Preparation of synthetic antigen and monoclonal antibody for indirect competitive ELISA of citrinin

Figures & data

Figure 1.  The molecular structure of CIT.

Figure 2.  Synthetic scheme for CIT-protein conjugate.

Figure 3.  UV spectra of CIT, KLH and CIT–KLH conjugate. (A) UV spectrum of CIT. (B) UV spectra of KLH and CIT–KLH conjugate

Figure 4.  IR spectra of CIT, KLH and CIT–KLH conjugate. (A) IR spectrum of CIT. (B) IR spectra of KLH and CIT–KLH conjugate.

Figure 5.  Competitive ELISA standard curve.

Figure 6.  Structures of four mycotoxins and three pigments used to test McAb specificity.

Table 1. Cross-reactivity of McAb.

Structurally related molecules	Cross-reactivity (%)
AFB1	0.01
OTA	0.01
DON	<0.001
ZEA	<0.001
Rubropunctatine	<0.001
Rubropunctamine	<0.001
Monascin	<0.001

Table 2. Determination of CIT in artificially contaminated red yeast rice samples.

Amount of citrinin added (ng/g)	N ^a	Amount of citrinin detected (ng/g)	Mean recovery (%)	Coefficient of variation (%)
1	3	0.92±0.07	92	7.6
10	3	9.19±0.63	92	6.8
100	3	89.23±8.06	89	9.0
^aNumber of the determinations.

Table 3. Comparison of immunogenicity of CIT conjugates prepared by different methods.

Method	Antigen	Coupling ratio	Antibody	Titer^a	IC50^b (ng/mL)	Detected limit^b (ng/mL)	Author
Bicoupling method	CIT–KLH	52.3	Mice antiserum	96,000	100	10	This study
	CIT–KLH	52.3	McAb	138,000	0.3	0.01	This study
	CIT–KLH	116	Mice antiserum	24,000	UI	UI	Liu et al. (2009)
	CIT–KLH	NR	Rabbit antiserum	NR	500	NR	Xiao et al. (1995)
Active ester method	CIT–BSA	7.76	Mice antiserum	40,000	UI	UI	Wang et al. (2010)
	CIT–KLH	202	Mice antiserum	16,000	UI	UI	Liu et al. (2009)
Formaldehyde condensation method	CIT–BSA	6.6	Mice antiserum	65,000	200	10	Wang et al. (2010)
	CIT–BSA	6.6	McAb	98,000	10	1	Wang et al. (2010)
	CIT–BSA	19.9	Rabbit antiserum	12,500	3.2–50	0.4	Kononenko & Burkin, (2007)
	CIT–BSA	NR	Egg Yolk-derived immunoglobulin	512	150	10	Duan et al. (2009)
	CIT–KLH	NR	Rabbit antiserum	11,000	6–40	1	Abramson et al. (1995)
	CIT–KLH	85	McAb	12,000	200	10	Liu et al. (2009)
Note: NR: not reported, UI: unattainable inhibition.
^aDetermined by the indirect non-competitive ELISA.
^bDetermined by the indirect competitive ELISA.