- Research article
- Open Access
Authentication of collagen VI antibodies
- Jamie Endicott1,
- Paul Holden2 and
- Jamie Fitzgerald1, 2Email authorView ORCID ID profile
- Received: 19 October 2016
- Accepted: 22 July 2017
- Published: 29 July 2017
Abstract
Background
Collagen VI is a ubiquitously-expressed macromolecule that forms unique microfibrillar assemblies in the extracellular matrix. Mutations in the COL6A1, COL6A2 and COL6A3 genes result in congenital muscular dystrophy, arguing that collagen is critical for skeletal muscle development and function. Antibodies against collagen VI are important clinical and diagnostic tools in muscular dystrophy. They are used to confirm genetic findings by detecting abnormalities in the distribution, organization and overall levels of collagen VI in cells and tissues isolated from patients.
Methods
Many antibodies have been raised against tissue-purified collagen VI and individual collagen VI chains, however few have been properly validated for sensitivity and chain specificity. To address this deficiency, we compared the ability of 23 commercially-available antibodies to detect extracellular collagen VI by immunohistochemistry on frozen tissue sections. To determine chain specificity, immunoblot analyses were conducted on cell lysates isolated from cells transfected with cDNAs for each individual chain and cells expressing all three chains together.
Results
Our analyses identified 15 antibodies that recognized tissue collagen VI by immunohistochemistry at varying intensities and 20 that successfully detected collagen VI by immunoblotting. Three antibodies failed to recognize collagen VI by either method under the conditions tested. All chain-specific antibodies that worked by immunoblotting specifically recognized their correct chain, and no other chains.
Conclusions
This series of side-by-side comparisons reveal at least two antibodies specific for each chain that work well for immunohistochemistry on frozen sections. This validation study expands the repertoire of antibodies available for muscular dystrophy studies caused by defects in collagen VI.
Keywords
- Collagen VI
- Muscular dystrophy
- Immunohistochemistry
Background
A major discussion point within the biomedical research community concerns experimental reproducibility. One recent study attempted to replicate the findings from 53 clinical experiments but found that results from just six of these studies could be independently confirmed [1]. While there are many reasons for this, a primary factor is the inadequate validation of antibodies leading to false positive (and negative) findings. Many antibodies in the public domain have been found to be poorly characterized. For example, a 2008 study found that only half of >6000 commercially-available antibodies uniquely recognized their stated targets [2]. The most common issue was poor specificity, where the antibody recognizes structurally-similar proteins in addition to the target protein. As the use of antibodies in research increases, it is critical that proper antibody authentication experiments are conducted. Funding bodies are also concerned about the apparent lack of experimental reproducibility. In the US, National Institutes of Health requires a validation plan to be provided for key research tools, including antibodies, in all grant applications from 2016 onwards (see http://grants.nih.gov/reproducibility/index.htm).
Collagen VI is present in the extracellular matrix (ECM) of skeletal muscle where it functions to anchor the basement membrane to underlying interstitial tissues. Five collagen VI chains have been described in human [3, 4]. Mutations in three of these (COL6A1, COL6A2 and COL6A3) result in two congenital muscular dystrophies demonstrating that collagen VI is critical for muscle development. These diseases are the relatively mild Bethlem myopathy (BM) and the more severe Ullrich congenital muscular dystrophy (UCMD) [5, 6]. Studies on skeletal muscle biopsies and cultured fibroblasts isolated from affected individuals have established that, in general, the severity of disease is determined by the type of mutation. The majority of disease-causing mutations are amino acid substitutions that typically result in reduced efficiency of collagen VI assembly, leading to reduced and/or aberrant deposition of collagen VI in the muscle ECM [7–10]. Antibodies are used to assess the consequences of mutant or absent protein on collagen VI assembly by immunoblotting, and on ECM deposition and organization by immunohistochemistry [9, 11–14]. Commonly-used antibodies include VI-26 (MAB3303) raised against tissue-purified collagen VI, 3C4/Mab1944 for the α3 chain, and 6A1-H200 for α1. Public discussion within the collagen VI community about the use of antibodies for the molecular and histological analysis of collagen VI mutations [15, 16] stimulated an effort to authenticate commonly-used antibodies. In this report we define chain specificity and relative sensitivities of 23 commercially-available collagen VI antibodies by immunohistochemistry and immunoblotting.
