Mycolic acids (MA) are major, species-specific lipid components of Mycobacteria and
related genera. In Mycobacterium tuberculosis, it is made up of alpha-, methoxy- and keto-
MA, each with specific biological functions and conformational characteristics. Antibodies in
tuberculosis (TB) patient sera respond differently towards the three MA classes and were
reported to cross-react with cholesterol. To understand the antigenicity and cholesterol
cross-reactivity of MA, we generated three different chicken -derived phage-displayed single-
chain variable fragments (scFv) that reacted similarly towards the natural mixture of MA,
but the first recognized all three classes of chemically synthetic MAs, the second only the
two oxygenated types of MAs and the third only methoxy MA. The cholesterol cross-reactivity
was investigated after grafting each of the three scFv types onto two configurations of
constant chain domains±CH1-4 and CH2-4. Weak but significant cross-reactivity with cholesterol
was found only with CH2-4 versions, notably those two that were also able to recognize
the trans-keto MA. The cholesteroid nature of mycobacterial mycolic acids therefore
seems to be determined by the trans-keto MA subclass. The significantly weaker binding to
cholesterol in comparison to MA confirms the potential TB diagnostic application of these
S1 Fig. Sequences of gallibody clones produced by antibody engineering. 12) Anti-MA 12,
16) Anti-MA 16, 18) Anti-MA 18, CH1-4 = full length constant region, CH2-4 = truncated constant region, VH = variable heavy chain, VL = variable light chain.
S2 Fig. SDS-PAGE analysis illustrating gallibody purification using Ni-NTA affinity columns.
A) 12CH1-4, B) 16CH1-4, C) 18CH1-4, D) 12CH2-4, E) 16CH2-4, F) 18CH2-4. Gel lanes 1)
Marker, 2) Culture supernatant, 3) Flow through 1, 4) Flow through 2, 5) Washes, 6) Elution
1, 7) Elution 2, 8) Elution 3, 9) Elution 4. Successful purification is demonstrated by the comparable
thickness of the 67 kDa band obtained with the culture supernatant (2) and the elutions
S1 Dataset. Experimental data used for producing Figs 3 and 4.
S2 Dataset. Experimental data used for producing Fig 5.
S3 Dataset. Experimental data used for producing Fig 6.