Preparation and applications of monoclonal antibodies to different collagen types

Clm Bloc/m, Vol. 21, pp. 111-115, 1988 Printed in Canada. All rights reserved.

Copyright

0009-9120188 $3.00 + .OO 0 1988 The Canadian Society of Clinical Chemists.

Preparation and Applications of Monoclonal Antibodies to Different Collagen Types RICHARD MAYNE Department of Cell Biology and Anatomy, University of Alabama at Birmingham, Birmingham, Alabama, 35294 Monoclonal antibodies have recently been developed against all of the major collagen types isolated from both human and other species. These antibodies have several potential advantages over polyclonal antibodies, and a brief survey will be made of the different antibodies that have so far been developed. In addition, various successful applications of these antibodies to biological investigations will be briefly discussed.

KEY WORDS: munolocalization;

monoclonal antibodies; rotary shadowing.

collagen

types; im-

E

ach collagen type usually gives only a weak immunological response when injected into rabbits or a variety of other species. Previous studies have shown that polyclonal antibodies with a high degree of specificity for a single collagen type can be prepared after a series of cross-adsorptions against all other known collagen types (1). However, this approach is becoming increasingly impractical as more collagen types are discovered. At present, at least 12 different collagen types are recognized and a brief summary of the field is presented in Table 1. More extensive reviews of the structure and function of each collagen type are presented elsewhere (2). Previous experience by several laboratories has shown that the noncollagenous extension peptides of both collagen and procollagen molecules are more immunogenic than the collagen triple helix. For example, polyclonal antibodies can be easily prepared against the amino-terminal propeptide of type III collagen. Such antibodies have clinical value, and numerous studies have used these antibodies to follow serum levels of the amino-terminal propeptide of type III collagen during the progression of chronic liver diseases involving fibrosis (3-7). Another approach to overcome the generally weak immunological response to collagen is to prepare a series of monoclonal antibodies against a highly purified preparation of a single collagen type. From this population only those antibodies are selected that demonstrate a high degree of specificity and a high affinity

Correspondence: Dr. Richard Mayne, Department of Cell Biology and Anatomy, Medical Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA. Manuscript received June 8, 1987; revised July 15, 1987; accepted July 20, 1987. CLINICAL BIOCHEMISTRY,

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for the initial immunogen. This approach has now been successfully used for most of the major collagen types, and a summary of the antibodies that have been prepared is presented in Table 2. Unfortunately, most of these antibodies usually show a high degree of speciesspecificity, and antibodies to chicken collagen consistently do not crossreact with mammalian species.

1988

Applications of monoclonal different collagen types

antibodies

to

Monoclonal antibodies to collagen have been used experimentally in several ways, some of which will now be briefly discussed. IMMUN~L~CALIZATION~FEACHCOLLAGENTYPEBY FLUORESCENCE MICROSCOPY

The initial characterization of each monoclonal antibody usually involves immunolocalization of the collagen by light microscopy. For example, a monoclonal antibody prepared against the cartilage-specific type II collagen should give immunofluorescent staining only of cartilagenous tissues (8). Similarly, an antibody to type X collagen should give fluorescent staining only of hypertrophic regions of cartilage (9,101. Sometimes however, the tissue distribution of a collagen is highly restricted and only limited amounts of the antigen are available. For type VII collagen, immunofluorescent staining with a monoclonal antibody has clearly demonstrated specific localization only to the basement membrane zone of the dermal-epidermal junction of the skin, and to the amniotic epithelial basement membrane of the chorioamnion (11). However, for two monoclonal antibodies prepared against type V collagen, very weak fluorescent staining of tissues was initially observed. Subsequently, it was found that prior treatment ofthe sections with dilute acetic acid resulted in a marked increase in fluorescent staining (12). This simple observation has led to many additional experiments indicating that type V collagen is contained within fibrils of type I collagen (13). Exposure of sections to acetic acid is considered to swell the fibrils and expose the epitope for type V collagen. These results also indicate that some caution must be exercised in using mono111

