Name: comparative analysis of human growth hormone in serum using spri, nano-spri and elisa assays
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Description: Watch this Scientific Journal Video about Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays at JoVE.com

NanoManufacturing Innovation Consortium for funding support.

1. Preparation of Solutions and Protein Samples for SPRi
<ol>
	<li>Prepare 100 ml of low salt phosphate buffered saline (PBS) solution containing 10 mM phosphate, 150 mM sodium chloride, and pH 7.4.</li>
	<li>Prepare 50 ml of high salt PBS solution containing 10 mM phosphate, 750 mM sodium chloride, and pH 7.4.</li>
	<li>Prepare 5 ml of 10 mM sodium acetate.</li>
	<li>Prepare a stock of 5 mg/ml bovine serum albumin (BSA) diluted in low salt PBS solution.</li>
	<li>Prepare a stock solution of recombinant human growth hormone (rhGH) at a concentration of 1 &#956;g/ml in low salt PBS solution.</li>
	<li>Prepare stock solutions of anti-rhGH and negative control antibody at a concentration of 100 &#956;g/ml.</li>
</ol>
2. Prepare SPRi Chip for Antibody Array
<ol>
	<li>Clean the chip by sonication in 120 ml of stabilized piranha solution (3:1 concentrated sulfuric acid to 30% hydrogen peroxide) for 90 min at 50 &#176;C and follow by rinsing and sonication with water for 5 min. Then, rinse chip with ethanol and dry with nitrogen stream.</li>
	<li>Place the gold chip in an UV/Ozone chamber for 30 min to remove any contaminants.</li>
	<li>Add 150 mg of 11-mercaptoundecanoic acid to 20 ml ethanol in a test tube that contains a stir bar and a tube cap.</li>
	<li>Place chip in the test tube and microwave tube/chip at 50 W and 50 &#176;C for 5 min.</li>
	<li>Rinse chip with ethanol and soak in ethanol for 5 min.</li>
	<li>Follow by a rinse with water and soak in water for 5 min.</li>
	<li>Add 150 mg of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide to 20 ml water in a test tube that contains a stir bar and a tube cap. Microwave chip as in step 2.4.</li>
	<li>Rinse chip with water and soak in water for 5 min.</li>
	<li>Add 150 mg of N-hydroxysuccinimide in 20 ml water in a test tube that contains a stir bar and a tube cap. Microwave chip as in step 2.4.</li>
	<li>Rinse chip with water and soak in water for 5 min.</li>
	<li>Prepare 250 &#181;M polyethylene glycol (PEG, 800 Da) in 20 ml water in a test tube that contains a stir bar and a tube cap. Microwave chip as in step 2.4. 
	Note: Microwaving PEG800 on the surface of the sensor is performed as a blocking step to prevent non-specific binding of serum proteins and quantum dot complexes later used in the experiment. This is achieved by PEG800 reacting with non-occupied bare gold sites on the chip.</li>
	<li>Rinse chip with water and soak in water for 5 min.</li>
	<li>Prepare 15 &#956;g/ml of anti-rhGH antibody solution and negative control solution of anti-Immunoglobulin G (anti-IgG) and add 10 &#181;l of each antibody solution to a well in the 384 well plate.</li>
	<li>Design the spotting pattern in the microarrayer (4 x 7 quadrants for each sample) and spot chip with antibodies using a 500 &#956;m Teflon tipped pin.</li>
	<li>Incubate spotted chip for 2 hr at RT and under a humid atmosphere of 75% or greater.</li>
	<li>Rinse and soak chip in water for 5 min. Then, dry chip with nitrogen stream.</li>
</ol>
3. SPRi Experiment Setup and Blocking
<ol>
	<li>Initialize the instrument and select directory. Select real time camera and start the syringe pump at 1 ml/min for 10 ml of degassed water. Insert chip into the instrument and keep injecting water until all bubbles are removed.</li>
	<li>After rinsing chip with 10 ml of water, start image acquisition, and follow by switching the buffer to low salt PBS and allow it to run at 1 ml/min for 5 ml and then slow flow rate down to 20 &#181;l/min for 20 min.</li>
	<li>Select the highly contrasted image, which shows the immobilized antibodies spotted onto the chip.</li>
	<li>Select and define the 400 &#181;m individual circular spots corresponding to the immobilized antibodies.</li>
	<li>After the instrument traces the plasmon curves, choose a working angle that has the highest slope in the reflectivity curves.</li>
	<li>Flow a running buffer at 20 &#181;l/min of low salt PBS through the system until the signal from all of the selected spots stabilizes.</li>
	<li>Load the BSA (100 &#181;g/ml) in running buffer into the sample loop (150 &#181;l) with the injection valve in the load position. Then switch the valve to the injection position to inject the solution to the flow cell. 
	Note: BSA is injected to covalently bind to the amine reactive surface, the remaining unbound BSA will wash off the surface through buffer rinse.</li>
	<li>Inject 150 &#181;l of a solution of 10 mM sodium acetate over the surface and follow by a 150 &#181;l injection of high salt PBS.</li>
	<li>Inject 150 &#181;l of ethanolamine (1 mM) to deactivate the amine reactive surface.</li>
	<li>Inject 150 &#181;l of 10 mM sodium acetate to wash the surface.</li>
	<li>Then, switch the running buffer to PBS with 50 &#181;g/ml BSA at a flow rate of 250 &#181;l/min for a total of 5 ml. 
	Note: 50 &#181;g/ml BSA is added to the running buffer for additional blocking of the surface by non-covalently occupying the non-specific sites and this is done to further reduce non-specific binding of serum proteins to the surface.</li>
	<li>Change flow rate to 5 &#181;l/min and allow it to stabilize for 20 min.</li>
</ol>
4. SPRi Detection of Human Growth Hormone
