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The sandwich ELISA is one of the Enzyme-linked immunosorbent Assay (ELISA) methods which are analytical techniques for the detection of various compounds/analytes in a sample in the biomedical and research setting.

In sandwich ELISA, unlike the other types of ELISA, two antibodies are employed: a capture antibody and a detection antibody having the antigen of interest bound in between, hence the name “sandwich”. The capture antibody is immobilized on a surface, while the detection antibody (conjugated to an enzyme or fluorophore label) is applied as a last step before quantitation. The two antibodies used in a sandwich ELISA must be paired and tested before use. This means that they are known to bind to different places on the target antigen. It is very important that the capture antibody and the detection antibody don’t affect the binding capability of one another.

Magnetic beads are one of the most promising nanomaterials that in recent years have attracted interest in a wide range of biomedical applications especially in diagnostic and analytical tests for the isolation and purification of the analyte/component of interest after detection.

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Principles of Sandwich ELISA

The detection antibody will bind to the target antigen in the sample and the linked enzyme will be able to act on the substrate and a signal will be produced. This signal can be a simple color change (chromogenic) or a light produced (chemiluminescence). The signal produced is directly dependent on the enzyme-substrate pair used for detection. If the antigen is not present in the sample, then the enzyme-linked detection antibody will not have anything to bind to and no signal will be produced. In the case of a fluorescence ELISA, the detection antibody will not be labeled with an enzyme, but will instead be labeled by a fluorophore. A fluorophore emits light after it is excited by incident light. So as to say, enzyme-substrate labels produce light or color as a result of the chemical interaction of the enzyme and substrate, but fluorescent labels require incident light in order to excite the electrons of the fluorophore into a higher energy state from which they can release energy and emit light. An ELISA reader is a versatile instrument that can be used to detect any of these labels, so a variety of labels are available and simple to use in sandwich ELISAs.

magnetic beads have been introduced and widely used as a solid phase in ELISA immunoassay techniques for the isolation and purification of components such as proteins, peptides, hormones etc. Using magnetic beads, the analyte of interest can be immobilized and collected easily by applying an external magnetic field which in turn improves following analytical sensitivity and detection time.

 

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Common enzyme-substrate pairs

Horse radish peroxidase (HRP) and alkaline phosphatase (ALP) are the two most common chromogenic enzymes used for ELISA assays; the former catalyzes the cleavage of its substrate hydrogen peroxide in the presence of a hydrogen donor to produce a color change, and the latter catalyzes the color change of P-Nitrophenyl-phospate (pNPP). The chromogenic sandwich ELISA can be quantified by measuring absorbance. Many chemiluminescent and fluorescent enzyme-substrate pairs are also commercially available and they require fluorimeters or luminometers for quantification.

 Application of the sandwich ELISA method

ELISA is widely used in clinical settings to test for a range of antibodies and antigens. The technique is used for allergy testing. The test will capture antibodies from blood samples towards specific foods or other typical allergens. ELISA can also be used to detect allergen proteins in food products for quality control. ELISA can be used to detect antibodies in blood or serum against viruses as well. As the human body fights infections from viruses, antibodies specific to that virus are made and circulated, and those antibodies serve as a biomarker for infection in ELISA techniques. ELISA has been used to detect well known diseases such as lyme, HIV, and zika.

With magnetic beads, homogeneous reactions are conducted with efficient heterogeneous isolation and purification of different components simultaneously such as nucleic acids, hormones, proteins, viruses, and even whole cells.

 Advantages and Disadvantages of sandwich ELISA

The advantage of the sandwich ELISA is how sensitive it can be. The sensitivity of an ELISA is described as the detection limit, or the lowest concentration of antigen/analyte at which still a signal can be detected from the assay. High specificity and affinity of the primary and secondary antibody pair can increase the sensitivity. Various types of detection methods pre-conjugated to the secondary antibody can also be used.

In sandwich ELISA it is important to ensure that the primary and secondary antibody pair have a highly specific interaction. This is considered a disadvantage of sandwich ELISA because the secondary antibody could potentially bind to the capture antibody used to capture antigen onto an ELISA plate which this in turn introduces potentially incorrect readout of the assay. The readout might be interpreted as a captured antigen but incorrectly giving a signal from a capture antibody-secondary antibody interaction. 

 Magnetic beads in Sandwich ELISA

Magnetic beads are made up of tiny (20 to 30 nm) particles of iron oxides, such as magnetite (Fe3O4), which give them superparamagnetic properties. Superparamagnetic beads exhibit magnetic behavior only in the presence of an external magnetic field which can be separated from the suspension, along with the analyte they are bound to. Since they don’t attract each other outside of a magnetic field, they can be used without any concern about unwanted clumping.

There are different types of magnetic beads to be modified with different surface coatings and chemistries for specific binding properties (based on the analyte/component of interest) which can be used for magnetic separation (isolation and purification) of a variety of biomolecules (e.g. nucleic acids, proteins, enzymes, oligonucleotides, sugars, antibodies) in an easy, effective, and scalable way. This ease-of-use makes them automation friendly and well suited for a range of applications, including sample preparation for next generation sequencing (NGS) and PCR, protein purification, molecular and immunodiagnostics, and even magnetic activated cell sorting (MACS), among many others.

 

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Lluis M. Martínez | SEPMAG Chief Scientific Officer

Founder of SEPMAG, Lluis holds a PhD in Magnetic Materials by the UAB. He has conducted research at German and Spanish academic institutions. Having worked in companies in Ireland, USA and Spain, he has more than 20 years of experience applying magnetic materials and sensors to industrial products and processes. He has filed several international patents on the field and co-authored more than 20 scientific papers, most of them on the subject of magnetic particle movement.

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