Various analytical methods have been developed for the detection and discovery of biomarkers of major human diseases. Among these methods, enzyme-linked immunosorbent assays (ELISA) is a sensitive and highly specific tool for quantifying clinical biomarkers. Biomarker analyses by ELISA often requires low sample volumes but often also require extraction to either concentration samples or eliminate matrix interference effects. Very often, it is the limited availability of sample together with the need for a reliable, cost and time-effective method makes immunoassays the preferred method. The concentrations of biomarkers present in a biological sample varies with age, sex, sample type and health or disease states.

A researcher is often faced with the question if sample extraction is required for determining concentration of an analyte of interest by ELISA. This is a routine question received by technical support and many researchers prefer to skip this step since this involves time and associated cost of reagent and extra steps in sample preparation. The expectation to obtain a readout by adding a small volume of sample in an ELISA workflow is understandable. In reality, biological samples are complex and biomarkers are often present at very low concentrations in circulation and measuring their concentrations in samples like serum, plasma, or saliva can present a particular challenge. In some cases, extraction can be needed to remove interfering proteins from the sample. Quite often the optical density readings in an ELISA falls below the detection limits of the assay. This is where sample extraction is required to enrich the concentration of the target analyte. Sample extraction is a procedure that would eliminate the effect of potentially interfering molecules, reduce matrix effects and concentrate and enrich the analyte from the sample. Samples return values are most meaningful when the sample run in an ELISA can fall on the standard curve and can demonstrate dilutional linearity. The goal of sample preparation is to improve sensitivity by eliminating interfering substances or concentrating a sample in order to achieve a detectable sample return value that is not only accurate but reproducible.

However, sample extraction protocols are often viewed as an intricate process requiring extensive chemistry background and many researchers consider this as unnecessary. Unfortunately, cost savings and sample preservation usually take precedence over the appropriate and necessary sample preparation. Furthermore, some laboratories are not equipped to work with extraction procedures requiring solvents, Solid-phase extraction (SPE) columns and speedvac centrifugal evaporators. It is hard to predict if extraction is required from samples before running an ELISA. Ideally, an initial serial dilution trial run with pooled samples is required in order to determine which dilution will not only give linearity but align the samples on a linear part of the standard curve in the assay used for sample analysis. The best dilution is selected where the readings falling on the mid-part of the standard curve is used for the next round of ELISA with individual samples. A variety of enrichment methods have been developed and these are widely used and can be found as suggested procedures in the ELISA assay manuals accompanying the assay or in publications where researchers are reporting on similar sample populations.

Free, Bound or Total Concentration

Proteins, hormones and macromolecules form complexes with specific binding proteins and this macromolecular binding is necessary for transportation from the site of synthesis to the site of action. Glucocorticoid and cortisol hormones have a specific binding globulin (transcortin) and the sex hormones also have a specific globulin which transport these steroids in human blood. Therefore, to extract steroids hormones from serum or plasma, it is necessary to disrupt the steroid-binding protein. A small percentage of biomarkers also exists in free form (unbound and bound forms) and there exists an equilibrium, which can easily shift when a sample is processed. Even simple dilution of a biological sample would theoretically lead to a shift of the equilibrium and a change in the free versus bound concentrations. The true free concentration of a protein in a complex biological matrix is, therefore, inherently difficult to establish. The researcher must have a clear understanding as to whether the assay used for sample analysis has an affinity or specification for bound analyte, free analyte, or a combination of both.

Solvent Extraction and Liquid–liquid extraction (LLE)

