero® Bioanalytics is a leading CRO offering GLP-compliant immunoassay services and specializing in real-time qPCR platforms on a contract basis. Accurate, verifiable and reproducible test results can streamline the nucleic acid drug development process, and important nucleic acid therapies will consequently earn FDA approval sooner, thus becoming available faster to those patients who stand to benefit the most from new pharmaceutical breakthroughs.
The Role of the Immunoassay in Bioanalytical Research
An immunoassay measures the amount of a targeted substance in solutions that may contain a number of elements. Immunoassays can determine whether specific analytes are present in a biological sample such as serum, and the quantity of the analyte if it is present. These types of tests depend on the specific binding ability of an antibody to join with individuals in limited groups of molecules. Molecules that bind to antibodies are known as antigens.
Immunoassays can target either contingent of the antibody and antigen duo. In the case of an antigen immunoassay, lab technicians can also, with correct preparation, employ the antibody that binds to the antigen as the analytical reagent, or substance used to quantify a chemical reaction.
An immunoassay, such as the real-time immuno PCR (iqPCR) or proximity ligation assay, serves to identify and then quantify the response, or chemical reaction, that results from target-specific binding. To simplify quantification, contemporary immunoassay protocol calls for detectable labels, such as fluorescent or phosphorescent dyes that illuminate the multiplication of analytes. Other quantification markers used in other types of nucleic acid therapy testing include radioactive elements used in a radioimmunoassay, magnetic particles, coenzymes, and several more.
These labels, or markers, make it possible for laboratory personnel to detect and quantify the occurrence of binding, either subsequent to the separation of free and bound reagents or during the binding itself. With the help of control calibrators containing specific analyte concentrations, technicians can determine the assay response of the test sample using comparative evaluation techniques as well as tools such as the calibration curve.
Because many nucleic acid therapies rely on the binding ability of microRNA or siRNA to target molecules, the role of immunoassay in detecting whether binding is taking place and the quantity of that occurence is critical in determining the efficacy of those therapies. Real-time immuno PCR (iqPCR) or the proximity ligation assay are examples of an immunoassay that, when carried out in a GLP-compliant facility, provides quantitative data and analysis that helps pharmaceutical firms streamline the development of therapies for chronic and life-threatening diseases.
An immunoassay can be an irreplaceable tool for detecting analytes in very low concentrations that other tests may not have the sensitivity to pinpoint. An immunoassay can identify hormones, proteins, markers that indicate the presence of tumors, and markers that are indicative of heart injury. Appropriate immunoassays can also detect antibodies produced in response to the onset of viral hepatitis, Lyme disease, and HIV.
Types of Immunoassay
ELISA, the term that CRO technicians use to describe the enzyme-linked immunoassay, is one category of nucleic acid therapy research that GLP facilities use to quantify certain molecules. The sandwich and competitive immunoassay belong in the ELISA lexicon, as well as the antigen-down assay. Because sandwich assays are often the most sensitive and reveal the most robust results, they represent the most commonly-used immunoassay in CROs facilities worldwide.
During the course of a competitive immunoassay, the antigen contained in an unknown sample competes with a known, labeled antigen in binding with antibodies. Technicians quantify the labeled antigen that bound with antibodies and calculate the concentration of the unknown antigen using an inverse formula, based on the logic that the greater the quantity of known antigen, the lesser the quantity of the competing, unknown antigen.
The noncompetitive immunoassay, also called a sandwich assay, involves two different antibodies that bind to different surfaces of the antigen. The technician attaches the target antibody to a solid surface, and then adds the primary antigen. In the next step, the technician adds a second antibody, which is called the detection antibody. This creates a “sandwich” of two antibodies with the antigen in the middle. The degree to which the antibody binds to the antigen often determines the sensitivity of the immunoassay as well as the precision of the test results. The detection antibody increases in direct proportion to the increasing concentration of the antigen, facilitating a higher response measurement. The sandwich assay utilizes detection antibodies attached to enzyme markers for the purposes of quantification, and the quantity measured will be proportional to the amount of the target antibody.
