Rapid detection of antibodies in sera using multiplexed self-assembling bead arrays - PubMed (original) (raw)
Comparative Study
Rapid detection of antibodies in sera using multiplexed self-assembling bead arrays
Jessica Wong et al. J Immunol Methods. 2009.
Abstract
Rapid detection of antibody immunity in serum or plasma, whether to pathogenic antigens, tumor antigens, or autoimmune antigens, is critical for diagnosis, monitoring, and biomarker assessment of the immune response. Individual or multiplexed ELISAs that use purified recombinant proteins are dependent on a priori protein purification, a labor-intensive process that may take months to obtain proteins of sufficient purity and stability for serologic assays. We developed a programmable multiplexed immunoassay for the rapid monitoring of humoral immunity using the Luminex suspension bead array platform. In this approach, epitope-tagged antigens (GST- or FLAG-tagged) are expressed using in vitro transcription and translation, and captured onto anti-epitope-coupled Luminex SeroMap beads. The antigen-loaded beads are mixed, serum is added, and human IgG is detected with standard secondary detection reagents. By coupling high-throughput DNA preparation of cDNA ORFs with antigen expression/capture, we demonstrate that 71/72 (98.6%) of GST-tagged proteins can be expressed and specifically detected on the bead ELISA. Detection of antibodies to the test viral antigen EBNA-1 in human sera is highly reproducible, with intra-assay variation of 3-8%, inter-assay variation of 5%, and with stability over 11 months. The specificity and limits of detection of the bead ELISAs for the tumor antigen p53 are comparable to both standard protein ELISAs and plate-based programmable (RAPID) ELISAs, and are also comparable to the detection of directly-conjugated p53 protein. Multiplexing a panel of analytes does not impair the sensitivity of antibody detection. Immunity to a panel of EBV-derived antigens (EBNA-1, EBNA-3A, EBNA-3B, and LMP-2) is specifically and differentially detected within healthy donor sera. This method allows for rapid conversion of ORFeome-derived cDNAs to a multiplexed bead ELISA to detect antibody immunity to both infectious and tumor antigens.
Figures
Figure 1. Schematic of the bead ELISA for the detection of antibodies in serum
The cDNAs encoding full-length antigens are individually expressed as GST-tagged fusion proteins using rabbit reticulocyte lysate. Recombinant proteins are captured onto anti-GST-coupled Luminex microspheres. The beads displaying different antigens are pooled and re-aliquoted prior to addition of human serum so that there are 5000 of each color/antigen bead per serum. Human IgG is detected with PE-conjugated secondary anti-IgG antibodies.
Figure 2. Protein expression, stability, and specificity of the bead ELISA
A) Specific detection of 72 captured GST-tagged tumor antigens on beads. DNA was prepared using a high-throughput robotic preparation method, expressed in vitro, bound to anti-GST-coupled beads, and detected using an anti-GST MAb. Background expression (10% above the average of non-antigen containing beads) is shown as a dotted line. B) Anti-GST-coupled beads stored over 11 months showed no change in the stability of bound IgG using donkey anti-goat IgG-PE antibody. C) EBNA-1 antibody-positive serum from a healthy donor was tested for anti-EBNA-1 antibodies using the bead ELISA. EBNA-1-GST and p21-GST fusion proteins were expressed by IVTT and bound to anti-GST-coupled Luminex beads. Serum was added in the titrations shown for either 30 minutes at rt or at 4°C overnight. P21-GST and no DNA controls were tested at 1:20 dilution. Incubation overnight increased the sensitivity of the assay 8-fold to >1:20,000, and optimal serum dilution for maximal detection was at 1:80–1:320.
Figure 2. Protein expression, stability, and specificity of the bead ELISA
A) Specific detection of 72 captured GST-tagged tumor antigens on beads. DNA was prepared using a high-throughput robotic preparation method, expressed in vitro, bound to anti-GST-coupled beads, and detected using an anti-GST MAb. Background expression (10% above the average of non-antigen containing beads) is shown as a dotted line. B) Anti-GST-coupled beads stored over 11 months showed no change in the stability of bound IgG using donkey anti-goat IgG-PE antibody. C) EBNA-1 antibody-positive serum from a healthy donor was tested for anti-EBNA-1 antibodies using the bead ELISA. EBNA-1-GST and p21-GST fusion proteins were expressed by IVTT and bound to anti-GST-coupled Luminex beads. Serum was added in the titrations shown for either 30 minutes at rt or at 4°C overnight. P21-GST and no DNA controls were tested at 1:20 dilution. Incubation overnight increased the sensitivity of the assay 8-fold to >1:20,000, and optimal serum dilution for maximal detection was at 1:80–1:320.
