Natural IgG Autoantibodies Are Abundant and Ubiquitous in Human Sera, and Their Number Is Influenced By Age, Gender, and Disease

<div><p>The presence of self-reactive IgG autoantibodies in human sera is largely thought to represent a breakdown in central tolerance and is typically regarded as a harbinger of autoimmune pathology. In the present study, immune-response profiling of human serum from 166 individuals via human protein microarrays demonstrates that IgG autoantibodies are abundant in all human serum, usually numbering in the thousands. These IgG autoantibodies bind to human antigens from organs and tissues all over the body and their serum diversity is strongly influenced by age, gender, and the presence of specific diseases. We also found that serum IgG autoantibody profiles are unique to an individual and remarkably stable over time. Similar profiles exist in rat and swine, suggesting conservation of this immunological feature among mammals. The number, diversity, and apparent evolutionary conservation of autoantibody profiles suggest that IgG autoantibodies have some important, as yet unrecognized, physiological function. We propose that IgG autoantibodies have evolved as an adaptive mechanism for debris-clearance, a function consistent with their apparent utility as diagnostic indicators of disease as already established for Alzheimer’s and Parkinson’s diseases.</p> </div

Representative images of human protein microarrays.

<p>(Left) Small portion of a protein microarray containing 9,480 native human proteins probed with blocking buffer and secondary antibody as a quality control. (Right) Protein microarray probed with an individual human serum sample under the same conditions. Manufacturer’s controls (white boxes), gradient of IgG positive controls (green boxes), anti-human IgG controls (blue boxes) and examples of immunopositive reactions (yellow boxes) are indicated. In this study, all arrays were scanned at PMT setting of 600. Under this condition, the relative fluorescence unit (RFU) at saturation is around 75000 as exemplified by IgG controls (green boxes). The RFUs of BSA negative controls are under 1000. The coefficient of variance between duplicate spots averaged 4.7% ∼ 8.8%.</p

Dot blot confirmation of serum IgG auto-reactivity.

<p>Two identified antigens of serum autoantibodies, PTCD2 and ICAM4, were spotted onto nitrocellulose and probed with serum identical to that used on the protein microarrays. Serial dilution with diminishing immunoreactivity confirmed the presence and activity of IgG autoantibodies in human sera.</p

Representative autoantibody data showing their relative abundance in healthy individuals.

<p>An example of the reactions of four highly prevalent autoantibodies in a population of healthy subjects is presented. The X axis indicates individual samples arranged in order of increasing age from left to right. Relative fluorescence units (RFUs) (Y axis) reflect the resulting antigen-antibody reaction detected on the protein microarrays. Four specific autoantigens [tripartite motif-containing 21 (TRIM21), APEX nuclease (apurinic/apyrimidinic endonuclease) 2 (APEX2), General transcription factor II-I and SERPINE1 mRNA binding protein 1 (SERBP1)] were chosen due to their high prevalence in > 60% of the healthy population. The clear lamination of signal intensities shown by each autoantibody-antigen reaction suggests comparable levels of these autoantibodies in the blood across the population</p

Effects of Age and Gender on Autoantibody Count.

<p>A total of 57 normal healthy controls were split into three age groups and two gender groups. The percentage of female subjects in each group is indicated. A Z-factor of greater than 0.4 was used to define a positive antibody response. The mean and standard deviation of antibody counts are shown. The difference of the antibody counts between indicated groups were tested by student t test and p value as shown.</p

Serum IgG autoantibodies are detected in all mammals tested.

<p><b>A</b>. Pig, human, and rat brain lysates were separated by SDS-PAGE and then probed with their respective species-specific serum as a primary antibody. Each species possessed IgG autoantibodies that bound to a variety of SDS-denatured brain self-antigens. <b>B</b>. Proteins from pig brain extract were probed with serum collected from a single pig at two timepoints, TP1 and TP2, spanning three months. The majority of the pig auto-reactive IgG antibodies remain the same.</p

Serum IgG autoantibodies consistently bind to diverse antigens in many human organs. A.

<p>Human liver, kidney, lung, and brain tissue lysates were separated by SDS-PAGE and then probed with human serum. As indicated by the many immunoreactive protein bands of differing molecular weights, serum IgG autoantibodies bind to a variety of common and organ-specific SDS-denatured antigens. <b>B</b>. In western blots, proteins from human brain extract were probed with human serum (HS-I and HS-II) as primary antibodies. Serum samples at three timepoints, T1, T2 and T3, were collected from the same individual (HS-I) over a period of four years. Serum samples T1’ and T2’ were collected from a different individual (HS-II) over a period of two years. Consistent patterns of serum IgG immunoreactivity were specific to individuals and appeared to remain stable over time.</p

Effects of Health Status on Autoantibody Count.

<p>Several diseased groups (Alzheimer’s disease, Parkinson’s disease, breast cancer and multiple sclerosis) and the corresponding age-and-gender matched controls were studied. The age (mean ± standard deviation), percentage of female subjects, number of positive autoantibodies (mean ± standard deviation) are shown for each group. The difference of the autoantibody counts between diseased and corresponding control groups were tested by student t test with p value shown.</p