Antibody Microarrays

Merrill Birkner ph296~December 1, 2003

Antibody (Ab) Microarray
• A complete microarray-based system for profiling protein expression in biological samples; used to compare two biological samples to measure the relative differences in protein expression. The microarray consists of hundreds of monoclonal antibodies covalently bound in an ordered layout to a glass slide.
– A protein which can be synthesized in pure form by a single clone (population) of cells. These antibodies can be made in large quantities and have a specific affinity for certain target molecules called antigens which can be found on the surface of cells and those that are malignant.

The array can be used as a means to correlate specific proteins with physiological or pathological process of interest, by comparing hundreds of proteins at a time. It is used for toxicity testing, disease investigation, and drug discovery.

Antigens

Antibodies

• Antigens: – Molecules that stimulate the production of specific antibodies and combine specifically with the antibodies produced. Most antigens are foreign to the blood and other bodily fluids. • Antibodies: – Antibody proteins (immunoglobulins) are found in the gamma globulin class of plasma proteins. There are five main subclasses : IgG, IgA, IgM, IgD, and IgE. (ex. Most antibodies in serum are from the class IgG).

Antibody Structure
• Consists of four interconnected polypeptide chains. Two heavy chains (H-chains) and joined to two shorter chains (L-chains).
• These four chains are arranged in the form of a „Y‟ ; with the stalk of the Y is called the “crystallizable fragment” and the top of the Y is known as the “antigen-binding fragment”.

Antigen/Antibody Interaction .

Serum samples). • Removal of unbound dye. • Labeling of extracted protein with fluorescent dyes Cy5 and Cy3 (direct labeling. • Scanning of the array and the analysis of the results.Ab Array Procedure • Extraction of total cellular protein from biological samples of interest (eg. paired Ab sandwich assay). direct labeling with hapten tag. . • Incubation of labeled protein with the array.

.

instead they provide a relative measure of protein abundance [i.• This procedure is a fluorescence-based analysis. all antibodies are printed and tested against their specific purified antigen (when available) and against cell lines and tissues samples (for quality control). covalently immobilized antibodies are used to capture fluorescently labeled antigens. • As part of array development. . • They do not measure absolute concentrations. and similar to the gene expression microarrays. thus ensuring that all proteins from the samples are represented in the reference. the abundance of protein in one sample as compared to another sample]. a pool of equal aliquots from each sample to be measured is used. • A reference pool is also used.e.

Enzyme linked immusorbent assay (ELISA) .

Direct Labeling with a hapten tag. C. Direct Labeling B.A. . Paired Ab sandwich assays.

Direct Labeling (w/ hapten tag) • A convenient method to measure multiple proteins in a complex mixture. Advantages: – Only one captured antibody per target is required. – Can label different samples with different tags and to co-incubate the samples on the same arrays. – Potential for disruption of antibody-antigen interactions if the labeling reaction severely alters an antigen’s binding site. as compared to the next method. . All proteins are labeled with either a fluorophore or a hapten tag such as biotin. • • Disadvantages: – Potential for a high background: all proteins are labeled from the sample.easier to expand detection to new targets for which matched antibody pairs may not be available. nonspecific binding or adsorption of these proteins to Ab could cause interference  reduce detection sensitivity or data accuracy. including high concentration proteins such as albumin in serum.

is incubated on the arrays. . – Sandwich assays are more sensitive than the direct labeling method because background is reduced through the specific detection of two antibodies instead of one. Advantages: – Quantification of the bound detection antibodies provides a measure of each antigens abundance. • • Disadvantages: – The development and validation of assays measuring many targets in parallel is difficult because of the cross reactivity and precipitation when using many detection antibodies. and a “cocktail” of detection antibodies. each antibody matched to one of the spotted antibodies.Dual Antibody Sandwich • Antibodies spotted onto microarray substrates capture specific antigens.

• These tests are often used to validate the microarray results . ELISA technology links a measurable enzyme to either an antigen or antibody.ELISA as a validation method • The Enzyme-Linked Immunoabsorbent Assay is serologic test used as a general screening tool for the detection of antibodies or antigens in a sample.

Ab/Ag interaction in ELISA wells .

Gene Expression vs. • In cases when there is a correlation between mRNA and protein abundance. • There are also many reasons for merely studying protein abundance. Ab Microarray • Gene expression. the correlation is often time shifted. – With these arrays it is now possible to compare changes in gene expression with changes in protein expression using similar technologies. does not necessarily correlate with changes in protein expression. in most cases. – This time shift is likely to be different for each mRNA-protein pair. .