Methods
Immunohistochemistry
Summary of collagen VI antibodies and experimental conditions
Antibody | Supplier | Cat number | Species | Immunogen | IHC dilution | IB dilution |
---|---|---|---|---|---|---|
Collagen VI VI-26 | Millipore | MAB3303 | mou mAb | Human-tissue not specified | 1:100 | 1:500 |
Collagen VI | Fitzgerald Ind. | 70R-CR009X | rab pAb | Human/bovine placenta | 1:100 | 1:1000 |
Collagen VI 172C2 | Santa Cruz | sc-47764 | mou mAb | Human-tissue not specified | 1:100 | 1:500 |
Collagen VI 5C6 | U. Iowa DSHB | – | mou mAb | Human fetal membranes | 1:100 | 1:500 |
Collagen VI | Millipore | AB7821 | rab pAb | Human placenta | 1:100 | 1:500 |
COL6A1 H200 | Santa Cruz | sc-20649 | rab pAb | α1 chain, amino acid 51-250 | 1:40 | 1:500 |
COL6A1 | Abcam | ab199720 | rab mAb | α1 chain, within aa 800 to C-term. | 1:400 | 1:2000 |
COL6A1 | Abcam | ab182744 | rab mAb | α1 chain within amino acid 1-250 | 1:100 | 1:1000 |
COL6A1 | Proteintech Gp | 17023-1-AP | rab pAb | Not available | 1:100 | 1:500 |
COL6A1 B4 | Santa Cruz | sc-377143 | mou mAb | α1 chain amino acid 53-86 | 1:40 | 1:500 |
COL6A2 | Proteintech Gp | 14853-1-AP | rab pAb | Not available | 1:100 | 1:500 |
COL6A2 EPR7889 | Abcam | ab180855 | rab mAb | α2 chain, amino acid 800-900 | 1:400 | 1:1000 |
COL6A2 | Abcam | ab172606 | rab mAb | α2 chain, amino acid 200-300 | 1:100 | 1:1000 |
COL6A2 D20 | Santa Cruz | sc-83607 | rab pAb | α2 chain, internal epitope | 1:40 | 1:500 |
COL6A2 H300 | Santa Cruz | sc-292186 | rab pAb | α2 chain, amino acid 241-540 | 1:40 | 1:500 |
COL6A2 K15 | Santa Cruz | sc-377143 | rab pAb | α2 chain, C-terminus | 1:40 | 1:500 |
COL6A2 B7 | Santa Cruz | sc-374566 | mou mAb | α2 chain, amino acid 241-540 | 1:40 | 1:500 |
COL6A3 G18 | Santa Cruz | sc-131139 | goat pAb | Not available | 1:40 | 1:500 |
COL6A3 3C4 | Millipore | MAB1944 | mou mAb | Not available | 1:100 | 1:1000 |
COL6A3 3C4 | Santa Cruz | sc-47712 | mou mAb | Not available | 1:100 | 1:250 |
COL6A3 H300 | Santa Cruz | sc-367543 | rab pAb | α3 chain, amino acid 301-600 | 1:40 | 1:500 |
COL6A3 H3-2 | Santa Cruz | sc-81766 | mou mAb | α3 chain, globular domains | 1:100 | 1:500 |
COL6A3 64C11 | Abcam | ab49273 | mou mAb | α3 chain, 295 aa C-term. frag | 1:100 | 1:1000 |
COL6A3 N12 | Santa Cruz | sc-131140 | goat pAb | Not available | 1:40 | 1:500 |
Cell transfections
cDNAs for each chain were PCR amplified from existing cDNA clones and subcloned into a mammalian expression vector (pCDNA6). COL6A1 and COL6A2 were full-length cDNA clones encompassing the N1 to C2 domains. The COL6A3 cDNA spanned the N6 to C5 domains and we have previously established that this cDNA assembles into multimeric collagen VI [17]. All clones were verified by Sanger sequencing.
HEK-293 cells (ATCC, CRL-1573) were independently transfected with cDNAs for the human COL6A1, COL6A2 and COL6A3 genes producing four cell lines that stably express the α1, α2 and α3 chains of collagen VI separately, and one line that expresses all three chains together. We have previously established that HEK-293 cells do not express detectable levels of COL6A1, COL6A2 or COL6A3 mRNA by RT-PCR (data not shown).
