Preparation of Monoclonal Antibody for Brevetoxin 1 and Development of Ic-ELISA and Colloidal Gold Strip to Detect Brevetoxin 1

Brevetoxin-1 (BTX-1), a marine toxin mostly produced by the dinoflagellatae Karenia brevis, has caused the death of marine organisms and has had numerous toxicological effects on human health. Hence, it is very necessary to develop a rapid, economical, and reliable immunoassay method for BTX-1 detection. In this study, two kinds of complete antigen were synthesized using the succinic anhydride and isobutyl chloroformate two-step methods. Conjugate BTX-1-OVA was used as an antigen for mice immunization, and BTX-1-BSA for measuring the titer of the produced antibodies. A hybridoma cell line 6C6 stably secreting monoclonal antibody (mAb) against BTX-1 was obtained by fusing SP2/0 myeloma cells with the spleen cells from the immunized mouse. The hybridoma 6C6 was injected into the abdomen of BALB/c mice to obtain ascites, and the anti-BTX-1 mAb was harvested from ascites by precipitation with caprylic acid/ammonium sulfate (CA-AS). The anti-BTX-1 mAb was identified as an IgG1 subtype, and the cross-reactivity results showed that anti-BTX-1 mAb was highly specific to BTX-1 with the affinity of 1.06 × 108 L/mol. The indirect competitive ELISA results indicated that the linear range for BTX-1 detection was 14–263 ng/mL with IC50 of 60 ng/mL, and a detection limit of 14 ng/mL. The average recovery rate from the spiked samples was 88 ± 2% in intra-assay and 89 ± 2% in inter-assay. The limit of detection (LOD) using the colloidal gold strip was 200 ng/mL with high specificity. Therefore, the anti-BTX-1 mAb can be used to detect BTX-1 in shellfish and other related samples.


Introduction
The toxic photosynthetic dinoflagellate Karenia brevis (K. brevis) is widely distributed in the Gulf of Mexico, and has also been indicated in other locations [1]. Proliferations of K. brevis occasionally generates large 'red tide' blooms. Natural toxins from these blooms cause toxicity to fish, other animals, and human health problems. Brevetoxins (BTXs), actively biosynthesized by K. brevis, are the first example of polyether compounds [2]. BTXs consist of more than nine congeners, which are divided into two types (types A and B) on the basis of their polyether backbone structure. The principal toxins

Screening and Characterization of Positive Hybridoma Cell Line
In this study, spleen cells from an immunized BALB/c mouse were fused with SP2/0 myeloma cells at the ratio of 10:1 by polyethylene glycol (PEG, 1450). After cell fusion, cells were cultured in HAT medium. After 9 d, the titer of culture supernatant was measured by indirect ELISA. After three times sub-cloning, a positive cell line named 6C6 secreting mAb against BTX-1 was obtained successfully (Figure 2A). In this study, PEG was used for cell fusion. However, the fusion rate of cells and the positive rate of fusion cells were low. There are many factors affecting the cell fusion, such as the growth state of myeloma cells, the concentration of the PEG, the ratio between spleen cells and myeloma cells, and the temperature of the medium. The good-growth state of SP2/0 myeloma cells is one of the most important factors that affects cell fusion. It is very harmful for cells with high concentration of PEG and long incubation time. In this study, 30% PEG was used for cell fusion, there is just 1 min for PEG incubation in the process of cell fusion, and good fusion results were obtained. The titer result showed that the OD value of blood samples from BALB/c mice immunized with BTX-1-OVA was significantly higher than that of the control ( Figure 1C).The result further suggested that BTX-1-OVA complete antigen was successful in inducing the antibody against BTX-1. Therefore, the spleen cells of these two mice were selected to perform cell fusion experiments.

