Synthetic Antigen Controls for Immunohistochemistry

Summary

This work documents a simple method to create synthetic antigen controls for immunohistochemistry. The technique is adaptable to a variety of antigens in a wide range of concentrations. The samples provide a reference with which to assess intra- and inter-assay performance and reproducibility.

Abstract

Immunohistochemistry (IHC) assays provide valuable insights into protein expression patterns, the reliable interpretation of which requires well-characterized positive and negative control samples. Because appropriate tissue or cell line controls are not always available, a simple method to create synthetic IHC controls may be beneficial. Such a method is described here. It is adaptable to various antigen types, including proteins, peptides, or oligonucleotides, in a wide range of concentrations. This protocol explains the steps necessary to create synthetic antigen controls, using as an example a peptide from the human erythroblastic oncogene B2 (ERBB2/HER2) intracellular domain (ICD) recognized by a variety of diagnostically relevant antibodies. Serial dilutions of the HER2 ICD peptide in bovine serum albumin (BSA) solution are mixed with formaldehyde and heated for 10 min at 85 °C to solidify and cross-link the peptide/BSA mixture. The resulting gel can be processed, sectioned, and stained like a tissue, yielding a series of samples of known antigen concentrations spanning a wide range of staining intensities.

This simple protocol is consistent with routine histology lab procedures. The method requires only that the user have a sufficient quantity of the desired antigen. Recombinant proteins, protein domains, or linear peptides that encode relevant epitopes may be synthesized locally or commercially. Laboratories generating in-house antibodies can reserve aliquots of the immunizing antigen as the synthetic control target. The opportunity to create well-defined positive controls across a wide range of concentrations allows users to assess intra- and inter-laboratory assay performance, gain insight into the dynamic range and linearity of their assays, and optimize assay conditions for their particular experimental goals.

Introduction

Immunohistochemistry (IHC) allows the sensitive and specific, spatially precise detection of target antigens in formalin-fixed, paraffin-embedded (FFPE) tissue sections. However, IHC staining results may be affected by multiple variables, including warm and cold ischemia time, tissue fixation, tissue pretreatment, antibody reactivity and concentration, assay detection chemistry, and reaction times¹. Accordingly, reproducible performance and interpretation of IHC reactions require rigorous control of these variables and the use of well-characterized positive and negative control samples. Frequently used controls include paraffin-embedded tissues or cultured cell lines known from independent analyses to express the antigen of interest, but such samples are not always available¹. Furthermore, the expression levels of the target antigens in tissues and cell line controls are generally understood only qualitatively and may be variable. Controls containing reproducible, precisely known concentrations of target antigen can assist in the optimization of IHC reaction conditions. A general method, adaptable to a variety of antigen types in a physiologically relevant range of concentrations to create synthetic IHC control samples, has been described by the authors². A detailed protocol is provided here for the creation and use of this type of standard.

Appropriate controls are essential for the valid interpretation of IHC assays^1,3,4. Tissues, cultured cells, and peptide-coated substrates have been employed as IHC controls according to the investigators' specific needs. The advantages and limitations inherent in using tissues as IHC controls have been extensively discussed^1,4. For many antibodies, appropriate controls can be chosen from selected normal tissues containing cell populations expressing the target antigen over a wide dynamic range. Tissue controls are less suitable when the target antigen is not well-characterized concerning expression site or abundance, or when potentially cross-reacting antigens are co-expressed in the same cells or tissue sites. In these contexts, blocks of cultured cell lines expressing the antigen of interest can be helpful. For providing further evidence of target specificity, cell lines can be engineered to over-or under-express target antigens. For example, such an approach was recently used to evaluate a variety of anti-PD-L1 assays using a tissue microarray of isogenic cell lines expressing a range of PD-L1 antigen⁵. Practical limitations to the routine use of cell line blocks include the cost and time needed to produce sufficient cell numbers and the fact that the expression of some antigens may not be reliably consistent, even within clonal cell lines⁶. Synthetic peptides are a third option for IHC controls for antibodies that recognize short linear epitopes. Steven Bogen and colleagues have published extensively on the use of peptides coupled to the surface of glass slides^7,8 and glass beads⁹. One study by this group demonstrated that quantitative analysis of peptide-based IHC controls could detect staining process variation missed by qualitative evaluation of tissue controls analyzed in parallel¹⁰. While standards using bead-based antigens could be widely applicable, many details are proprietary to the authors, limiting widespread adoption.