• Biomarkers in cancer are potentially valuable for early detection. . or as surrogate markers for drug response. • These microarrays increase the number of proteins that can be conveniently measured. staging of patients. classification of patients.Ab Microarrays & Cancer Research • Information from protein profiling experiments may reveal associations between proteins or groups of proteins and disease states or experimental conditions. therefore taking advantage of the benefit of using combined markers in diagnostics.

• Work continues on the optimization of various aspects of the protocols. such as substrates for Ab attachment.• Important in this field because there is a low volume requirement and the multiplex detection capability of microarrays make optimal use of precious clinical samples. the methods of Ab attachment. . wash conditions. etc. Ab buffers and concentrations.

.. Proteomics 2003. H. Teh. Miller.Antibody microarray profiling of human prostate cancer sera: Antibody screening and identification of potential biomarkers. Kwekel. J. Zhou. 56-63..B.. Butler. 3. R. J. Burke. Cavallo... J. B. . Haab.. E. B.

autoimmune diagnostics. and Ab-based detection of multiple antigens. • • . Ab & protein microarrays can have many applications including protein profiling of cancer tissue. by chemical cross–linking to derivatized glass surfaces.Background • Protein Biomarkers in the serum hold great promise for noninvasive disease detection and classification. protein interaction screening. Hydrogels recently have also been introduced as a protein microarray substrate. • Ab can be immobilized by adsorption to poly-L-lysin membranes. Certain parts of the Ab microarray technology have not been perfected: – An optimized protein immobilization method is needed that retains native structure and reactivity and decreases nonspecific protein adsorption.

• Previous work in the development of the antibody microarray methods made use of solutions of known target antigen concentrations to characterize antibody performance.– Another important issue is to create an efficient method of validating antibody performance in the microarray assay. • This is often very expensive and the antigens are often unavailable. .

2. – Hypothesis: a statistical filter could identify antibody measurements that are consistent with specific and quantitatively accurate antigen binding. by analyzing the relative protein abundances in serum samples from prostate cancer patients and controls. . & polyacrylamide-based hydrogels on glass. 3.Goals 1. Establish an efficient method to screen antibodies for those that are functional in the microarray assay. This hypothesis is tested by comparing microarray measurements to ELISA tests. Compare two surfaces and antibody immobilization schemes: poly-Llysine coated glass with a second photoreactive cross linking layer. Demonstrate the use of this technology to screen serum samples for potential biomarkers.

2 ng/mL median: 0. – PSA (prostate-specific antigen) concentration 2. TX.Serum samples • 33 males with prostate cancer ages 39-85 at the Methodist Hospital Houston.2-3. prior to commencement of radiotherapy.5-335 ng/mL (from ELISA) median: 6.85 ng/mL .4 ng/mL – Histological grades of cancer tissue samples ranged from a Gleason combined scores of 6-9 • 20 serum samples taken from healthy males aged 30-69 – Normal PSA levels 0. USA.

– A reference pool is a pool of equal aliquots from each sample. . • The serum samples and reference pool were diluted and mixed with the Cy5 or Cy3.Microarray preparation • The microarrays were deposited on two different types of substrates: the poly-L-lysine (HSBA) and the hydrogel [details found in paper]. thus ensuring that all proteins from the samples are represented in the reference.

The ratio of the net signal from the sample-specific channel to the net signal from the reference specific channel was calculated for each antibody spot. • . • • • • Antibodies that did not have good measurements in at least 75% of the samples were removed from subsequent analysis. The permutation t-test was calculated using the program Cluster Identification Tool. • The local background in each color channel was subtracted from the signal at each antibody spot (spots with defects or no detectable signal removed).Data analysis and statistics. Ratios were log transformed & median centered. The resulting ratios were multiplied by a normalization factor for each array (next slide) Hierarchical clustering and visualization were performed using Cluster and Treeview. ratios from replicate antibody measurements in the same array were averaged.

calculated by: N= (SIgG / μIgG)/RIgG SIgG = the ELISA-measured IgG concentration of the serum sample on that array. μIgG = the mean ELISA-measured IgG concentration of all of the samples. RIgG = the average ratio of the replicate anti-IgG antibody spots on a particular array.Normalization Method • The resulting ratios were multiplied by a normalization factor for each array N. .

Combine one of each of these samples and scan. . With this method potential variability is eliminated because each protein sample labeled with each dye.Internal Normalization 4 slides per sample were created (label sample A with Cy3 and Cy5 and sample B with Cy3 and Cy5). determine the signal ratios of the 2 slides (Cy5/Cy3).

