Technique / Proteomics and protein biochemistry / Protein Array Analysis

We have demonstrated a microarray format immunoassay using HydroGel-coated slides. HydroGel is a porous substrate based on a polymer matrix that provides a three-dimensional hydrophilic environment similar to free solution suitable for biomolecular interactions. This substrate has been used to develop fluorescence-based multiplexed cytokine immunoassays. Forty-three monoclonal antibodies (mAbs) of cytokines and chemokines are printed at a volume of 350 pL per spot using a PerkinElmer BioChip Arrayer. Cytokines that are captured by the arrayed mAb are detected using another biotinylated mAb, following by the addition of a Texas red conjugated streptavidin. The fluorescent images of arrays are recorded using a PerkinElmer ScanArray 5000 confocal slide scanner and quantitated using PerkinElmer QuantArray software. Experiments demonstrate that 43 cytokines can be simultaneously screened and quantitated in conditioned culture media, cell lysate, and human plasma. Using this chip, we have examined cytokine expression in breast cancer cells and identified the chemokines associated with human cervical cancers.

To reduce the huge labor-cost in the screening in traditional monoclonal antibody generation, We established a new system for monoclonal antibody generation integrating with protein array. BALB/c mice were immunized by eight recombinant proteins respectively, and the positive hybridoma cells were obtained by cell fusion and ELISA screening. All the eight kinds of positive hybridoma cells were mixed, cloned, screened by protein array, and definite dilution cloned. RESULTS: 175 single cell clones were obtained by complex cloning, and 119 of those were positive clones. Then 8 positive cell lines were generated by the following 2 rounds definite dilution cloning. By comparing with the traditional method, we got 8 monoclonal antibodies using the combined protein array screening and multiplex cloning method in 1 cycle, and fewer amounts of antigens were used. As a result, the combined protein array and multiplex cloning method could be used as an economical, rapid and simple tool applying in high throughput monoclonal antibody generation.

Protein patterning on solid surfaces is a topic of significant importance in the fields of biosensors, diagnostic assays, cell adhesion technologies, and biochip microarrays. In this letter, we have established a novel, rapid method for the fabrication of a "protein recording material", which enables us to spatiotemporally regulate the recording, reading, and erasing of a fluorescent protein array as information by a photochemical technique. A photolinker that we synthesized here was used to control the protein array spatiotemporally. The recording process was almost completed after 1 min of photoirradiation to read a clear pattern consisting of a specific protein-ligand complex with high spatiotemporal resolution. The erasing of the protein array was then achieved by photoirradiation onto the entire patterned surface.