Solvent extraction is a classical analytical technique which is a relatively fast and easy technique that is widely used because of its sensitivity to recover quantities from small volume samples. Solvent extraction operates on the principle of differential solubilities. Solvent extraction from liquid samples is Liquid–liquid extraction (LLE) and is a traditional method of selectively extracting a specific sample based on its partition between immiscible aqueous and organic solutions. In a typical LLE experiment, an aqueous sample is mixed with an apolar, nonmiscible solvent. After combining the two liquids in the vial, the contents are shaken vigorously to aid mixing of the two liquids. Liquid droplets are formed as the two solvents are not miscible. These are allowed to coalesce which may require centrifugation and the two bulk phases are separated from each other. Polar and apolar analytes are separated to a large degree: the polar species concentrate in the aqueous phase and the apolar species in the organic phase. The phase in which the analyte is dissolved is collected (e.g., by pipetting that phase into another vial). The organic phase is then evaporated and the resulting extract is reconstituted in assay buffer. The efficiency of an extracting solvent depends on the affinity of the compound for this solvent, as measured by the partition coefficient. Some examples of various approaches are smaller peptides like Bradykinin requiring a mixing of blood samples with chilled ethanol or Arg-Vasopressin, a well-studied hormone marker, which is extracted in a mixture of butanol: di-isopropyl ether as part of their extraction procedure. The general disadvantages with LLE is the reservation to use organic solvents to minimize environmental pollution. The protocol for disposal or evaporation is a concern to biology laboratory personnel.

Type of ELISA	Sample	Solvent used LLE
Arg8-Vasopressin	Serum and Plasma	Mixture of butanol and diisopropyl ether. Acetone and Petroleum ether
Corticosterone	Serum and Plasma	Ethyl Acetate
Progesterone	Serum and Plasma	Diethyl Ether
Testosterone	Serum and Plasma	Diethyl Ether
Cortisol	Serum and Plasma	Diethyl Ether

Table 1: . Some examples of solvent used to extract specific markers

Solid Phase Extraction (SPE)

Solid Phase Extraction (SPE) is a method for rapid sample preparation in which a solid stationary phase is typically packed into a syringe barrel and used to selectively extract, concentrate, and purify target analytes prior to analysis. Extraction of compounds is achieved on the basis of their chemical and physical properties which determines their distribution between a mobile liquid phase and a solid stationary phase. This method combines the separation power of classical liquid chromatography with a low cost, convenient, disposable column. It is very effective and is a more direct, economical, and reproducible process than traditional LLE. During the last two decades, SPE has steadily gained acceptance within the analytical community and is now rapidly replacing LLE as the sample preparation technique. SPE sorbents are available in a wide range of surface chemistries, pore sizes, particle sizes, and base supports such as silica, alumina polymers. The key element to any SPE product is the sorbent. The physiochemical properties of the sorbent determine extraction efficiency and the overall quality of the separation.

Steps in SPE consist of 4 basic steps: equilibrating the column, sample loading for binding to the sorbent, washing and eluting. After binding of the molecules with the correct properties on a solid matrix, the remaining compounds are washed away and the bound molecules are eluted by changing the mobile phase into the elution solvent. Following elution, solvent is evaporated in a centrifugal vacuum evaporator and sample dissolved in a small volume of assay buffer.

Solid-phase extraction columns are single use and they are usually disposed after each sample. The advantage of SPE is one-step elution. 3 ml micro columns with 200 mg matrix are well suited for sample extraction for ELISA. The most commonly used SPE material used in extraction of proteins and peptides is reversed-phase such as C4 or C18. Biomarkers like Oxytocin, Prostaglandin E2, Aldosterone are extracted on C18 column from biological samples. Either liquid-liquid extraction or solid phase extraction methods can be used for estradiol-17Β concentration in serum and plasma samples.

Type of ELISA	Sample	Type column Used in SSE
Angiotensin I	Serum and Plasma	C18 or phenylsilica
Oxytocin	Serum and Plasma	C 18 column
Prostaglandin E2	Serum and Plasma	C 18 column

Table 2: Some examples of Specific columns used for Analyte extraction by SPE

Enzo Life Sciences offers an extensive range of ready-to-use ELISA kits for a variety of research fields. Our kits well renowned and are highly published. Enzo Life Sciences has decades of experience developing and optimizing commercial immunoassays to meet the ever-changing needs of our scientific community. We provide a broad range of immunoassay kits and other research supplies for wildlife endocrinology, immunology, cancer, neuroscience and other research fields, as well as industrial applications such as bioprocess monitoring. For more information on assay validation please download our ELISA E-book or contact our Technical Support Team for further assistance.

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