How Neogen's Test Kits Work
Overview
Many of Neogen's food safety kits use immunoassay tachnology to rapidly detect target substances. The easy to use and interpret immuoassays rely on test devices coated with antibodies specifically created to capture a target substance that may be present in a sample. Neogen's superior antibodies, the most sensitive and specific available, set Neogen's products apart from all other immunoassay test kits.
Neogen uses one of three formats in its immunoassay-competitive direct enzyme-linked immunsorbent assay ( CD-ELISA) in a microwell format, sandwich enzyme-linked immunosorbent assay (S-ELISA) in a microwell, or lateral flow immunochromatic assay. Each uses antibodiy-coated test devices and color change in response to the addition of a sample to indicate a positive or negative reult for the presence of a target substance.
Neogen's GeneQuence and GENE-TRAK tests use highly-specific sandwich DNA hybridization technology to capture any present ribosomal RNA (rRNA) of a target foodborne pathogen in a sample. As with an immunoassay, a substrate is then used to produce a color change to indicate the presence of a target organism. Neogen's ISO-GRID and NEO-GRID tests use grid membrane filtration to capture target organisms on a membrane. which is then placed on an organism-specific agar plate. Target organisms are the detected and/or enumerated following incubation.
Competitive Direct Wnzyme-Linked Immunosorbent Assays (CD-ELISAs)
(All mycotoxin and histamine microwell test kits)
Each test kit contains antibody-coated microwells with antibodies specific to the kit's target substance.
First, samples and controls are added ot their respective test wells. Next, an enzyme conjugate (target substance chemically linked with an enzyme) is added. The samples/controls and conjugate are mixed and transfered to antibody wells where they complete for the antibody binding sites. The more target substance in the sample, the less conjugate the binds in the wells.
After an incubation, the wells are washed to remove all unbound materials.
A substate, which changes color in the presence of the conjugate, is then added to the wells. During an incubation, blue color develops in proportion to the amount of conjugate versus target substance in the well. The more conjugate bound, the more blue color that develops, indicating less substance present.
Results are read visually in a screening format-the less blue color, or more red, the more target substance detected. In a quantitative format, results are obtained by measuring the wells' color change in a microwell reader and comparing the readings against a standard curve.
1. Microwells are coated with antibodies
specific to the target substance
2. Conjugate competes with target substance/
controls for antibody binding site
3. Conjugate and target substance/controls
remain bound in wells
4. Substrate is added to produce a color change
5. Result are read visually or in a reader -
the less blue color, or more red, the move
target substance detected
Sadwich Enzyme-Linked Immumosorbent Assay (S-ELISAs)
(All food allergen and GeneQuence E.coli 0157:H7 microwell test kits)
Each test kit contains antibody-coated wells with antibodies specific to the kit's target substance.
First, samples and controls are added to their respective wells. During an incubation, the target substance binds to the antibodes. The wells are washed to remove all unbound materials.
An enzyme conjugate (antibody chemically linked with an enzyme) is then add to all wells. During an incubation, conjugate binds to the already bound target substence, forming a "sandwich." The more target substance in the wells, the more conjugate that binds in the wells.
The well are washed again to remove all unbound conjugate.
A Substrate, which changes color in the presence of the conjugate, is then added to the wells. During an incubation, color develops in proportion to the amount of conjugate in the wells. The more conjugate, the more color, and the more target substance that is detected.
Results are read visually in a screening format-the more blue color, the more target substance detected, In a quantitative format, results are obtained by measuring the wells' color change in a microwell reader and comparing the readings a standard curve.
1. Microwells are coated with antibodies
specific to the target substance
2. Samples and controls are added to their
respective wells
3. Conjugate is added, which binds to ready
bound target substance
4. Substrate is added to produce a color change
5. Results are read visually or in a reader -
the more blue color, the more target substance
detected
Lateral Flow Immunochromatic Assays
(All pathogen, allergen, mycotoxins, GMO and ruminant test kits in the lateral flow format)
First, a portion of enrichede or extracted sample is placed into the device's sample port. The sample is then wiched through the reagent zone, which contain antibodies specific for the target substance conjugated to colored oarticles. If the target substance s present, it will bind to the particle conjugated antibodies.
