Antibody Screening and Pair Selection using the Bio-Plex® Multiplex Immunoassay System


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The HuCAL® antibody generation process can be tailored to select antibodies that are best suited for applications for use on a bead-based multiplex platform, such as the Bio-Rad Bio-Plex Multiplex System instruments, or other platforms using Luminex xMAP technology. As part of a custom antibody project, we can also use multiplex technology for the high-throughput screening of large numbers of antibodies in order to select the best pairs for immunoassays.

Application specific primary screening

When primary screening is carried out on antigen-coated beads, rather than antigens immobilized on microtiter plates, the likelihood of selecting antibodies suitable for use in applications on the Bio-Plex System, or other multiplex instrumentation is increased. The high-throughput screening is performed using antibody-containing E. coli lysates in 384-well format. The throughput is the same as for primary screening by ELISA, where the antigen is absorbed to the bottom of microtiter plates.

Primary screening example data

Panning was performed on a customer supplied viral protein antigen coupled to Bio-Plex beads. Primary screening in 384-well format was performed on the Bio-Plex 3D Suspension Array System. One hundred and thirty nine antibody candidates were identified that showed binding to the bead-coupled antigen of interest but did not recognize two control antigens (BSA and a closely related viral protein), also coupled to beads. Figure 1 shows the results as a 3D column chart. Twenty hits were sequenced, 8 unique antibodies were found, and all hits were confirmed in quality control ELISA.

Antibody generation project primary screening with Bio-Plex 3D

Fig. 1. Results of primary screening using the Bio-Plex 3D Suspension Array System.

High-throughput identification of antibody pairs

We have developed a method for automated coupling of HuCAL technology generated antibodies to Bio-Plex microspheres, enabling high-throughput identification of antibody pairs on the Bio-Plex 3D System. The multiplex nature of the technology enables identification and titration of antibody pairs simultaneously, on the same plate. Further confirmation of the pairs selected using the Bio-Plex System can be done using standard ELISA in microtiter plates, if desired.

Antibody pair screening and titration example data

The multiplex nature of the Bio-Plex 3D System technology allows the simultaneous identification and titration of antibody pairs on the same plate. It is possible to screen 30 x 30 pairs as 10-point titrations per 384-well plate (Figure 2).

Titration of antibody pairs using Bio-Plex 3D System.

Fig. 2. Ten-point titrations of 12 x 12 anti-etanercept antibody pairs using the Bio-Plex 3D System. Capture and detection antibody concentrations are kept constant, the etanercept antigen is titrated to generate a ten-point standard curve.


  • Primary screening using the Bio-Plex 3D System increases the likelihood of identifying antibodies suitable for use on this and similar bead-based multiplex platforms
  • Automated coupling of antibodies to beads allows high-throughput screening for sandwich ELISA antibody pairs (up to 30 x 30 pairs in one plate, including titration)
  • Simultaneous identification and titration of antibody sandwich pairs can be performed on the same 384-well plate
  • Using the Bio-Plex 3D System enables a more than 6-fold increase in the number of antibody pairs screened per 384-well plate compared to a standard ELISA method
  • Assay-specific variations can occur, therefore thorough antibody pair characterization is recommended for the application of choice

Your technical sales specialist can guide you through the details of the service we offer, and provide a quote tailored to your needs.