Kinetics of immunoassays with particles as labels: effect of antibody coupling using dendrimers as linkers

Abstract

Received 5 DOI:In this communication, we report on poly(amido amine) dendrimers (PAMAM) as coupling agents for recombinant single-chain (ScFv) antibodies to nanoparticle (NP) labels, for use in immunoassay. We present a simple theory for the kinetics of particle capture onto a surface by means of an antibody-antigen reaction, in which the important parameter is the fraction of the particle surface that is active for reaction. 10 We describe how increasing the generation number of the linking dendrimer significantly increased the fraction of the NP surface that is active for antigen binding and consequently also increased the assay kinetic rates. Use of dendrimers for conjugation of the NP to the antibody resulted in a significantly higher surface coverage of active antibody, in comparison with mono-valent linker chemistry. As a direct consequence, the increase in effective avidity significantly out-weighed any effect of a decreased 15 diffusion coefficient due to the NP, when compared to that of a molecular dye-labelled antibody. The signal to noise ratio of the G4.5 dendrimer-sensitised nanoparticles out-performed the dye-labelled antibody by approximately four-fold. Particle aggregation experiments with the multi-valent antigen CRP demonstrated reaction-limited aggregation whose rate increased significantly with increasing generation number of the dendrimer linker.

Synthesis and characterisation of far-red fluorescent cyanine dye doped silica nanoparticles using a modified microemulsion method for application in bioassays

“…Changing chemical properties of the surfactants in the micelle, such as charge and packing, provide additional control over NP nucleation pathway and size [16,17]. Silica nanoparticles prepared using the microemulsion method are monodispersed and have been used extensively in immunoassays where they exhibit good reproducibility [16,18,19]. Furthermore, hydrophilic cancer drugs have been loaded into silica NPs for site-specific drug delivery in the treatment of tumour cells [20].…”

TIRF microscopy as a screening method for non-specific binding on surfaces

“…We have recently reported that fluorescent nanoparticles are very useful as labels in immunoassays to achieve improvements both in assay kinetics and sensitivity [7][8][9]. However, these particles typically have a negatively charged surface due to surfactants used to stabilize them and avoid aggregation [10,11].…”

“…TIRF allows surfaceparticle interactions to be investigated, as it excites only particles within the evanescent field of the excitation light, providing surface specificity and improving signal to noise compared with conventional microscopy [5]. In this work, we use TIRF in a novel way to screen surfaces for the levels of non-specific binding in a particular system.We have recently reported that fluorescent nanoparticles are very useful as labels in immunoassays to achieve improvements both in assay kinetics and sensitivity [7][8][9]. However, these particles typically have a negatively charged surface due to surfactants used to stabilize them and avoid aggregation [10,11].…”

Polymer-antibody fragment conjugates for biomedical applications

“…ScFvshave also been tethered tonanoparticle labels via PAMAM dendrimers using amine coupling chemistry, with the scFv complex exhibiting an approximately four-fold higher signal to noise ratio than the dye-labelled scFv in immunoassays [165]. As discussed above (Section 3.2), the conjugation of anti-hen egg white lysozyme scFvs to NHS-activated poly(N-isopropylacrylamide) has been used to develop a novel strategy to achieve affinity separation of biomolecules in solution as opposed to on solid supports traditionally used in immunoaffinity chromatography applications [146].…”