<ol>
	<li>Prepare a solution of a set concentration of rhGH (30,000 pg/ml; 250 pg/ml; 25 pg/mL; 2.5 pg/ml and 0.25 pg/ml) in 10% serum and diluted in PBS containing 1 mg/ml BSA.</li>
	<li>Prepare a 2:1 solution by adding 1 &#181;l of biotin labeled anti-rhGH detection antibodies (6 &#181;M) to 3 &#181;l of streptavidin coated near infrared quantum dots (1 &#181;M) in a 0.5 ml microcentrifuge tube and incubate for 30 min for effective coupling.</li>
	<li>Inject 150 &#181;l of the hGH spiked human serum solution in the injection loop. This results in a strong increase in signal followed by a slow drop from the non-specifically bound serum rinsing off the surface.</li>
	<li>Once the signal stabilizes, wash the surface with a solution of 450 mM sodium chloride added to the running buffer.</li>
	<li>Dilute the solution of anti-rhGH detection antibodies and quantum dots to a concentration of 10 nM (2:1) with running buffer (PBS with 50 &#181;g/ml BSA) and inject into the flow cell.</li>
</ol>
5. SPRi Data Analysis
Note: Following the injection of the rhGH spiked human serum and quantum dot enhancer, an increase in reflectance change occurs due to the amplified shift of the plasmon curves. This results in a difference image showing a gray scale image correlating to the signal of on the chip.
<ol>
	<li>Import the data into a data analysis program. Plot the SPRi signal (% Reflectivity) versus time (s) and determine the difference between the anti-rhGH and negative control antibody spots after the high salt wash.</li>
</ol>
6. ELISA Protocol (Day 1)
<ol>
	<li>Store all assay components included in the rhGH ELISA kit at 2-8 &#176;C. This includes the anti-rhGH antibody coated well plates, standards (0-5) in sheep serum, controls (1 &#38;2) in human serum, conjugate buffer, (200x) wash buffer, chromogen TMB (Tetramethylbenzidine) and stop reagent.</li>
	<li>Setup the reagents and follow the methods as described in the commercial ELISA kit protocol.</li>
	<li>First, allow the anti-rhGH antibody coated 96-wells to warm to RT prior to opening the foil package.</li>
	<li>Then remove the desired number of 8-well strips for assay and reseal the foil package containing the remaining wells and store at 2-8 &#176;C.</li>
	<li>Prepare standards by reconstitution in 2 ml of distilled water for standard #0, in 1 ml of distilled water for standards (1-5), and in 1 ml of distilled water for the controls (1&#38;2). Below is a table of the corresponding concentrations of the standards and controls (Table 1):</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Standards</td>
			<td>Concentration (ng/ml)</td>
		</tr>
		<tr>
			<td>0</td>
			<td>0</td>
		</tr>
		<tr>
			<td>1</td>
			<td>0.17</td>
		</tr>
		<tr>
			<td>2</td>
			<td>0.94</td>
		</tr>
		<tr>
			<td>3</td>
			<td>2.63</td>
		</tr>
		<tr>
			<td>4</td>
			<td>10.4</td>
		</tr>
		<tr>
			<td>5</td>
			<td>26.9</td>
		</tr>
		<tr>
			<td>Control #1</td>
			<td>1.22 &#177; 0.33 ng/ml</td>
		</tr>
		<tr>
			<td>Control #2</td>
			<td>4.97 &#177; 1.25 ng/ml</td>
		</tr>
	</tbody>
</table>
Table 1. Concentrations of the standards and controls included in the commercial ELISA kit.
<ol start="6">
	<li>Prepare the samples which consist of the rhGH hormone in 10% human serum at the following concentrations (Table 2):</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample</td>
			<td>Concentration (ng/ml)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>0.025</td>
		</tr>
		<tr>
			<td>2</td>
			<td>0.2</td>
		</tr>
		<tr>
			<td>3</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>4</td>
			<td>2.5</td>
		</tr>
		<tr>
			<td>5</td>
			<td>10</td>
		</tr>
		<tr>
			<td>6</td>
			<td>30</td>
		</tr>
	</tbody>
</table>
Table 2. Concentrations of the rhGH samples prepared in 10% Serum.
<ol start="7">
	<li>Add 50 &#956;l of each standard, control and sample to each well (in triplicates). Seal the wells and incubate with the standards, controls, and rhGH samples O/N at 4 &#176;C under gentle shaking.</li>
</ol>
7. ELISA Protocol (Day 2)
<ol>
	<li>Remove the plate from the shaker and allow it to warm to RT (15-20 min) prior to proceeding with the assay.</li>
	<li>Remove the Anti-rhGH-HRP, conjugate buffer, wash buffer (200x), chromogen TMB (tetramethylbenzidine), and stop reagent from the refrigerator and allow warming to RT.</li>
	<li>Reagent preparation: (according to manufacturer&#39;s specifications)
	<ol>
		<li>Dilute the Anti-rhGH-HRP 40x with the conjugate buffer. Prepare the wash buffer by diluting 200x with distilled water.</li>
	</ol>
	</li>
	<li>Add 50 &#956;l&#160;of Anti-rhGH-HRP conjugate into each well, seal the wells and incubate at RT for 30 min while gently shaking.</li>
	<li>Decant the solution from the wells and invert the plate and tap dry onto an absorbent tissue. Then add 200 &#956;l of wash buffer into each well.</li>
	<li>Decant the wash solution and tap dry onto an absorbent tissue. Repeat this step 3 times.</li>
	<li>Add 100 &#956;l of Chromogen into each well within 15 min following the wash step. Incubate for 30 min at RT in the dark while gently shaking. The solutions turn from colorless to blue.</li>
	<li>Add 100 &#956;l of the stop reagent into each well. The solutions turn from blue to yellow. Immediately, read the absorbance of each well at 450 nm using a microplate reader.</li>
	<li>Plot the standard curve for the provided standards (0-5). Then plot the optical density of the rhGH in 10% serum samples versus the concentration of each sample.</li>
</ol>