Another approach to IHC standards incorporates target antigens into artificially created protein gels. This concept was first demonstrated by Per Brandtzaeg in 1972 in a study in which normal rabbit serum was polymerized using glutaraldehyde¹¹. Small blocks of the resulting gel were then soaked for 1-4 weeks in solutions containing the immunoglobulin antigens of interest at various concentrations. After alcohol fixation and paraffin embedding, sections of the resulting controls were shown to stain with intensities corresponding to the logarithm of the antigen solutions in which they had been soaked. Later, investigators prepared glutaraldehyde conjugates of specific amino acids in dilute BSA or brain homogenate solutions as positive controls in immune-electron microscopy studies^12,13. More recent work investigated the use of gels made from formaldehyde-fixed protein solutions as surrogates for FFPE tissue in mass spectrometry analysis¹⁴. Another recent work investigated the structure and properties of gels formed by heating human or bovine serum albumin solutions at various concentrations and pH¹⁵. These authors found that serum albumin forms three types of gels differing in mechanical elasticity, secondary structure preservation, and fatty acid-binding capability depending on the experimental conditions. Together, these papers demonstrate the general feasibility of the approach employed here. Protein solutions of defined composition create tissue-like gels that can be further processed, sectioned, and stained using routine histological methods.

This protocol describes the formation of a synthetic IHC control made from bovine serum albumin (BSA) polymerized with heat and formaldehyde. The gels can incorporate a wide variety of antigens, including full-length proteins, protein domains, and linear peptides, as well as non-protein antigens including oligonucleotides². This demonstration uses an example antigen a linear peptide encoding the C-terminal 16 amino acids of the human ERBB2 (HER2/neu) protein TPTAENPEYLGLDVPV-COOH (see Table of Materials). This sequence includes the epitopes recognized by three commercially available, diagnostically relevant antibodies including the Herceptest polyclonal reagent (ENPEYLGLDVP) and the monoclonal antibodies CB11 (AENPEYL) and 4B5 (TAENPEYLGL) (see Table of Materials)¹⁶.

The method demonstrated here employs readily available reagents using processes and techniques familiar to any practicing histology laboratory. The most significant limitation is the need to identify and purchase the target antigens, which can be accomplished in many cases at a relatively modest cost. Because these synthetic controls are of wholly defined composition and made with simple methods, they can be implemented in many laboratories with reproducible results. Their use may facilitate the objective, quantifiable evaluation of IHC staining results and allow greater intra- and inter-laboratory reproducibility.

Protocol

1. Preparation of stock solution and tools

1. Prepare 20 mL of a 25% w/v BSA solution by mixing 5 g BSA powder in 14 mL of PBS, pH 7.2 in a 50 mL conical tube until evenly dispersed. Vortex as necessary to disperse the BSA powder.
   1. Keep the solution overnight at 4 °C to allow complete dissolution. Adjust the final volume to 20 mL with PBS to make a 25% w/v stock solution.
2. Prepare 20 mL of a 31.3% w/v BSA solution by mixing 6.26 g BSA powder in 13 mL of PBS, pH 7.2 in a 50 mL conical tube until evenly dispersed. Keep the solution overnight at 4 °C to allow complete dissolution. Adjust the final volume to 20 mL with PBS to make a 31.3% w/v stock solution.
3. Preheat a heat block to 85 °C.
   NOTE: The protocol below creates peptide/BSA gels with volumes of 1.26-1.4 mL formed in 1.5 mL microcentrifuge tubes. To use smaller volumes, for example, when antigen stocks are limiting, prepare the gels in PCR tubes and use a thermocycler set to 85 °C as a heat block.
4. Test that the BSA/formaldehyde mixture forms a gel as expected by mixing 700 µL of 25% BSA solution with 700 µL of 37% formaldehyde. Mix well by pipetting up and down 5 times within 5-10 s. Avoid creating air bubbles.
   CAUTION: Concentrated formaldehyde is toxic; use with appropriate safety precautions.
5. Immediately after mixing the BSA and formaldehyde solutions, place the closed microcentrifuge tube in a heat block at 85 °C for 10 min. Remove the tube from the heat block and allow it to cool. Confirm that the gel has formed as expected.

