Applications ELISA & Serial Dilution 2018-12-04T04:45:26+00:00

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ELISA assays are now optimized using Dynamic Devices Lynx liquid handing robot platform configured with the f8 Fixed-8 VVP, i8 Independent VVP or 96 VVP Pipetting Tools. With the ability of independently controlling each tip with individual volumes and volume verification, an entire 96 well assay plate may be diluted and mixed in a single step.

The 96 VVP Pipetting Tool can be used to dilute assay plates for an ELISA assay in multiple high throughput ways:

  1. An entire 96 well compound plate can be diluted with different volumes or a single reagent as conjugate, substrate, or stop with recorded verified volumes for EVERY transfer.
  2. A 384 well assay plate may be diluted in 5 steps with a single aspiration and 4 dispenses since the head may multi-pipet different volumes over multiple plates.
  3. A serial diluted assay plate may be created by picking up either single rows of columns of tips to run the serial dilution over a single plate.

In addition, the f8 Fixed-8 VVP and i8 independent VVP Pipetting Tools may also perform any of the above tasks with the same performance and validation and is required for applications like hit picking for a secondary assay when individual well and / or tube access is required.

LabAuto 2010 Liquid Handling Robot ELISA System Display:

elisa labauto px600

elisa labauto read wash px400

Automated Liquid Handling Robot Configuration:

  1. Oasis LM 1200 with 96 Head and Advanced Gripper
  2. BioTek Washer
  3. BioTek Reader
  4. D2 Reagent Dispenser
  5. D2 Incubator
  6. Oasis Deck Extension

The Liquid Handling Robot ELISA system configuration can be slightly modified to accomplish the following assays requiring incubation, washing or reading:

  • Enzyme-linked immunosorbent assays (ELISAs)
  • Enzyme immunoassays (EIAs)
  • Radio immunoassays (RIA) without on-line radioactive detection
  • Fluorescence immunoassays (FIA)

Enzyme-linked Immunosorbent Assays (ELISAs) combine the specificity of antibodies with the sensitivity of simple enzyme assays by using antibodies or antigens coupled to an easily-assayed enzyme. ELISAs can provide a useful measurement of antigen or antibody concentration. There are two main variations on this method: The ELISA can be used to detect the presence of antigens that are recognized by an antibody or it can be used to test for antibodies that recognize an antigen.
An ELISA is a five-step procedure

  1. Coat the microtiter plate wells with antigen
  2. Block all unbound sites to prevent false positive results
  3. Add antibody to the wells
  4. Add anti-mouse IgG conjugated to an enzyme
  5. Reaction of a substrate with the enzyme to produce a colored product, thus indicating a positive reaction

There are many different types of ELISAs. One of the most common types of ELISA is “sandwich ELISA.”

Automated Radioimmunoassays

Radioimmunoassay (RIA):
RIAs are commonly used to measure the levels of hormones in blood and tissue fluids while ELISA assays are commonly used in viral diagnostics. For both these methods one needs a pure preparation of a know antibody or antigen, or both, in order to standardize the assay. In RIA for an antigen, pure antibody against that antigen is radioactively labeled, for the ELISA, an enzyme is linked chemically to the antibody. With either of these assays, the liquid handling steps are similar so that most systems may be designed to run multiple assays of either type.

Dynamic Devices has developed a fully automated liquid handling robot for Radioimmunoassay (RIAs) from its history with motion control and liquid handling. This flexible and scalable RIA workstation is configured with shielded radiation containers (lead lined), standard reagent reservoirs, microplate positions, microplate washer and a microplate reader.

To gain high throughput from this liquid handling system, it is configured with a 96 channel head that has the ability to pick up rows or columns for standards and controls addition to the plate, serial dilutions and flexible pipetting. In this system, a single pipet tip is attached and removes an aliquot of radioactive labeled compound and adds it to a trough to dilute the radioactive-tag for a single plate. The 96 head then attached 8 tips and draws out a row of aliquots to add to the microplate in multi-dispensing mode. In this way, the 96 head may make multiple copies of the solution or sample plate as opposed to using an 8 channel arm to continually work in a lower throughput fashion.

RIA System
d2 ria assay 1

d2 ria assay 2

Automated Liquid Handling Robot System Configuration:

  1. Oasis LM 1200 with 96 Head and Advanced Gripper
  2. BioTek Washer
  3. PE MicroBeta Reader
  4. BioTek Synergy Reader
  5. Cytomat Incubator & Storage

The RIA automated liquid handling robot configuration can be slightly modified to accomplish the following assays requiring incubation, washing or reading:

Enzyme-linked immunosorbent assays (ELISAs)
Enzyme immunoassays (EIAs)
Radioimmunoassays (RIA) without on-line radioactive detection
Fluorescence immunoassays (FIA)

d2 ria assay deck

Serial Dilutions

Serial dilution pic1

Serial Dilutions are easy on the Dynamic Devices Liquid Handling Robot Platform. You may first use the 96 head to transfer diluent to a destination plate, this diluent can be a different volume for each row or the same across the plate depending on your dilution scheme. You have the choice of using our 96 standard head and a single row of tips for the different volumes or our 96 VVP head where the volume may be different in each tip or row. If your samples are starting out in microplates or microtube format, the 96 channel head can attach a single row of tips and remove 8 samples to transfer to the dilution plate. The head then mixes the sample and diluent by aspirating and dispensing, and transfers the next volume to the next row and mixes. This process can be repeated as many times as necessary or a new sample row can be placed on the plate.

The head can also transfer the first or final row in the plate from a different source if standards and controls are necessary for an assay.

The liquid handling robot also has the ability and deck capacity to make each plate its own serial dilution, where all the samples are transferred into diluent at once, mixed and transferred again to another microplate for the next series of dilutions. In this process, throughput is increased by handling 12 rows at a time as opposed to one. It is also possible to transfer 11 rows, or 88 samples, if standards and controls need to be added to each assay plate.

For further optimized serial dilutions, our 96 VVP Head can dispense different volumes of dilution buffer in each well (96 in a single pass) and then use a row of single tips to transfer 8 different volumes of samples to the plate. The 96 VVP Head then continually dilutes the samples across the plate using 8 individual volumes for every transfer leaving the plate with 8 different serial dilutions according to a spreadsheet or operator input.