This work was supported by the Special Funds for Fundamental Research Funds of institutions of higher-learning affiliated with central departments. We appreciate the support of Classical Prescription Basic Research Team of the Beijing University of Chinese Medicine.

All of the procedures performed in this study were approved by the Ethics Review Committee at the Beijing University of Chinese Medicine (approval number 2017BZYYL00120).
1. Preparation and Characterization of Colloidal Gold
NOTE: For colloidal gold synthesis, as colloidal gold is easily adsorbed on the inner wall of the vessel and is prone to precipitation by impurities, the vessel for synthesis and storage of colloidal gold should be thoroughly clea...

<ol>
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In this work, we present a protocol for the preparation of mAbs against natural product-derived small molecules. The essential steps and the matters needing attention in the procedure have been outlined, and we have demonstrated the utility of this protocol using the small molecule SSD as an example. Example spectra, TEM images, quantitative results and methodological investigations are shown in representative data. Hence, we have demonstrated that the colloidal gold production, AuNP-mAb conjugation and strip assembly st...

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost and rapid detection. The nitrocellulose membrane as the carrier and colloidal gold as markers of immune chromatography rapid diagnostic reagents are the most commonly used POCT (point of care testing) method, and the testing scope of the project is wider. From their original application in monitoring during pregnancy, their use has been extended to monitor blood coagulation state1,2, myocardial injury3, veterinary medicine4, pesticide residues5, infectious diseases6 and drug concentrations. More types of samples can be assessed, including urine, saliva, whole blood, serum and other body fluids7,8,9.
In recent years, numerous novel assays have been developed for detecting biomarkers in the diagnosis of disorders, including HPLC, UPLC, LC-MS and ELISA, which are sensitive and accurate, credible and specific. However, these methods require sophisticated instrumentation, complex preprocessing and time-consuming treatments9. Hence, developing a more rapid and convenient point-of-care diagnostic strategy for the self- and real-time detection of medicinal active compounds is urgent10,11.
The popularity of ICSs, especially for common tests, is driven by their ease of use, as they do not require professionals or elaborate instrumental setups12. In other words, people who do not have special training can operate strips or self-tests13. The results of the test can be obtained in 5 minutes, which means it can be used for site inspections14. Moreover, according to our calculations, the cost of strips could be lower than 1 RMB15, which means that the tests are inexpensive to promote16. Hence, the ICS is a relatively accurate, simple, and inexpensive disposable device. ICSs based on colloidal gold17,18 are also applied in rapid COVID-19 detection.
The principle of ICS can be divided into sandwich ICS and competitive ICS. Figure 1A is a schematic diagram of the sandwich ICS, which is mainly used for detecting macromolecular substances such as proteins, including tumor markers, inflammatory factors, and human chorionic gonadotropin (HCG, early pregnancy antigen). In this method, paired antibodies targeted at different epitopes of the antigen are used, and the capture antibody is dried on the NC membrane as a test line. Labeled antibody is dried on the conjugate pad, and secondary antibody is used as the control line.
Figure 1B is a schematic diagram of competitive ICS, which is mainly used to detect small molecule substances (MWCO &#60; 2000 Da). The coating antigen is fixed on the NC membrane as a test line, and the labeled antibody is dried on the conjugate pad. During detection, the sample and labeled antibody flow through the detection line under capillary action, and the coated antigen competitively binds free antigen in the sample and develops a red color on the detection line.
Recently, we described the procedure of monoclonal antibody generation against natural products19. In this work, we developed a novel lateral flow immunoassay based on the prepared anti-SSD mA20 for rapid, on-site detection. The results indicate that the immunochromatography assay is an indispensable and convenient tool for detecting natural product-derived compounds.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62754/62754fig01.jpg" /> 
Figure 1 Schematic diagram of the immunochromatography assay (A) Sandwich immunochromatographic test strips. (B) Indirect competitive immune chromatographic test strips. This figure has been modified from Zhang et al., 201821. <a href="https://www.jove.com/files/ftp_upload/62754/62754fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chloroauric acid solution (HAuCl4)</td>
			<td>Tianjin Fu Chen Chemical Reagents Factory</td>
			<td>JY-SJ102</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumin</td>
			<td>AMRESCO</td>
			<td>332</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge tube 15 mL</td>
			<td>Corning</td>
			<td>430645</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge tube 50 mL</td>
			<td>Corning</td>
			<td>430828</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA plates, 96 well</td>
			<td>NUNC</td>
			<td>655101</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Sinopharm</td>
			<td>H5072</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass fibre membranes</td>
			<td>Jieyi</td>
			<td>XQ-Y6</td>
			<td></td>
		</tr>
		<tr>
			<td>goat-anti-mouse IgG antibody</td>
			<td>applygen</td>
			<td>C1308</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membranes</td>
			<td>Millipore</td>
			<td>millipore 180</td>
			<td></td>
		</tr>
		<tr>
			<td>ovalbumin</td>
			<td>Beijing BIODEE</td>
			<td>5008-25g</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG20000</td>
			<td>Sigma Aldrich</td>
			<td>RNBC6325</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette 10mL</td>
			<td>COSTAR</td>
			<td>4488</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette 25mL</td>
			<td>FALCON</td>
			<td>357525</td>
			<td></td>
		</tr>
		<tr>
			<td>semi-rigid PVC sheets</td>
			<td>Jieyi</td>
			<td>JY-C104</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td>Beijing Chemical Works</td>
			<td>C1034</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium periodate</td>
			<td>Sinopharm Chemical</td>
			<td>BW-G0008</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-GMBS</td>
			<td>Perbio Science Germany</td>
			<td>22324</td>
			<td></td>
		</tr>
		<tr>
			<td>TipOne Tips 1,000 &#181;L</td>
			<td>Starlab</td>
			<td>S1111-2021</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Solarbio</td>
			<td>77-86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN 20</td>
			<td>Solarbio</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of a lateral flow immunochromatographic strip for rapid and quantitative detection of small molecule compounds