Figure 2. Protein expression, stability, and specificity of the bead ELISA
A) Specific detection of 72 captured GST-tagged tumor antigens on beads. DNA was prepared using a high-throughput robotic preparation method, expressed in vitro, bound to anti-GST-coupled beads, and detected using an anti-GST MAb. Background expression (10% above the average of non-antigen containing beads) is shown as a dotted line. B) Anti-GST-coupled beads stored over 11 months showed no change in the stability of bound IgG using donkey anti-goat IgG-PE antibody. C) EBNA-1 antibody-positive serum from a healthy donor was tested for anti-EBNA-1 antibodies using the bead ELISA. EBNA-1-GST and p21-GST fusion proteins were expressed by IVTT and bound to anti-GST-coupled Luminex beads. Serum was added in the titrations shown for either 30 minutes at rt or at 4°C overnight. P21-GST and no DNA controls were tested at 1:20 dilution. Incubation overnight increased the sensitivity of the assay 8-fold to >1:20,000, and optimal serum dilution for maximal detection was at 1:80–1:320.
Figure 3. p53-specific IgG from human sera are specifically detected
A) Detection of antibodies to p53 by bead ELISA (Luminex) and standard ELISA in a breast cancer patient serum with p53 antibodies. B) Detection of p53 antibodies in the same serum with IVTT-generated p53 is comparable to that with recombinant p53 protein directly captured onto beads.
Figure 3. p53-specific IgG from human sera are specifically detected
A) Detection of antibodies to p53 by bead ELISA (Luminex) and standard ELISA in a breast cancer patient serum with p53 antibodies. B) Detection of p53 antibodies in the same serum with IVTT-generated p53 is comparable to that with recombinant p53 protein directly captured onto beads.
Figure 4. Detection of p53 antibodies by bead ELISA (Luminex) and RAPID (96-well format) ELISA
A) Quantification of p53 antigen detection by bead ELISA and RAPID ELISA. P53-GST fusion protein was expressed by bead ELISA and RAPID ELISA, and detected with titrations of an anti-p53 MAb. B) i–iv. Detection of anti-p53 antibodies by bead ELISA and RAPID ELISA from 4 breast cancer patient sera containing p53 autoantibodies.
Figure 4. Detection of p53 antibodies by bead ELISA (Luminex) and RAPID (96-well format) ELISA
A) Quantification of p53 antigen detection by bead ELISA and RAPID ELISA. P53-GST fusion protein was expressed by bead ELISA and RAPID ELISA, and detected with titrations of an anti-p53 MAb. B) i–iv. Detection of anti-p53 antibodies by bead ELISA and RAPID ELISA from 4 breast cancer patient sera containing p53 autoantibodies.
Figure 5. Multiplexed detection of antibodies in patient sera
The p53 autoantibodies were detected by bead ELISA using p53-loaded anti-GST beads either in singleplex or in multiplex with 7 other antigen-loaded beads, including a vector control, BCL2, CDK4, CHEK2, p21, PCNA, and PKB . The p53 autoantibody detection was not diminished by the addition of other antigen-loaded beads.
Figure 6. Multiplexed detection of antibodies by bead ELISA
CDK4, p53, BCL2, and PCNA antigens and a vector control were individually expressed by IVTT as GST fusion proteins, bound to anti-GST beads, and pooled. A) Within the pooled beads, individual antigens could be specifically detected with monoclonal antibodies specific to PCNA, BCL2, p53, and CDK4 at a 1:400 dilution. B) The monoclonal antibodies specific for each antigen were then pooled in different combinations: mix 2 (BCL2 and p53 Mab’s), mix 3 (PCNA, BCL2, and CDK4 Mab’s), and mix 4 (all 4 Mab’s). Each antigen could be specifically detected within the pooled antigens without loss of sensitivity or specificity.
Figure 7
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