PRACTICAL SANDWICH ELISA KIT FOR
CIGUATOXIN “CTX1B”

Takeshi Sato 1 , Mariko Yoshimura 2 , Masako Hayakawa 2 , Takeshi Tsumuraya 3 and Masahiro Hirama 3

1 Cell Science Inc., Aoba-ku, Sendai 989-3212, Japan
2 Wako Pure Chemical Industries, Ltd., Chuo-ku, Tokyo 103-0023, Japan
3 Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Osaka 599-8531, Japan

Abbreviations

ELISA (Enzyme Linked Immuno-Sorbent Assay), CTX (Ciguatoxin), AP (Alkaline Phosphatase), PNPP (Disodium p-Nitrophenyl Phosphate)

Introduction

Prof. Hirama and his coworkers of Tohoku University in Japan chemically synthesized four congeners of CTXs (CTX3C, CTX1B, 51-hydroxy-CTX3C, and 54-deoxy-CTX1B), which are mainly present in the Pacific Ocean. Subsequently, using synthetic hapten-KLH conjugates as antigens, Prof. Tsumuraya produced several kinds of monoclonal antibodies that strongly bind to the right side or the left side of CTXs molecule, and they successfully developed a highly sensitive
immunochemical detection method.

Based on these researches, we have commercialized a new ELISA kit “CTX-ELISA 1B” to detect and quantify CTX1B, which is likely to be most abundant in the Pacific Ocean, at the detection range of 0.2 ~ 0.0005 ppb. Here we show the ELISA kit “CTX-ELISA 1B” and illustrate how to use it for an analysis of CTX1B in the flesh of a ciguatoxic fish.

Materials and Methods

“CTX-ELISA 1B” KitContents of the Kit

1. Antibody coated 96 well plate “CTX1B” 2
2. Wash buffer x20 conc. “W-1” 50ml 1
3. STD/Sample dilute “D-1” 50ml 1
4. Anti-CTX1B-AP dilute “D-2” 50ml 1
5. AP Substrate dilute “R-1” 50ml 1
6. CTX1B Standard DMSO solution 1
7. AP-conjugated “Anti-CTX1B–AP” 1
8. Plate cover sheet 6

Table 1. AP-substrates usable in this kit and their detection condition

Summary of assay procedure

1. As described in the protocol of “CTX-ELISA 1B” Kit, prepare washing solution, test sample, CTX standard solution.
                               ⇓
2. Add 100 µL test sample and CTXs standard solution into each well.
                                                                                ⇓  Incubate for 30 minutes at 37˚C or at room temp.
3. Aspirate and wash 3 times with 200~300 µL/well of wash buffer.
                               ⇓
4. Add 100 µL Anti-CTXs-AP solution into each well.
                                                                               ⇓ Incubate for 30 minutes at 37˚C or at room temp.
5. Aspirate and wash 3 times with 200~300 µL/well of wash buffer.
                               ⇓
6. Prepare AP-Substrate solution, and add 100~200 µL/well.
                                                               ⇓ Incubate at 37˚C.
7. Read Absorbance or Fluoresence Intensity within 30~45min.

Fig.1 Comparative test of blocking materials “Synthetic Polymer and Bovine Serum

Albumin (BSA)” that suppress non-specific binding of CTX1B to the plastic surface.

1. CTX1B solution of 250pg/ml was injected into a micro-test-plate pretreated with Synthetic Polymer or BSA, and incubated for 30 minutes.

2. After washing the plate, Anti-CTX1B–AP solution was added into each well, and incubated for 30 minutes.

3. After washing, added AP-substrate PNPP into each well to measure nonspecific binding of CTX1B.

4. Absorbance at 405nm was measured on each time.

Results

1. Synthetic polymer strongly blocked non-specific binding of CTX1B and/or Anti-CTX1B–AP, and kept low absorbance for long time.
2. BSA that was used as a blocking reagent generally, did not adequately block the increase of background color.
3. The synthetic polymer effectively inhibited the nonspecific binding of Anti-CTX1B–AP and CTX1B to the plastic surface, and exerted excellent suppressing effect of background coloration. Synthetic polymer is extremely useful for improving the detection sensitivity.

Fig.2 Specificity of the “CTX-ELISA 1B” Kit.

Three CTXs, “CTX1B, CTX3C and 51-OH-CTX3C” were separately assayed with “CTX-ELISA 1B” Kit, and the amount of CTXs were visualized using PNPP as substrate. Standard curve was prepared by measuring the absorbance at 405nm.

Photograph of micro test plate after 45min incubation

Standard curve of CTX1B, CTX3C and 51-OH-CTX3C

Results

“CTX-ELISA 1B” Kit can detect only CTX1B but not CTX3C and 51-OH-CTX3C.
* The “CTX-ELISA 1B” kit can specifically detect CTX1B.

Fig.3 Comparative test of detection range for CTX1B by colorimetry and fluorometry.

