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WO1990005304A1 - Analyses structurelles et enzymatiques sur des glycocomposes, leur emploi dans le diagnostic du cancer, kits utilises dans les analyses et dispositif de prelevement d'un echantillon de secretion via un orifice du corps - Google Patents

Analyses structurelles et enzymatiques sur des glycocomposes, leur emploi dans le diagnostic du cancer, kits utilises dans les analyses et dispositif de prelevement d'un echantillon de secretion via un orifice du corps Download PDF

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Publication number
WO1990005304A1
WO1990005304A1 PCT/DK1989/000265 DK8900265W WO9005304A1 WO 1990005304 A1 WO1990005304 A1 WO 1990005304A1 DK 8900265 W DK8900265 W DK 8900265W WO 9005304 A1 WO9005304 A1 WO 9005304A1
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WIPO (PCT)
Prior art keywords
ligand
labelled
sample
wga
altered
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PCT/DK1989/000265
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English (en)
Inventor
Torben Falck ØRNTOFT
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Oerntoft Torben Falck
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Publication of WO1990005304A1 publication Critical patent/WO1990005304A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57469—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving tumor associated glycolinkage, i.e. TAG

Definitions

  • This invention relates to a method of detecting and quantifying altered saccharide structures on glyco ⁇ proteins, glycolipids, poly- and oligosaccharides (in the following termed glycocompounds) or a glycosyltransferase responsible for such altered saccharide structures.
  • This method is useful for the diagnosis of disease which leads to altered glycosyiation of glycocompounds in the body, and in particular for the diagnosis of cancer.
  • the in- vention also relates to kits for use in carrying out the method, and to a device for taking a sample of secretion via a body opening and for storing and processing the sample.
  • oligosaccharide chains are a) used by the cells, as the chains are incorpotated into glyco- proteins and glycolipids and thereby become part of cell membranes, b) exported out of the cells as a secretion product like intestinal secretion, secretion of the cervix uteri, glycoprotein hormones, serum glycoproteins etc. , c) stored in the cells as oligosaccharide chains.
  • oligosaccharide chains are important for a large number of physiological processes as they regulate the turnover of glycoprotein hormones, the binding of bacteria to cells, the specificity of receptors, and often are in ⁇ volved in the tertiary structure of glycoproteins. In ad ⁇ dition, they are often strongly immunogenic, exemplified by the blood group ABH related antigens, that are all oligosaccharides. When the behaviour of cells is changed due to disease, this is often associated with a change in the synthesis of oligosaccharide chains, especially in the most terminal portions. This has been demonstrated in diseases like alcoholic liver cirrhosis, pancreatitis, colitis and especially in cancer of various organs.
  • This change in the synthesis of oligosaccharide chains is caused by a change in the relative activity of the gene-encoded glycosyl- transferases that form the oligosaccharide chains.
  • certain cells loose activity of a transferase, and, hence, the oligosaccharide chain-elongation is stopped, and a precursor structure is accumulated.
  • this precursor structure is an antigen it ⁇ self that can be recognized by antibodies and lectins, a disease associated oligosaccharide is produced.
  • the disease process may lead to increased activity of glycosyltransferases and the formation of longer or more branched structures than hitherto produced by the cells. This happens in the distal colon where ABH blood group antigens are synthesized in rectal carcinomas but not in normal mucosa.
  • the terminal part of the oligosaccharide chain This is the part of the chain which protrudes from a glycoprotein or glycolipid, whereas the opposite end is bound to the protein or lipid.
  • the terminal part may therefore be regarded as the variable part of the oligosaccharide chain, whereas the non-terminal part of the chain may be regarded as the constant part, which is rarely changed during disease.
  • the terminal part is very often glycosylated according to the genetic make-up of the individual e.g. blood group antigens. Single cells are capable of producing a wide range of glycoproteins. Some of these may be structural compounds, others receptors or hormones.
  • glyco-portion the oligosaccharide chains
  • the glyco-portion the oligosaccharide chains
  • the glyco-portion the oligosaccharide chains
  • the glyco-portion the oligosaccharide chains
  • the glyco-portion the oligosaccharide chains
  • mannose sugars e.g. lactoseries chains with repeating Gal01-3GlcNAc sequences, or repeating GlcNAc or mannose sugars.
  • a disease associated loss or gain of glycosyltransferase activity is leading to a change in terminal oligosaccharides structures, it may, thus, affect the glycosyiation of several different molecules in the same way.