Immunostaining transfected cells
HEK-293 cultures expressing each chain individually were grown to confluency in Dulbecco’s modified Eagles medium (GIBCO) supplemented with 10% fetal calf serum (GIBCO) in 4-well chamber slides. Cells were incubated overnight in media supplemented with 0.25 mM sodium ascorbate to facilitate collagen biosynthesis. Cells were fixed in 2% paraformaldehyde, permeabilized with 0.1% v/v Triton-X100 (Sigma), and blocked with 10% normal goat serum (Abcam, ab156046) in 1× PBS. Primary and secondary antibodies were added at the same concentration and incubation times as for the immunohistochemical studies (see Table 1).
Cell lysate preparation
Transfected HEK-293 cells were grown to confluence in DMEM with 10% (v/v) fetal calf serum, then supplemented with 0.25 mM sodium ascorbate for 24 h. Media was removed and the cells gently washed with 1× PBS to remove residual media. Lysates were prepared by scraping the adherent cell layer into 1% Nonidet-P40 (Sigma) in 1× PBS with EDTA-free protease inhibitor cocktail (Roche). Lysates were incubated under agitation for 24 h at 4 °C, centrifuged to pellet insoluble material, and the supernatants collected. To ensure equal gel loading, sample protein concentration were determined using a BCA assay kit (Thermo Scientific) and aliquots were resolved under reducing (20 mM DTT) and non-reducing conditions by SDS-PAGE. Gels were either 6% (w/v) polyacrylamide made in-house or 4–15% (w/v) polyacrylamide gradient gels purchased from Bio-Rad.
Immunoblotting
For immunoblotting, collagen chains resolved by SDS-PAGE were transferred onto PVDF membranes (Millipore), then blocked for 1 h at room-temperature in 4% nonfat dried milk in 0.1% PBS/tween-20. Blots were probed individually with collagen VI antisera, some chain-specific and some raised against tissue-purified collagen VI. Antibody dilutions are listed in Table 1. Bound primary antibody was detected with either anti-goat, -rabbit, or—mouse fluorescently-labeled secondary IgG. Blots were visualized on the Odyssey CLx imager (LiCor).
All immunoblot and immunohistochemistry experiments were repeated at least once to confirm results.
Results
To assess the relative efficiency of collagen VI antibodies to detect collagen VI in muscle, cross and longitudinal frozen sections of normal human skeletal muscle were immunostained with a panel of 23 commercially-available collagen VI antibodies. Primary antibodies were detected by fluorescently-labelled secondary antibodies. Experimental conditions were identical except for antibody dilutions, which were selected based on manufacturers guidelines, and choice of secondary antibody which was dictated by the species the primary antibody was raised in.
Summary of immunohistochemistry and immunoblot results
Antibody | Cat number | Signal by IHC | α1 lysate | α2 lysate | α3 lysate | α1 α2 α3 lysate | Figures |
---|---|---|---|---|---|---|---|
COL6A1 H200 | sc-20649 | ++ | ++ (R) | − | − | + | |
COL6A1 | ab199720 | +++ | +++ (R)/+++ (NR) | − | − | + | |
COL6A1 | ab182744 | ++ | ++ (R) | − | − | + | |
COL6A1 | 17023-1-AP | +++ | +++ (R)/+++ (NR) | − | − | + | |
COL6A1 B4 | sc-377143 | − | +++ (R) | − | − | + | |
COL6A2 | 14853-1-AP | ++ | − | ++ (R)/+ (NR) | − | nd | |
COL6A2 EPR7889 | ab180855 | +++ | − | +++ (R) | − | − | |
COL6A2 | ab172606 | ++ | − | + (R) | − | nd | |
COL6A2 D20 | sc-83607 | +++ | − | +++ (R) | − | ++ | |
COL6A2 H300 | sc-292186 | ++ | − | + (R) | − | + | |
COL6A2 K15 | sc-377143 | +++ | − | + (R) | − | + | |
COL6A2 B7 | sc-374566 | − | − | +++ (R)/+ (NR) | − | ++ | |
COL6A3 G18 | sc-131139 | ++ | − | − | + (NR) | + | |
COL6A3 3C4 | MAB1944 | +++ | − | − | + (R)/++ (NR) | − | |
COL6A3 3C4 | sc-47712 | − | − | + | nd | ||
COL6A3 H300 | sc-367543 | +++ | − | − | − | − | |
COL6A3 H3-2 | sc-81766 | − | − | − | − | − | |
COL6A3 64C11 | ab49273 | − | − | − | − | − | |
COL6A3 N12 | sc-131140 | − | − | − | − | nd | |
Collagen VI VI-26 | MAB3303 | +++ | − | − | − | + | |
Collagen VI | 70R-CR009X | +++ | − | − | ++ (NR) | +++ | |
Collagen VI 172C2 | sc-47764 | − | − | − | ++ (NR) | − | |
Collagen VI 5C6 | − | − | − | − | + | ||
Collagen VI | AB7821 | − | − | Non-sp. | Non-sp. | ++ |
Chain specificity was also confirmed for three antibodies by immunocytochemistry on transfected cells (Additional file 1: Figure S1). These antibodies were; ab199720 for α1, ab180855 for α2 and 70R-CR009X for α3. While these cells do not produce a collagen VI extracellular matrix because they lack the other chains necessary for heterotrimeric assembly, they are used in the current study to confirm chain specificity as indicated by immunoblot data.
