Using HydroSpeed™ Plate Washer and Infinite® F50 Absorbance Reader for Fast and Efficient Processing of ELISA Assays

Introduction

This article describes the results of a successful investigation of Tecan’s advanced HydroSpeed plate washer and Infinite F50 absorbance reader by means of a HBs Ag (hepatitis B viral surface antigen) assay. An example of a qualitative, one step ‘sandwich’ type enzyme immunoassay, the HBs Ag assay can be used to detect HBs Ag in human plasma or serum (1).

Tecan’s advanced HydroSpeed plate washer

Tecan’s Infinite F50 absorbance reader

Hepatitis B virus (HBV) causes hepatitis B, which is an infectious disease. When HBV infects the human liver, it causes an inflammation. The virus is transmitted by exposure to infected body fluids such as infected blood. HBV infects one third of the world’s population.

The HBs Ag in human plasma or serum is detected by the HBs Ag assay. It is the first marker to appear post infection, and can be detected two or three weeks prior to the first biological and clinical symptoms of the disease(1). The detection of HBV is a routine yet important clinical test that is carried out worldwide, as hepatitis B is a serious transfusion hazard.

To prevent HBs Ag transmission during a transfusion, it is important that it is detected at the time of blood donation.

The new HydroSpeed plate washer from Tecan has advanced features for many applications including magnetic bead separation, vacuum filtration, and washing of ELISA- and cell-based assays in 96- and 384-well plate format.

The HydroSpeed’s automated Anti-Clogging function provides a reliable operation, and during idle time between plates, prevents needle blockage that is often caused by crystallization of the wash buffer.

Tecan’s Infinite F50 absorbance reader is an advanced 8-channel absorbance microplate reader that delivers accurate, fast, and reproducible measurements, and has also set a new benchmark in innovative and compact design. The Infinite F50 is ideal for various ELISA applications as it works with Tecan’s user-friendly control and data analysis software, Magellan.

For ELISA-based assays, the Infinite F50 absorbance reader and HydroSpeed plate washer are a perfect combination, providing accurate data acquisition/analysis and high throughput capacity and satisfying the key requirements for research and clinical laboratories.

Materials and methods

Instruments

Infinite F50 absorbance reader

HydroSpeed plate washer is fitted with the 96-HT or 96-indexing wash head for ELISA and cell washing

Microplate

NUNC® MaxiSorp 96-well strip plate (provided with the kit)

Reagents

HBs Ag ELISA kit

Assay procedures

A minute part of the surface antigen of the HBV at low sample concentrations is detected by the HBs Ag ELISA. The ELISA assay employs tetramethylbenzidine (TMB) and an alkaline wash buffer for detecting color. Detailed information and an explanation of the principle of this ELISA assay is given in the assay description(1).

The manufacturer’s assay manual was followed for performing the assay, using the kit’s positive and negative controls as well as 76 negative and 15 positive patient serum samples, as shown in Figure 1.

The HydroSpeed plate washer fitted with the 96i wash head or the 96-HT wash head was used to perform all washing steps, and the Infinite F50 absorbance reader was used to perform the readout. Figure 2 and Table 1 show complete information on the washing program and the measurement settings.

Figure 1. Plate layout. NC1-4 = negative control; PC1 = positive control; Pos = positive patient samples; Neg = negative patient samples.

Figure 2. Wash program for the HydroSpeed plate washer equipped with the 96-HT wash head. For the 96i wash head the same program with a wash rate of 180 μl/s was used.

Measurement settings and wash program

Table 1. Measurement parameters for Infinite F50 absorbance reader

Measurement parameters

Plate definition file

NUN96ft.pdfx

Wavelength

450 nm

Reference wavelength

620 nm

 

Results and discussion

Interpretation of assay results (according to the kit manual):

  1. All negative control values (NC1 - NC4) should provide an absorbance value of ≤ 0.08 OD
  2. All positive controls should provide an absorbance value of ≥ 1.0 OD
  3. Cut-off value: NCmean + 0.05
  4. Calculation of ratio: OD of sample / OD of cut-off

Ratio > 1: sample is positive

Ratio < 0.9: sample is negative

Ratio > 0.9 and < 1: sample is negative but has to be tested again

The raw data and final cut-off results, acquired from Magellan software by means of the HydroSpeed plate washer with the 96-HT wash head, are shown in Figures 3 and 4. All positive controls and all negative controls show absorbance values above 1.0 OD and absorbance values below 0.08 OD, respectively.

No false positive or false negative results were produced as all positive and negative patient samples were accurately detected. No retesting was done on any of the samples. Results for the 96i wash head were similar to the data listed above (results not shown).

Figure 3. Magellan - raw data.

Figure 4. Magellan - cut-off results.

Note: All positive patient samples were used with no pre-dilution of the probe. Thus, raw data values are expressed as ‘Overflow’ (OD > 4). Consequently, these wells were not marked red, though positive samples are typically indicated as such.

Conclusion

This article presented results that clearly show that the Infinite F50 absorbance reader and the HydroSpeed plate washer are ideal for quick and efficient processing of ELISAs, such as a HBs Ag assay.

The HydroSpeed plate washer provides excellent washing performance, and enables handling of critical samples, such as the undiluted serum probes used in this investigation. High throughput ELISA washing is also provided by the HydroSpeed plate washer, thanks to the 96-channel wash head.

This matches the concept of the Infinite F50 absorbance reader, which is integrated with an 8-channel absorbance optic that allows ultra-fast absorbance measurements of less than 20 seconds for a 96-well plate.

Literature

  1. www.biorad.com

List of Abbreviations

  • HBs Ag             surface antigen of Hepatitis B virus
  • NC                   negative control
  • Neg                  negative patient samples
  • PC                    positive control
  • Pos                  positive patient samples
  • TMB                 tetramethylbenzidine
  • OD                   optical density
  • HT                    high throughput

Acknowledgements

We would like to express our thanks to Mag. Dr. Tobias Kiesslich and Josef Lang (Department of Internal

Medicine I, Paracelsus Medical University and SALK, Salzburg, Austria) for their collaboration and for performing these experiments.

About Tecan

Tecan

Tecan is a leading global provider of automated laboratory instruments and solutions. Their systems and components help people working in clinical diagnostics, basic and translational research and drug discovery bring their science to life.

In particular, they develop, produce, market and support automated workflow solutions that empower laboratories to achieve more. Their Cavro branded instrument components are chosen by leading instrumentation suppliers across multiple disciplines.

They work side by side with a range of clients, including diagnostic laboratories, pharmaceutical and biotechnology companies and university research centers. Their expertise extends to developing and manufacturing OEM instruments and components, marketed by their partner companies. Whatever the project – large or small, simple or complex – helping their clients to achieve their goals comes first.

They hold a leading position in all the sectors they work in and have changed the way things are done in research and development labs around the world. In diagnostics, for instance, they have raised the bar when it comes to the reproducibility and throughput of testing.

In under four decades Tecan has grown from a Swiss family business to a brand that is well established on the global stage of life sciences. From pioneering days on a farm to the leading role our business assumes today – empowering research, diagnostics and many applied markets around the world


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Last updated: Jun 14, 2017 at 5:56 AM

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