The target substance-antibody-particle complax then leaves the reagent zone and travels through the membrane into the device's test zone. The test zone contains anti-target substance antibodies that capture the complex, and display a visible colored line. (Note: For mycotoxinsonly, the presence of a predetermined level of target substance will result in no visible line in the test zone. With mycotoxins, the device's test zone captures colored particle-conjugated antibodies not carrying the target substance complex.) The remainder of the sample contonues to migrate to the end of the membrane where it is deposited into the waste reservoir.
The reagent zone also contains a control immune complex that iseluted by the sample solution regardless of the presence of the target substance. The control conjugate migrates through the membrane to the control zone where it forms a visible colored line. Regardless of the presence or absence of the target substance, the control line will form in the control zone to ensure the test is working properly.
Sandwich DNA Hybridization Assay
(GENE-TRAK pathogen test kits in dipstick and microwell formats, and GeneQuence test systems n icrowell format, excliding GeneQuence E.coli 0157:H7)
Each test kit contains capture and detector DNA probe specific to ribosomal RNA (rRNA) of the target organism and a coated solid phase (dipstick or microwell).
First, a portion of the enrichment culture is placed into a test tube. A lysis reagent is added, which disrupts the cell and releases the nucleric acid target molecules. A portion of the lysed sample is then transferred to a microwell and the probe reagents are added.
(In the dipstrick format assay, thepeople reagents are added to the tube with the lysed sample, followed by introduction of the coated dipstrick to the tube.)
The probe reagents consist of: 1) an 
oligonucleotide capture probe
specific to rRNA sequences of the target organism and labeled at the 3' end with polydeoxyadenylic acid (poly vdA); and 2) an oligonucleotide detector probe also specific to rRNA sequences of the target organism and labeled at the 5' end with the enzyme horseradish peroxidase (HRP).
The hybridization reaction is then allowed to proceed for the hour. If the target rRNA is present in the sample, both probe will hybridize to their complementary sequences on the target molecule. The resilting complex is captured onto the solid phase coated with polydeoxythymidylic acid (poly dT), which is complementary to the poly dA portion of the capture probe. Unbound probe is then washed away, and a substrate of HRP is added.
Following a short incubation, blue color indicates the presence of hybridized detertor probe in the complex and thus the presence of rRNA from the target organism. Results are determined spectrophotometrically at 450 nm. An absorbance value in excess of a predetermined threshold indicates a positive test result.
Hydrophobic Grid Membrane Filtration
(ISO-GRID and NEO-GRID test kits)
ISO-GRID/NEO-GRID tests are based on the concept of hydrophobic grid membrane filtration. The filter membrane is embrossed with hydrophobic ink to produce a grid containing 1600 indivudual squares. The ink' hydrophobic nature contains the growth of an organism isolated during the filtration process within the square of capture.
Samples are first diluted in a sterile buffer. The ISO-GRID and/or NEO-GRID filtration unit is then placed onto a manifold connected to a vacuum source, and a sterile filter membrane is placed in the ISO-GRID filtration unit (NEO-GRID already contains a sterile filter membrane). Using the vacuum source. a portion of the diluted sample is filtered through the filter membrane.
The membrane is then transferred to an agar plate prepared with a medium formulated to promote/differentiate the growth of the target organism. Follwing incubation, the plate's squares containing presumptively positive colonies are counted to yield a most probable number (MPN) result.
ATP Sanitation Monitoring
(AccuPoint ATP Sanitation System)
An adenosine triphosphate (ATP) sanitation system utilizes ATP bioluminescence to determine the cleanliness of the test samples. ATP is a chemical compound found in all living cells or cells that cells that were once living, including bacteria, food dabris, yeast and mold. Bioluminescence is a chemical reaction that produces light, such as the light produced by fireflies.
ATP bioluminescence accurs when ATP from a sample comes into contact with luciferase, and luciferin. The amount of light emitted in this reaction is directly proportional to the amount of ATP detected in a sample. The more ATP detected on a food contact surface, the more light produced.
After a sample is taken, the sampler is pressed into its cartridge, breaking the cartridge's seals and initiating the mixing of any presence ATP with luciferase and luciferin. The cartridge also contains a buffering solution to help flush any ATP from the sampling pad and to counteract the effects of sanitizers. The light-producing reaction takes place within the cartridge and a luminometer measures the amount of light produced. The light measurement then displayed on a LCD display in relative light units (RLU).
With our Access sampler, the buffering solution is contained in a plastic tube. After a sample is taken, the user snaps the tube open by depressing a tab at the top of the sampler, This opens the tube to allow the liquid inside to drain onto the sampling pad.
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