MAYNE TABLE 1 Summary of Different Collagen Types

Chains

Chain organization of triple helix

I

al(I), c~2(I)

[al(I)]2a2(I)

II

c~l(II)

[~l(II)]3

III

~l(III)

[~l(III)]3

IV

~I(IV), a2(IV)

V

al(V), ~2(V), a3(V)

VI

al(VI), c~2(VI), a3(VI)

Type

VII VIII

[c~l(IV)]2c~2(IV) [al(V)]2a2(V) al(V)c~2(V)~3(V) ~I(VI)c~2(VI)c~3(VI)

~I(VII) cd (VIII), ~2(VIII)?

[~I(VII)]~ Unknown

IX

~I(IX), ~2(IX), a3(IX)

~I(IX)a2(IX)a3(IX)

X

~l(X)

[~l(X)]3

XI

al(XI), ~2(XI), a2(XI)

~I(XI)a2(XI)a3(XI)

XII

~1 (XII)

[~I(XII)]3

clonal antibodies for fluorescent staining. It is possible t h a t the single epitope t h a t each monoclonal antibody recognizes m a y be easily m a s k e d in specific tissues or locations. One monoclonal antibody which potentially m a y have considerable diagnostic value was recently prepared

Function Fibril formation (skin, tendon, bone, etc.) Fibril formation (cartilage, vitreous humor, etc.) Fibril formation (skin, blood vessels, major organs). Forms cofibrils with type I collagen Structural meshwork of all basement membranes Uncertain. Small fibrils or incorporated into larger fibrils of types I and III collagen Microfibrils of most connective tissues Anchoring fibrils of skin, etc. Unknown Synthesized by vascular and corneal endothelial cells Forms cofibrils with type II collagen in cartilage Unknown. Found only in hypertrophic cartilage Uncertain. May be present within fibrils of type II collagen Unknown. Small amounts are found in embryonic tendon. Closely related to type IX collagen.

against the carboxy-terminal propeptide of type I procollagen (14). This antibody gives extensive intracellular staining of cells t h a t are actively synthesizing collagen, and the antibody has already proved useful in identifying the collagen-producing fibroblasts in patients suffering from fibrotic lung disease.

Figure 1--Electron microscopic visualization of type IX collagen after rotary shadowing in the presence of monoclonal antibody 2C2. Panel A: The pepsin-resistant fragment of type IX collagen called HMW. Panel B: The intact type IX molecule as secreted by a suspension culture of embryonic chick chondrocytes. Note that the fragment HMW is missing both a collagenous extension at one end of the molecule and also a knob at the other end of the molecule. Arrows indicate antibody molecules. Bar = 100 nm. (For further details see ref. 46). 112

CLINICAL BIOCHEMISTRY, VOLUME 21, APRIL 1988

MONOCLONAL ANTIBODIES TO COLLAGEN TABLE2 Monoclonal Antibodies to Different Collagen Types Collagen Type I

Species Human Sheep

Chicken II

Human Chicken

III

Human Human

IV

Human Mouse Chicken

V VI

Rat Chicken Human

Chicken VII

Human

VIII IX

Not described Chicken

X

Chicken

XI XII

Not described Not described

Specificity (ref.) Carboxyl terminus of type I procollagen (14) Amino terminal cleavage site of type I procollagen (31) Triple helical domain (32) Triple helical domain (29,33) Triple helical domain (8,30) Procollagen (34) Triple helical domain (19,35) Pepsin-resistant fragments (20,21,36-39) Major noncollagenous (NC1) domain (40) Pepsin-resistant fragments (22,41) Triple helix (42) Triple helix (12,43) Identified for both the intact molecule and pepsinresistant fragments (44) Pepsin-resistant fragment (45) Pepsin-resistant fragment (11) Pepsin-resistant fragment (46) Triple helical domain (9)

