Antibody-based sex determination of human skeletal remains

Abstract

Assignment of biological sex to skeletal remains is critical in the accurate reconstruction of the past. Analysis of sex-chromosome encoded AMELX and AMELY peptides from the enamel protein amelogenin underpins a minimally destructive mass spectrometry (MS) method for sex determination of human remains. However, access to such specialist approaches limits applicability. As a convenient alternative, we generated antibodies that distinguish human AMELX and AMELY. Purified antibodies demonstrated high selectivity and quantitative detection against synthetic peptides by ELISA. Using acid etches of enamel from post-medieval skeletons, antibody determinations corrected osteological uncertainties and matched parallel MS, and for Bronze Age samples where only enamel was preserved, also matched MS analyses. Toward improved throughput, automated stations were applied to analyze 19th-century teeth where sex of individuals was documented, confirming MS can be bypassed. Our immunological tools should underpin development of routine, economical, high-throughput methods for sex determination, potentially even in a field setting.

Keywords: Immunology; Methodology in biological sciences; Paleobiology.

Graphical abstract

Figure 1

Amelogenin peptide sequences and antibody detection by dot-blot (A) Sequence alignment of endogenous sex-specific human amelogenin peptides AMELX and AMELY. Asterisks indicate sequence identity, “-” indicates gap introduced in the alignment, M[Ox] represents oxidized methionine, numbering relates to amino acid residues in full-length amelogenin sequences. (B) Representative dot-blot indicating affinity purified antibody detection of appropriate synthetic peptides at 10ng sensitivity, with no visual evidence of cross-reactivity of anti-AMELX antibody against AMELY peptide, or vice versa (detection antibodies shown on the right-hand side). “50 + 50” indicates a mixture of AMELX and AMELY peptides.

Figure 2

ELISA detection of synthetic amelogenin peptides (A) Representative ELISAs demonstrating appropriate detection of synthetic AMELX (red lines) and AMELY (blue lines) peptides by affinity-purified antibodies as indicated, down to ∼100pg antigen, again with limited cross-reactivity against the “wrong” peptide. Data are represented as mean ± SEM. (B) Bar graph demonstrating the quantitative nature of ELISA allows distinction of mixtures of synthetic AMELX and AMELY peptides at various ratios as indicated (500pg total peptide e.g., 1:1 represents 250pg of each peptide). Y axis indicates ratios of observed ELISA values, determined by separately probing the same peptide mixtures with α-AMELX or α-AMELY antibodies (e.g., for 5:1, X:Y, α-AMELX value divided by α-AMELY value).

Figure 3

Sex determination of post-medieval human teeth using manual ELISA (A) AMELX/AMELY (X/Y) ratios detected by ELISA for enamel extracts from four post-medieval human teeth (12.5% of each total enamel extract loaded per well, in duplicate). (B) As for (A) with an additional four post-medieval human teeth with lower sample load (6.25% of each total enamel extract loaded per well, in duplicate). Male or female assignments, based on ratios, are indicated. (C) Representative extracted ion chromatograms showing the MS/MS transitions m/z 540.3 → 879.5 (AMELX, black) and m/z 440.2 → 645.4 (AMELY, red) demonstrating that LU7 and LU8 are samples from a male and a female, respectively.

Figure 4

Sex determination of Bronze Age human teeth using manual ELISA AMELX/AMELY (X/Y) ratios detected by ELISA for enamel extracts from seven Bronze Age human enamel samples with “high” (>100pg) endogenous AMELX detection (12.5% of each total enamel extract loaded per well, in duplicate). Male or female assignments, based on ratios, are indicated.

Figure 5

Sex determination of 19^th century human teeth using automated ELISA AMELX/AMELY (X/Y) ratios detected by ELISA using automated stations for enamel extracts from six 19^th century human enamel samples (12.5% of each total enamel extract loaded per well, in duplicate). Male or female assignments, based on ratios, are indicated.

Figure 6

Statistical analyses of ELISA data (A) Boxplot showing median differences between AMELX/AMELY (X/Y) ratios determined using manual ELISAs (data for post-medieval and Bronze Age teeth samples combined). ∗∗∗, p ≤ 0.001, two-tailed Mann-Whitney U-test. Data are represented as median with error bars indicating 5–95 percentile. (B) Bar graph showing differences in mean AMELX/AMELY (X/Y) ratios determined using automated ELISA (19^th century teeth samples). ∗, p ≤ 0.05, two-tailed t-test. Data are represented as mean ± SEM.