クジラミオグロビンの検出のための金コロイドベースのイムノクロマト法テストストリップの開発

The overall goal of this colloidal gold immunochromatographic test strip is to differentiate the myoglobin of cetaceans from that of seals and other animals. This method can provide rapid and on-the-spot screening for cetacean meat, thereby restraining its illegal trade and consumption. The main advantage of this technique is that a successful result can be observed directly in five to ten minutes after homogenizing 03 gram of muscle, with outstanding specificity and sensitivity.

So this method can provide rapid detection of cetacean meat in the field. It can also be applied to other meat such as horse meat, or pork. To design synthetic peptides for raising monoclonal antibodies with reactivity toward the myoglobin of cetaceans, the amino acid sequences of myoglobin antigenic reactive regions from various animals are analyzed.

Begin by retrieving from GenBank the amino acid sequences of myoglobin from tuna, chicken, ostrich, domestic mammals, seal, and 18 species of cetaceans. Next, align the sequences using the proper software. Launch the Alignment Explorer by selecting the Align Edit/Build Alignment on the launch bar.

Select Create New Alignment, and click OK.A dialog will appear asking, are you building a DNA or Protein Sequence alignment? Click the button labeled Protein, select Data Open Retrieve sequences from File, and select sequence file. Select the Edit Select All menu command to select all sites for every sequence in the data set for creating a multiple sequence alignment.

Select Alignment Align by ClustalW from the main menu. Select BLOSUM as the protein weight matrix and then click the OK button. The third step is to analyze the sequence alignment.

Focus on five antigenic reactive sites and find the fragment conserved among cetaceans. An asterisk symbolizes consensus in the alignment and indicates a position that has a single, fully-conserved residue. Two conserved fragments in cetaceans were found.

Candidate sequence fragments are then synthesized and used to raise monoclonal antibodies. Begin this procedure by determining the optimal pH for the colloidal gold solution defined as the minimum pH that keeps the monoclonal antibody and colloidal gold solution mixture red in color for two hours. Add 0.15 micrograms of purified detecting monoclonal antibody to 100 microliters of colloidal gold solution with pH values varying from five to nine.

For the purposes of this demonstration, pH six and pH eight are tested. pH eight was considered the optimal pH by this optimization step. Next, determine the optimal concentration of the monoclonal antibody by adding various amounts of purified detecting monoclonal antibody to 100 microliters of colloidal gold solution at pH eight.

The optimum concentration, which is six micrograms per milliliter in this study, will keep the color of the mixture red with no black precipitate. Based on the results from the optimization steps, add 60 micrograms of purified detecting monoclonal antibody drop-wise to 10mL of colloidal gold solution. Emulsify the mixture gently at room temperature for ten minutes.

Add two milliliters of a 5%BSA solution in PBS to the mixture and emulsify gently at room temperature for 15 minutes to reduce background interference. Centrifuge the mixture at 10, 000 times g for 30 minutes at four degrees Celsius. After centrifugation, carefully remove the supernatant with unconjugated antibody and suspend the resulting pellet in 4mL of PBS-T containing 1%BSA and 0.1%Tween-20.

Repeat the centrifugation and suspension once or twice. Suspend the final precipitates in 1mL of PBS-T and store at four degrees Celsius until use. A graphic of the immune strip design is shown here.

The dimensions of the cardboard, pads, and nitrocellulose membrane are listed in the protocol text. To prolong storage life, the immune strips should be prepared and assembled in a low-humidity laboratory environmental condition. Use a micropipette to add the colloid gold-labeled monoclonal antibody solution to the conjugate pad to saturate it and then dry the pad at 37 degrees Celsius for one hour before assembling.

Once the conjugate pad is dry, use double-sided tape to first paste the nitrocellulose membrane onto the cardboard. Using an immunostrip printer, distribute the specific antigen capturing monoclonal antibody and the rapid anti-mouse IGG on the test zone and the control zone, respectively, on the nitrocellulose membrane. Maintain a distance of greater than five millimeters between the two zones.

Then paste the conjugate pad on one side of the nitrocellulose membrane, and the absorbent pad on the other side, overlapping the pads on each side by about two millimeters. Place the sample pad over the conjugate pad by two millimeters and paste it on the cardboard. Use a paper cutter to create six-millimeter-wide strips.

Pack the strips in an aluminum foil bag with dessicant and store at four degrees Celsius until use. To prepare a sample for the cross-reactivity test, add 1mL of PBS containing 0.1%BSA to 0.03 grams of raw muscle. And use a bamboo stick or a grinding rod to homogenize the sample.

Hold the immune strip by the end opposite to the test area and dip the sample pad part into the specimen for five to ten minutes. Observe the result directly. Test various muscle samples in triplicate.

The T test line is designed to show a positive signal when both monoclonal antibodies detect cetacean myoglobin. Because the myoglobin from non-cetacean animals can only be detected by one of these two monoclonal antibodies, the test line shows a negative result when muscle samples from other animals are tested. The C control line should always show a positive result because rapid anti-mouse IGG binds the colloid gold-labeled detection antibody.

A failed result in the Control line indicates that the quality of the materials on the strip is poor. Western blood analysis using hybridoma supernatants shows that the CGF5H9 monoclonal antibody detects cetaceans and other mammals as a single stained band at a predicted molecular weight of approximately 17kDa. The common Minke whale shows a comparatively fainter band than the bands of other cetaceans.

Bands are absent for tuna, seal, and chicken. Whereas a band at about 50kDa is observed for pig. Identical results are obtained by dot blood analysis.

Although Western blood analysis using the CSF1H13 monoclonal antibody shows multiple nonspecific bands for pig and tuna, CSF1H13 is highly specific because it reacts only with cetaceans. And seal has a band at a predicted molecular weight of approximately 17kDa. Identical results are obtained by dot blood analysis.

Results from Indirect ELISA of muscle extracts of different species using purified CGF5H9 demonstrate positive signals for cow, goat, dog, rabbit and cetaceans, a weak positive signal for pig, and negative signals for tuna, chicken, and seal. CSF1H13 shows high affinity towards cetaceans and seals. Both monoclonal antibodies can react strongly with all four cetacean species which are from different families, indicating their broad reactivity to diverse cetacean species.

The specificity of the immune colloidal gold strip is shown by these representative results. The use of non-cetacean muscle samples results in only a single band at the control line. However, when cetacean muscle samples are used, the signal bands are present at both the test line and control line.

Once mastered, the preparation of labeled antibodies and this construction of strips can be done in four hours if it's performed properly. When attempting this procedure, it's important to remember to design the peptide carefully. Use several methods for screening suitable monoclonal antibodies.

And use a high quality of colloidal gold to prepare labeled antibodies. Following these procedures, other methods like PCR-based DNA analysis can be performed in order to answer additional questions, like the cetacean species and from where it came. The CSF1H13 monoclonal antibody which is strong and exhibits an affinity towards only marine mammals can be employed in various formats of ELISA that offer many other opportunities for studying the biological, physiological and chemical aspects of myoglobin in marine mammals.

After watching this video, you should have a good understanding of how to develop specific monoclonal antibodies and subsequently apply them over quick test strip.