2. Preparation and dilution of peptides

1. Obtain 5-20 mg of lyophilized peptide of the desired sequence.
   NOTE: The C-terminal 16 amino acids of the human ERBB2 intracellular domain recognized by 4B5 is TPTAENPEYLGLDVPV-COOH.
   1. Add 4 amino acids to the N-terminus, acetyl-YGSG, and C-terminus, GSGC-amide to facilitate cross-linking of the peptide to BSA and provide spacing between the BSA molecule and the peptide epitope.
      NOTE: The complete sequence is: acetyl-YGSGTPTAENPEYLGLDVPVGSGC-amide.
   2. If desired, use other N- and C-terminal amino acid sequences to extend the core peptide epitope.
      NOTE: The impact of different sequences varies with different antibody/epitope combinations. The addition of the C-terminal peptide reduces the binding of some antibodies to C-terminal epitopes. In such cases, omit this sequence.
   3. Confirm that peptides from commercial sources are supplied at >95% purity, the composition of which is confirmed by HPLC and mass spectrometry analysis.
2. Calculate the necessary volumes for the 5x (1.25 E-2 M) peptide stock solutions. Referring to Table 1, columns C-E, enter values for the antigen molecular weight (g/mole), percent antigen purity (0-100), and antigen mass (mg).
   NOTE: The volume of solvent (in µL) to resuspend the sample to achieve a stock solution of 1.25 E-2 M is 800 x antigen molecular weight x percent antigen purity/antigen mass.
   1. Prepare and clearly label eight 1.5 mL microcentrifuge tubes.
      NOTE: The tubes will contain 5x peptide stock in a solvent, 1x peptide stock in a solvent, five 10x serial dilutions of 2.5 E-4 M to 2.5 E-8 M peptide/BSA/formaldehyde gel, and a negative control gel containing BSA/formaldehyde lacking added antigen. All gel samples look identical. When preparing multiple sets of peptide dilutions at one time, take care to label and identify all tubes and processing cassettes correctly. Use color-coded microcentrifuge tubes and processing cassettes where possible to minimize misidentification.
3. Prepare a 5x peptide stock solution at 1.25 E-2 M by resuspending the entire mass of lyophilized peptide (20 mg for the ERBB2 peptides) in 60 µL of the appropriate solvent.
   NOTE: In this example, dimethylformamide (DMF) was added directly to the vendor's container.
   1. Inspect the solution to ensure that the peptide is completely dissolved. If necessary, add additional solvent and/or sonicate the sample until the peptide is completely dissolved, taking care not to exceed the volume calculated in Table 1 for the 5x peptide stock.
      CAUTION: DMF is toxic; use with appropriate precaution.
      NOTE: Depending on the amino acid sequence, and the corresponding hydrophobicity and charge, peptides may be soluble in DMF, dimethyl sulfoxide (DMSO), pure water, or dilute solutions of acetic acid, formic acid, or ammonium bicarbonate. Peptide characteristics may be calculated using a variety of online tools¹⁷. Some peptide vendors may suggest solvents appropriate for specific sequences.
   2. Add solvent as necessary to bring the volume of the 5x peptide stock to the final volume calculated in Table 1. Vortex for 5 s and centrifuge at room temperature at 5000 x g for 5 s. Peptide stock solutions can be stored at -80 °C.
4. Referring to Table 2, Column C, prepare 150 µL of 1x peptide stock solution (2.5 E-3 M) by diluting 30 µL of the 5x peptide stock into 120 µL of solvent (DMF this example). Vortex for 5 s and centrifuge at room temperature at 5000 x g for 5 s.
5. Referring to Table 2, Column D, prepare 700 µL of 5 E-4 M peptide/BSA solution (Dilution 1) by diluting 140 µL of 1x peptide stock into 560 µL of 31.3% BSA/PBS, pH 7.2. Vortex for 5 s and centrifuge at room temperature at 5000 x g for 5 s.
   NOTE: The final BSA concentration of this solution is 25% (w/v).
6. Referring to Table 2, Columns E-H, prepare four successive 10x serial dilutions of the 5 E-4 M peptide/BSA stock by adding 70 µL of peptide/BSA solution to 630 µL of 25% BSA/PBS, pH 7.2. Vortex for 5 s and centrifuge at room temperature at 5000 x g for 5 s.
   NOTE: After this step, there will be five 10-fold serial dilutions of peptide (5 E-4 M to 5 E-8 M) in 25% BSA/PBS, pH 7.2. The first four samples will contain 630 µL. The last sample will contain 700 µL.
7. Referring to Table 2, Column I, prepare a negative control BSA sample containing 700 µL of 25% BSA/PBS, pH 7.2 (Figure 1A).