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M.G. Sandros is a consultant for Horiba Scientific. She has received honoraria for participation in oral presentations from: Horiba Scientific and Syngenta. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript

Irregular levels of hGH, a naturally occurring hormone, have been linked to numerous medical disorders that affect human growth and development. Moreover, exogenous administration of rhGH is commonly used by athletes, even though it is forbidden, as a doping agent to enhance their performance. Challenges in detecting rhGH misuse result from the difficulty in distinguishing exogenous hGH from endogenous form. As such, the current approved technique for detecting exogenous hGH relies on measuring the ratio of the 22 kDa hGH isoform in relation to the 20 kDa isoform. Since the isoform test demands for the measurement of multiple hGH isoforms simultaneously within a short period of time in a wide range of concentrations, therefore, we considered the SPRi platform as a perfect match. In addition, endogenous hGH level fluctuate to a very low level (0.03 ng/ml) in the bloodstream therefore the detection system must be able to measure this range comfortably with high specificity. As a result, we also investigated in this study the potential of Nano-SPRi as a diagnostic tool for hGH and compared it directly with SPRi and the classical immunoassay ELISA.
Based on results obtained from this study, the main advantage of the SPRi and Nano-SPRi method is that rhGH concentrations can be measured in a quicker manner in comparison to the more conventional method ELISA. A standard duration for measuring rhGH levels in one sample with the direct detection method was 1 hr whereas the Nano-SPRi required 2 hr due to the additional steps in the process. Overall, with SPRi and Nano-SPRi experiments, before the injection of a sample, a calibration step is highly recommended. In addition, the injection of a crude sample like human serum results in some non-specific interactions as a result it is imperative to inject a high salt wash buffer to only reveal specific interactions. It is also worth noting that a wash step is absolutely necessary after the introduction of blocking molecules to the sensor surface, to remove unbound molecules. As for ELISA time requirements are far greater (~16-18 hr) for the analysis of one sample. A longer incubation time is needed as the sensitivity of the assay is enhanced especially for this study, as the focus was to compare the lower limit of detection.
The choice of surface chemistry will vary from one application to another and this could be realized as one of the limitation of the SPRi technique. In this study, a wide range and combination of chemical linkers and blocking molecules were assessed to achieve the right combination in order to observe optimal binding efficiency of rhGH to the sensor surface. For example, in this study, the combination of BSA and PEG served well in minimizing non-specific interactions. However, in a previous study17, where the capture ligand was an aptamers, PEG alone served as the best blocking molecule. The variables that affect binding efficiency of analyte to ligand are also dependent on pH, buffer and temperature. Therefore, with any application, these variables need to be optimized. In addition, it is critical to determine the optimal spotting concentration of the ligand to the chip surface. A titration experiment with a range of concentrations of immobilized ligands is performed before initiating the study. As for ELISA, a crucial step in the procedure was to decant the wash buffer from wells by tapping the microplate as this ensured no residual liquid is leftover. Removing the buffer wash with pipette was not sufficient as any residual liquid interfered with the signal reading of the target sample.
In reference to sensitivity, ELISA (1 ng/ml) is comparable to SPRi (3.61 ng/ml) but nano-SPRi (9.20 pg/ml) improves sensitivity by three orders of magnitude, thereby enabling measurements at the lower biological levels of rhGH 0.03 ng/ml. As we previously reported16,17, the signal enhancement imparted by the NanoEnhancers is attributed to a mass loading effect and the strong coupling that exists between NIR fluorophores and propagating surface plasmons for gold film nanostructures. Even though, Nano-SPRi adds an extra step to the procedure, this level of sensitivity can widen the applications of SPRi technology in various outlets.
SPRi provides scientists a full kinetic profile (KD, Ka and Kd) of antibody/rhGH interaction whereas ELISA can report only affinity values. The coefficient of variation (CV) was below 10% for SPRi (4.1%) and ELISA (6.5%), suggesting good reproducibility. The ELISA and SPRi affinity values are different because the capture antibodies immobilized on the sensor chip are different from antibodies immobilized in the ELISA 96-well plate. As for Nano-SPRi a higher CV value (20%) was observed. There are several parameters that can contribute as a source of errors for CV determination. For example, with the Nano-SPRi experiment a much lower concentration of analyte is being measured, the addition of NanoEnhancers adds another step in the procedure and the experiment was performed manually. A very good correlation between Nano-SPRi and ELISA was achieved for the detection of rhGh in spiked human serum. Finally, ELISA can be a reliable technique however the method itself is time consuming which makes it difficult to use in situations requiring real time monitoring and multiplexing, as it is the case with hGH. In addition, a more attractive feature that was not investigated directly in this study that SPRi offers over ELISA, is the ability to measure hundreds of interactions simultaneously in real time. Therefore in the future, Nano-SPRi method will be assessed to detect multiple biomarkers simultaneously in real time (multiplexing) present in serum at various concentrations in order to examine its potential as a viable clinical diagnostic tool.

