Conjugate preparation is significantly modified from previous work [11
]. Specifically, TTX binds to Bovine Serum Albumin (BSA; Sigma; A7906-50 G) with formaldehyde and the BSA will tether TTX (a small hydrophilic molecule) to the plate. Because the commercial anti-TTX antibodies were created against a keyhole limpet cyanin (KLH) conjugate, the antibodies do not cross-react with BSA in the final assay [12
]. Seven-hundred μL TTX (Sigma; T5651) at 1 mg/mL, 300 μL sodium acetate buffer (1 N; adjusted to pH 7.4 using 0.05 N acetic acid; Sigma; S7670), 179 μL of BSA at 33.6 mg/mL, and 41 μL of 37% formaldehyde (Fisher Scientific; AC11969) are added drop-wise to an amber glass vial (conjugate is light sensitive), in that order, and vortexed. TTX is soluble at a pH of 4-5, however previously reported methodology states that TTX should be dissolved at a pH of 7.4 in sodium acetate buffer [11
]. We utilized1 mg TTX lyophilized in 5 mg citrate buffer and dissolved in 1 ml of ddH2
O, which yielded the appropriate pH, with no consequences to the efficacy of the conjugate. The conjugate solution is then incubated in a shaker for three days at 37°C. Following incubation, the solution is transferred to dialysis tubing and dialyzed over a three day period at 4°C against four equally spaced 1 L-changes of phosphate buffered saline (PBS; Fisher Scientific; BP665-1). The concentration is then determined by spectrophotometry (NanoDrop ND-1000 Spectrophotometer; at 280 nm). Finished conjugate may be stored at 4°C and does not need to be lyophilized [as in [13
Optimal conjugate concentration is determined by running plates of standard curves with serial dilutions of the conjugate. Excessively concentrated conjugate results in high variation due to nonspecific binding [17
]. Others [11
] reported 2 μg/mL concentrations for anti-TTX antibodies (Hawaii Biotech) and 10 μg/mL BSA-TTXF conjugate to coat the plate. We found that using a 2 μg/mL solution of conjugate and consequently, a lower concentration of antibodies, can be used saving materials, eliminating nonspecific binding, decreasing variation, and improving the fit and accuracy of the standard curve. For each new lot of antibody purchased and used, the appropriate concentration of antibodies will have to be optimized using standard solutions of TTX and testing serially diluted anti-TTX antibodies. Both primary and secondary antibodies can be stored at 4°C or -20°C between uses.
Extraction of TTX and preparation of standards
TTX is extracted by previously described methods [18
]. Briefly, filtrates may be stored at -80°C for up to 5 yr. without degradation of TTX (CT Hanifin pers comm.). Standards are prepared using 1 mg TTX lyophilized in citrate buffer (Ascent Scientific; Asc-055) dissolved in 1 mL of a 1% solution of BSA diluted in PBS. The linear range of the curve is quite large (see results), so we use standard concentrations of 10, 50, 100, 300 and 500 ng/mL diluted in 1% BSA-PBS from the 1 mg/mL stock solution for each assay. In cases where the samples are not diluted by at least 1:2, standards are prepared by diluting in 0.1 M acetic acid rather than the 1% BSA solution. We have found that the absorbance values for acetic acid are slightly different than those of 1% BSA solution. Using acetic acid as the background for samples that are not diluted compensates for this, and does not alter the accuracy of the standard curve. All standards, samples, and stock solutions should be stored at -80°C between uses with little affect due to freeze/thaw of solutions.
Assays are run in 96-well microtiter plates (Nunc MaxiSorp, Fisher Scientific; 439454). The first of three controls is a blank and does not receive any sample, standard, or antibody (Figure ). The second is a positive control that tests the efficacy of the alkaline-phosphatase labeled goat anti-mouse IgG+IgM (H+L) secondary antibodies (Jackson ImmunoResearch; 115-055-044). The third is a negative control, in which 1% PBS-BSA is used as a sample with no TTX. In cases where acetic acid is used to prepare standards, 0.1 M acetic acid is the negative control. This assay is very sensitive to temperature changes and should be run at approximately 25-30°C. We also report here, for the first time, that small pigment molecules not excluded during the extraction process can add to the absorbance and thus interfere with TTX quantification. Additionally, there may be non-specific secondary binding in some cases, which may give false positive results. These issues are easily circumvented by running controls of each extract with no anti-TTX antibody to measure baseline absorbance for each sample, which will be subtracted from the absorbance of the quantified sample.
A typical template used for plates. Each sample starts with the letter S. Samples run without primary antibodies are used to eliminate any background noise caused from the sample itself.
The assay: (1) Each plate is coated with 100 μL conjugate diluted in PBS (2 μg/mL). The plate is incubated for one hour at room temperature (RT), and washed three times with 250 μL of PBS-T (500 μL Tween-20 (Fisher Scientific; 23336-2500) per 1 L PBS) buffer using a plate washer (Bio-Rad model 1575; may also be performed by hand). (2) We next block the plate using 200 μL of 1% PBS-BSA, incubate for one hr at RT, and again wash the plate. (3) Fifty-μL of standards or samples are added to wells in triplicate. Samples should be diluted to within the range of the standard curve (preliminary data may be collected to determine proper dilutions). (4) Fifty-μL of anti-TTX antibodies diluted to the optimal concentration (0.391 μg/ml in our case) are added to all sample and standard wells except individual extract controls, incubated one hr at RT, and washed. (5) One hundred μL of anti-mouse IgG + IgM antibodies (H + L) are added to all wells except the positive and blank controls, incubated one hr at RT, and washed. (6) Fifty μL of secondary antibodies are added to the positive control wells, and 200 μL of a 1 mg/mL pNPP solution (diluted in diethanolamine buffer: 400 mL ddH2O, 52.22 g diethanolamine (Sigma; D8885-500 G), adjusted to pH 9.80 with concentrated HCl, 0.051 g MgCl2 (Fisher Scientific; AC41341-0025)) is added to all of the wells. The plate is protected from light and incubated at RT for 10 minutes. (7) The plate is then read in a Bio-Rad xMark Microplate Spectrophotometer (any standard absorbance reader with the appropriate filter is sufficient) at an absorbance of 405 nm. Readings are taken every 5 minutes following the initial 10-minute reading.
The mean, standard deviation, and coefficient of variation (CV) for each of the standards are calculated. To back-calculate standard concentrations, mean absorbance values of the standards are plotted against the log of the known concentration for each standard. The time frame with the best standard predictions (least summed variance from known values; usually highest r2 value of the regression line) is selected for sample quantification. The best time frame is usually 20-45 min. Sample values outside the range of the standard curve are diluted and re-run, re-assayed as a more concentrated extract, or reported as either below detection limit (BDL) or above detection limit (ADL) depending on whether they fall above or below the curve.. The concentrations for any samples are adjusted via dilution factor. The mean value for the negative control (BSA or acetic acid), or preferably individual extract controls, should be subtracted from the mean values of the unknowns to eliminate background noise for the most accurate final concentration.