3. Preparing BSA-peptide gels

1. Confirm that the heat block or thermocycler is stable at 85 °C.
2. Refer to Table 3, Columns B-E. Working one sample at a time, add to the first 25% BSA/peptide sample (Dilution 1) 630 µL of 37% formaldehyde. Mix well by pipetting up and down 5 times within 5-10 s. Avoid creating air bubbles.
   CAUTION: Concentrated formaldehyde is toxic; use with appropriate safety precautions.
   1. After mixing the peptide/BSA and formaldehyde solutions, place the closed microcentrifuge tube in a heat block at 85 °C for 10 min.
      NOTE: Mix the BSA-peptide solution and formaldehyde thoroughly, but do not spend more than 10 s pipetting the mixture before placing the sample on heat. Since formaldehyde cross-linking begins immediately, the gel may form differently if the procedure is varied for different samples. The final BSA concentration in these gels is 12.5% (w/v). Final BSA concentrations less than 10% may yield gels that do not solidify; final BSA concentrations greater than 16% may produce gels more brittle and difficult to section after processing.
   2. Repeat steps 3.2 and 3.2.1 for each of the dilutions 2-4.
   3. Repeat steps 3.2 and 3.2.1 for dilution 5, but add 700 µL of 37% formaldehyde, a volume equal to the 700 µL of BSA-antigen solution.
   4. Refer to Table 3 column Column G; repeat steps 3.2 and 3.2.1 for the negative control sample, adding 700 µL of 37% formaldehyde, a volume equal to the 700 µL of negative control BSA solution.
3. Remove the tubes from the heat block after 10-12 min. The heating time should be as consistent as possible for each sample. Allow the gels to cool on the benchtop for 5-10 min (Figure 1B).
4. Using a clean, flexible disposable laboratory spatula, remove the gel sample in one piece from the microcentrifuge tube, and place it in a sealed container containing at least 15 mL of neutral buffered formalin (NBF), using a separate container of NBF for each sample.
   1. Alternatively, cut off the bottom of the microcentrifuge tube with a new single edge razor blade, and push the gel out from the bottom with air or a suitable probe (Figure 1C-G).
      ​NOTE: The solidified formaldehyde/BSA gels can remain in the microcentrifuge tubes at room temperature for up to 24 h. Leaving the gels in the microcentrifuge tube for more than 24 h can cause them to become brittle and more difficult to process and section.

4. Trimming, processing, and embedding BSA gels

1. Trim the gel cone into cylindrical discs approximately 5 mm thick using a clean single edge razor (Figure 1H,I). Wrap the discs in a biopsy wrap, placing one larger gel disc into one cassette (to be used in the pilot study in step 5), and the remaining gel discs together into a second cassette (Figure 2A,E) for use in tissue microarray (TMA) construction in step 6. Place the wrapped gel discs in clearly labeled tissue processing cassettes.
   1. Place the cassetted gels in at least 15 mL of 10% NBF per gel sample before processing, using a separate container of NBF for each sample. Gels can remain in 10% NBF for 6-48 h.
2. Process the gels in an automated histology tissue processor, following a large tissue schedule with pressure and vacuum. Each step takes 1 h: 10% NBF, 70% ethanol, 95% ethanol (repeat two times), 100% ethanol (repeat two times), xylenes (repeat three times), paraffin at 60 °C (repeat three times).
   NOTE: For investigators choosing to process samples manually, follow the same sequence of reagents and times.
3. When the sample processing is completed, remove the cassettes from the tissue processor and move them to the paraffin embedding center.
4. Unwrap gels from the biopsy wrap and embed the gels in paraffin. For each sample, embed one disk of gel in a small 15 mm x 15 mm mold (Figure 2B-D), and the remaining gel discs together in a second larger mold (Figure 2F-H). The first block with one sample will be used to test the peptide gel in a pilot study. The second block can be used for TMA construction.

