PRACTICAL SANDWICH ELISA KIT FOR
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
ELISA (Enzyme Linked Immuno-Sorbent Assay), CTX (Ciguatoxin), AP (Alkaline Phosphatase), PNPP (Disodium p-Nitrophenyl Phosphate)
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
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.
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.
“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
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
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.