Screening and Characterization of Positive Hybridoma Cell Line
In this study, spleen cells from an immunized BALB/c mouse were fused with SP2/0 myeloma cells at the ratio of 10:1 by polyethylene glycol (PEG, 1450). After cell fusion, cells were cultured in HAT medium. After 9 d, the titer of culture supernatant was measured by indirect ELISA. After three times sub-cloning, a positive cell line named 6C6 secreting mAb against BTX-1 was obtained successfully (Figure 2A). In this study, PEG was used for cell fusion. However, the fusion rate of cells and the positive rate of fusion cells were low. There are many factors affecting the cell fusion, such as the growth state of myeloma cells, the concentration of the PEG, the ratio between spleen cells and myeloma cells, and the temperature of the medium. The good-growth state of SP2/0 myeloma cells is one of the most important factors that affects cell fusion. It is very harmful for cells with high concentration of PEG and long incubation time. In this study, 30% PEG was used for cell fusion, there is just 1 min for PEG incubation in the process of cell fusion, and good fusion results were obtained.
Hybridoma cell line 6C6 was chosen for chromosome analysis to further identify whether hybridoma 6C6 was from the fusion of SP2/0 myeloma cell and spleen cell. The average chromosome number of SP2/0 myeloma cell and spleen cell were 62-70 and 38-40, respectively [10]. The chromosome number of the hybridoma 6C6 was counted as 104 ( Figure 2B), indicating that cell fusion was successfully carried out.
HAT medium. After 9 d, the titer of culture supernatant was measured by indirect ELISA. After three times sub-cloning, a positive cell line named 6C6 secreting mAb against BTX-1 was obtained successfully (Figure 2A). In this study, PEG was used for cell fusion. However, the fusion rate of cells and the positive rate of fusion cells were low. There are many factors affecting the cell fusion, such as the growth state of myeloma cells, the concentration of the PEG, the ratio between spleen cells and myeloma cells, and the temperature of the medium. The good-growth state of SP2/0 myeloma cells is one of the most important factors that affects cell fusion. It is very harmful for cells with high concentration of PEG and long incubation time. In this study, 30% PEG was used for cell fusion, there is just 1 min for PEG incubation in the process of cell fusion, and good fusion results were obtained. The isotype of the positive mAb against BTX-1 was identified by subtype kit (IgM, IgA, IgG1, IgG2a, IgG2b, IgG3), and the result indicated that the subtype of 6C6 positive clone belongs to IgG1 subtype ( Figure 2C).

Purification of Anti-BTX-1 mAb and Titer Analysis
Hybridoma cell line 6C6 were cultured and injected into the abdominal cavity of BALB/c mice to prepare more mAb. The anti-BTX-1 mAb was obtained from ascites and purified by caprylic/ammonium sulfate precipitation (CA-AS) method. The purified anti-BTX-1 mAb was analyzed by SDS-PAGE, and the result indicated that the purified antibody has one clear heavy chain at 50 kDa and the light chain at 26 kDa ( Figure 3A). The titer of the purified anti-BTX-1 mAb was measured by indirect ELISA, and the result showed that the titer was above 3.2 × 10 4 ( Figure 3B). The result indicated that the antibody was successfully purified with high titer, and could have potential to develop kit for BTX-1 detection. The purified anti-BTX-1 mAb was saved in a refrigerator at −20 • C for a long time, and the titer of the purified anti-BTX-1 mAb was determined by indirect ELISA at the same concentration, and the result indicated that the antibody could retain activity for at least four months ( Figure 3C). Hybridoma cell line 6C6 was chosen for chromosome analysis to further identify whether hybridoma 6C6 was from the fusion of SP2/0 myeloma cell and spleen cell. The average chromosome number of SP2/0 myeloma cell and spleen cell were 62-70 and 38-40, respectively [10]. The chromosome number of the hybridoma 6C6 was counted as 104 ( Figure 2B), indicating that cell fusion was successfully carried out.
The isotype of the positive mAb against BTX-1 was identified by subtype kit (IgM, IgA, IgG1, IgG2a, IgG2b, IgG3), and the result indicated that the subtype of 6C6 positive clone belongs to IgG1 subtype ( Figure 2C).

Purification of Anti-BTX-1 mAb and Titer Analysis
Hybridoma cell line 6C6 were cultured and injected into the abdominal cavity of BALB/c mice to prepare more mAb. The anti-BTX-1 mAb was obtained from ascites and purified by caprylic/ammonium sulfate precipitation (CA-AS) method. The purified anti-BTX-1 mAb was analyzed by SDS-PAGE, and the result indicated that the purified antibody has one clear heavy chain at 50 kDa and the light chain at 26 kDa ( Figure 3A). The titer of the purified anti-BTX-1 mAb was measured by indirect ELISA, and the result showed that the titer was above 3.2 × 10 4 ( Figure 3B). The result indicated that the antibody was successfully purified with high titer, and could have potential to develop kit for BTX-1 detection. The purified anti-BTX-1 mAb was saved in a refrigerator at −20 °C for a long time, and the titer of the purified anti-BTX-1 mAb was determined by indirect ELISA at the same concentration, and the result indicated that the antibody could retain activity for at least four months ( Figure 3C).