5. Pilot evaluation of the peptide dilution series

1. For each peptide dilution series, plan to create two glass slides containing a total of 6 separate sections: one section from each of the five dilution series samples, plus one section from the BSA-only negative control sample.
   1. Onto the first glass slide, cut one 4 µm thick section from each of the smaller blocks containing one gel disc with the three highest peptide concentrations (2.5 E-4 M to 2.5 E-6 M).
   2. Onto a second slide, cut one 4 µm thick section from each block with the two lowest peptide concentrations (2.5 E-7 M and 2.5 E-8 M) and one section from the BSA-only control block. Record the order of the samples on the slides.
      NOTE: Expect the paraffin-embedded gels to cut smoothly, producing uniform sections without fragmentation, tearing, or chattering artifact. If particular paraffin-embedded gel samples are difficult to section, briefly soak the block face in ice-cold distilled water before sectioning. If necessary, experiment with different soaking times or with different solutions (e.g., ammonia water).
   3. After sectioning, dry the slides at room temperature (about 23 °C) for 24 h followed by 60 °C for 30 min.
2. Stain the two slides prepared for each peptide with the desired antibody according to standard IHC protocols.
   NOTE: The primary antibody on-slide concentration used for rabbit monoclonal 4B5 in this demonstration was 1.5 ug/mL.
   1. Expect to see a relatively uniform signal within each gel section, with the different gel samples showing a range of signal intensity corresponding to the peptide dilutions.
3. If the results for the pilot study are satisfactory, construct a TMA from the gel donor blocks containing different concentrations of peptide antigen, as described in the next steps of the protocol.

6. BSA gel TMA construction

1. Construct a tissue microarray containing duplicate 1 mm diameter cores from donor blocks containing BSA gel alone and BSA gels containing all five dilutions of ERBB2 peptide.
   NOTE: If desired, include BSA gels containing the same five dilutions of a non-target peptide as additional negative controls. If desired, include cores of representative ERBB2-expressing cell lines as positive controls.
2. Cut 4 µm thick sections of the TMA and stain with 1.5 ug/mL of anti-ERBB2/HER2/neu rabbit monoclonal 4B5 (see Table of Materials) according to laboratory-standard protocols.
3. Assess the resulting stain intensity qualitatively by inspection or quantitatively by digital image scanning and analysis (Figure 3A,B).
   NOTE: As digital image analysis is not the focus of this protocol, these steps are left for the reader to perform according to their preference.

Results

Peptides should dissolve entirely in an appropriate solvent at room temperature to form an optically clear solution. If visible particulate material is still present after 30-60 min, it may be helpful to add additional volumes of the original solvent or an alternative solvent not exceeding the intended volume of the 5x peptide stock solution calculated in Table 1. Likewise, the combined peptide/BSA solution should remain translucent (Figure 1A).

P...

Discussion

This method allows the user to create uniform samples of known composition and antigen concentration as standards in IHC reactions, using materials and techniques familiar to most histology laboratories. The most crucial step is to identify the epitope to which the antibody of interest binds. This protocol describes using a linear peptide antigen from the ERBB2/HER2 ICD. The same protocol can be used to form BSA gels containing oligonucleotides, fluorescent labels, protein domains, or full-length proteins. This latter ap...

Materials

Name	Company	Catalog Number	Comments
Anti-HER2/neu clone 4B5	Ventana	5278368001
Biopsy Wraps	Leica	3801090
Bovine Serum Albumin, ultra pure	Cell Signaling Technology	BSA #9998
50 mL Conical Tube	Corning	352070
Disposable base mold (15 mm x 15 mm)	Fisher	22-363-553
Disposable base mold (24 mm x 24 mm)	Fisher	22-363-554
Disposable spatula	VWR	80081-188
Eppendorf Thermomixer	Eppendorf	22331
37% Formaldehyde	Electron Microscopy Sciences	15686
ERBB2 / HER2 peptide			UniProt P04626-1; a.a. 1240-55
Leica Autostainer XL	Leica	ST5010
Magnetic Stir Bar
NanoZoomer 2.0 HT whole slide imager	Hamamatsu
10% Neutral Buffered Formalin	VWR	16004-128
Nuclease-free microfuge tubes 1.5 mL
Paraplast paraffin	Leica	39601006
Peptide parameter calculator	Pep-Calc17	https://www.pep-calc.com/
Peptide suppliers	ABclonal Science		Users should contact peptide vendors for details of mass, purity and cost.
	Anaspec Peptide		Users should contact peptide vendors for details of mass, purity and cost.
	CPC Scientific		Users should contact peptide vendors for details of mass, purity and cost.
	New England Peptide		Users should contact peptide vendors for details of mass, purity and cost.
Phosphate Buffered Saline pH 7.2
Reagent Alcohol	Thermo Scientific	9111
Single Edge Razor	VWR	55411-050
Superfrost Plus positively charged microscope slides	Thermo Scientific	6776214
TMA Tissue Grand Master	3DHISTECH
Xylenes	VWR	89370-088