Antibodies raised against tissue-purified collagen VI
Validation of antisera raised against tissue-purified collagen VI. Antibody clone, supplier and catalogue number are listed down the left-hand side. Staining was conducted on cross- (A, E, I, M, Q) and transverse (B, F, J, N and R) sections of normal human skeletal muscle using dilutions described in Table 1. Blots containing lysates from cells expressing α1, α2 and α3 chains run under reducing (boxed first three lanes) and non-reducing (boxed second set of lanes) conditions are shown in C, G, K, O and S. Each blot was probed with antibodies indicated on left at the dilutions shown in Table 1. Blots from cell lysates (non-reduced) expressing multimeric collagen VI expressing all three chains were also probed (D, H, L, P and T). Asterisks denote high molecular weight collagen VI band. Migration position of molecular weight markers (in kDa) is shown between each pair of immunoblots
Antibodies directed against individual collagen VI chains
α1(VI)
Detection of the α1 chain of collagen VI. Immunostaining was conducted on cross- (A, E, I, M, Q) and transverse (B, F, J, N and R) sections of normal human skeletal muscle. Antibodies are listed on left. Blots containing reduced and non-reduced sets of lysates from cells expressing α1, α2 and α3 chains individually (C, G, K, O and S) were probed with each α1 antibody. Blots containing lysates from cells expressing multimeric collagen VI containing all three chains were resolved under non-reducing conditions and probed (D, H, L, P and T). Migration position of molecular weight markers (in kDa) is shown between each pair of immunoblots
In transfected cells, ab199720 gave a strong signal in cultures of cells expressing α1, but not in cells expressing α2 or α3 chains (Additional file 1: Figure S1, panels A, F and K). This confirms the immunoblot finding (see Fig. 2G) that this antibody specifically recognizes the α1 chain.
α2(VI)
Detection of the α2 chain. Immunostaining was conducted on cross- (A, D, H, K, O, S and W) and transverse (B, E, I, L, P, T and X) sections of skeletal muscle. Blots containing reduced and non-reduced sets of lysates from cells expressing α1, α2 and α3 chains individually (C, F, J, M, Q, U and Y) were probed with each α2 antibody. Blots containing multimeric collagen VI expressing all three chains and electrophoresed under non-reducing conditions were probed (G, N, R, V and Z). Migration position of molecular weight markers (in kDa) is shown
α3(VI)
Detection of α3. Immunostaining was conducted on cross- (A, E, I, M, Q and T) and transverse (B, F, J, N, R and U) sections of skeletal muscle. Blots containing reduced and non-reduced sets of lysates from cells expressing α1, α2 and α3 chains individually (C, G, K, O, S and V) were probed for α3. Blots from cell expressing multimeric collagen VI expressing all three chains resolved under non-reducing conditions were probed (D, H, L, and P). Migration position of molecular weight markers (in kDa) is shown between each pair of immunoblots
Comparison of 3C4/MAB1944 antisera. Lysates from HEK-293 cells transfected with COL6A3 cDNA were immunoblotted using two different lots of MAB1944 from Millipore (A, B) and 3C4 from Santa Cruz (C) under reducing (Red) and non-reducing conditions (NR). Molecular weight markers, in kDa, are on left. Arrow showing migration position of α3 chain is shown on right
Controls
Control experiments where sections were stained using rabbit, mouse and goat IgGs at dilutions of 1:100 did not detect any collagen VI (Additional file 2: Figure S2, panels A–F). Similarly, blots containing lysates from triple transfected cells probed with mouse IgG and normal rabbit and goat serum failed to detect significant bands (G–I). A faint band at 70 kDa is present on the blot probed with normal rabbit serum but this is smaller than the 140 kDa α1 and α2 bands and 260 kDa α3 bands and represents background (asterisk in panel H).
