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

0009-9120188 $3.00 + .OO Copyright 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 investiga- tions will be briefly discussed.

KEY WORDS: monoclonal antibodies; collagen types; im- munolocalization; rotary shadowing.

E ach collagen type usually gives only a weak im- munological response when injected into rabbits or

a variety of other species. Previous studies have shown that polyclonal antibodies with a high degree of spec- ificity 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 dis- covered. At present, at least 12 different collagen types are recognized and a brief summary of the field is pre- sented in Table 1. More extensive reviews of the struc- ture and function of each collagen type are presented elsewhere (2). Previous experience by several labora- tories 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 col- lagen. Such antibodies have clinical value, and nu- merous studies have used these antibodies to follow serum levels of the amino-terminal propeptide of type III collagen during the progression of chronic liver dis- eases involving fibrosis (3-7).

Another approach to overcome the generally weak immunological response to collagen is to prepare a se- ries of monoclonal antibodies against a highly purified preparation of a single collagen type. From this popu- lation only those antibodies are selected that demon- strate 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, VOLUME 21, APRIL 1988

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 pre- pared is presented in Table 2. Unfortunately, most of these antibodies usually show a high degree of species- specificity, and antibodies to chicken collagen consis- tently do not crossreact with mammalian species.

Applications of monoclonal antibodies to different collagen types

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 an- tibody usually involves immunolocalization of the col- lagen 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 dem- onstrated specific localization only to the basement membrane zone of the dermal-epidermal junction of the skin, and to the amniotic epithelial basement mem- brane of the chorioamnion (11). However, for two mono- clonal antibodies prepared against type V collagen, very weak fluorescent staining of tissues was initially ob- served. Subsequently, it was found that prior treatment ofthe sections with dilute acetic acid resulted in a marked increase in fluorescent staining (12). This simple ob- servation has led to many additional experiments in- dicating 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 mono-

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TABLE 1 Summary of Different Collagen Types

Type Chains

Chain organization of

triple helix Function

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) [c~l(IV)]2c~2(IV)

V al(V), ~2(V), a3(V) [al(V)]2a2(V) al(V)c~2(V)~3(V)

VI al(VI), c~2(VI), a3(VI) ~I(VI)c~2(VI)c~3(VI)

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

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.

clonal antibodies for fluorescent staining. I t is possible tha t the single epitope tha t each monoclonal antibody recognizes m a y be easily masked in specific t issues or locations.

One monoclonal antibody which potentially may have considerable diagnostic value was recently prepared

agains t the carboxy-terminal propeptide of type I pro- collagen (14). This ant ibody gives extensive intracel- lular s ta ining of cells tha t are actively synthesizing collagen, and the ant ibody has already proved useful in identifying the collagen-producing fibroblasts in pa- t ients 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).

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MONOCLONAL ANTIBODIES TO COLLAGEN

TABLE 2 Monoclonal Antibodies to Different Collagen Types

Collagen Type Species Specificity (ref.)

I Human Carboxyl terminus of type I procollagen (14)

Sheep Amino terminal cleavage site of type I procollagen (31)

Chicken Triple helical domain (32)

II Human Triple helical domain (29,33)

Chicken Triple helical domain (8,30)

III Human Procollagen (34) Human Triple helical

domain (19,35) IV Human Pepsin-resistant

fragments (20,21,36-39)

Mouse Major non- collagenous (NC1) domain (40)

Chicken Pepsin-resistant fragments (22,41)

V Rat Triple helix (42) Chicken Triple helix (12,43)

VI Human Identified for both the intact molecule and pepsin- resistant fragments (44)

Chicken Pepsin-resistant fragment (45)

VII Human Pepsin-resistant fragment (11)

VIII Not described IX Chicken

X Chicken

XI Not described XII Not described

Pepsin-resistant fragment (46)

Triple helical domain (9)

IMMUNOLOCALIZATION OF COLLAGENS BY

ELECTRON MICROSCOPY

Several groups have successfully used monoclonal antibodies for electron microscopic immunolocaliza- tion, the procedure usually involves a second antibody coupled to colloidal gold. For example, by this proce- dure, 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 inde- pendent 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

type VII collagen forms the anchoring fibrils of the der- mis (11), and that there is an extended network of these fibrils to small plaques of amorphous material contain- ing 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 per- formed with a monoclonal antibody of the IgM class against type III collagen (19). This antibody is suffi- ciently 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 con- cluded that all collagen fibrils of dermis are probably mixed fibrils containing both type I and type III col- lagens.

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 vis- ualized on the electron microscope after low-angle ro- tary 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 mono- clonal 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 colla- genase 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 gen- erated after collagenase cleavage. By using the mono- clonal 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 ep-

CLINICAL BIOCHEMISTRY, VOLUME 21, APRIL 1988 113

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itopes along the type II molecule are involved in the arthritogenic response. In one series of experiments in which human type II collagen was used as the immu- nogen, all of the antibodies were of the IgG2a class and one monoclonal antibody by itself was reported to in- duce a mild form of arthritis when injected into a mouse (29).

Conclus ions

Sufficient experience has now been gained in the preparation of monoclonal antibodies to different col- lagen types tha t it seems likely that high affinity mono- clonal antibodies will eventually be available for each collagen type. As described earlier, these antibodies have several advantages over polyclonal antibodies in many areas of biological research, and may eventually prove useful in several clinical applications.

Acknowledgements

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.

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