Human growth hormone (hGH) is a 191 amino acid peptide (22 kDa) produced by the pituitary gland and directly released into the bloodstream. Interactions between the hypothalamic peptide growth hormone-releasing hormone (GHRH) and somatotropin induce pulsatile secretions of hGH. As a result, levels of hGH vary from highs in the 50-100 ng/ml to lows in the 0.03 ng/ml range1. Deficiency or excess of hGH in the body can provoke a wide range of abnormal physiological symptoms. For example, excess levels of hGH can lead to gigantism2 and diabetes3. Depleted levels of hGH cause low blood sugar in newborns, and weak bone density and depression in adults4.
The administration of the recombinant form of hGH (rhGH) improves lean muscle mass while reducing body fat. As such, this substance became the drug of choice for professional and amateur athletes as it improves physical strength that confers an advantage in competitive sports. rhGH is banned by the World Anti-Doping Agency (WADA)5,6 and much effort by international researchers has been focused on developing tests that can detect its presence or anabolic effect.
Enzyme-linked immunosorbent assay (ELISA) has been the preferred method for the determination of hGH in whole blood7. Although, ELISA is a reliable technique offering good sensitivity and selectivity, it is relatively time- and labor-intensive. In addition, ELISA relies on the indirect detection of hGH by employing enzymatic tags. In contrast, surface plasmon resonance (SPR) permits detection of hGH directly without the use of labels in real time. The detection principle behind SPR involves a sensing surface consisting of a prism that is coated with a thin metal layer (gold or silver); when a monochromatic polarized light interacts with the metal surface, &#34;surface plasmons&#34; are generated. The binding of an analyte to a surface receptor immobilized on the metal surface perturbs the resonance conditions resulting in a shifted resonance dip, which can then be correlated to the analyte concentration. SPR-based biosensors are now commercially available that offer a real-time, label free technique to monitor biomolecular binding events and biochemical reactions8-10. More recently, SPRi was developed in response to the need for multiplexing (i.e.,&#160;monitoring multiple binding events simultaneously), which was not possible in classical SPR biosensors. Thus, SPRi has emerged as a tool to monitor several binding events simultaneously. Current SPRi systems are based on microscopic imaging of a surface which is excited with light at a specific angle and wavelength10. The image is then captured onto a charge-coupled device (CCD) array.
To date, there have been a few SPR-based assays developed to detect hGH11-14. One particular strategy, known as the isoform method15, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22-kDa endogenous levels drop after exogenous rhGH administration. Recently, de Juan-Franco et al.11 reported on the development of a SPR-based immunosensor for the selective detection of the 22 kDa and 20 kDa hGH isoforms in human serum samples. Monoclonal antibodies specific to each isoform were immobilized directly on the gold sensor permitting the measurement of both isoforms simultaneously in a single injection with a limit of detection at 0.9 ng ml-1. Alternatively, SPR has been used to screen antibodies with high specificity to hGH13. If the concentration of target analyte falls below the SPRi system&#39;s limit of detection (&#60;nM), one has to resort to amplifying the SPRi signal via the utilization of nanoparticles (Nano-SPRi). Such SPR-based amplification has been well documented in the literature16-19 for various types of analyte and surfaces.
In this work, the analytical potential of SPRi and Nano-SPRi based biosensors was examined, particularly for the detection of rhGH in spiked human serum, and comparison of its detection capability directly to ELISA. The following parameters will be reviewed and considered: detection time, sensitivity, kinetic profile, reproducibility and specificity.