Discussion
To provide a meaningful assessment of the relative usefulness of collagen VI antibodies for immunohistochemistry 23 collagen VI antibodies from Abcam, Santa Cruz, ProteinTech Group and Millipore were tested for reactivity on human muscle sections under standard conditions. Since aberrant collagen VI tissue deposition can be caused by mutations in three genetically-distinct collagen VI chains and it would be valuable to know whether any antibody cross-reacted with the other structurally-similar chains, each antibody was tested for chain specificity by immunoblot analyses and, for three antibodies, immunocytochemistry. Comparative testing revealed that 15 out of 23 antibodies detected pericellular collagen VI on human skeletal muscle sections. The best antibodies specific for each chain are: ab199720 and 17023-1-AP for α1, ab180855 and 14853-1-AP for α2, 3C4/MAB1944 and sc-367543 for α3 (see Table 2). The only two antibodies raised against tissue collagen VI that detected multimeric collagen VI by immunocytochemistry were MAB3033 and 70R-CR009x.
14 out of 18 chain-specific antibodies recognized their target collagen VI chains although with a wide range of sensitivities. Notably, all 14 antibodies demonstrated chain specificity with each antisera recognizing a single collagen VI chain and none appeared to cross-react with the other collagen VI chains. However, good specificity within the collagen VI family does not exclude the possibility that these antibodies bind to other collagens or non-collagenous proteins. Each distinct chain was recognized by at least two different antibodies with high sensitivity (Table 2). The finding that antisera against α1 and α2 primarily recognizes the reduced form of the chain suggests that these chains form intra- or inter-chain disulfide bonds that nominally mask the epitopes, and complete reduction of disulfides is essential for optimal antibody recognition. Epitopes within high molecular weight collagen VI were detected by multiple antibodies with the three strongest signals being ab182744 against α1, and sc-374566 and sc-83607 against α2.
There has been discussion in the collagen VI literature regarding the use of VI-26/MAB3303 and 3C4 for immunostaining patient muscle tissue and fibroblasts [15, 16]. It has been reported that VI-26 is less sensitive at detecting collagen VI in muscle and fibroblast preparations compared to 3C4 leading to an underestimation of the amount of collagen VI degradation [23]. Our data indicates that they recognize different forms of collagen VI; VI-26 detects high-molecular weight, (presumably) triple helical collagen VI but not any individual chains (Fig. 1C, D) whereas 3C4 detects the α3 chain and not collagen VI multimers (Fig. 4G, H). These antibodies clearly recognize different epitopes and may detect different sub-populations of collagen VI fibrils in the ECM. We suggest staining using more than one antibody when detecting collagen VI by immunohistochemistry.
Conclusions
This study expands the list of collagen VI antibodies that can be used in immunoblotting for the analysis of collagen VI on patient samples. Antibodies with high specificity and sensitivity are available for each collagen VI chain. Most α1 and α2 antisera perform best on reduced material; in contrast the majority of α3 antisera tested performed better on non-reduced samples. Several antibodies recognize multimeric collagen VI with the strongest signal from the 70R-CR009X antibody.
There have been calls to standardize antibody production, characterization and use [24, 25]. It is recommended that journals insist that authors be more transparent and consistent with reporting antibody details [26]. We echo this sentiment and reiterate the need to include basic information when reporting antibody experiments by including dilutions used, vendor and catalogue/clone numbers, and species antibody was raised in. In addition, it is essential to include controls such as isotype controls for monoclonal antibodies, pre-immune serum controls for polyclonal antisera and blocking peptide controls for peptide antibodies, if possible.
Declarations
Authors’ contributions
JE conducted the immunohistochemical, immunocytochemical and the immunoblotting analyses. PH conducted the immunoblotting analyses. JF wrote the manuscript with input from JE and PH. All authors read and approved the final manuscript.
Acknowledgements
The authors would like to thank LaToya Jackson for providing paraffin sections.
Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its additional information files. All antibodies are commercially available.
Consent for publication
Informed consent was obtained from all tissue donors.
Ethics approval and consent to participate
Research on human material was conducted in accordance with the Declaration of Helsinki. Human tissue was collected from the Michigan Society of Histotechnologists who have the authority to supply researchers with human material.
Funding
This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number R01AR055957 (to J.F.).
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Authors’ Affiliations
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