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.