<table><tbody><tr><td>Somatotropin Growth Hormone (GH1) capture antibody&amp;nbsp;</td><td>Acris Antibodies</td><td>DM1015</td><td></td></tr><tr><td>Bovine Serum Albumin&amp;nbsp;</td><td>Fisher Bioreagents&amp;nbsp;</td><td>BP671-10</td><td></td></tr><tr><td>Biotin labeled Somatotropin Growth Hormone (GH1) detection antibody&amp;nbsp;</td><td>Acris Antibodies</td><td>AM09304BT-N</td><td></td></tr><tr><td>1-ethyl-3-(3-dimethylaminopropyl) carbodiimide&amp;nbsp;</td><td>TCI America</td><td>D1601</td><td></td></tr><tr><td>Ethanolamine</td><td>Acros Organics&amp;nbsp;</td><td>141-43-5</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Chemicals&amp;nbsp;&amp;nbsp;</td><td>BP2818-4</td><td></td></tr><tr><td>Human HGH ELISA Kit&amp;nbsp;</td><td>invitrogen&amp;nbsp;</td><td>KAQ1081</td><td></td></tr><tr><td>Human Serum&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>H4522&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Rabbit IgG Antibody&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>I5006</td><td></td></tr><tr><td>11-mercaptoundecanoic acid&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>450561</td><td></td></tr><tr><td>Nanostrip&amp;nbsp;</td><td>Cyantek&amp;nbsp;</td><td></td><td></td></tr><tr><td>N-hydroxysuccinimide&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>130672</td><td></td></tr><tr><td>Phosphate Buffer Saline</td><td>Invitrogen&amp;nbsp;</td><td>00-3002</td><td></td></tr><tr><td>Polyethylene glycol (800 Da)&amp;nbsp;</td><td>Sigma Aldrich&amp;nbsp;</td><td>729108</td><td></td></tr><tr><td>Recombinant Human Growth Hormone&amp;nbsp;</td><td>Calbiochem</td><td>869008</td><td></td></tr><tr><td>Sodium Acetate</td><td>Sigma Aldrich&amp;nbsp;</td><td>127-09-3</td><td></td></tr><tr><td>Sodium Chloride&amp;nbsp;</td><td>Fisher Chemicals&amp;nbsp;&amp;nbsp;</td><td>7647-14-5</td><td></td></tr><tr><td>Streptavidin coated near infrared quantum dots&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>Q10171MP</td><td></td></tr><tr><td>UV/Ozone Procleaner</td><td>Bioforce Nanosciences&amp;nbsp;</td><td></td><td></td></tr><tr><td>Microwave reactor&amp;nbsp;&amp;nbsp;</td><td>CEM Corporation</td><td></td><td>Discover system&amp;nbsp;</td></tr><tr><td>SPRi-Arrayer&amp;nbsp;&amp;nbsp;</td><td>LabNext Xactll Microarray System&amp;nbsp;</td><td></td><td></td></tr><tr><td>SPR biochip</td><td>HORIBA&amp;nbsp;</td><td></td><td></td></tr><tr><td>SPRi-Lab+</td><td>HORIBA</td><td></td><td></td></tr><tr><td>Synergy Mx Multimode Microplate reader&amp;nbsp;</td><td>BioTek&amp;nbsp;</td><td></td><td></td></tr><tr><td>ScrubberGen</td><td>HORIBA</td><td></td><td>Data Analysis software&amp;nbsp;</td></tr></tbody></table>