Specialty and Affinity of the Anti-BTX-1 mAb
The affinity constant of 6C6 mAb against BTX-1 was 1.06 × 10 8 L/mol ( Figure 4A), suggesting that the anti-BTX-1 mAb is highly sensitive to BTX-1. Competitive ELISA was performed to analyze the specificity of the anti-BTX-1 mAb produced by positive clone 6C6, and the ELISA result indicated

Specialty and Affinity of the Anti-BTX-1 mAb
The affinity constant of 6C6 mAb against BTX-1 was 1.06 × 10 8 L/mol ( Figure 4A), suggesting that the anti-BTX-1 mAb is highly sensitive to BTX-1. Competitive ELISA was performed to analyze the specificity of the anti-BTX-1 mAb produced by positive clone 6C6, and the ELISA result indicated that 6C6 was highly specific to BTX-1 without any reaction to other complete antigens ( Figure 4B). At the same time, anti-BTX-1 mAb showed no cross-reactions between BTX-2, BTX-3, and other marine toxins ( Figure 4C) (Table S1). Therefore, it could be further used to develop an ELISA kit for BTX-1 detection.

Standard Curve and Samples Detection by ic-ELISA
The 6C6 mAb was used to establish a standard curve for BTX-1 detection by competitive inhibition ELISA. In the study, BTX-1 was diluted in PBS or matrix, and methanol-water (5:5, v/v) was used as extraction solution. A standard curve prepared by diluted matrix was compared to a standard curve prepared in PBS, and the two curves showed little difference ( Figure 5A), confirming that the matrix was minimized. The relationship between concentration of BTX-1 and inhibition value was analyzed using OriGinpro 8. As shown in Figure 5A, the logistic curve equation was y = 0.08779 + (0.098917 − 0.08779)/[1 + (x/58.00982) 1.58278 ], with a correlation coefficient (R 2 ) of 0.98078. The half inhibitory concentration (IC50) of BTX-1 binding to anti-BTX-1 mAb was 60 ng/mL, and the linear range to detect BTX-1 was 14-263 ng/mL, which defined as the concentration of BTX-1 toward from 20% to 80% inhibition ratio. The linear equation is y = 47.197x − 34.411, with a correlation coefficient (R 2 ) of 0.0.9719 ( Figure 5B), and the detection limit (LOD) was 14 ng/mL.
Recovery and coefficient of variation (CV) were measured by the ic-ELISA, and shellfish samples without any contamination were spiked with different concentrations of BTX-1. The result showed that the recovery rates were ranged from 84.21 ± 2.15% to 93.11 ± 1.26% with the average of 88 ± 2%, and the variation coefficient is 1.35-2.97% (average 2%) in intra-assay, while the recovery rates were ranged from 85.55 ± 2.05% to 91.55 ± 1.03% with the average of 89 ± 2%, and the variation coefficient is 1.01-4.74% (average 2%) in inter-assay (Table S2). Meanwhile, Shellfish samples (razor, clam The specificity analysis of the purified antibody. Different kinds of complete antigen were coated, and the antibody did not react with other antigens; (C) cross-reactivity of anti-BTX-1 mAb to other toxins was determined by ic-ELISA. The antibody did not have the cross-reaction with other marine toxins.

Standard Curve and Samples Detection by ic-ELISA
The 6C6 mAb was used to establish a standard curve for BTX-1 detection by competitive inhibition ELISA. In the study, BTX-1 was diluted in PBS or matrix, and methanol-water (5:5, v/v) was used as extraction solution. A standard curve prepared by diluted matrix was compared to a standard curve prepared in PBS, and the two curves showed little difference ( Figure 5A), confirming that the matrix was minimized. The relationship between concentration of BTX-1 and inhibition value was analyzed using OriGinpro 8. As shown in Figure 5A