The sandwich hybridization ligand binding assay is a specific type of drug test that CROs frequently perform for pharmaceutical developers. Originally created for diagnostic purposes for the detection of pathogens, CROs currently employ this hybridization assay most frequently to further the development of nucleic acid therapies. Requiring the use of a capture probe and a detection probe that contain complementary sequences to hybridize a single strand nucleic acid molecule, this immunoassay provides critical quantification data that reveals how effectively the proposed therapy binds with the target molecule.
Real-time immuno PCR (iqPCR) – quantitative immuno polymerase chain reaction – is a particular assay that is at least ten times more sensitive than the most commonly-used ELISA immunoassay formats. As such, the immuno PCR (iqPCR) is the perfect test platform for identifying difficult to detect analytes such as peptide and protein markers. Because these markers can be indicative of the presence of illegal doping drugs in an athlete’s system, the ability to detect them in minute concentrations is essential to fair sportsmanship in both professional and amateur arenas worldwide. Also able to identify mycotoxin in food, or microbial pathogens for the purpose of diagnosis, real-time immuno PCR (iqPCR) is an invaluable component to the tool kit of GLP-compliant CROs. Real-time immunoassays like the immuno PCR (iqPCR) assay are most convenient for the detection of any immune response to novel drugs in antibody detection assays (ADA).
The proximity ligation assay is a ultrasensitive variant of the immuno PCR (iqPCR), which allows also for real-time quantification of peptide or protein therapeutics in biological samples, but may exhibit a superior immunoassay sensitivity. In contrast to a typical immuno PCR (iqPCR) assay, two different antibodies bind to the antigen, most preferred recognizing distinct epitopes. Both antibodies bear a short DNA tag, which might be in proximity to each other upon binding to the antigen. If so, a DNA ligase generates a covalent link between both DNA tags, which afterwards can be detected in an immuno PCR (iqPCR) assay. Because only ligation products are detected in the real-time qPCR, this kind of immunoassay is expected to provide an excellent assay sensitivity.
An antigen-down immunoassay, also called an immunometric assay, is a testing process which calls for binding the antigen to a solid surface rather than to an antibody. The laboratory technician coats the surface of a plate with the antigen, and then applies a biological sample, such as human serum, to the coated surface. Antibodies, contained in the sample, bind to the antigen coating. Then, the technician adds a detection-enabled, species-specific antibody to the plate that makes it possible to quantify the results of the binding process.
The radioimmunoassay, another testing format, requires specialized facilities for safety purposes, since radioactivity is present. The enzyme immunoassay is currently the format of choice in many Japanese labs. In addition, many CLOs most frequently employ the non-competitive immunoenzymometric assay (IEMA), utilizing chemiluminescent detection materials to quantify enzyme activity levels.
No matter the type of immunoassay to be performed, certain starting parameters provide a basis for gleaning accurate data and reproducible results from any immunoassay format.
To begin with, FDA-sanctioned Good Laboratory Practice compliance should be a given in any contract research organization facility. Immunoassay and other nucleic acid therapy testing should comply with GLP guidelines such as:
• Standardized and GLP-certified laboratory operating procedures
• Standardized and GLP-certified statistical procedures when evaluating data
• GLP-compliant instruments at proscribed calibrations
• GLP certification of reagents and ancillary materials
• GLP certification of facilities
• Standardized and GLP-certified procedures for tracking specimens and samples
While many commercially-produced laboratory testing kits and formats are available that answer a wide spectrum of immunoassay requests, a reputable and experienced CRO, such as Accelero® Bioanalytics, has the ability to customize a particular protocol to the specification nucleic acid therapy development clients.
By following Good Laboratory Practice (GLP) guidelines during all testing procedures, from using the right instruments to timing each step of the immunoassay precisely, a CRO can invariably provide accurate test results and verifiable, reproducible data that play a critical role in streamlining the drug approval process for pharmaceutical developers.