comparative analysis of human growth hormone in serum using spri, nano-spri and elisa assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The performance of SPRi and Nano-SPRi (SPRi employing the NanoEnhancers) was compared with ELISA for the detection of rhGH in a complex environment. The differences in the setup of these methods are described here briefly. For SPRi (direct detection, Figure 1), the capture antibody is immobilized on the surface and then the sample is injected and binding of analyte to the sensor surface is measured directly in real time and label-free manner. However, with Nano-SPRi (Figure 1), after the analyte binds to the sensor surface, a consecutive injection is followed with quantum dots coated with detection antibodies to amplify the SPRi signal.
<img alt="Figure 1" src="/files/ftp_upload/53508/53508fig1.jpg" /> 
Figure 1. A schematic representation comparing direct-mode (SPRi) and amplified-mode (Nano-SPRi) of detection of rhGH in crude samples. Ligands (rhGH or IgG specific antibodies) were immobilized in an array format on the SPRi biochip. Target protein (rhGh) spiked in human serum introduced to the sensor surface are directly detected (SPRi) and sequentially highlighted with NanoEnhancers (Nano-SPRi). <a href="https://www.jove.com/files/ftp_upload/53508/53508fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
As for ELISA, the multiwell plates arrive already pre-functionalized with capture antibody and then sample is introduced, the analyte of interest will bind. A detection antibody is introduced followed by substrate addition. The optical density is then measured at 450 nm. In this study, a commercial ELISA kit (Figure 2) was used to measure rhGH spiked in 10% human serum.
<img alt="Figure 2" src="/files/ftp_upload/53508/53508fig2.jpg" /> 
Figure 2. A schematic representation of ELISA assay procedure. rhGH protein (blue ovals) is introduced to wells that have been pre-functionalized with monoclonal antibodies (yellow) specific to rhGH. Non-specific interactions are eliminated by rinsing the wells with wash buffer followed by the introduction of a detection antibody prefunctionalized with horseradish peroxidase (HRP, purple). The solution will change in color after adding the substrate tetramethylbenzidine (TMB, gold). <a href="https://www.jove.com/files/ftp_upload/53508/53508fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 3 represents the titration curve of rhGH spiked in 10% human serum and is plotted against the obtained OD at 450 nm. A good linear response was observed and the limit of detection was determined to be 1 ng/ml. The coefficient of variation (CV) was 6.5% suggesting good reproducibility.
<img alt="Figure 3" src="/files/ftp_upload/53508/53508fig3.jpg" /> 
Figure 3. ELISA data Analysis. Concentration of rhGH spiked in serum is plotted against the obtained OD at 450 nm. <a href="https://www.jove.com/files/ftp_upload/53508/53508fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Next, the detection of rhGH spiked in human serum was assessed with SPRi. Direct detection of rhGH resulted with the corresponding concentration gradient curve (Figure 4), each point represents the average value of the reflectivity difference calculated from three SPRi kinetic curves for each concentration. The limit of detection (LOD) was determined to be 3.61 ng/ml. The SPRi direct detection assay was highly reproducible as the CV of the assay was only 4.1%.
<img alt="Figure 4" src="/files/ftp_upload/53508/53508fig4.jpg" /> 
Figure 4. Direct SPRi detection of hGH spiked in 10% human serum. The resultant normalized SPRi kinetic plot after the injection of various amounts of hGH spiked in human serum followed by the injection of a high salt buffer (dashed vertical line) to remove non-specific interactions . A concentration gradient curve representing the binding of various amounts of hGH spiked in human serum to the sensor surface that has been prefunctionalized with biotinylated hGH-specific-Antibody . <a href="https://www.jove.com/files/ftp_upload/53508/53508fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To increase the sensitivity of the SPRi biosensor, NanoEnhancers (QDs pre-functionalized with detection antibodies) are sequentially introduced to the sensor surface in order to highlight the presence of rhGH spiked in human serum. After background subtraction, the NanoEnhancers were able to amplify the biosensor response up to 7.9%, however, with minimal signal change (Immunoglobulin G (IgG) -specific antibody, 0.38% change in reflectivity; Figure 5 on controlled regions of interest. Imaging of the sensor surface revealed that only regions of interest that have rhGH-specific antibodies immobilized experience the largest contrast change corroborating directly with the kinetic sensorgram response.
<img alt="Figure 5" src="/files/ftp_upload/53508/53508fig5.jpg" /> 
Figure 5. Detection of rhGH using a sandwich assay spiked in 10% human serum. SPRi kinetic plot after the injection of rhGH (30 ng/ml) in buffer (10 mM PBS, 150 mM sodium chloride, pH = 7.4) onto a pre-functionalized chip with 11-mercaptoundecanoic acid / rhGH-specific antibody and control IgG-antibody then blocked with BSA followed by the addition of detection antibody-coated quantum dots (NanoEnhancers). <a href="https://www.jove.com/files/ftp_upload/53508/53508fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To demonstrate the practicability of the Nano-SPRi biosensor, the measuring range of rhGH in crude samples was assessed. An extended working range from 30,000 pg/ml to 0.25 pg/ml resulted as a response to the addition of NanoEnhancers (Figure 6). It is worth noting that each point on the titration curve is averaged from three independent experiments. Consequently, the lower limit of detection was calculated to be 9.20 pg/ml and the coefficient of variation was 20%.
<img alt="Figure 6" src="/files/ftp_upload/53508/53508fig6.jpg" /> 
Figure 6. Nano-SPRi detection of hGH spiked in human serum. Normalized SPRi kinetic plot representation of hGH_specific_Anti-QDs-amplified signal for human serum samples spiked with different concentrations of hGH. A vertical dashed line (grey) represents the injection point of the running buffer. (b) A concentration gradient curve representing the binding of NanoEnhancers (hGH_specific_Anti-QDs) after the injection of various amounts of hGH spiked in human serum to the sensor surface that has been prefunctionalized with 11-mercaptoundecanoic acid /hGH-specific antibody. <a href="https://www.jove.com/files/ftp_upload/53508/53508fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Next, the change that occurs in the SPR reflectivity curve as a function of the concentration was compared between the direct and the NanoEnhancer bioassay method (Figure 7). For the direct detection technique between 25 and 0.25 pg/ml, the signal started to plateau and this is not surprising as these concentrations fall below the LOD of 3 ng/ml (Figure 7). Similarly, for the amplified technique, concentrations below the LOD of 9.2 pg/ml signal started to plateau and showed virtually no variation.
<img alt="Figure 7" src="/files/ftp_upload/53508/53508fig7.jpg" /> 
Figure 7. A comparative analysis of SPRi with Nano-SPRi. This bar graph depicts the percent change in reflectivity (%R) after introduction of rhGH spiked in human serum (direct detection) followed by the injection of NanoEnhancers (amplified detection) for 30,000 pg/ml, 250 pg/ml, 25 pg/ml, 2.5 pg/ml and 0.25 pg/ml. <a href="https://www.jove.com/files/ftp_upload/53508/53508fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Furthermore, the specificity of the Nano-SPRi biosensor was assessed. Insulin-like growth factor-1 (IGF-1) spiked in human serum was injected as a control since hGH stimulates the secretion of IGF-1 through the growth hormone receptor on the hepatocyte membrane. After the injection of the IGF-1 in serum, the NanoEnhancers were sequentially injected and the SPR signal response did not show any specific binding (Figure 8). However, when rhGH spiked in serum sample was injected to the very same spot functionalized with rhGH antibody, signal enhancement was observed. In conclusion, the Nano-SPRi platform has demonstrated excellent specificity and selectivity for rhGH.
<img alt="Figure 8" src="/files/ftp_upload/53508/53508fig8.jpg" /> 
Figure 8. Assessment of Nano-SPRi selectivity towards rhGH. The Nano-SPRi biosensor response after the injection of rhGH (black) and IGF-1 (red) in spiked serum. <a href="https://www.jove.com/files/ftp_upload/53508/53508fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A correlation analysis was performed using the Pearson correlation coefficient to determine the correlation between the SPRi signal intensity and ELISA optical density values (Figure 9). A p-value &#60;0.01 was considered significant. As illustrated in this graph, there is a good correlation between the rhGH levels in spiked human serum measured by SPRi and ELISA. The r-value was 0.9263 for 9 different samples.
<img alt="Figure 9" src="/files/ftp_upload/53508/53508fig9.jpg" /> 
Figure 9. Pearson correlation analysis between ELISA and Nano-SPRi. The plot correlates Nano-SPRi signal intensity (y-axis) with ELISA optical density (x-axis) values. (Pearson correlation coefficient n = 9, r = 0.9263, p = 0.00000183). <a href="https://www.jove.com/files/ftp_upload/53508/53508fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The equilibrium dissociation constant (KD) was determined using the graphpad software for ELISA. The calculated KD value was approximately 79 nM (Table 3). The on (Ka) and off (Kd) rates could not be determined by ELISA. However, using the data analysis software, the direct detection method resulted with KD value of 23 pM using the molecular mass of 22 kDa that corresponds to one rhGH molecule. The on and off rates were calculated to be 6.1 x107 M-1s-1 and 1.33 x 10-3 sec-1, respectively. This inherently translates that 0.13% of the rhGH and antibody complexes decay per second. As for the amplified SPRi experiment, a stronger overall interaction was observed between NanoEnhancers and rhGH as the calculated binding affinity was determined to be 4.3 pM. In addition, a stronger association rate was observed for NanoEnhancers and rhGH than capture antibody/rhGH, however the dissociation rate suggests that 0.26% of NanoEnhancer/rhGH/Capture antibody decay per second.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Method</td>
			<td>KD (Affinity)</td>
			<td>Ka &#160;(on-rate)</td>
			<td>Kd &#160;(off-rate)</td>
			<td>LOD</td>
		</tr>
		<tr>
			<td>ELISA</td>
			<td>79.45 x 10-9 M</td>
			<td>NA</td>
			<td>NA</td>
			<td>1 ng/ml</td>
		</tr>
		<tr>
			<td>SPRi</td>
			<td>23.2 x 10-12 M</td>
			<td>6.1 x 107 M-1sec-1&#160;</td>
			<td>1.33 x10-3 sec-1</td>
			<td>3.61 ng/ml</td>
		</tr>
		<tr>
			<td>Nano-SPRi</td>
			<td>4.33 x 10-12 M</td>
			<td>7.54 x108 M-1sec-1&#160;</td>
			<td>2.62 x10-3 sec-1</td>
			<td>0.0092 ng/ml</td>
		</tr>
	</tbody>
</table>
Table 3. Full kinetic data analysis. Evaluation of the affinity, on-rate and off-rate of the antibody responses using graphpad (ELISA) and data analysis (SPRi and Nano-SPRi) software. The determination of the avidity using the molecular mass of 22 kDa that corresponds to one rhGH molecule was chosen. The limits of detection were determined for all three studies using excel.

