Table 4.

Studies identifying presence of autoantibodies in individuals with ASD.

Subject details	Methods	Summarized findings	References
48 autism (5.9 ± 3.9 years) 19 CDD (7.0 ± 2.4 years) 14 PDD-NOS (4.8 ± 3.9 years) 9 LKS (7.4 ± 2.3 years) 37 epilepsy (5.9 ± 3.8 years) 29 HC (4.3 ± 2.0 years) 21 NNI (4.2 ± 2.6 years)	Serum ELISA measurements of BDNF, IgG/IgM auto-Abs to BDNF, endothelial cells, MBP, and histones Note: Subject numbers indicate total included in study. Actual numbers varied slightly depending on assay	↑ BDNF in ASD, CDD compared to HC and NNI ↑ anti-BDNF IgM and IgG in autism, CDD and epilepsy compared to HC ↑ IgM to endothelial cells in autism, CDD, PDD-NOS, and epilepsy compared to HC and NNI ↑ IgG to endothelial cells in autism and PDD-NOS compared to HC ↑ IgM and IgG to MBP in autism, CDD, PDD-NOS, and epilepsy compared to both HC and NNI, LKS not elevated	Connolly et al., 2006
29 ASD (3–12 years) 9 SIB (4–8 years) 13 TD (9–17 years)	Serum ELISA and Western blot reactivity to human brain	↑ reactivity to 100 kDa epitope in caudate putamen and prefrontal cortex in ASD ↑reactivity to 73 kDa epitope in cerebellum and cingulate gyrus in ASD and SIB	Singer et al., 2006
63 ASD (2–15 years) 63 TD (2–14 years) 25 SIB (1–13 years) 21 DD (2–5 years)	Western blot of plasma reactivity to adult human hypothalamus and thalamus protein extracts	↑ reactivity to 52 kDA thalamus and hypothalamus proteins in ASD ↑ reactivity to 3 hypothalamus proteins (42–48 kDa MW)	Cabanlit et al., 2007
11 ASD 9 SIB (>6 years)	72-h neuronal culture analyzed for effect of ASD sera on differentiation of NPCs by immunoblotting, morphometry, and immunocytochemistry	Treatment with ASD sera: ↓ NPC proliferation ↑ cell migration ↑small cells with processes ↑ length and number of processes ↑ synaptogenesis	Mazur-Kolecka et al., 2007
33 ASD (7.3 ± 3.0 years) 26 regressive autism (6.7 ± 2.7 years) 25 TD (8.9 ± 3.4 years) 24 Tourette syndrome (10.0 ± 2.6 years)	Plasma ELISA and Western blot reactivity to MBP	↑ auto-Abs to MBP found in regressive autism compared to classic (infantile) autism and Tourette syndrome subjects.	Libbey et al., 2008
63 ASD (2–15 years) 63 TD (2–14 years) 25 SIB (1–13 years) 21 DD (2–5 years)	Western blot of plasma reactivity to human cerebellar protein extracts. Cerebellar-specific auto-Abs detected by IHC of Macaca fascicularis monkey cerebellum.	↑auto-Abs to 52 kDa cerebellar protein in ASD ↑“intense immunoreactivity” to Golgi cells of the cerebellum in ASD, associated with auto-Abs to 52 kDa cerebellar protein	Wills et al., 2009
37 ASD (1–12 years) 37 TD (1–14 years)	Measured effect of ASD sera on cell response to oxidative stress via immunoblotting, morphology, immunofluorescence, apoptosis, and proliferation assays.	Oxidative stress reduced proliferation in differentiating NPCs treated with TD sera. Effect was not as prominent with ASD sera, indicating an altered response to oxidative stress.	Mazur-Kolecka et al., 2009
20 ASD (3.0 ± 0.4 years) 12 TD (3.0 ± 1.2 years)	Taqman Real time PCR to detect serum mtDNA. Serum ELISA analysis to detect mtDNA antibodies	↑ extracellular mtDNA in ASD ↑ anti-mtDNA auto-Abs (type 2) in ASD	Zhang et al., 2010
277 ASD (70 BAP) 189 TD (2–5 years)	Western blot for child plasma reactivity to Rhesus macaque cerebellum protein medley	↑auto-Abs to 45 kDa protein in ASD ↑auto-Abs to 62 kDa protein in BAP Increases in either auto-Ab was associated with lower adaptive and cognitive scores, increased aberrant behaviors.	Goines P. et al., 2011