Construction and Characterization of Colloidal Gold Strip Test
The schematic diagram for constructing colloidal gold strip was shown in Figure 6A. There will be two lines in the absence of BTX-1 in the sample solution as a negative control. On the contrary, if the sample solution has enough BTX-1, the test line will disappear and only one control line exists on the control zone. The reason was that BTX-1 will bind with antibody-gold nanoparticle conjugates, so it makes the antibody-gold nanoparticle conjugates fail to bind with the BTX-1-BSA that was coated onto the Millipore 135 NC membrane (Jieyi biotech Co., shanghai, china). Thus, it is shown as a positive control. If there is one test line or no line, it indicates an invalid result.
Different kinds of toxins, including BTX-1, BTX-2, BTX-3, okadaic acid (OA), domoic acid (DA), saxitoxin (STX), tetrodotoxin (TTX), and conopeptide (CTX), were used to identify the cross-reactivity of the test strip. The result from Figure 6B showed that there were two lines with the existence of other toxins and only one control line with the existence of BTX-1. This result indicated that the colloidal gold strip test based on anti-BTX-1 mAb had high specificity to BTX-1. Different concentrations of BTX-1 solution (0-250 ng/mL) were used to determine the limit of the colloidal gold strip. The results showed that the limit of detection (LOD) of the strip for BTX-1 was 200 ng/mL ( Figure 6C), and the test line will disappear totally if the concentration was above this level. Even though the LOD of the colloidal gold strip was higher than that of ELISA, the colloidal gold strip is more convenient, easy to operate, and requires a shorter time than ELISA.

Real Sample Assay with Colloidal Gold Strip
Four kinds of real sample solutions were used to identify whether the collected samples had BTX-1 or not. The result showed that the color density of four test lines of sample solution were the same as the negative control ( Figure 6D), indicating that there was no BTX-1 in these four samples. Therefore, this anti-BTX-1 mAb with high affinity and specificity can develop the ELISA kit for detection of BTX-1 in real samples. It also has the potential to develop the colloidal gold rapid diagnostic stripe. Recovery and coefficient of variation (CV) were measured by the ic-ELISA, and shellfish samples without any contamination were spiked with different concentrations of BTX-1. The result showed that the recovery rates were ranged from 84.21 ± 2.15% to 93.11 ± 1.26% with the average of 88 ± 2%, and the variation coefficient is 1.35-2.97% (average 2%) in intra-assay, while the recovery rates were ranged from 85.55 ± 2.05% to 91.55 ± 1.03% with the average of 89 ± 2%, and the variation coefficient is 1.01-4.74% (average 2%) in inter-assay (Table S2). Meanwhile, Shellfish samples (razor, clam mussel, oyster, and scallop) were obtained from a market, and the shellfish sample extracts were diluted 20-fold with phosphate-buffered saline and determined by ic-ELISA. As the results showed in Table S3, there was no BTX-1 in these samples.

Construction and Characterization of Colloidal Gold Strip Test
The schematic diagram for constructing colloidal gold strip was shown in Figure 6A. There will be two lines in the absence of BTX-1 in the sample solution as a negative control. On the contrary, if the sample solution has enough BTX-1, the test line will disappear and only one control line exists on the control zone. The reason was that BTX-1 will bind with antibody-gold nanoparticle conjugates, so it makes the antibody-gold nanoparticle conjugates fail to bind with the BTX-1-BSA that was coated onto the Millipore 135 NC membrane (Jieyi biotech Co., shanghai, china). Thus, it is shown as a positive control. If there is one test line or no line, it indicates an invalid result.
Different kinds of toxins, including BTX-1, BTX-2, BTX-3, okadaic acid (OA), domoic acid (DA), saxitoxin (STX), tetrodotoxin (TTX), and conopeptide (CTX), were used to identify the cross-reactivity of the test strip. The result from Figure 6B showed that there were two lines with the existence of other toxins and only one control line with the existence of BTX-1. This result indicated that the colloidal gold strip test based on anti-BTX-1 mAb had high specificity to BTX-1. Different concentrations of BTX-1 solution (0-250 ng/mL) were used to determine the limit of the colloidal gold strip. The results showed that the limit of detection (LOD) of the strip for BTX-1 was 200 ng/mL ( Figure 6C), and the test line will disappear totally if the concentration was above this level. Even though the LOD of the colloidal gold strip was higher than that of ELISA, the colloidal gold strip is more convenient, easy to operate, and requires a shorter time than ELISA.

Conclusions
In summary, a hybridoma cell line 6C6 stably secreting mAb against BTX-1 was obtained. The titer of the antibody was more than 1:32,000 and the affinity of the mAb was 1.06 × 10 8 L/mol. ELISA and colloidal gold strip assays for BTX-1 were developed based on the mAb against BTX-1. The linear range of ELISA to detect BTX-1 was 14-263 ng/mL with IC50 of 60 ng/mL, and the LOD was 14 ng/mL. The average recovery rate of ELISA from the spiked samples is 88 ± 2% in intra-assay and 89 ± 2% in inter-assay. The LOD for colloidal gold strip assay was 200 ng/mL. All these results indicated that the anti-BTX-1 mAb excreted by hybridoma 6C6 could be used to detect BTX-1 in shellfish and other related samples.