Watch this Scientific Journal Video about Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays at JoVE.com

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

comparative-analysis-human-growth-hormone-serum-using-spri-nano-spri

Research

JoVE Journal

Biology

16.4K Views.  University of North Carolina at Greensboro. The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

Video: Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

Cancer Research

High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT).
Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2R&alpha;5, TNFR16, HGF7, IL-88, elafin2, and REG3&alpha;3 (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials.
This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.

High throughput validation of multiple candidate biomarkers can be performed by sequential ELISA in order to minimize freeze/thaw cycles and use of precious plasma samples. Here, we demonstrate how to sequentially perform ELISAs for six different validated plasma biomarkers1-3 of graft-versus-host disease (GVHD)4 on the same plasma sample.

Unbiased discovery proteomics strategies have  the potential to identify large numbers of novel biomarkers that can improve  diagnostic and prognostic ...

Medicine

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 &#181;m) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.

A selective and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method coupled with an efficient solid phase extraction on a mixed-mode cation-exchange (MCX) 96-well microplate was developed for the measurement of free 3-nitrotyrosine (3-NT) in human urine with high throughput, which is suitable for clinical applications.

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

Chemistry

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Bioengineering

A RAPID Method for Blood Processing to Increase the Yield of Plasma Peptide Levels in Human Blood

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the correct determination of a peptide&#39;s concentration, it should be stable during blood processing. However, this is not the case for several peptides which are quickly degraded by endogenous peptidases. Recently, we developed a blood processing method employing Reduced temperatures, Acidification, Protease inhibition, Isotopic exogenous controls and Dilution (RAPID) for the use in rats. Here, we have established this technique for the use in humans and investigated recovery, molecular form and circulating concentration of food intake regulatory hormones. The RAPID method significantly improved the recovery for 125I-labeled somatostatin-28 (+39%), glucagon-like peptide-1 (+35%), acyl ghrelin and glucagon (+32%), insulin and kisspeptin (+29%), nesfatin-1 (+28%), leptin (+21%) and peptide YY3-36 (+19%) compared to standard processing (EDTA blood on ice, p&#160;&#60;0.001). High performance liquid chromatography showed the elution of endogenous acyl ghrelin at the expected position after RAPID processing, while after standard processing 62% of acyl ghrelin were degraded resulting in an earlier peak likely representing desacyl ghrelin. After RAPID processing the acyl/desacyl ghrelin ratio in blood of normal weight subjects was 1:3 compared to 1:23 following standard processing (p&#160;= 0.03). Also endogenous kisspeptin levels were higher after RAPID compared to standard processing (+99%, p&#160;= 0.02). The RAPID blood processing method can be used in humans, yields higher peptide levels and allows for assessment of the correct molecular form.

The RAPID blood processing method can be used in humans and yields higher peptide levels as well as allows for assessment of the correct molecular form. Therefore, this method will be a valuable tool in peptide research.

