High-affinity recombinant full-length antibody-based immunochromatographic strip assay for rapid and reliable detection of pyraclostrobin residues in food samples

Figures & data

Figure 1. Chemical structures of pyraclostrobin (A) and its hapten (B).

Table 1. Primer sequences for VH and VL gene amplification.

Primer name^a	Number	Oligo sequences^b
VHF	1	ATGAACTTYGGGYTSAGMTTGRTTT
	2	ATGTACTTGGGACTGAGCTGTGTAT
	3	ATGAGAGTGCTGATTCTTTTGTG
	4	ATGGATTTTGGGCTGATTTTTTTTATTG
VHR	/	ACTAGTCGACATGGRATGGASCKKIRTCTTTMTCT
VLF	1	ACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCT
	2	ACTAGTCGACATGGATTTWCARGTGCAGATTWTCAGCTT
	3	ACTAGTCGACATGGTYCTYATVTCCTTGCTGTTCTGG
	4	ACTAGTCGACATGGTYCTYATVTTRCTGCTGCTATGG
VLR	/	ACTGGATGGTGGGAAGATGGA

^aVHF/R are the primers of heavy chains with IgG subtype; VLF/R are the primers of light chains with κ subtype.

^bDegenerate primer code: R = A/G, M = A/C, K = G/T, S = C/G, W = A/T.

Figure 2. Partial constructs of the expression vector and the illustration of homologous recombination.

Table 2. Primers of PCR progress for obtaining homologous recombination fragments.^a

Primer name^b	Oligo sequences
B174001HF	GTCCTCCTGACTGGGGTGAGGGCCGAAGTGGAGCTGGTCGAGTCTGGG
B174001HR	GACAGATGGGGGTGTCGTTTTAGCTGAGGAGACTGTGAGAGTGGTGCC
B174001LF	GTCCTCCTGACTGGGGTGAGGGCCGACATTGTGCTGACCCAATCTCCA
B174001LR	TACAGTTGGTGCAGCATCTGCTCTTTTGATTTCCAGCTTGGTGCCTCC

^aPrimer sequences: Homologous arms-Variable regions of light or heavy chain of PY-C7.

^bB174001HF/R are the primers of VH; B174001LF/R are the primers of VL.

Figure 3. (A) Schematic illustration of the detection procedure by GNPs-ICS. (B) The decision criterion of GNPs-ICS assay. * (B) Negative samples (-), positive samples (+) and strongly positive samples (++) judged by naked eyes.

Table 3. The ratio of gradient elution of pyraclostrobin detection by LC-MS/MS in strawberry.

Time (min)	Flow rate (mL min^−1)	0.1% formic acid	methyl alcohol
0	0.3	80%	20%
1	0.3	80%	20%
5	0.3	10%	90%
8	0.3	10%	90%
9.5	0.3	80%	20%
10	0.3	80%	20%

Figure 4. (A) The pyraclostrobin-specific mAbs secretory capacity of resuscitated PY-C7 cell line in different monoclonal cell wells. (B) WB results of light or heavy chains of positive and negative monoclonal cell lines.

Figure 5. (A) PCR amplification of variable region fragments of PY-C7-mAb with homologous arms. (B) SDS-page of the full-length rAb expressed in mammalian cells HEK293(F). * (A) M’: 5000 KD Marker in actual map; M: standard 5000 KD Marker; (B) M’: 200 KD Marker in actual map; M: standard 200 KD Marker.

Figure 6. The affinities of PY-C7-mAb and PY-C7-rAb binding pyraclostrobin.

Figure 7. GNPs-mAb/rAb-ICS assay for testing gradient-spiked pyraclostrobin standard solution. * The concentrations of pyraclostrobin standard were, in order, 0, 0.01, 0.02, 0.05, 0.10, 0.20, 0.40 mg L⁻¹.

Figure 8. Pyraclostrobin, kresoxim-methyl, azoxystrobin, difenoconazole, each at a concentration of 0.2 mg L⁻¹, were tested by GNPs-ICS assays based on the mAb and the rAb.

Figure 9. The visual results of recovery tests of pyraclostrobin in strawberry, banana and Chinese cabbage samples detected by GNPs-mAb/rAb-ICS assays.

Table 4. Results of repeated recovery tests of pyraclostrobin in spiked food samples detected by GNPs-mAb/rAb-ICS assays.

Sample	Spiked (mg kg^−1)	Visual detection (n = 3)
		GNPs-mAb-ICS			GNPs-rAb-ICS
Strawberry	0.25	–^a	–	–	–	–	–
	1.00	–	+^a	+	++^a	++	++
	2.00	++	++	++	++	++	++
Banana	0.25	–	–	–	–	–	–
	1.00	+	+	+	++	++	++
	2.00	++	++	++	++	++	++
Chinese cabbage	0.25	–	–	–	–	–	–
	1.00	+	+	+	++	++	++
	2.00	++	++	++	++	++	++

^aNegative samples (–), positive samples (+) and strongly positive samples (++) judged by naked eyes.

Table 5. The detection of pyraclostrobin residue in actual strawberry samples by GNPs-ICS assay and LC-MS/MS.

Samples no.	S1	S2	S3	S4	S5	S6	S7	S8	S9	S10
LC-MS/MS (mg kg^−1)	ND^a	ND	ND	0.15	1.427	4.287	ND	0.02	ND	ND
GNPs-ICS	–^a	–	–	–	+^a	++^a	–	–	–	–

^aNot detectable (ND), negative samples (–), positive samples (+) and strongly positive samples (++).