Materials
Brevetoxins were purchased from Taiwan Algal Science Inc.

Real Sample Assay with Colloidal Gold Strip
Four kinds of real sample solutions were used to identify whether the collected samples had BTX-1 or not. The result showed that the color density of four test lines of sample solution were the same as the negative control ( Figure 6D), indicating that there was no BTX-1 in these four samples. Therefore, this anti-BTX-1 mAb with high affinity and specificity can develop the ELISA kit for detection of BTX-1 in real samples. It also has the potential to develop the colloidal gold rapid diagnostic stripe.

Conclusions
In summary, a hybridoma cell line 6C6 stably secreting mAb against BTX-1 was obtained. The titer of the antibody was more than 1:32,000 and the affinity of the mAb was 1.06 × 10 8 L/mol. ELISA and colloidal gold strip assays for BTX-1 were developed based on the mAb against BTX-1. The linear range of ELISA to detect BTX-1 was 14-263 ng/mL with IC 50 of 60 ng/mL, and the LOD was 14 ng/mL. The average recovery rate of ELISA from the spiked samples is 88 ± 2% in intra-assay and 89 ± 2% in inter-assay. The LOD for colloidal gold strip assay was 200 ng/mL. All these results indicated that the anti-BTX-1 mAb excreted by hybridoma 6C6 could be used to detect BTX-1 in shellfish and other related samples.

Materials
Brevetoxins were purchased from Taiwan Algal Science Inc.

Preparation and Analysis of Complete Antigens
BTX-1-OVA and BTX-BSA complete antigens were prepared by two-steps approach (succinic anhydride method and isobutyl chloroformate method) according to the guidebook [16]. Agarose gel electrophoresis (0.8%, non-denaturing) was used to verify whether the conjugation was successful or not [17].

Production of Monoclonal Antibody
BTX-1-OVA was used as an immune antigen. BTX-1-OVA was emulsified with an equal volume of Freund's complete adjuvant, and then injected into female BALB/c mice (6-8 weeks old) at multiple sites subcutaneously. After eight injections, the titer of the antiserum from the immunized mouse was tested by indirect ELISA [17]. Indirect ELISA steps re described as follows: After coating with BTX-1-BSA antigen, plates were washed with phosphate buffer solution (PBS) and blocked with 5% PBSM (PBS containing 5% defatted milk, 200 µL/well) at 37 • C for 2 h. Then, 100 µL serially-diluted antiserum was added into the plates and incubated for 1 h at 37 • C. Subsequently, plates were washed three times with PBS and PBST (PBS containing 0.5% Tween 20), respectively, and incubated with 100 µL HRP conjugated goat anti-mouse IgG (1:8000 dilution) per well for 1 h at 37 • C. Following, the plates were washed again, and then TMB was added into the well and incubated for 10 min at 37 • C. After that, 2 M H 2 SO4 was added into the wells to stop the reaction, and the absorbance was measured immediately at 450 nm by a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).
Spleen cells were isolated and collected from the immunized mice with high titer serum, and then fused with SP2/0 myeloma cells at the ratio of 10:1 by polyethylene glycol (PEG, 1450). Cell fusion was carried out according to the guidebook [18]. The hybridoma was subcloned by the limiting dilution method [19].

Analysis of Hybridam Antibody
Chromosome number of the hybridoma cell was analyzed according to the guidebook [20]. Chromosome of hybridoma cell was counted after the treatment of colchicine. Hybridoma cells were treated with 0.4 µg/mL colchicine and harvested by centrifugation, and then resuspended in 10 mL of 0.075 mol/L potassium chloride hypotonic solution. Cells were gathered again by centrifugation and then resuspended with stationary liquid (methanol: acetic acid, v/v 3:1). The cells were dropped on the glass, and stained with Giemsa stain (St. Louis, MO, USA). Then, the chromosomes can be observed by microscope.
The subtype of the mAb against BTX-1 was tested with a Mouse Monoclonal Antibody Subtyping Kit (IgG1, IgG2b, IgG2a, IgG3, IgA, IgM). Six kinds of antibody subtyping reagents were diluted with PBS, and the solution was dropped into plate for incubation at 37 • C for 1 h following the same procedure as indirect ELISA.

