Development of a specific monoclonal antibody-based icELISA for detection of arecoline in traditional Chinese medicines and fresh areca nuts

Figures & data

Figure 1. The synthesis procedure for the antigen.

Figure 2. The results of molecular weight determination by MALDI-TOF/TOF-MS. The value marked near the peak is the molecular weight. (A) OVA; (B) coating antigen (arecaidine-OVA); (C) KLH; (D) immunogen (arecaidine-KLH).

Table 1. Cross reactivity of arecoline and related alkaloids to the mAb-A5H12 in the icELISA.

Compound	Structure	IC50 (ng/mL)	CR (%)
Arecoline		67.9	100
Arecaidine		>10000	<1
Guvacoline		>10000	<1
Guvacine		>10000	<1
Nicotinic acid		>10000	<1
Trigonelline		>10000	<1
Salicylic acid		>10000	<1
Benzoic acid		>10000	<1
Picolinic acid		>10000	<1
Nicotinamide		>10000	<1

Figure 3. Standard inhibition curve for arecoline by icELISA. Each point represents the average data for three determinations ± standard deviations. B₀ and B denote the absorbance at 450 nm in the absence and presence of arecoline, respectively. The R² value is 0.99,917.

Table 2. The effects of methanol content in sample diluent in icELISA.

Methanol content	IC50 (ng/mL)	R^2
0%	67.9	0.998
5%	74.7	0.992
10%	76.9	0.999
20%	113.4	0.992

Table 3. The recoveries of arecoline determined by icELISA in spiked areca nut leaves, pericarp and endosperm samples (n = 3).

Samples	Spiked concentration (mg/g)	Arecoline icELISA (mg/g)	Mean recovery (%)	CV (%)
Areca leaves^a	0	5.06 ± 0.07^c	–	1.38
	1.5	6.51 ± 0.18	99.29	2.81
	3	8.18 ± 0.13	101.49	1.60
	5	9.99 ± 0.02	99.30	0.18
Areca nut pericarp^b-1	0	1.89 ± 0.10	–	0.16
	1	2.93 ± 0.14	101.39	0.14
	5	6.78 ± 0.11	98.42	0.05
	10	13.01 ± 0.58	109.36	0.13
Areca nut pericarp^b-2	0	1.35 ± 0.10	–	6.44
	1	2.56 ± 0.36	108.95	14.4
	5	5.67 ± 0.62	104.37	5.85
	10	12.56 ± 0.65	110.64	7.99
Areca nut endosperm^b	0	12.43 ± 0.80	–	7.18
	1	15.18 ± 2.17	113.08	14.04
	5	17.62 ± 1.03	101.11	9.33
	10	26.02 ± 2.08	116.02	5.21

- unavailable.

^a From Haikou, Hainan, China.

^b From Danzhou, Hainan, China.

^c Data are average values ± standard deviations (SD)

Table 4. Arecoline concentrations in traditional Chinese medicines tested by icELISA and HPLC (n = 3, the receipts of these medicines are shown in supplementary table 1).

Samples	icELISA (mg/g)	HPLC (mg/g)
Bing Lang Si Xiao Pills	0.69 ± 0.13^a	0.66 ± 0.0005
Jin Sang Li Yan Capsules	0.12 ± 0.02	0.15 ± 0.0006
Kai Xiong Shun Qi Pills	0.94 ± 0.11	1.03 ± 0.002
Shu Gan Jian Wei Pills	0.61 ± 0.17	0.49 ± 0.001
Wei Su Particles	0.53 ± 0.11	0.52 ± 0.0004

^a Data are average values ± standard deviations (SD)

Table 5. Arecoline concentrations of fresh areca nuts tested by icELISA and HPLC (n = 3).

Samples	icELISA (mg/g)	HPLC (mg/g)
Areca leaves^a-1	5.11 ± 0.14^c	5.12 ± 0.07
Areca leaves-2	5.48 ± 0.05	5.39 ± 0.08
Areca nut pericarp^b	2.25 ± 0.16	2.74 ± 0.07
Areca nut endosperm^b	14.78 ± 3.99	10.09 ± 0.09
Areca female flowers^b	2.47 ± 0.49	1.87 ± 0.01
Areca male flowers^b	1.76 ± 0.42	1.75 ± 0.04
Areca old leaves^b	0.61 ± 0.09	0.68 ± 0.03

^a From Haidian campus of Hainan University, Haikou, Hainan, China.

^b From Danzhou campus of Hainan University, Danzhou, Hainan, China.

^c Data are average values ± standard deviations (SD)

Figure 4. Validation between the results of icELISA and HPLC for (A) five TCMs and (B) fresh tissues from areca trees.