Ultrasonic Sample Prep for ELISA Assays
ELISA – Enzyme-Linked Immunosorbent Assay
ELISA stands for enzyme-linked immunosorbent assay and is a widely used biochemical analysis technique of the category of ligand-binding assays. In ELISA, a liquid sample is added onto a stationary solid phase with special binding properties. Normally, the stationary solid phase is applied as coating to a well plate or ELISA plate. Then, various liquid reagents are sequentially added, incubated, and washed, so that finally an optical change (e.g., color development by the product of an enzymatic reaction) occurs in the final liquid in the well. The optical change allows to measure the quantity of the analyte by a so-called quantitative “reading”. For the quantitative reading, a spectrophotometer, fluorometer, or luminometer is used to detect and measure the intensity of the transmitted light. The sensitivity of detection is influenced by the amplification of the signal during the analytic reactions. Since enzyme reactions are well investigated and reliable amplification processes, enzymes are used to create the signal. The enzymes are linked to the detection reagents in fixed proportions to allow accurate quantification, which explains also the name “enzyme-linked” immunosorbent assay.
As ELISA assays are performed in microtiter plates / 96-well plates, it is known as plate-based assay technique and is used e.g. in clinical in-vitro diagnostic, research, drug development etc. for detecting and quantifying antibodies, peptides, proteins, and hormones.
The ELISA technique is frequently used as a diagnostic tool in medicine, biotech, plant pathology and is also an important quality control measurement in multiple industries.
Ultrasonic Sample Prep before ELISA
Before the ELISA assay can be performed, the samples require steps of preparation such cell lysis and extraction of intracellular proteins, DNA, RNA etc. The advantage of ultrasonic cell lysis and protein isolation is its high efficiency, reliability and reproducibility. All of these factors are important in order to obtain high-quality diagnostics and research results
- Homogeneous sample treatment
- Complete lysis
- Complete protein extraction (e.g. antibodies, DNA)
- Optimal adaptation to cell type
- For any sample size
- Automatic data protocoling on SD-card
Protocol for Pre-ELISA Ultrasonic Cell Lysis
- For Cell Cultures: Before the ultrasonic cell lysis, centrifuge cells for 5 mins at 270 x g in a microcentrifuge. Remove the supernatant by aspiration and resuspend cells in 30 – 100 μL of RIPA buffer. Then, incubate the cell pellet on ice for 30 min.
- Now, the cell sample is ready for ultrasonic lysis:
Use a probe-type ultrasonicator (e.g. UP200Ht with S26d2 probe) or an ultrasonic multi-sample device (e.g. VialTweeter for simultaneous sonication of up to 10 vials or the UIP400MPT for microtiter plates / 96-well plates) depending on the amount of samples you need to prepare.
For the probe-type sonication of a single sample, place the cells in 1.5 mL microcentrifuge tubes.
- Pre-set your ultrasonic duration, total energy input, cycle mode and/or temperature limits in the digital menu of the ultrasonicator. This ensures highly reliable sonication and repeatability.
- Inset the sonotrode and switch the ultrasonic device on. Gently move the micro-tip of the ultrasonic probe through the sample to sonicate the sample uniformly.
For most cells, ultrasonic lysis will be completed after 2 -4 cycles of 10 sec sonication.
- After sonication, remove the sonotrode from the sample. The samples should be incubated on ice for 5 min. Then, centrifuge at 10,000 x g for 20 min to pellet the debris. Transfer the supernatants to a new microcentrifuge tube. Label the analytes and store at -20°C.
- The ultrasonic sonotrode can be cleaned by wiping it properly with alcohol or sonicate in a beaker filled with alcohol, e.g. 70% ethanol. All ultrasonic probes made from titanium are autoclavable.
For Tissue Homogenates:
- Rinse the tissue with ice-cold PBS (0.01M, pH=7.4) to remove excess hemolysis blood thoroughly.
- Weigh the tissue (kidney, heart, lung, spleen etc.) and macerate it into small pieces, which are homogenized in PBS. The volume of PBS required is related to the weight of the tissue. As a rule of thumb, 1g of tissue requires approx. 9mL PBS. It is recommended to add some protease inhibitor to the PBS. (RIPA or hypotonic lysis buffer containing protease and phosphatase inhibitor cocktail can be used alternatively.)
