Epitope specificity of monoclonal and polyclonal antibodies to human elastin.

Polyclonal (pAb) and monoclonal (mAb) anti-human aorta elastin antibodies were reacted with a series of overlapping hexapeptides along the human tropoelastin sequence covering exons 2-7 and 23-36 from the N-terminus to the C-terminus, advancing 1 amino acid residue each time. ELISA indicated reactive epitopes. mAb A2.1 recognized sequences containing Ala-Lys, mAb G8.1, A7.1 and pAb, hydrophobic sequences. None of them reacted with the hexapeptide VGVAPG, or with desmosine or isodesmosine. pAb L85 reacted with a His-containing sequence coded in exon 26A. pAb kappaE(L), kappaE(S) and L85 reacted with the Cys-containing sequence of exon 36. A synthetic 14-residue peptide containing the three proximal tyrosines coded in exon 13 did not react with any of the antisera tested. It appears therefore that the most frequently recognized epitopes are hydrophobic sequences. One polyclonal antibody detected several isoforms of tropoelastin in the medium of cultured vascular smooth muscle cells. Monoclonal and polyclonal antibodies stained elastic fibers on tissue sections, suggesting that the epitopes recognized are available on the native fibers for reaction with the antibodies.

The technical problems shoot for the production of anti low molecular weight hapten antibody.

Four kinds of small molecular weight haptens, Desmosine, Adriamycin, 4'-carboxycotinine and 5'-Fluorouracil were used as the immunizing antigens to obtain rabbits polyclonal antisera. All of these haptenes were conjugated to Bovine Serum Albumin (BSA) using 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDCl) reagent. The functional amino or/and carboxyl group in hapten molecules was utilized for conjugation with BSA by EDCl reagent. The hapten conjugated with BSA was mixed with Freund's Complete or Incomplete Adjuvant, and immunized to rabbits abdominal subcutaneous regions. More than 3 times immunized rabbit's antisera were evaluated the titer, and the specificity was investigated by ELISA method. The specific antisera against desmosine and adriamycin were produced. Anti-desmosine antibodies had no cross-reaction to molecular structure resemble pyridinoline. Anti-adriamycin antibodies reacted well to adriamycin and it could distinguish the differences of -epi type of adriamycin. The ELISA assay systems used here had such sensitivity. However the specificity possessing antisera against 4'-carboxycotinine and 5-Fluorouracil could not produce even though the antisera titers were elevated. The reasons why and what kinds of problems were there are discussed in this paper. For the production of the specific affinity possessing antisera, the length and distance between the carrier protein and the hapten and the shape of the functional groups were the important factors for production of the specific antisera which recognize free form hapten molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Polyclonal anti-desmosine antiserum is specific to desmosine molecule: no cross-reaction to pyridinoline.

Inhibition ELISA assay was used to examine the cross-reaction of the polyclonal anti-desmosine antiserum produced in rabbit against molecules possessing a "pyridinium ring" as their core structure i.e. isodesmosine, pentasine, pyridinoline and 2'-deoxypyridinoline, highly purified with column chromatography, and structurally unrelated substances, i.e. cysteic acid, taurine and 2-aminopyridine (core structure of desmosine). No cross-reaction was observed to the pyridinoline, 2'-deoxypyridinoline possessing "pyridinium ring" and derived from collagen cross-links, structurally unrelated cysteic acid and taurine, nor core structure of 2-aminopyridine. The antiserum specifically recognized the molecules derived from the elastin cross-links. Using the ELISA assay system with antisera and amino acids analysis, 10 micrograms of desmosine were extracted from 1.0 mg of the hydrolysate of commercial elastin.

Experience with an enzyme-linked immuno sorbent assay for the quantitation of urinary desmosine.

An enzyme-linked immuno sorbent assay has been set up for the quantitation of the elastin crosslink desmosine. With the assay desmosine could be detected in the range 0.01-10 ng (the amount is corresponding to 0.4-400 ng/ml) in standard solutions. One hundred percent crossreactivity with isodesmosine was found. The low titer rabbit antiserum used in this inhibition immunoassay was less suitable for the measurement of desmosine in urinary hydrolysates, because a number of unknown urinary substances interfered with the measurement. Interfering substances could not be removed completely with a column purification method. Future research is needed to determine the applicability of the higher titer anti-desmosine antiserum from a second rabbit.

Radioimmunoassay for desmosine.

A radioimmunoassay was developed for the determination of desmosine. Desmosine conjugated to albumin was injected into rabbits which developed useful titers of serum antibodies after six months. A radioactive probe was prepared with desmosine using [125I]-Bolton-Hunter reagent. Bound desmosine was separated from free desmosine by cellulose acetate filter binding. The sensitivity of the assay is 1-50 picomoles of desmosine. The antibody is highly selective for desmosine, reacting less than 1% with other known crosslinks. Some prepurification may be necessary with complex samples which contain trace amounts of elastin peptides.