An ELISA also exploits the binding reaction between binding partners, one of which is labelled to allow detection. Many immunoassays are based on the reaction between enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (AP), and a substrate, e.g. tetramethyl benzidine (TMB) or p‐nitrophenylphosphate. The reaction leads to the formation of a coloured product which can be readily detected spectrophotometrically. Other enzymes have been developed which give fluorescent products, and the fluorescence intensity is measured instead. The amount of analyte present in the sample can be determined by reading off a calibration curve generated using a series of standard solutions. ELISA assays can be very sensitive.
The commercial ELISA kit used in this work for the detection of domoic acid was an inhibition assay, where free domoic acid in the sample competed with domoic acid‐ conjugated protein coated on to a solid phase, typically a plastic well, for binding to anti‐domoic acid antibody (a polyclonal ovine anti‐domoic acid antibody) free in solution. The solution containing domoic acid was incubated in the wells with the anti‐domoic acid antibody‐horseradish peroxidase conjugate. The wells were washed, and the amount of antibody bound to the well was measured by incubation with a substrate which gave a blue product upon reaction with the horseradish peroxidase enzyme. Addition of acid stopped the reaction and induced a colour change in the product which could be measured spectrophotometrically.
Chapter 3
Surface
Plasmon
Resonance
of
Estrone
Analogues
The main focus and central theme of this study has been to probe means of improving the sensitivity of surface plasmon resonance (SPR) assays. One aim of the work described in this chapter was to investigate the effect of the linker length and characteristics on binding response using estrone as a model compound. The oligo(ethylene glycol) linker was appended to estrone glucuronide, and to a short acyclic linker already appended to estrone, and the binding response and sensitivity of the two estrone derivatives compared to the estrone derivatives without the oligo(ethylene glycol) linker. A second aim was to probe signal enhancement by secondary antibodies and gold nanoparticles, using estrone as a model system and, using these strategies, to develop an assay which could detect the steroid in the pg/mL range necessary for analyses of wastewaters. Estrogenic compounds were chosen, because the steroid estrone (E1) is an important reproductive hormone and because highly sensitive methods of detection are required for analyses of wastewaters.
The sensitivity of a surface plasmon resonance (SPR) with an inhibition immunoassay is limited by the amount of antibody used – an assay with a lower antibody concentration added to analyte solutions will have a lower limit of detection. The level of antibody added to an analyte solution must be high enough so that sufficient un‐bound (so‐called “residual”) antibody remains in solution, which can then bind with the binding partner immobilised on the sensor surface and generate a signal. Methods which enhance the size of the signal allow the use of smaller amounts of antibody, producing a lower limit of detection.
One potential method of sensitivity improvement probed with the estrone system was signal enhancement using secondary antibodies and gold nanoparticles.
Estrone is a relatively small molecule, which generates a modest surface plasmon resonance response upon binding to a sensor surface, so a competitive inhibition assay (refer to Chapter 2) was designed. In this type of assay, a known, constant amount of anti‐estrone antibody is added to a solution of estrone (in the glucuronide form) and the residual un‐bound antibody in solution binds to the estrone derivative attached to the sensor surface. In this way, the higher molecular‐ weight antibody binds to the sensor surface rather than the very much smaller steroid, and a larger surface plasmon resonance response is generated. Using a second binding event, where a secondary antibody binds to the primary antibody already bound to the sensor surface will generate a second response.
Gold nanoparticles have been attached to the antibodies to amplify the surface plasmon resonance signal. The signal enhancement is thought to be due to two effects – not only does the high mass of the nanoparticle increase the signal size, but also the nanoparticle itself undergoes surface plasmon resonance and reinforces the signal. Gold nanoparticle signal enhancement has been used in several surface plasmon resonance assays [83, 84, 114, 134, 135]. Some earlier workers have attached a gold nanoparticle to a primary antibody to obtain signal amplification. In this work the nanoparticle was attached to the secondary antibody in the expectation of exploiting both of the enhancement effects mentioned above. Inserting an oligo(ethylene glycol) (OEG) linker between a steroid and the sensor surface (or between a steroid and the protein which is then attached to the sensor surface) has been shown to improve greatly antibody binding in the case of progesterone [76]. Oligo(ethylene glycol) (OEG) linkers are hydrophilic, so much so that their use in a conjugate to elicit an immune response for antibody generation has been found to reduce the antibody production, by effectively covering the protein with a hydrophilic shell of oligo(ethylene glycol) [136, 137]. In biosensing, OEG linkers can prevent protein absorption, avoiding fouling of the sensor surface [138‐140]. This ability to hinder unwanted protein adsorption has been used to good effect in self‐assembled monolayers [141‐143]. Linker insertion is thought to
aid antibody binding by orientating the steroid into the flow of aqueous buffer. The assumption has been that intermediate linkers diminish potential steric obstruction when a relatively large antibody binds to much smaller surface‐bound partner. Insertion of a linker and gold nanoparticle signal enhancement of primary antibody binding has been exploited in the analysis of estradiol [78] and insertion of a linker and secondary antibody signal enhancement was used to enhance sensitivity in the analysis of the steroid hormone cortisol [82]. Many workers have prepared gold nanoparticles using the classical methods of Frens [144] and Turkevich [145]. Commercial gold nanoparticles were used in all work described here to minimise the possibility of aggregation of the gold. Aggregation could potentially compromise the performance of the micro‐fluidics in the surface plasmon resonance instrument. In this chapter, the investigations of effects of gold nanoparticle enhancement and linker chemistry on the surface plasmon resonance detection of estrone glucuronide are described. The estrone sensing elements used were in the form of ovalbumin protein conjugates immobilised on to carboxymethylated dextran surfaces (Figure 9). O O R H N O O O H N O HO2C O HO HO HO O O HO HO HO2C HO R2 = R1 = H N O O O O H N O HO2C H N O R4 = R3 = O O R OVA R1 = (8) E1G C24H30O8 Mol. Wt.: 446.49 R2 = (9) E1C3, C5 C28H39NO5 Mol. Wt.: 469.61 R3 = (10) E1G, OEG C38H56N2O13 Mol. Wt.: 748.86 R4 = (11) E1C3, OEG C36H54N2O9 Mol. Wt.: 658.82 R1 = (12) E1G‐OVA R2 = (13) E1C3, C5‐OVA
R3 = (14) E1G, OEG‐OVA
R4 = (15) E1C3, OEG‐OVA
HO2C
Figure 9: The estrone derivatives and their corresponding ovalbumin conjugates used in sensor surfaces for