IMMUNOLOCALIZATION OF COLLAGENS BY ELECTRON MICROSCOPY

Several groups have successfully used monoclonal antibodies for electron microscopic immunolocalization, the procedure usually involves a second antibody coupled to colloidal gold. For example, by this procedure, it was demonstrated that type VI collagen forms microfibrils in the extracellular matrix of both tendon (15) and cornea (16), and that these fibrils are independent of the major collagen fibrils. Each monoclonal antibody to type VI collagen showed periodic binding along the fibril that was consistent with the known location of its epitope and with models proposed earlier for the assembly of the monomers of type VI collagen to form a fibril (17). Monoclonal antibodies to type VII collagen have also been used in similar experiments to demonstrate that CLINICAL BIOCHEMISTRY, VOLUME 21, APRIL 1988

type VII collagen forms the anchoring fibrils of the dermis (11), and that there is an extended network of these fibrils to small plaques of amorphous material containing type IV collagen (18). In these experiments, it was also possible to demonstrate that the site of binding of the antibody along the fibril was entirely consistent with the known location of the epitope for each antibody as determined by rotary shadowing. Recently, a series of innovative experiments was performed with a monoclonal antibody of the IgM class against type III collagen (19). This antibody is sufficiently large that it can be observed in the electron microscope binding along all the collagen fibrils of the dermis in a periodic manner consistent with the known location of its epitope. From these results, it was concluded that all collagen fibrils of dermis are probably mixed fibrils containing both type I and type III collagens. ELECTRON MICROSCOPIC LOCALIZATION OF THE EPITOPE FOR EACH MONOCLONAL ANTIBODY AFTER ROTARY SHADOWING

Several monoclonal antibodies to different collagen types have sufficiently high affinities that the location of each epitope along the collagen molecule can be visualized on the electron microscope after low-angle rotary shadowing with platinum. For type IV collagen, several monoclonal antibodies were prepared against pepsin-resistant fragments and then used to locate these fragments in tetramers of type IV collagen (20-24). In general, the results of these experiments indicate that there is a single type IV collagen molecule of chain composition [al(IV)]2~2(IV), and that four identical molecules overlap in an antiparallel arrangement to form the domain called 7S. Similar experiments were also performed for type IX collagen in which a monoclonal antibody was prepared against a pepsin-resistant fragment called HMW. This antibody was then used to identify the intact type IX molecule as secreted by a suspension culture of chondrocytes (Figure 1). Rotary shadowing was also used to investigate the location of the two cleavage sites for vertebrate collagenase in type X collagen (25). A monoclonal antibody was used which binds both to the triple helix of intact type X collagen, and also to the major fragment generated after collagenase cleavage. By using the monoclonal antibody to orient the fragments, it was possible to determine the location of the two cleavage sites along the triple helix of the molecule. MONOCLONAL ANTIBODIES TO TYPE I I COLLAGEN AND ARTHRITIS

Several groups have shown that injection of type II collagen into some highly inbred strains of rats and mice results in the development of a form of arthritis (26-28). In order to analyze the humoral response to type II collagen in the arthritis-susceptible DBA/1 strain of mice, two groups have now prepared a series of monoclonal antibodies from these animals (29,30). The results indicate that only a limited number of ep113

MAYNE itopes along the type II molecule are involved in the arthritogenic response. In one series of experiments in which h u m a n type II collagen was used as the immunogen, all of the antibodies were of the IgG2a class and one monoclonal antibody by itself was reported to induce a mild form of arthritis when injected into a mouse (29).

13.

14.

Conclusions Sufficient experience has now been gained in the preparation of monoclonal antibodies to different collagen types t h a t it seems likely t h a t high affinity monoclonal antibodies will eventually be available for each collagen type. As described earlier, these antibodies have several advantages over polyclonal antibodies in m a n y areas of biological research, and m a y eventually prove useful in several clinical applications.

15.

16.

Acknowledgements

17.

Original research was supported by National Institutes of Health Grant AM 30481. The author thanks Pauline M. Mayne for her assistance in the preparation of this manuscript.

18. 19.

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