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, physiological index and clinical biomarker detection. In this protocol, we provide a detailed description of the steps to develop a rapid, sensitive and quantitative lateral-flow immunoassay (using AuNPs as a marker and mAbs as a probe). The procedure describes the preparation and characterization of colloidal gold, synthesis of the AuNP-mAb conjugate, assembly of the immunochromatographic strip, and methodological investigation of the assay. The results showed that the final strips can be further utilized for the rapid and convenient self-diagnosis of a small molecule, which may provide an alternative tool in the rapid and precise analysis of physiological and biological indices.

Characterization of colloidal gold 
The prepared colloidal gold solutions were claret red. TEM analyses were used to determine the morphology and shape of AuNPs (Figure 2A-D). Figure 2A and Figure 2B reveal that the particles are polyhedral in shape and uniformly distributed. The average diameter of AuNPs was found to be approximately 14 nm (Figure 2C). A high-reso...

Watch this Scientific Journal Video about Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds at JoVE.com

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, ...

The procedure describes the preparation of colloidal gold and assembly of the immunochromatographic strips. Firstly, chloroauric acid and sodium citrate were used for the synthesis of colloidal gold. Sodium citrate was added to chloroauric acid solution and boiled to obtain colloidal gold with appropriate particle size.Synthesis, the colloidal gold antibody conjugate. After centrifugation, colloidal gold particles were dissolved in appropriate solution and then resuspended to obtain colloidal gold solution. Submerge the glass fiber membrane into the conjugation to prepare the conjugate pad.Drop artificial antigen and secondary antibodies on the NC membrane as T line and C line. Attach the NC membrane, absorbent membrane, sample pad, and the glass fiber membrane into the PVC board respectively. Cut the assembled paper board into 3.5 millimeters wide strip.Confect full percent gold chloride acid solution and 1%sodium citrate solution respectively. Turn on the magnetic stirrer and place the flask on a mixer. Add 120 milliliters distilled water into a round-bottom flask and heat it to boiling on a thermostatic magnetic stirrer.Keep boiling and quick add 0.5 milliliters of full percent chloroauric acid and five milliliters of 1%sodium citrate. Continue heating for five to 10 minutes. Observe the color of the solution changing from pale yellow to wine red.Turn off the power of the thermostatic magnetic mixer, cool to room temperature and the place the mixture into a clean bottle. Store at four centigrade. The gold nanoparticles shows the Tyndall effect.The size and the morphology of the gold nanoparticles were determined by ultraviolet spectroscopy and the transmission electron microscope imaging. Add 100 microliters sodium chloride solution to eight tubes. Adjust the colloidal gold solutions to pH 5 to 12 using 0.1 mol per liter potassium carbonate.Add 100 microliters of colloidal gold solution to eight tubes containing sodium chloride. Stand for post several minutes after blending. Observe the color change of each tube solution and record the tube that remains red.Choose the pH value with the lowest amount of added potassium carbonate and a stable solution color as the optimum pH value for preparing conjugate. Adapted the same approach. Observed the color change and record the tube that remains red.Choose the antibody amount with the lowest concentration of mAB and a stable solution color as the optimum mAB amount. Take 10 milliliters colloidal gold solution and use 0.1 mol per liter potassium carbonate to adjust the solution to the optimal pH value. Slowly add SSd mABs with the appropriate concentration and shake at room temperature for 30 minutes.Centrifuge the mixture at 2000 RPM for 20 minutes at four centigrade. Remove the precipitate containing impurities of the precipitated colloidal gold. Centrifuge the mixture at 10, 000 RPM for 40 minutes.Discard the