54 ASD 54 TD (4–11 years)	Serum anti-ganglioside M1 Abs were measured by ELISA	↑ antiganglioside M1 auto-Abs in ASD, especially in severe compared to mild or moderate autism.	Mostafa and Al-Ayadhi, 2011
86 ASD (2.0–5.6 years) 43 TD (2.3–4.7 years)	IHC for plasma reactivity to sections of macaque monkey brain (methods similar to Wills et al., 2009), results compared to behavioral assessments.	No differences in rate of plasma immunoreactivity to cerebellar Golgi neurons and other neural elements in ASD vs. TD, however immunoreactivity associated with worsening behavior and higher multiple CBCL scores.	Rossi et al., 2011
7 ASD with reactivity (2.5 to 7 years) 7 ASD with no reactivity 6 TD with no reactivity (2.5 to 8 years)	IHC: follow up of subgroup of ASD children from previous study (Wills et al., 2009) with reactivity to cerebellar 52-kDa protein and to Golgi cell region of the cerebellum. IHC to detect plasma immunoreactivity in the maqaque and male mouse brains.	Reactivity seen in previous study identified as GABAergic interneurons (based on co-localization of staining to calcium-binding proteins). Reactivity extended to other regions of the brain with slight preponderance to superficial layers of the cortex.	Wills et al., 2011
80 ASD 80 TD (6–12 years)	Indirect immunofluorescence used to measure serum anti-neuronal antibodies	↑ anti-neuronal auto-Abs in ASD, associated with increased severity of autism and seen more frequently in females ASD (90 vs. 53.3%, P = 0.001).	Mostafa and Al-Ayadhi, 2012b
50 ASD 30 TD (5–12 years)	Serum ELISA measurements of 25-hydroxy vitamin D and anti-MAG autoAbs	↓ 25-hydroxy vitamin D in ASD ↑ anti-MAG auto-Abs in ASD 25-hydroxy vitamin D levels negatively correlated with CARS scores and anti-MAG auto-Abs	Mostafa and Al-Ayadhi, 2012a
54 ASD 22 DD 33 TD (2–5 years)	Plasma ELISA measurements of anti-cardiolipin, anti-phosphoserine, and anti-β-glycoprotein 1 auto-Abs	↑ auto-Abs to cardiolipin, phosphoserine, and β-glycoprotein 1 in ASD compared to TD and DD controls, significantly associated with worsening behaviors.	Careaga et al., 2013
42 ASD 42 TD (6–11 years)	Serum ELISA measurement of human anti-MBP Abs. Severity of ASD and manifestation of allergic/asthma symptoms compared to results.	↑ auto-Abs to MBP and MAG in ASD, regardless of allergies. Severity of autism was also found to be associated with increased allergies.	Mostafa and Al-Ayadhi, 2013
93 ASD (2.9–17.4 years)	Patented process of identifying FRA: incubated serum with folate receptors then added radio-labeled folic acid. HPLC measurement of 5-MTHF in the CSF.	↑ prevalence of FRA in ASD sera. Blocking FRA correlated with CSF 5-MTHF concentrations in 16 children. Treatment with folinic acid improved attention, language and communication, and repetitive behaviors, with “moderate to much” improvement seen in 1/3 of children treated.	Frye et al., 2013
75 ASD (2–22 years) 30 DD (1–18 years)	Patented process of identifying FRA: incubated serum with folate receptors then added radio-labeled folic acid.	↑ prevalence of FRA in ASD vs. DD ↑ prevalence of FRA in parents of ASD vs. DD, suggesting familial autoimmune component to ASD	Ramaekers et al., 2013