- Depending on the tissue size, a short vortex treatment (approx. 1-2 min. in 15 sec. pulses) can be helpful to pre-treat the tissue.
- Mount a micro-tip, e.g. S26d2, to your ultrasonicator. Place the sample tube with the tissue in an ice bath.
- Sonicate the sample with your ultrasonicator, e.g. UP200St (80% amplitude) for 1 min. in pulse mode (15sec on, 15sec pause). Keep the sample in the ice bath.
- The homogenates are then centrifuged to obtain specific pools (cytosolic, nuclear, mitochondrial or lysosomal) in order to enrich the protein for analysis. By centrifuging the sample for 5 minutes at 5000×g, the supernatant is retrieved.
Reliable Temperature Control during Sonication
Temperature is a crucial process-influencing factor that is especially important for the treatment of biological samples, e.g. to prevent thermal degradation of proteins. As all mechanical sample preparation techniques, sonication creates heat. However, the temperature of the samples can be well controlled when using the Hielscher Ultrasonics devices. We present you various options to monitor and control the temperature of your samples whilst preparing them with the a probe-type ultrasonicator or the VialTweeter pre-analytically.
- Monitoring the sample temperature: All Hielscher digital ultrasonic processors are equipped with an intelligent software and a pluggable temperature sensor. Plug the temperature sensor into the ultrasonic device (e.g., UP200Ht, UP200St, VialTweeter, UIP400MTP) and insert the tip of the temperature sensor in one of the sample tubes. Via digital coloured touch-display, you can set in the menu of the ultrasonic processor a specific temperature range for your sample sonication. The ultrasonicator will automatically stop when the max temperature is reached and pause until the sample temperature is down to the lower value of the set temperature ∆. Then the sonication starts automatically again. This smart feature prevents heat-induced degradation.
- Regarding the ultrasonic multi-sample unit VialTweeter, the titanium block, which hold the sample tubes, can be pre-cooled. Put the VialTweeter block (only the sonotrode without transducer!) into the fridge or freezer to pre-cool the titanium block helps to postpone temperature rise in the sample. If possible, the sample itself can be pre-cooled too.
- Use an ice bath or dry ice to cool during sonication. Place your sample tube(s) during sonication into an ice bath. For the VialTweeter, use a shallow tray filled with dry ice and place the VialTweeter on the dry ice so that heat can rapidly dissipate.
Customers worldwide use the Hielscher probe-ultrasonicators as well as the multi-sample sonication units VialTweeter and UIP400MTP for their daily sample preparation work in biological, biochemical, medical and clinical laboratories. The intelligent software and temperature control of the Hielscher processors, temperature is reliably controlled and heat-induced sample degradation avoided. Ultrasonic sample preparation with Hielscher ultrasonic solutions delivers highly reliable and reproducible results!
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Literature / References
- Brandy Verhalen , Stefan Ernst, Michael Börsch, Stephan Wilkens (2012): Dynamic Ligand-induced Conformational Rearrangements in P-glycoprotein as Probed by Fluorescence Resonance Energy Transfer Spectroscopy. J Biol Chem. 2012 Jan 6;287(2): 1112-27.
- Claudia Lindemann, Nataliya Lupilova, Alexandra Müller, Bettina Warscheid, Helmut E. Meyer, Katja Kuhlmann, Martin Eisenacher, Lars I. Leichert (2013): Redox Proteomics Uncovers Peroxynitrite-Sensitive Proteins that Help Escherichia coli to Overcome Nitrosative Stress. J Biol Chem. 2013 Jul 5; 288(27): 19698–19714.
- Elahe Motevaseli, Mahdieh Shirzad, Seyed Mohammad Akrami, Azam-Sadat Mousavi, Akbar Mirsalehian, Mohammad Hossein Modarressi (2013): Normal and tumour cervical cells respond differently to vaginal lactobacilli, independent of pH and lactate. ed. Microbiol. 2013 Jul; 62(Pt 7):1065-1072.
- Nico Böhmer, Andreas Dautel, Thomas Eisele, Lutz Fischer (2012): Recombinant expression, purification and characterisation of the native glutamate racemase from Lactobacillus plantarum NC8. Protein Expr Purif. 2013 Mar;88(1):54-60.