supernatant. The precipitate is colloidal gold mAB conjugate. Add Tris hydrochloric acid, BSA, Tween 20, and PEG 20, 000, then blend well to prepare resuspension buffer.Add the resuspension buffer and dissociate the precipitate. The membrane material strip, including glass cellulose membrane, polyester cellulose membrane, and the PVC board. The membrane material, need to track and evaluate by sterile microscope to get rid of the inhomogeneity.First, place the NC membrane on the PVC board two centimeters from the edge of the section end of the board. Second, drop two milligrams per milliliter SSd-BSA on the NC membrane at two centimeters from the upper edge as a test line and drop 1.5 milligrams per milliliter gold anti-mouse IgG on the NC membrane at two centimeters from the lower edge as a control line. Attach the absorbent pad to the PVC sheet above the NC membrane and overlap with the NC membrane by two millimeters.Trim the glass fiber membrane to five centimeters long and two centimeters wide and use as a conjugate pad. Submerge the glass fiber membrane into the gold nanoparticles mAB conjugate solution. Soak for a while to make the gold nanoparticles mAB absorption.Dry the wide membrane in the incubator at 37 degree. Paste the pretreated conjugate pad under the NC membrane and the overlapping length with the NC membrane should be 0.1 centimeters. Trim the Fusion3 membrane into 1.8 centimeters long and 3.5 centimeters wide and use as the sample pad.Then, place the sample pad on the PVC board and overlap with the conjugate pad by two millimeters. Cut, assemble the paper board into 3.5 millimeter wide strips using a cutting machine and compact using a batch lamination system. Finally, place the test strips into the shell.Seal them in a aluminum foil bag containing desiccant and the store away from light. The ICS is now assembled. Drop 50 microliters of sample solution onto the sample hole to observe the chromatography process.Next, analyze the strips using a portable strip reader. The machine can provide the ratio of the test line to the control line. Evaluate the specificity, sensitivity, repeatability, and stability of the ICS test.The prepared colloidal gold solutions were read. Transmission electron microscope analyzes revealed that the particles were polyhedral in shape and were uniformly distributed with a 14 nanometer average diameter. High-resolution transmission electron microscope images showed a continuous fringe pattern with a spacing of 0.117 nanometer of gold nanoparticles.Ultraviolet visible absorption indicated that different sizes of colloidal gold particles can be obtained by changing the proportion of citrate sodium and chloroauric acid. In the quantitative experiment, the strips with the test results of saikosaponin D were scanned using a portable ICS reader. We calculated a standard curve by relating non-concentrations of saikosaponin D to the corresponding absorption at each concentration.After methodological investigation, the method can be applied for quantitative and semi-qualitative detection of small molecule compounds. This procedure includes preparation of colloidal gold, synthesis of gold nanoparticles mAB conjugate, assembling the strip and methodological investigation. This work provides a detailed protocol for development of immunochromatographic strip for use in rapid and quantitative detection of small molecule.After watching this video I hope you have a good understanding for preparation of the immunochromatographic strip. Thank you for watching and good luck for your experiments.

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JoVE Journal

Biochemistry

6.7K visualizações.  Beijing University of Chinese Medicine. O procedimento descreve a preparação do ouro coloidal e a montagem das tiras imunocromatográficas. Em primeiro lugar, o ácido cloroauúrico e citrato de sódio foram usados para a síntese de ouro coloidal. O citrato de sódio foi adicionado à solução de ácido cloroaurico e fervido para obter ouro coloidal com tamanho de partícula adequado.Síntese, o anticorpo de ouro coloidal conjugado. Após a centrifugação, partículas de ouro coloidais foram dissolvidas em solução apr...