The simplest immunoassay procedure is called immunoprecipitation. This test measures the amount of precipitate that forms after the antibody and antigen have incubated and bonded as an insoluble aggregate.
The particle immunoassay involves the linkage of several antibodies to a particle, which in turn binds several antigen molecules at one time. Because this particular assay significantly accelerates the visible reaction, the technician performing the particle immunoassay can quickly quantify antibodies that are indicative of such diseases as mononucleosis and rheumatoid arthritis.
When the union of an antibody and antigen form non-precipatory complexes, the lab technician may elect to utilize a testing platform known as immunonephelometry, in which technicians use a piece of equipment called a nephlelometer to quantify instead the incident light that the binding process emits.
The radioimmunoassay employs radioactive isotopes as detection labels attached to either the antibody or the antigen to facilitate a highly sensitive and simplified signal detection protocol. While offering the primary advantages of accuracy and speed, the radioimmunoassay format presents risks to human safety that are associated with the utilization of radiation. In addition, this type of testing is more expensive than alternative assay formats, and requires special facility licensing.
The enzyme immunoassay (EIA) is a viable alternative to the radioimmunoassay, offering a similarly high degree of sensitivity without the health risk of radioactive substances. An enzyme immunoassay relies on the use of an enzyme label attached to either the antibody or the antigen. Perhaps the most widely-used EIA format in CROs across the globe today to detect infectious disease is ELISA.
The fluorescent immunoassay (FIA) uses a fluorescent label to optimize the lab’s quantification of binding activity. More sensitive than EIA testing, the FIA is often the test method of choice for some types of drug development.
Finally, the chemiluminescent immunoassay makes use of molecule markers that emit light when activated by chemical activity. Technicians use a light meter to measure test results.
Factors that Affect Test Results
Although the immunoassay is typically a highly sensitive, specific test that produces reliable results, certain factors may adversely affect the reliability of the data. In the laboratory, substandard storage of samples or faulty preparation can cause false positive or negative results. The use of deteriorated reagents can nullify test data. During the immunoassay step-by-step process, improper washing technique between steps can also produce false data.
By selecting a reputable, GLP-compliant contract research facility, pharmaceutical developers can be assured that samples are properly stored and treated, reagents are robust and each immunoassay is meticulously carried out according to the highest possible standards. Only a CRO that is GLP compliant can consistently provide the most accurate, reproducible results, keep verifiable records, and interpret data in accordance within professional parameters.
Setting Up the Sandwich Immunoassay
Precision and strict adherence to standard protocols distinguishes a GLP-compliant facility from a non-compliant facility, as a review of the basic preparations required for a sandwich immunoassay illustrates.
Initially, a CRO undertaking an immunoassay should establish criteria for the success of the proposed test and make sure that adequate supplies of reagents, testing equipment, and other critical materials are readily available. The well-organized, GLP-certified facility will have the detailed supply lists, high quality, calibrated equipment and qualified staff on hand to streamline the pre-testing process.
Prior to progressing to the actual immunoassay procedure, technicians will take care of a few preliminaries, such as:
1. The initial development of immunoassay parameters: In order to design the basics of the sandwich immunoassay, the lab technician must determine which of two antibodies should be designated primary and which should be the secondary, detection antibody. This step requires experimentation carried out under GLP guidelines.
2. The identification of the matrix effect of the sample type on the testing method: For example, serum matrix is a complex substance that can significantly affect testing methods. Depending on the sample to be tested, the lab technician must modify the testing protocol, within the specifications of GLP, so that the immunoassay is effective.
3. Address any changes that need to be made to ensure that the immunoassay is as effective as possible to achieve the data needed: The tech determines the optimal conditions for variables such as the incubation process, the buffers used, as well as the prime antibody concentrations that must all effectively interact to produce the desired data. Only GLP-certified personnel have the knowledge, training and experience to make these determinations.
Once the test process is underway, Good Laboratory Practice (GLP) standards inform many of the procedures necessary to achieve the most reliable results possible. Precise incubation times within a specific temperature range can be critical to creating reproducible immunoassay results. Washing the plate surfaces with appropriate buffer solutions the requisite number of times at specific junctures in the immunoassay process can also ensure consistent results among identical tests.
