Table 3.

Characterization of VHH against small molecular weight compounds

Analyte (MW)	Immunogen- protein (animal)	Expression system (Yield %)	Elution conditions	Temperature stability (range)	Formats and uses	Comments
Azo dyes [11, 70] RR6 (733.38) RR120 (1469.98)	RR6-BSA (llama) RR120-BSA	E coli Yeast	no panning, screened supernatants of randomly picked E coli colonies	4 °C to 90 °C	Crystallography Resonant mirror biosensor (Kd 18 – 83 nM)	Stable in up to 4M ammonium thiocyanate or 50% ethanol. DNA shuffling used to alter affinity, specificity and yield in yeast
Picloram [16] (241.46)	None (llama)	E.coli TG1 (free VHH and VHH-Phage)	EB20 [50 mM Tris– acetate (pH 7.5), 150 mM NaCl, 20 mM EDTA, 50 µg/mL Saccharomyces cerevisiae RNA]		SPR analysis, phage ELISA	Failed in phage- system, used ribosomal display. Best clones contained mutations introduced during PCR steps.
Auxin [17] (indole-3-acetic acid) (175.19)	None (llama)	E.coli TG1 (~1 mg/L)	0.1 M triethylamine		SPR analysis, CDR shuffling, pentamerization	SPR with immobilized pentamerized sdAbs allow direct observation of analyte-Ab interaction (Kd).
Caffeine [18] (194.19)	caffeine carboxylate-KLH (llama and alpaca)	E. coli	0.1 M triethylamine	>90% recovery after 90°C	Patented lateral flow devices, immunoaffinity chromatographic separation and detection	Application to real samples: commercial beverages
Methotrexate [22] (454.44)	MTX-BIP (llama)	E. coli (2 – 20 mg/L)	0.1 M triethylamine pH 11.0	Not tested	SPR (Kd 29 – 515 nM; Kd competitive assay, 80 nM)	Utilized CDR grafting to explore mechanism of binding. VHH crystallized
15- Acetyldeoxynivalenol [25] (338.35)	15-DON-BSA (llama)	E. coli (3–6 µg/L)	triethylamine	Not tested	fluorescence polarization, direct ELISA, SPR	Used nested PCR, made monomer/pentamer
Ochratoxin A [26] (403.813)	OTA-KLH (llama)	E.coli BL21(DE3)ply sS	panning 2× against OTA-OVA, 1× against OTA-KLH	Not demonstrated	fused to AP	Application to real cereal samples
Triclocarban [29] (315.58)	TCC-Thyr (llama)	E.coli BL21(DE3)	eluted with target analyte, decreasing concentrations	some inactivated at 85 °C; others remained active after 1h at 100 °C	none	VHH that had additional disulfide bonds did not perform the best in thermal stability study.
3-Phenoxybenzoic acid [31] (214.22)	3-PBA-Thyr (alpaca)	E.coli TOP10F’	eluted with target analyte, decreasing concentrations	Not demonstrated	none	Spike-recovery validation
2,2’,4,4’- Tetrabromodiphenyl ether [32] (485.79)	BDE-9-KLH (BDE-C1) (alpaca)	E.coli TOP10F’	eluted with target analyte, decreasing concentrations	95 °C for 10, retained >50% of its activity; 95 °C for 60, retailed >25% of its activity	biosensors, electrochemical and chip[34]
Tetrabromobisphenol A [35] (543.9)	T5-Thy (alpaca)	E.coli TOP10F’	eluted with target analyte, decreasing concentrations	90 °C heat for 10 mins 80% activity; 90 °C heat for 90 mins 20% activity	fused to AP, improved stability[38]
Aflatoxin B1 [39] (312.27)	AFB1-BSA (alpaca)	E.coli TOP10F’	panned with AFB1- BSA, eluted with target analyte, decreasing concentrations	Nb26 clone: ≈ 70% activity, Nb28 clone: ≈ 70% activity at 85 °C for 1h		Spike-recovery validation

Abbreviations used: BSA, bovine serum albumin; Pic, picloram hapten; CDR, complementarity determining region; OVA, ovalbumin; KLH, keyhole limpet hemocyanin; MTX, methotrexate hapten; DON, deoxynivalenol hapten; TNB, trinitrobenzene hapten; OTA, ochratoxin hapten; TCC, triclocarban hapten; Thyr, thyroglobulin; AP, alkaline phosphatase; 3-PBA; 3-phenoxybenzoic acid hapten; BDE-9, brominated diphenyl ether hapten 9; AFB₁, aflatoxin B₁ hapten; BIP, blue carrier immunogenic protein.