  • the carbohydrate portion of glycoproteins and glycolipids are changed in various disease states like cancer and in ⁇ flammatory diseases (ref. 1). These changes can be de ⁇ tected by the use of carbohydrate specific ligands like lectins or mono/polyclonal antibodies raised against these carbohydrate structures.
  • carbohydrate specific ligands like lectins or mono/polyclonal antibodies raised against these carbohydrate structures.
  • mucins are high molecular weight glycoproteins consisting of more than 40% carbo ⁇ hydrate, the rest being a peptide backbone, from which the carbohydrate oligosaccharide chains jut out
  • the carbohydrate determinants on glycoproteins and especially those on mucins have gained interest in recent years as colorectal tumor markers. They have been in ⁇ vestigated by histochemical methods as mentioned above, and especially as serological tumor markers. However, like CEA, they have the drawback that when the epitope they detect is highly tumor associated (very specific), the number of false negative colorectal carcinoma bearing patients is too high (low sensitivity). In addition the principle has the general drawback that is seems that tumors have to be invasive before the antigens occur in the circulation. It would be much better to detect carcinomas at an earlier stage in their development.
  • Shamsuddin et al. (5) developed a non-quantitative galactose oxidase test on rectal mucus, removed from the patient by a gloved finger. This test detected all cancer patients but also reacted with 8.5% of non-carcinoma patients giving false positive results. Patients with in- flammatory bowel diseases were not investigated.
  • I invented a new method of detecting and quantifying altered saccharide structures on glycoproteins, glyco ⁇ lipids, poly- and oligosaccharides in a body fluid which is characterized by contacting the body fluid with a first ligand binding to the non-terminal (constant) portion of the saccharide structures and with a second ligand binding to a specifically altered terminal (variable) portion of the saccharide structures, but not to the normal terminal portion, or binding to the normal terminal portion of the saccharide structures, but not to the specifically altered terminal portion, one of said ligands being immobilized on a solid phase, and the other being labelled, and there ⁇ after detecting labelled immobilized glycocompounds and/or determining the amount of labelled ligand bound to im ⁇ mobilized glycocompounds or the amount of labelled ligand not bound to immobilized glycocompounds.
  • Another aspect of the invention is a method of detecting and quantifying a glycosyltransferase enzyme producing altered saccharide structures on glycoproteins, glyco ⁇ lipids, poly- and oligosaccharides in a body fluid or a cell or tissue homogenate, which is characterized by contacting the body fluid or homogenate with an im ⁇ mobilized sugar chain which is an acceptor substrate for the enzyme and with a nucleotide-sugar which is a donor substrate for the enzyme, adding a labelled ligand binding to the saccharide structure of the coupling product, and thereafter detecting labelled immobilized coupling product and/or determining the amount of labelled ligand bound to immobilized coupling product or the amount of labelled ligand not bound to immobilized coupling product.
  • the body fluid to be assayed is conveniently selected from the group consisting of rectal, vaginal, uterine cervical and pulmonary secretions. Most conveniently the body fluid is rectal secretion. In these circumstances the pre ⁇ dominant glycocompounds in the secretion are mucins.
  • a special device for taking samples of secretion via a body opening it is expedient to use a special device according to the inven- tion, which is characterized in that it comprises a con ⁇ tainer and a rod which is provided at one end portion thereof with an absorbing material fixed to the rod within the end portion in question, and which slidably extends through a cover means at the other end portion, said cover means being adapted to be fixed in an opening of the con ⁇ tainer.
  • a sample of secretion absorbed in the ab- sorbing material is squeezed out into a liquid in the con ⁇ tainer by displacement of the rod with respect to the cover means so that the absorbing material is compressed against the inner side of the cover means of the con ⁇ tainer, which may be filled with a buffer liquid for this purpose, e.g. pH neutral isotonic phosphate-buffered brine to which a bacteriostatic (e.g. antibiotics) or bacteri ⁇ cidal (e.g. antibiotics or sodium azide) agent is optio ⁇ nally added.
  • a buffer liquid for this purpose, e.g. pH neutral isotonic phosphate-buffered brine to which a bacteriostatic (e.g. antibiotics) or bacteri ⁇ cidal (e.g. antibiotics or sodium azide) agent is optio ⁇ nally added.
  • the absorbing material opposite the end portion may be fixed to the rod in a rupturable manner so that when the rod is displaced outwardly with respect to the cover means, the absorbing material is com- pressed against the inner side of the cover means for squeezing the sample of secretion out of the absorbing ma ⁇ terial.