A CRO team that demonstrates a willingness to go to the extra mile in regards to the record-keeping, facility expense, and time commitment of compliance with Good Laboratory Practice can be relied upon to be just as conscientious in all of its professional endeavors. Pharmaceutical development firms seeking to outsource nucleic acid immunoassay work should always verify that their CRO of choice is GLP compliant.
GLP Standards for Performance
In the GLP facility, standards governing instruments used for immunoassay procedures ensure uniform and high-quality performance. The equipment that the technicians intend to use to quantify the immunoassay results must be pre-tested for linearity and performance. The instruments should be freshly calibrated prior to use, following the manufacturer’s recommended procedures. Instrument wavelengths and settings should be verified as accurate and appropriate prior to use. Depending on the type of marker involved in the immunoassay, equipment may include a fluorescence microplate reader or a chemiluminescence microplate reader, each of which should be tested and verified for accuracy.
Analyte—a substance that is being analyzed
Antibody—immunoglobin molecule that reacts to and with specific antigens
Antigen—a molecule that binds to an antibody
Antigen-down immunoassay—one of three ELISA testing formats, involving the binding of an antigen to a solid plate rather than to an antibody
Assay—a set of procedures toward the goal of quantification
Calibration curve—a method used in analytical chemistry to quantify a substance in an unknown sample by comparing it to standard samples of predetermined concentration
CRO—contract research organization, ideally GLP-approved, that accepts outsourced lab work from drug development firms
Competitive immunoassay— one of three ELISA formats, also called a competitive binding assay, that is based on the completion of labeled and unlabeled ligand for a limited field of antibody binding locations
ELISA—enzyme-linked immunoassay that relies on the antibody’s ability to bind to the structure of a specific molecule
Enzyme immunoassay—an immunoassay that utilizes an antibody labeled with an enzyme marker
Fluorescent immunoassay—an immunoassay that employs fluorescent markers to facilitate quantification
GLP—Good Laboratory Practice, a set of guidelines defined by the Food and Drug Administration to standardize laboratory procedures
Immunoassay—a biochemical test based on the innate ability of an antibody to bind exclusively to one or an extremely limited group of molecules for the purposes of identification and quantification
Immunoprecipitation—a type of drug testing that measures the precipitant emitted once the incubation and bonding of the antigen and antibody are complete
Immunonephelometry—when the antibody/antigen binding process does not emit precipitates, the technician may use a nephlelometer to quantify the light emitted by the antigen/antibody bond Ligand—antigen
Ligand binding assay—a test that quantifies reactions with binding reagents, largely utilized in the development of nucleic acid therapies
MicroRNA—short stranded ribonucleic acid molecules that bind with target molecules for therapeutic purposes
Noncompetitive immunoassay—another term for the sandwich assay
Nucleic acid therapy—drugs based on the ability of microRNA, siRNA and other nucleic acids to bind effectively with target molecules that cause disease
Particle immunoassay—a type of testing that involves linking multiple antibodies to a particle, which then binds with numerous antigen molecules
Quantification—the process by which a CLO measures the reaction levels of an antibody and antigen during an immunoassay
Radioimmunoassay—a testing method that uses radioactive isotopes as detection markers
Reagents—those substances added to a system to cause a chemical reaction or to identify whether a reaction will occur
Real-time qPCR—quantitative immune polymerase chain reaction; a type of lab test frequently used in the development of new nucleic acid therapies
Sandwich assay—the most frequently-used and powerful of three ELISA testing formats, in which the subject analyte is bound on each side by one of two antibodies, called the detection and the capture antibodies
Serum—an almost clear body fluid prepared from blood
siRNA—small interfering RNA that inhibits the function of target genes
Substrate—a molecule that an enzyme acts upon
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More details on bioanalytical ligand binding immunoassays in drug development are kindly presented in our ligand binding assay album.