Development of an antigen microarray for high throughput monoclonal antibody selection

Abstract

Monoclonal antibodies are valuable laboratory reagents and are increasingly being exploited as therapeutics to treat a range of diseases. Selecting new monoclonal antibodies that are validated to work in particular applications, despite the availability of several different techniques, can be resource intensive with uncertain outcomes. To address this, we have developed an approach that enables early screening of hybridoma supernatants generated from an animal immunised with up to five different antigens followed by cloning of the antibody into a single expression plasmid. While this approach relieved the cellular cloning bottleneck and had the desirable ability to screen antibody function prior to cloning, the small volume of hybridoma supernatant available for screening limited the number of antigens for pooled immunisation. Here, we report the development of an antigen microarray that significantly reduces the volume of supernatant required for functional screening. This approach permits a significant increase in the number of antigens for parallel monoclonal antibody selection from a single animal. Finally, we show the successful use of a convenient small-scale transfection method to rapidly identify plasmids that encode functional cloned antibodies, addressing another bottleneck in this approach. In summary, we show that a hybrid approach of combining established hybridoma antibody technology with refined screening and antibody cloning methods can be used to select monoclonal antibodies of desired functional properties against many different antigens from a single immunised host.

Keywords: High throughput; Hybridoma; Monoclonal antibodies; Protein microarray.

Fig. 1

Establishment of the optimal printing and storage conditions for biotinylated antigen arrays in streptavidin-coated 96-well plates. (A) Schematic representation of a 96 well plate with a 5 × 5 antigen array. (B–L) A purified biotinylated antibody (OX68-bio) was spotted in streptavidin-coated 96 well plates. (B and C) Detection with either a non-fluorescent alkaline phosphatase-conjugated secondary antibody followed by a precipitating colourimetric NBT/BCIP substrate (B); and, with a fluorescent Alexa 568-conjugated secondary antibody (C). (D) Different concentrations of OX68-bio, 1 = 0.5; 2 = 0.25; 3 = 0.1; 4 = 0.05 μg/μl were arranged vertically, and in quadruplicate horizontally. (E) Different amounts of Tween 20 added prior to printing, 1 = 10%; 2 = 1%; 3 = 0.1%; 4 = None. (F) Different amounts of glycerol added prior to printing, 1 = 10%; 2 = 1%; 3 = 0.1%; 4 = 1% Tween 20. (G) Incubation of different concentrations of spotted antibody (as in (D)) for 2 h at room temperature with a 4% formalin solution. Storage of a printed plate for 2 days at room temperature (H), and at −20 °C (I), prior to antigen detection. Examples of: a 25-spot array using 0.4 mm pins, 0.66 mm pitch (J); a 53-spot array using 0.2 mm pins, 0.45 mm pitch (K), and a 101-spot array using 0.2 mm pins, 0.32 mm pitch. In panels D–L printed antibody was detected with an Alexa-488 conjugated secondary antibody.

Fig. 2

Antigen microarrays enable early screening of antibody specificity. (A) Twenty different recombinant biotinylated zebrafish cell surface and secreted proteins were purified and arrayed as shown, together with three control spots (No. 21). (B) An example of a positive, specific staining signal on antigen 19 (Fgfr1). (C) An example of a cross-reactive antibody that bound two paralogous antigens arrayed in position 10 (Pcam) and 18 (Ncam). (D) An antibody recognising the recombinant protein tags common to all arrayed proteins. (E) A smaller array with 11 different antigens and four control spots that was used to identify antibodies against: (F), antigen 4 (Robo1); (G), antigen 7 (Brevican), and (H), antigen 8 (Ncam2).

Fig. 3

Small scale transfection of pooled plasmids facilitates the detection of those plasmids encoding functional recombinant antibodies. Plasmid DNA was purified from 96 bacterial colonies and pools of eight clones from each column were made. (B) All twelve pools were transfected into small HEK293 cell cultures and the resulting supernatants tested by ELISA, positive pools were from columns 1, 3, 4, 8 and 9. Positive (+ve) control is the anti-Cd4 antibody used to detect the Cd4-tag on the immobilised recombinant antigen. (C) Transfections and ELISAs from individual clones from columns 4 and 8 were repeated to identify plasmids 4E and 8C as encoding functionally-positive antibodies.