Research in the field of food intake regulation is gaining importance. This often includes the measurement of peptides regulating food intake. For the ...

Immunology and Infection

Using Extraordinary Optical Transmission to Quantify Cardiac Biomarkers in Human Serum

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor analytes against the background of human serum are crucial.
Nanoimprinting lithography (NIL) was used to fabricate, at a low cost, sensing areas as large as 1.5 mm x 1.5 mm. The sensing surface was made of high-fidelity arrays of nanoholes, each with an area of about 140 nm2. The great reproducibility of NIL made it possible to employ a one-chip, one-measurement strategy on 12 individually manufactured surfaces, with minimal chip-to-chip variation. These nanoimprinted localized surface plasmon resonance (LSPR) chips were extensively tested on their ability to reliably measure a bioanalyte at concentrations varying from 2.5 to 75 ng/mL amidst the background of a complex biofluid-in this case, human serum. The high fidelity of NIL enables the generation of large sensing areas, which in turn eliminates the need for a microscope, as this biosensor can be easily interfaced with a commonly available laboratory light source. These biosensors can detect cardiac troponin in serum with a high sensitivity, at a limit of detection (LOD) of 0.55 ng/mL, which is clinically relevant. They also show low chip-to-chip variance (due to the high quality of the fabrication process). The results are commensurable with widely used enzyme-linked immunosorbent assay (ELISA)-based assays, but the technique retains the advantages of an LSPR-based sensing platform (i.e., amenability to miniaturization and multiplexing, making it more feasible for POC applications).

This work describes a nanoimprinting lithography method to fabricate high-quality sensing arrays that work on the principle of extraordinary optical transmission. The biosensor is low-cost, robust, easy to use, and can detect cardiac troponin I in serum at clinically relevant concentrations (99th percentile cutoff &#8764;10-400 pg/mL, depending on the assay).

For a biosensing platform to have clinical relevance in point-of-care (POC) settings, assay sensitivity, reproducibility, and ability to reliably monitor ...

Detection and Quantification of Calcitonin Gene-Related Peptide (CGRP) in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay

Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide that plays a putative role in the pathophysiology of migraine headaches and may be a candidate for biomarker status. CGRP is released from neuronal fibers upon activation and induces sterile neurogenic inflammation and arterial vasodilation in the vasculature that receives trigeminal efferent innervation. The presence of CGRP in the peripheral vasculature has spurred investigations to detect and quantify this neuropeptide in human plasma using proteomic assays, such as the enzyme-linked immunosorbent assay (ELISA). However, its half-life of 6.9 min and the variability in technical details of assay protocols, which are often not fully described, have yielded inconsistent CGRP ELISA data in the literature. Here, a modified ELISA protocol for the purification and quantification of CGRP in human plasma is presented. The procedural steps involve sample collection and preparation, extraction using a polar sorbent as a means of purification, additional steps to block non-specific binding, and quantification via ELISA. Further, the protocol has been validated with spike and recovery and linearity of dilution experiments. This validated protocol can theoretically be used to quantify CGRP concentrations in the plasma of individuals not only with migraine, but also with other diseases in which CGRP may play a role.

Detection and Quantification of Calcitonin Gene-Related Peptide CGRP in Human Plasma Using a Modified Enzyme-Linked Immunosorbent Assay

Published data pertaining to calcitonin gene-related peptide (CGRP) concentrations in human plasma are inconsistent. These inconsistencies may be due to the lack of a standardized, validated methodology to quantify this neuropeptide. Here, we describe a validated enzyme-linked immunosorbent assay (ELISA) protocol to purify and quantify CGRP in human plasma.

Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide that plays a putative role in the pathophysiology of migraine headaches and may be a ...

Biochemistry

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 &#176;C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 &#176;C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...

Development and Validation of an Ultrasensitive Single Molecule Array Digital Enzyme-linked Immunosorbent Assay for Human Interferon-&#945;

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ImmunoSorbent Assay (ELISA) assay. This system enables the quantification of human IFN-&#945; protein with unprecedented sensitivity, and with no cross-reactivity for other species of IFN.
The first key step of the protocol is the choice of the antibody pair, followed by the conjugation of the capture antibody to paramagnetic beads, and biotinylation of the detection antibody. Following this step, different parameters such as assay configuration, detector antibody concentration, and buffer composition can be modified until optimum sensitivity is achieved. Finally, specificity and reproducibility of the method are assessed to ensure confidence in the results. Here, we developed an IFN-&#945; single molecule array assay with a limit of detection of 0.69 fg/mL using high-affinity autoantibodies isolated from patients with biallelic mutations in the autoimmune regulator (AIRE) protein causing autoimmune polyendocrinopathy syndrome type 1/autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APS1/APECED). Importantly, these antibodies enabled detection of all 13 IFN-&#945; subtypes.
This new methodology allows the detection and quantification of IFN-&#945; protein in human biological samples at attomolar concentrations for the first time. Such a tool will be highly useful in monitoring the levels of this cytokine in human health and disease states, most particularly infection, autoimmunity, and autoinflammation.

Here we present a protocol to describe the development and validation of a single molecule array digital ELISA assay, which enables the ultra-sensitive detection of all IFN-&#945; subtypes in human samples.

The main aim of this protocol is to describe the development and validation of an interferon (IFN)-&#945; single molecule array digital Enzyme-Linked ...

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved F&#246;rster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved F&#246;rster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

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