20 ASD (1.4–5 years) 18 TD (1.4–4.4 years)	Immunoblotting and immunocytochemistry to detect serum auto-Abs against differentiating NPCs	↑ auto-Abs against human neuronal progenitor cell proteins of 55, 105, 150, and 210 kDa molecular weights in ASD subjects compared to controls. Strongest reactivity noted in NPCs expressing Tuj1.	Mazur-Kolecka et al., 2014
100 ASD 100 TD (4–11 years)	ELISA measurement of serum anti-ds-DNA Abs. Immunofluorescence measurement of serum antinuclear Abs.	↑ anti-ds-DNA and anti-nuclear auto-Abs in ASD Presence of anti-ds-DNA auto-Abs positively associated with a family history of autoimmunity.	Mostafa et al., 2014
355 ASD 142 SIB (2–47 years, mean age: 9.06)	Western blot plasma reactivity to homogenized Rhesus macaque brain tissue and human adult cerebellum	Plasma reactivity at 45 and 62 kDa brain proteins associated with autism severity and larger head circumference. 45 kDa reactivity associated with cognitive impairment/lower VABS scores while 62 kDa reactivity associated with stereotypies.	Piras et al., 2014
60 ASD 60 TD (3–12 years)	ELISA measurement of serum anti-nucleosome-specific antibodies.	↑ anti-nucleosome-specific auto-Abs in ASD, associated with family history of autoimmunity.	Al-Ayadhi and Mostafa, 2014
55 ASD (3–12 years) 25 TD (4–12 years)	ELISA measurement of plasma levels of anti-endothelial cell antibodies	↑anti-endothelial cell auto-Abs in children with autism compared to healthy controls, associated with autism severity.	Bashir and Al-Ayadhi, 2015
62 ASD (4–11 years) 62 TD (5–12 years)	ELISA measurement of serum ENA-78 and anti-neuronal auto-antibodies	↑ anti-neuronal auto-Abs in ASD ↑ENA-78 (neutrophil-recruiting chemokine CXCL5) associated with increases in anti-neuronal auto-Abs	Mostafa and Al-Ayadhi, 2015
40 ASD/FRAA-(7.0 ± 3.3 years) 16 ASD/FRAA blocking + (6.4 ± 3.0 years) 48 ASD/FRAA binding + (7.3 ± 3.1 years)	Measured redox, methylation, vitamins and immune biomarkers using various assays and compared to behavioral assessments	↓ 3-Chlorotyrosine (a marker of inflammation) was in those positive for blocking FRAs Presence of blocking FRAs in ASD associated with less severe ASD symptoms compared to ASD negative for these FRAs	Frye et al., 2016

ASD, autism spectrum disorders; CDD, childhood disintegrative disorder with regression after age 2 years; PDD-NOS, pervasive developmental disorder-not otherwise specified; LKS, Landau-Kleffner syndrome; HC, healthy children; NNI, children with non-neurologic illnesses; ELISA, enzyme-linked immunosorbent assay; BDNF, brain-derived neurotrophic factor; MBP, myelin basic protein; SIB, typically-developing sibling; TD, typically developing child; kDa, kilodalton; DD, non-ASD developmentally delayed; NPCs, human neuronal progenitor cells; MEF, myelin-enriched fraction of the brain; AEF, axolemma-enriched fraction of the brain; IHC, immunohistochemistry; auto-Abs, autoantibodies; PCR, polymerase chain reaction; mtDNA, mitochondrial DNA; BAP, broader diagnosis of autism spectrum disorder; CBCL, The Child Behavior Checklist; MAG, myelin-associated glycoprotein; FRA, folate receptor antibodies; 5-MTHF, 5-methyltetrahydrofolate; CSF, cerebrospinal fluid; Tuj1, neuron-specific Class III β-tubulin; ds-DNA, double-stranded DNA; VABS, Vineland Adaptive Behavior Scales; ENA-78, Epithelial cell-derived neutrophil-activating peptide-78; CXCL5, C-X-C motif chemokine 5.