CTX1B of 0.1 ~ 250 pg/ml were measured by calorimetry and fluorometry.
PNPP and Atophos Ⓡ AP substrate were used as AP-substrates for colorimetry and fluorometry, respectively, and compered detectable range between both procedures.

Colorimetry by PNPP

Fluorometry by AtoPhos Ⓡ AP substrate

Results

1. “CTX-ELISA 1B” Kit is usable for both of colorimetry and fluorometry.
2.Detection limits by colorimetry and fluorometry are about 5 pg/ml (0.005ppb) and 0.5 pg/ml (0.0005ppb), respectively.
3. The fluorometry is 10 times more sensitive than the colorimetry, and sufficiently
exceeds the FDA guidance level (0.01ppb).

Fig.4 Detection of CTX1B containing in the ciguatoxic fish by “CTX-ELISA 1B”

Tentative procedure for preparing fish flesh extract.

1. Put 5 g of fish flesh in a glass test tube, add 5 mL of methanol, and then mince it finely with ultra-sonic or glass homogenizer and so on.
2.Centrifuge at 15,000 rpm for 10min, collect methanol layer into glass tube and then remove methanol/water by evaporation in vacuum.
3. Add 1 mL of DMSO and dissolve the residue by vortex for a few minutes.
4. Centrifuge and take the supernatant in a test tube as a test sample, and store in refrigerator until use.

Calibration curve of CTX1B

Measurement of fish flesh extract

Results 

Approximately 2.34 ng/ml of CTX1B was detected from the
methanol extract of 5 g ciguatoxic fish flesh by “CTX-ELISA
1B” Kit. Therefore, the amount of CTX1B contained in the test fish was calculated to be about 468 pg/g.

Conclusion

The newly developed ELISA kit “CTX-ELISA 1B” can detect CTX1B, which is likely to be most abundant in the Pacific Ocean, at the wide range of 0.2~0.0005ppb. This “CTX-ELISA 1B” is the first investigation tool that canspecifically, quantitatively, and practically detect CTX1B. Since the detection sensitivity is much superior to the FDA guidance level of 0.01ppb, this kit will be useful not only for prevention of Ciguatera food poisoning (CFP) but also for the epidemiological and physiological studies.
Currently, we are also developing other CTX-ELISA kits, which can detect other CTXs. These new kits will be soon available from LINKSEAS TRADING CO.,LTD of Taiwan.

PRACTICAL SANDWICH ELISA KIT FOR
CIGUATOXIN “CTX1B”

Takeshi Sato 1 , Mariko Yoshimura 2 , Masako Hayakawa 2 , Takeshi Tsumuraya 3 and Masahiro Hirama 3

1 Cell Science Inc., Aoba-ku, Sendai 989-3212, Japan
2 Wako Pure Chemical Industries, Ltd., Chuo-ku, Tokyo 103-0023, Japan
3 Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Osaka 599-8531, Japan

Abbreviations

ELISA (Enzyme Linked Immuno-Sorbent Assay), CTX (Ciguatoxin), AP (Alkaline Phosphatase), PNPP (Disodium p-Nitrophenyl Phosphate)

Introduction

Prof. Hirama and his coworkers of Tohoku University in Japan chemically synthesized four congeners of CTXs (CTX3C, CTX1B, 51-hydroxy-CTX3C, and 54-deoxy-CTX1B), which are mainly present in the Pacific Ocean. Subsequently, using synthetic hapten-KLH conjugates as antigens, Prof. Tsumuraya produced several kinds of monoclonal antibodies that strongly bind to the right side or the left side of CTXs molecule, and they successfully developed a highly sensitive
immunochemical detection method.

 

 

 

 

 

Based on these researches, we have commercialized a new ELISA kit “CTX-ELISA 1B” to detect and quantify CTX1B, which is likely to be most abundant in the Pacific Ocean, at the detection range of 0.2 ~ 0.0005 ppb. Here we show the ELISA kit “CTX-ELISA 1B” and illustrate how to use it for an analysis of CTX1B in the flesh of a ciguatoxic fish.

Materials and Methods

“CTX-ELISA 1B” Kit

 

             

 

 

 

 

 

Contents of the Kit
1. Antibody coated 96 well plate “CTX1B” 2
2. Wash buffer x20 conc. “W-1” 50ml 1
3. STD/Sample dilute “D-1” 50ml 1
4. Anti-CTX1B-AP dilute “D-2” 50ml 1
5. AP Substrate dilute “R-1” 50ml 1
6. CTX1B Standard DMSO solution 1
7. AP-conjugated “Anti-CTX1B–AP” 1
8. Plate cover sheet 6

Table 1. AP-substrates usable in this kit and their detection condition

 

Summary of assay procedure
1. As described in the protocol of “CTX-ELISA 1B” Kit, prepare washing solution, test sample, CTX standard solution.
                               ⇓
2. Add 100 µL test sample and CTXs standard solution into each well.
                               ⇓  Incubate for 30 minutes at 37˚C or at room temp.
3. Aspirate and wash 3 times with 200~300 µL/well of wash buffer.
                               ⇓
4. Add 100 µL Anti-CTXs-AP solution into each well.
                               ⇓ Incubate for 30 minutes at 37˚C or at room temp.
5. Aspirate and wash 3 times with 200~300 µL/well of wash buffer.
                               ⇓
6. Prepare AP-Substrate solution, and add 100~200 µL/well.
                               ⇓ Incubate at 37˚C.
7. Read Absorbance or Fluoresence Intensity within 30~45min.

