Tracking humoral responses using self assembling protein microarrays - PubMed (original) (raw)

. 2008 Oct;2(10-11):1518-27.

doi: 10.1002/prca.200800034. Epub 2008 Sep 10.

Karen S Anderson, Jacob V Raphael, Eugenie Hainsworth, Sahar Sibani, Wagner R Montor, Marcin Pacek, Jessica Wong, Mariam Eljanne, Martin G Sanda, Yanhui Hu, Tanya Logvinenko, Joshua Labaer

Affiliations

Tracking humoral responses using self assembling protein microarrays

Niroshan Ramachandran et al. Proteomics Clin Appl. 2008 Oct.

Abstract

The humoral immune response is a highly specific and adaptive sensor for changes in the body's protein milieu, which responds to novel structures of both foreign and self antigens. Although Igs represent a major component of human serum and are vital to survival, little is known about the response specificity and determinants that govern the human immunome. Historically, antigen-specific humoral immunity has been investigated using individually produced and purified target proteins, a labor-intensive process that has limited the number of antigens that have been studied. Here, we present the development of methods for applying self-assembling protein microarrays and a related method for producing 96-well formatted macroarrays for monitoring the humoral response at the proteome scale. Using plasmids encoding full-length cDNAs for over 850 human proteins and 1700 pathogen proteins, we demonstrate that these microarrays are highly sensitive, specific, reproducible, and can simultaneously measure immunity to thousands of proteins without a priori protein purification. Using this approach, we demonstrate the detection of humoral immunity to known and novel self-antigens, cancer antigens, autoimmune antigens, as well as pathogen-derived antigens. This represents a powerful and versatile tool for monitoring the immunome in health and disease.

Copyright © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Figures

Figure 1

Figure 1

Development of RAPID 96-well format ELISA assay for detection of autoantibodies in serum. A. Schematic of RAPID ELISA. cDNA encoding full-length antigens are expressed as tagged fusion proteins using rabbit reticulocyte lysate. The proteins are captured by anti-tag antibodies in individual wells. Human serum containing antigen-specific antibodies is added and detected with HRP-conjugated secondary anti-IgG antibodies. B. Specific detection of anti-p53 autoantibodies by RAPID-NAPPA. Three C-terminal GST-tagged antigens, p21, myc, and p53, were expressed and captured in wells with anti-GST antibodies. The GST fusion protein was detected with an anti-GST detection antibody (left), demonstrating protein expression. Human sera positive for anti-p53 antibodies were added (right) and human IgG was detected. No added DNA was the negative control. C and D. RAPID ELISA shows comparable sensitivity to standard protein ELISA for the detection of anti-p53 antibodies. P53 antibody-positive breast cancer patient sera were titrated, and antibodies were detected by RAPID-NAPPA (C) and a standard p53 recombinant protein ELISA (D). Internal positive and negative control sera were titrated in duplicate on every standard ELISA assay, with the negative control O.D.(450nm)<0.3.

Figure 2

Figure 2

Autoantibodies to p53 are detected by protein microarray (NAPPA) and RAPID (96-well format) ELISA. A. Detection of p53-positive antibodies by p53 standard ELISA and by NAPPA in cancer patient sera. 35 breast cancer patient sera were screened for anti-p53 autoantibodies by p53 standard ELISA using recombinant p53 protein, and by NAPPA. The signals on the NAPPA arrays were normalized to the 50% trimmed mean (mean of the middle 50% of protein spots). Antibodies to p53 antigen were detected in two patient sera by both assays. B. i. Quantification of p53 antigen detection by RAPID ELISA and NAPPA microarrays. P53-GST fusion protein was expressed by RAPID ELISA and NAPPA, and detected with titrations of an anti-p53 MAb (D01, Sigma). Both methods detected 10 fg/μL of antibody, with a linear range of 20 pg/μL-30 fg/μL. Serum signals greater than 110% of a feature with no antigen were considered positive on the NAPPA arrays. B. ii–vi. Detection of anti-p53 antibodies by RAPID ELISA and NAPPA microarrays from 5 human cancer patient sera containing p53 autoantibodies.

Figure 3

Figure 3

Optimizing the detection of bona fide but rare antibody signals on NAPPA. Because many autoantibodies can be of low concentration and low affinity in patient sera, we tested the effect of serum mixing, incubation time, and incubation temperature on the detection of the known viral antigen EBNA-1 and to an antigen, NAP1L3, in prostate cancer sera. A. At both 1:300 (top) and 1:8100 (bottom) serum dilutions, bound IgG antibodies are more readily detected with an overnight incubation on NAPPA (right) than with a one hour incubation (left). Antibody detection correlates with protein expression, but only during extended incubations and with mixing does the reaction achieve equilibrium. B. Mixing of the sera increases detection of tumor antigen-specific antibodies. Healthy control sera (left two panels) and prostate cancer serum (right two panels) were diluted at 1:600 and incubated on the protein microarrays overnight at 4°C, with or without mixing as shown. EBNA-specific responses are shown in blue, p53 specific response are shown in red squares, and the prostate tumor antigen NAP1L3 is shown in red circles. Response to other genes on the array is shown as purple triangles and the trimmed mean (25–75%) signal is shown as a red line. By mixing the sera, detection of NAP1L3 is specifically enhanced over background.

Figure 4

Figure 4

Detection of autoantibodies by NAPPA ELISA is highly reproducible. Over 600 individual c-terminal GST tagged proteins were expressed in microarray format, Antigen expression across the slide was confirmed by anti-GST antibody (left). Slides containing anchored cDNAs were stored at room temperature, and proteins were expressed on consecutive days. Human IgG registration spots are visible as diagonal duplicates across the slide. P53 antigen was specifically detected in duplicate using human sera containing anti-p53 antibodies. The coefficients of variation for antibody detection were 7% (within day) and 11% (between days). The CV's were calculated based on the means of the replicate spots on each slide.

Figure 5

Figure 5

Proof of concept detection of antibodies in several disease states. A. Detection of immunodominant pathogen-derived antigens. A selected set of 264 genes predicted to be outer membrane proteins from the pseudomonas genome was expressed by NAPPA and probed with control serum or serum derived from an infected patient. A well documented response to the pseudomonal antigen, fliC, was specifically detected in the patient sera. B. The Francisella tularensis proteome was expressed on a NAPPA array. The array was probed with sera from a mouse infected with a live attenuated strain and from a control mouse. The immune responses were detected with an anti-mouse antibody. C. An array expressing 655 human genes in duplicate was probed with sera from a healthy control and from a patient with prostate cancer. Antibodies to the common viral antigen EBNA-1 are readily detected in both sera, but antibodies to Bcl2 are only detected in the prostate cancer patient serum. D. An array expressing 659 human genes was probed with sera from a diabetic patient and a nondiabetic control. Serum response to a well known antigen related to IDDM, GAD65, was observed only in the patient sera.

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