Purification of mAb and Titre of the Antibody
Mature female BALB/c mice were injected with hybridoma cells (5 × 10 6 cells) suspended in RPMI 1640, and ascites fluid was collected from abdomen swelled mice through the needle of a 20 mL injector after 8 d injection. The antibody was purified from ascites by caprylic/ammonium sulfate precipitation method [21], and the purified antibody was analyzed by SDS-PAGE [22]. The titer of the purified anti-BTX-1 mAb was measured by indirect ELISA. The detailed information about indirect ELISA was the same as describe above.
The affinity of monoclonal antibody against BTX-1 was determined by indirect ELISA. Different concentrations of complete antigen BTX-1-BSA (10, 5, 2.5, 1.25 µg/mL) were coated into the plate, and the later procedures were the same as the indirect ELISA described above. The absorbance value of each well was measured at 450 nm by a microplate reader after the reaction was stopped by 2 M H 2 SO4. The curve diagram was made and the affinity constant of the antibody was calculated.

Standard Curve and Real Samples Detection by ic-ELISA
Firstly, BTX-1-BSA antigen was added into plates and blocked with PBSM (PBS containing 5% defatted milk), and different concentrations of BTX-1were mixed with the anti-BTX-1 antibody and added into plates. Then HRP and TMB were added into the plate subsequently, and the absorbance of each well was measured at 450 nm after the reaction was stopped by 2 M H 2 SO 4 . The standard curve was made and analyzed using OriGinpro 8 (OriginLab, Northampton, MA, USA). The linear range to detect BTX-1 was defined as the concentration of BTX-1 toward from 20% to 80% inhibition [23]. The matrix effect was minimized by diluting the samples before the ELISA assay, and matrix interference was measured by comparing a standard curve prepared in PBS buffer alone [19].
A recovery study was carried out to determine the efficacy of the standard curve. Different shellfish samples were purchased from local markets, and 10 g of each sample was ground into homogenization. The extraction solution of samples was used in the ELISA assay [17]. The recovery and coefficient of variation values were determined by the spiked samples with different concentrations of BTX-1 with six repeats. Then, the concentration of BTX-1 was detected by the developed ic-ELISA.

Construction and Characterization of Strip Test
Colloidal gold nanoparticles with a mean particle diameter of 40 nm were used to produce antibody-colloidal gold probes in our study. Then, well-dispersed colloidal gold particles were conjugated with anti-BTX-1 mAb. There are four parts in the strip test, including the sample, conjugate, absorbent pads, and nitrocellulose (NC) membrane with test and control zones [24]. The BTX-1-BSA conjugate was coated onto the Millipore 135 NC membrane as a test line, and HRP-labeled rabbit anti-mouse IgG antibody was coated onto the Millipore 135 NC membrane as a control line. Colloidal gold-antibody conjugates were applied on the treated conjugate pad at a proper spray rate. Finally, four sections of the strip were assembled and stored at room temperature until use.
The competitive immunoassay was performed on the strip test to identify the cross-activity of the colloid gold strip. Different kinds of toxins including BTX-1, BTX-2, BTX-3, okadaic acid (OA), domoic acid (DA), saxitoxin (STX), tetrodotoxin (TTX), and conopeptide (CTX) were used to react with the colloidal gold-BTX-1 mAb conjugate which was pipetted onto the conjugate pad. The detection results could be observed by the naked eye after the mixtures moved forward to the nitrocellulose membrane for incubation for 10 min at room temperature. To evaluate the sensitivity of the strip test, different concentrations of BTX-1 were applied to the sample pad of individual test strips, so that they would flow along the nitrocellulose strip, and then a visible limit of detection (LOD) that resulted in the disappearance of a red band on the test line would be determined [25].

Assay of BTX-1 in Samples with a Colloid Gold Strip
Different kinds of real samples were pretreated as above, and 100 µL of extracted solution was used to react with the colloidal gold-BTX-1 mAb conjugate which was pipetted onto conjugate pad [26]. The detection results could be observed by the naked eye after the mixtures moved forward to the nitrocellulose membrane. After reaction for 5-10 min, the result could be determined whether the related sample contained BTX-1 or not. If both the test and control lines turn red on the NC membrane, the sample is recorded as negative. When the control line but not the test line was colored red, it is considered as positive. In any assay, a red color band should always appear on the control line to make sure the strip test is working properly.