Fig.1 Comparative test of blocking materials “Synthetic Polymer and Bovine Serum
Albumin (BSA)” that suppress non-specific binding of CTX1B to the plastic surface.

1. CTX1B solution of 250pg/ml was injected into a micro-test-plate pretreated with Synthetic Polymer or BSA, and incubated for 30 minutes.
2. After washing the plate, Anti-CTX1B–AP solution was added into each well, and incubated for 30 minutes.
3. After washing, added AP-substrate PNPP into each well to measure nonspecific binding of CTX1B.
4. Absorbance at 405nm was measured on each time.

 

 

 

 

 

 

 

 

Results

1. Synthetic polymer strongly blocked non-specific binding of CTX1B and/or Anti-CTX1B–AP, and kept low absorbance for long time.
2. BSA that was used as a blocking reagent generally, did not adequately block the increase of background color.
3. The synthetic polymer effectively inhibited the nonspecific binding of Anti-CTX1B–AP and CTX1B to the plastic surface, and exerted excellent suppressing effect of background coloration. Synthetic polymer is extremely useful for improving the detection sensitivity.

Fig.2 Specificity of the “CTX-ELISA 1B” Kit.

Three CTXs, “CTX1B, CTX3C and 51-OH-CTX3C” were separately assayed with “CTX-ELISA 1B” Kit, and the amount of CTXs were visualized using PNPP as substrate. Standard curve was prepared by measuring the absorbance at 405nm.

Photograph of micro test plate after 45min incubation

 

 

 

 

 

 

Standard curve of CTX1B, CTX3C and 51-OH-CTX3C

 

Results

“CTX-ELISA 1B” Kit can detect only CTX1B but not CTX3C and 51-OH-CTX3C.
* The “CTX-ELISA 1B” kit can specifically detect CTX1B.

Fig.3 Comparative test of detection range for CTX1B by colorimetry and fluorometry.

CTX1B of 0.1 ~ 250 pg/ml were measured by calorimetry and fluorometry.
PNPP and Atophos Ⓡ AP substrate were used as AP-substrates for colorimetry and fluorometry, respectively, and compered detectable range between both procedures.

Colorimetry by PNPP

 

 

 

 

 

 

 

Fluorometry by AtoPhos Ⓡ AP substrate

 

Results

1. “CTX-ELISA 1B” Kit is usable for both of colorimetry and fluorometry.
2.Detection limits by colorimetry and fluorometry are about 5 pg/ml (0.005ppb) and 0.5 pg/ml (0.0005ppb), respectively.
3. The fluorometry is 10 times more sensitive than the colorimetry, and sufficiently
exceeds the FDA guidance level (0.01ppb).

Fig.4 Detection of CTX1B containing in the ciguatoxic fish by “CTX-ELISA 1B”

Tentative procedure for preparing fish flesh extract.

1. Put 5 g of fish flesh in a glass test tube, add 5 mL of methanol, and then mince it finely with ultra-sonic or glass homogenizer and so on.
2.Centrifuge at 15,000 rpm for 10min, collect methanol layer into glass tube and then remove methanol/water by evaporation in vacuum.
3. Add 1 mL of DMSO and dissolve the residue by vortex for a few minutes.
4. Centrifuge and take the supernatant in a test tube as a test sample, and store in refrigerator until use.

Calibration curve of CTX1B

 

Measurement of fish flesh extract

 

Results 

Approximately 2.34 ng/ml of CTX1B was detected from the
methanol extract of 5 g ciguatoxic fish flesh by “CTX-ELISA
1B” Kit. Therefore, the amount of CTX1B contained in the test fish was calculated to be about 468 pg/g.

Conclusion

The newly developed ELISA kit “CTX-ELISA 1B” can detect CTX1B,
which is likely to be most abundant in the Pacific Ocean, at the wide range of 0.2~0.0005ppb. This “CTX-ELISA 1B” is the first investigation tool that can specifically, quantitatively, and practically detect CTX1B. Since the detection sensitivity is much superior to the FDA guidance level of 0.01ppb, this kit will be useful not only for prevention of Ciguatera food poisoning (CFP) but also for the epidemiological and physiological studies.

Currently, we are also developing other CTX-ELISA kits, which can detect
other CTXs. These new kits will be soon available from LINKSEAS TRADING
CO.,LTD of Taiwan.