Prep cell protein fractionation and two-dimensional (2D) gel. (a) Ponceau stained nitrocellulose membrane containing samples from every sixth fraction collected from Prep Cell separation of Rhesus fetal brain protein. (b) Western blots of the membranes in a probed with maternal plasma reactive against the target bands. (c) Duplicate 2D gels were run with target region 30–40 kDa, 39–50 kDa and 60–85 kDa optimized Prep Cell fractions. The left column shows chemiluminescent images of 2D western blot probed with diluted plasma from mothers reactive against each of the antigens. Circles on the gels in the right column represent spots of reactivity between maternal antibodies and cognate antigens on the 2D blots on the left that were used to guide spot picking.

Western blots of candidate antigens. (a) Purified lactate dehydrogenase (LDH, containing A and B subunits) or recombinant full-length Cypin, Y-box-binding protein (YBX1), collapsin response mediator protein (CRMP) 1, CRMP2 and stress-induced phosphoprotein 1 (STIP1) proteins were probed with plasma from mothers of children with autism (AU) or mothers of typically developing (TD) controls diluted to 1:400, P indicates polyclonal antibody positive controls. Arrows indicate location of band(s) of interest. Note the different patterns of reactivity to LDH A and B subunits in lanes 1–3 of the LDH panel. This reactivity is further characterized in panel b where the blot of recombinant human glutathion-S-transferase (GST)-tagged LDHA (A), recombinant human GST-tagged LDHB (B) or purified native human LDH (Enz) was probed with maternal plasma diluted to 1:400, demonstrating variable reactivity to the LDH subunits. Also of note, as demonstrated in , some mothers of typically developing children have antibodies to the individual proteins and it is reactivity to the specific antigen combinations that confers specificity for MAR autoantibodies.

Blocking western blot. Maternal plasma samples incubated without any target antigen (U–unblocked) or blocked with lactate dehydrogenase (LDH), collapsin response mediator protein (CRMP) 1, stress-induced phosphoprotein 1 (STIP1; mother with reactivity to all three proteins), CRMP2 (mother who is CRMP1− and CRMP2 +), Y-box-binding protein (YBX1) and Cypin (mother with reactivity to Cypin, STIP1 and CRMP1) were used to probe a western blot containing Rhesus macaque fetal brain. The pure recombinant proteins were incubated with the plasma sample diluted 1:400 overnight, centrifuged and applied to the membrane blot containing fetal monkey brain. Arrowheads indicate location of the target antigen removed by overnight pre-incubation. Note that only the specific band(s) is/are removed following pre-incubation with each individual target protein indicating no cross-reactivity between the candidate antigens.

Schematic model depicting the neurodevelopmental roles for each of the maternal autoantibody target proteins. Disruption of protein function or quantity may negatively alter neurodevelopment, potentially leading to the core features associated with autism. There is likely an additive effect as illustrated by the autism spectrum disorder specificity of the various autoantibody combinations. CRMP, collapsin response mediator protein; LDH, lactate dehydrogenase; STIP1, stress-induced phosphoprotein 1; YBX1, Y-box-binding protein.