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IMMUNORADIOMETRIC ASSAY (IRMA) OF GASTRIC MUCINS

Mucins are highly glycosylated (>80% of sugar) large molecular weight glycoproteins (>106 Daltons). Their oligosaccharide chains are attached to the peptide core (rich in serine and threonine) via O-glycosidic linkages composed of N-acetyl-glucosamine, N-acetyl-galactosamine, galactose, fucose and sialic acid. These cabohydrates are also found on the N-glycosylated glycoproteins and consequently cannot be regarded as specific for mucin components.
Mucin has been assayed using radiolabelled glucosamine. However, this method can only be used in an in vitro system of mucin synthesis. In this method, the radiolabelled glucosamine is incorporated in high molecular component isolated from the supernatant of mucus secreting epithelium (cell line or primary culture). Such a technique characterizes generally the high molecular weight glycoproteins containing glucosamine and does not detect specifically mucins.
Moreover, in no case it is possible to characterize the tissue specificity of these mucins. Indeed, the immunochemists have clearly demonstrated using antibodies the existence of different mucins according to their tissu and cell origins. To develop a technical assay specific for mucins, antibodies against their peptide core must be used. Indeed, antibodies against the saccharide moieties immunoreact against blood group related antigens expressed in other glycoconjugates (N-glycosylated glycoproteins and glycolipids).

A radio-immunometric assay method using monoclonal antibodies against M1 epitopes (M1 antigens) which are associated with the peptide core of gastric mucins. Polystyrene stars provided by Cis, Bio International (France) were used as a solid support. The star-shape of this solid support increases greatly the surface area for fixation of antibodies and hence the immunoreactive surface area.

The method is as following :

  1. The stars are washed with distilled water containing O.15M Nacl for 15 min and then rinsed twice with distilled water for 15 min with shaking.
  2. Incubate the stars under shaking for 2h with an anti-M1 monoclonal antibody (1-13M1): 10 µg/ml of 1-13M1 IgG1 (purified using Protein A Sepharose). Two stars per mililiter is recomeanded for an optimum coating (first layer).
  3. Add to the reaction medium bovine serum albumin (final concentration: 1%) and incubate overnight at 4°C.
  4. Dry the stars in an oven at 37°C. These stars can be conserved for several months at 4°C.
  5. Each star is to be jammed into the bottom of a special tube provided by Cis, Bio International (France) and incubated for 24h. with either different known quantities of M1 units of gastric mucins (standard curve) or biological samples of unknown mucin concentrations (2nd layer).
  6. Wash 3 times with Phosphate buffer Saline (PBS) containing 0.1% Tween-20.
  7. Incubate the stars for 24h with a mixture of 7 different anti-M1 monoclonal antibodies labelled with 1'125I (third layer).
  8. Wash 3 times with PBS-Tween-20 and count the radioactivity in each tube in a gamma counter.
The standard curve is prepared using a solution of gastric mucins containing a large amount of M1 antigens obtained from an ovarian mucinous cyst of pure endocervical type. A solution of this cyst fluid of 10 µg/ml of protein contains arbitrarily 10,000 Units of M1/ml. The assay is relevant in the linear portion of the curve between 100 and 1000 units M1/ml. For higher concentration, the biological samples have to be diluted. For the assays in biological fluids, it is important to add a cocktail of anti-protease reagents at a final concentration: aprotine 2 µg/ml (Sigma), pepstatine 1 µg/ml (ICN Biochemocals), 0.25 mg/ml of AEBSF (4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride - ICN Biochemicals) and 1 mM pCMPSA (p-chloromercuri-phenylsulfonic acid - Sigma).
This IRMA technique has been applied to cell culture supernatants of Capan-1 or HT29-S-B6 cells to study their mucin secretion after treatment with pharmacologic reagents. This technique has also been used to detect mucin in the pancreatic cyst in order to diagnose the mucinous pancreatic cyst which is regarded as precancerous and is to be removed by surgery.


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