Interference from Heterophilic Antibodies in Amyloid-β Oligomer ELISAs

Abstract

Amyloid-β (Aβ) oligomers of different sizes and forms have recently been the focus for many Alzheimer's disease (AD) researchers. Various immunoassays have been used to detect low concentrations of these elusive Aβ species in different forms of human samples using little or no sample dilutions. However, the possibility that positive results may be caused by interference from heterophilic antibodies (HA) is often overlooked. HA, which recognize immunoglobulins from other species, are present in human plasma and cerebrospinal fluid (CSF) and may cause interference in sandwich immunoassays like enzyme-linked immunosorbent assays (ELISAs) by cross-binding the capture and detection antibodies of the assay. They thus may generate a false positive signal. Here we show that when assessing the Aβ oligomer content in plasma samples from 44 individuals with a sandwich ELISA, none of the 21 positive signals remained when the assay was repeated in the presence of factors blocking HA. Similarly, in CSF samples from 104 individuals, the signals from the 22 positive samples were strongly reduced when analyzed after anti-HA treatment. Taken together, HA interference is a problem that needs to be addressed when measuring low levels of an antigen in human plasma and CSF samples.

Specificity and sensitivity of the Abeta oligomer ELISA

“…Xia et al (2009), using this ELISA to measure A␤ oligomer levels in plasma specimens from AD patients and control subjects, reported A␤ oligomers in 19 of 36 plasma samples from AD patients and 3 of 10 control subjects. However, as pointed out by Sehlin et al (2010), the possibility that heterophilic antibodies in the plasma samples may have resulted in a false positive signal in the study by Xia et al (2009) by crosslinking the capture and reporter antibodies was not examined. Sehlin et al (2010) found that 21 plasma samples which had tested positive with the ELISA subsequently tested negative when the assay was repeated with a diluent which blocked heterophilic antibody activity.…”

“…However, as pointed out by Sehlin et al (2010), the possibility that heterophilic antibodies in the plasma samples may have resulted in a false positive signal in the study by Xia et al (2009) by crosslinking the capture and reporter antibodies was not examined. Sehlin et al (2010) found that 21 plasma samples which had tested positive with the ELISA subsequently tested negative when the assay was repeated with a diluent which blocked heterophilic antibody activity. The study by Xia et al (2009) also used this ELISA to measure soluble and insoluble (guanidine HCl-extracted) A␤ oligomers in brain specimens.…”

How many biomarkers to discriminate neurodegenerative dementia?

“…Indeed, it is worth noting that the variability of the results is very high among the many studies on oligomeric aggregates in CSF. Actually, in the majority of these studies, Ab and aSyn oligomers have been measured using immunometric or cytofluorimetric assay, which can give false positive results 152,159 due to the presence of interfering factors, such as heterophilic antibodies 160 , or the presence of RIPA buffer 151,153 . Therefore, the search for CSF oligomers may be very promising for the diagnosis and the comprehension of pathological pathways of neurodegenerative diseases, but a great effort is required to validate a reliable method of analysis.…”

Increased Detection of Aβ Oligomers in the Cerebrospinal Fluid of Alzheimer s Disease: Fact or Artifact?

“…Moreover, Sehlin et al [10] cast doubt as to whether the reported increased levels of Aβ oligomers in the CSF by Georganopoulou et al and Fukumoto et al [5,6] are true or due to the interference of the heterophilic antibodies (HA) in the detection of Aβ oligomer with ELISAs [10]. HA, i.e.…”

“…naturally occurring human antibodies to immunoglobulins of animal origin, can be source of interference in immunometric routine assays. Sehlin et al [10] indeed studied the Aβ oligomer content with a sandwich ELISA in CSF samples from 104 individuals, and showed that the Aβ oligomer signals from the positive samples were strongly reduced when analyzed in the presence of factors blocking HA. It is worth noting that sick and hospitalized patients have higher levels of HA, so that these individuals may have an increased risk of generating a false positive response in any sandwich immunoassay [10,11].…”