Results • Serum & control samples were analyzed using a two-color comparative fluorescence assay on microarrays containing 184 different antibodies spotted in quadruplicate. . • Another 13 antibodies targeted 9 proteins that have been detected in the serum of cancer patients. • 40 of the antibodies targeted 32 unique proteins that are typically found in the serum of healthy serum samples. and the rest of the antibodies targeted normally intracellular proteins.

reflecting decreased detection limits as a result of higher S/N ratio measurements. – The fluorescent signal from the Ab on the hydrogels had an average six-fold higher S/N ratio than the corresponding antibodies on the other surface. – The Ab showed measurable signal above background using the hydrogels (78 Ab) as compared to the other surface (23 Ab). more consistent background than the other surface. – The hydrogel allowed weak detection from the greater number of Ab. there were 4 microarray experiments per sample. . • The hydrogel substrate generally produced a lower.Goal #1 • The samples were repeated twice on the 2 types of slides (internal normalization).

they examined the ability of the statistical filter based on the correlation of the data from reversed labeling experiments to distinguish between reliable and unreliable microarray measurements. • Using ELISA measurements as standards. • Developed an effective an efficient method to screen antibodies for those that function well in the microarray assay. .Goal #2 • Define a statistical test that could filter the Ab measurement for those that are consistent with specific and accurate antigen binding. • They examined the overall variation in the reproducibility of Ab measurements from the 2 different surfaces after reverse labeling (mentioned before).

These were combined and clustered. . The color and intensity of each square represents the relative protein binding of the sample versus the reference. HSBA sampled labels in red. – There are 4 slides: hydrogel sample in red.• First. in order to view patterns of similarity between sets of microarray measurements. and HSBA labeled in green. hydrogel sample in green. average linkage hierarchical clustering is used. • Ab measurements that reproduce well between the different experiments are clustered together. – Each colored square represents one Ab measurement from one array.

.

• In order to assess the degree to which the correlation parameter predicted specific and accurate antigen detection. • For these 7 antibodies a high inter-experiment set of correlations predicted a good agreement between the microarray and ELISA measurements. microarray measurements from 7 of the Ab were compared to ELISA measurements for the corresponding antigens. For both surfaces progressively fewer Ab exceeded the threshold as the threshold was increased. both for hydrogel and HSBA. – They found no examples of Ab measurements that have high interexperiment set correlation but poor agreement with ELISA measurements! . – The Pearson correlation of measurements between the reverselabeled experiment set was calculated.• The correlation of measurements from replicate data sets as an initial screen to identify “reliable” antibodies.

.

.

because the microarray measurements exceeding this threshold agreed well with the ELISA measurements. only Ab that passed the stringent correlation threshold for inclusion were used in the following analysis. • In order to estimate the significance of the association between expression patterns and sample groups (cancer and normal) permutation t-tests were used. – Determines the statistical significance of each gene’s discrimination using a user defined segregation of samples. – They used a correlation threshold of 0.Goal #3 : Detection of biomarkers… • As a result of the previous analysis. .7.

It is the probability under the null hypothesis that the test statistic is at least as extreme as Tg. • Standard t-tests assume normally distributed data in each class and equal variance within classes. . This test will be more accurate than the normal t-test for non-normal distributions and small samples. • The p-value pg is given as the fraction of permutations producing a test statistic that is at least as extreme as the observed one.Permutation t-test • Estimate the distribution of the t-test statistics under the null hypothesis by permutation of the sample labels.

000007 <0.00006 <0. CIT identified: – vWF.15 -0. Marker P-value Correlation w/ PSA vWF IgM ACT Villin IgG <0.01 0. and IgG with pvalues below 0.36 -0.001 <0.16 .18 -0. Villin.01. alpha-anti-chymotrypsin.19 -0.• When applied to the discrimination of cancer patients from the controls.001 <0. IgM.

• None of these markers significantly correlated with PSA. • IgM and IgG were lower and vWF higher in cancer patients & therefore similar to previous studies. • Since none of the proteins correlated with PSA. .• Hemoglobin was also discriminated but was found to be an artifact of hemolysis of controls. and all varied independently of PSA. they could potentially bolster diagnostic accuracy if used in conjunction with PSA.

• Using the D/S/A algorithm to analyze the data. (Data: Van Andel Inst. • Further development in this technology will have significant utility in medical diagnostics as well as broader clinical and research application. Brian Haab) . • Larger studies are needed to further examine the relationship between serum proteins and prostate cancer. Dr. Michigan.Remarks/Future work….

Sign up to vote on this title
UsefulNot useful