Facts Worth Knowing
Types of ELISA
There are several types of ELISA, which are distinguished by their functioning principle. They are known as direct ELISA, indirect ELISA, sandwich ELISA, competitive ELISA, and reverse ELISA. Below, we present you an overview over the various ELISA types and their main characteristics and differences.
ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result, whilst in quantitative ELISA the optical density (OD) of the sample is compared to a standard curve, which is typically a serial dilution of a known-concentration solution of the target molecule.
The direct ELISA is the most simple assay form of Elisa, where only an enzyme-labeled primary antibody is used and secondary antibodies are not required. The enzyme-labeled primary antibody binds directly to the target, i.e. antigen. The buffered antigen solution is added to each well of a microtiter plate (usually 96-well plates, ELISA-plates), where adheres to the plastic surface through charge interactions. When the enzyme linked to the primary antibody reacts with its substrate, it produces a visible signal that can be measured via spectrophotometer, fluorometer, or luminometer.
For the indirect ELISA test, both a primary antibody and a secondary antibody are required. However, in contrary to the direct ELISA, not the primary antibody, but the secondary antibody is labeled with an enzyme. The antigen is immobilized to the well plate and bound by the primary antibody. Subsequently, the enzyme-labeled secondary antibody binds to the primary antibody. Finally, the enzyme linked to the secondary antibody reacts with its substrate to produce a visible signal that can be detected.
Whilst in direct and indirect ELISA tests, the antigen is immobilized and coated to the surface of the well plate, in the sandwich ELISA the antibody is immobilized to the plastic surface of the ELISA-plate. The immobilized antibodies in sandwich ELISA are known as capture antibodies. Additionally to the capture antibodies, in sandwich ELISA also so-called detection antibodies are required. Detection antibodies include an unlabeled primary detection antibody and an enzyme-labeled secondary detection antibody.
Stepwise, the antigen of interest binds to the capture antibody immobilized to the plate. Then, the primary detection antibody binds to the antigen. Afterwards, the secondary detection antibody binds to the primary detection antibody. In the final reaction step, the enzyme reacts with its substrate to produce a visible signal that can be detected optically.
Competitive ELISA, also known as inhibition ELISA, is the most complex ELISA type because it involves the use of an inhibitor antigen. Each of the three formats, direct, indirect, and sandwich ELISA, can be adapted to the competitive ELISA format. In competitive ELISA, the inhibitor antigen and the antigen of interest compete for binding to the primary antibody.
For competitive ELISA, unlabeled antibody is incubated in the presence of its antigen, i.e. sample. These bound antibody/antigen complexes are then added to an antigen-coated well.
The plate is washed, so that unbound antibodies are removed. Competitive ELISA has its name due to the fact that the more antigen is in the sample, the more antigen-antibody complexes are formed. This means, there are less unbound antibodies available to bind to the antigen in the well and the antigens must compete for an available antibody. A secondary antibody, matching the primary antibody, is added. This second antibody is linked to the enzyme. When the substrate is added, the remaining enzymes produce a chromogenic or fluorescent signal.
At this point, the reaction is stopped to avoid the eventual saturation of the signal.
Some competitive ELISA kits include an enzyme-linked antigen instead of an enzyme-linked antibody. The labeled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). The less antigen in the sample, the more labeled antigen is retained in the well and the stronger the signal.
Reverse ELISA does not use well plates, but leaves the antigens suspended in the test fluid. The reverse ELISA test measures the amount of bound antibody via antigen. It was developed specifically to detect and investigate the West Nile virus envelope protein and how it is able to find virus-specific antibodies.
Enzymatic Marker Used for ELISA
The list below gives the most common enzymatic markers used in ELISA assays, which allow the results of the assay to be measured upon completion.
- OPD (o-phenylenediamine dihydrochloride) turns amber to detect HRP (Horseradish Peroxidase), which is often used to as a conjugated protein.
- TMB (3,3′,5,5′-tetramethylbenzidine) turns blue when detecting HRP and turns yellow after the addition of sulfuric or phosphoric acid.
- ABTS (2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) turns green when detecting HRP.
- PNPP (p-Nitrophenyl Phosphate, Disodium Salt) turns yellow when detecting alkaline phosphatase.