  • the absorbing material may consist of a suitable foamed plastic which is preferably surrounded by a net-shaped sleeve, which is adapted to protect the ab ⁇ sorbing material against lateral compression, but allows said axial compression of the absorbing material with a view to squeezing the sample of secretion out into the collection liquid.
  • fig. 1 is a perspective view of an embodiment of a sample- taking device according to the invention.
  • fig. 2 shows a container for the sample-taking device according to the invention
  • fig. 3 shows how the sample material is squeezed out of the absorbing material in the sample-taking device according to the invention.
  • the sample-taking device shown in fig. 1 comprises a rod 2 of a suitable foamed plastic and with a handle 4. Inside an outer rounded end portion 6 of the rod 2, it is sur ⁇ rounded by an absorbing material 8, e.g. foamed rubber or foamed plastic, which is in turn surrounded by a helical net-shaped sleeve 10 of plastics connected with the end portion 6.
  • the net sleeve 10 serves to prevent lateral compression of the absorbing material 8, so that the sample material is prematurely squeezed out of the mate ⁇ rial 8, which is temporarily fixed to the rod 2 opposite the end portion 6.
  • the net sleeve 10 may be com ⁇ pressed axially together with the foamed rubber material 8, which thus acts like a sponge.
  • the sample-taking device of the invention more ⁇ over comprises a container 14 which is provided at an opening 16 with internal threads 18 for fixing the cover means 12 in a liquid-tight manner to the container 14 by means of external threads 20 on the cover means 12. Also a separate closing plug 22 is associated with the container 14, said plug being used for liquid-tight sealing of the container 14 - prior to and optionally also after sample- taking - the container 14 preferably containing a suitable liquid, such as pH neutral isotonic phosphate buffered brine, destilled water or the like in which the sample ma- •terial may be steeped.
  • a suitable liquid such as pH neutral isotonic phosphate buffered brine, destilled water or the like in which the sample ma- •terial may be steeped.
  • the sample-taking device of the invention is used in the following manner, there being referred to the taking of a rectal mucus sample in this example:
  • the rod 2 with the foam rubber sponge 8 is preferably supplied as a sterile unit in a sealed plastics bag - and preferably some cream or vaseline is applied to the end portion 6 to facilitate insertion of the rod 2 through the sphincter of the anus.
  • the container 14 contains a suit ⁇ able collection liquid, optionally admixed with a bacteri ⁇ cidal agent, the container 14 being closed and sealed by means of the closing plug 22.
  • the end portion 6 of the rod 2 with the foam rubber sponge 8 is inserted into the rectum, and the rod 2 is pulled out very carefully after some time.
  • the closing plug 22 is removed from the container 14, and the rod 2 with the foam rubber sponge 8, which now contains a mucous sample, is placed in the container 14, and the cover means 12 is screwed tightly into the container opening 16 by means of the threads 18, 20, following which the container 14 is shaken and turned thoroughly to wash out the mucus sample from the sponge 8.
  • the closed container 14 containing the mucus sample sus ⁇ pended in the liquid can now be passed on for more de- tailed laboratory examination. If the sample cannot be analysed immediately, it may be stored in the container 14 - optionally frozen.
  • the mucus sample can advan ⁇ tageously be prepared additionally right away, in that the container 14 contains a suitable detergent solution, e.g. containing "Triton® X-100", it being possible to use the actual container 14 during the subsequent determination of the mucus sample 14 for activity of transferases and/or quantitative analysis of carbohydrate structures.
  • a suitable detergent solution e.g. containing "Triton® X-100"
  • the example below refers to cancer in the colon, but also other organs may be examined by the described method, e.g. the cervix uteri, the urinary system and their glands as well as neck and lungs; of course, the actual sample- taking device is to be modified depending upon the con ⁇ crete organ to be examined.
  • the intestinal secretion is collected from the sigmoideum of the patient either using a gloved finger or a similar oblong instrument or by ano/rectoscopy (telescope exami ⁇ nation of the rectum with a tubular instrument having a length of 5-50 cm).
  • ano/rectoscopy the sample material is collected e.g. directly on a piece of wadding or another suitable aid which is inserted into the intestine, which should preferably have been cleaned of faeces some hours before by means of enema.
  • the finger-collec ⁇ tion the finger is simply wiped in a piece of wadding or the like.
  • the mucus sample may then be stored frozen on the wadding, or the mucus sample may be washed from the wadding by means of a buffer, e.g. pH neutral isotonic phosphate buffered brine, and then the mucous sample is spun down by centrifugation (e.g. 800 x g). The super ⁇ natant is then removed, and the precipitated and washed mucus sample may be stored by freezing to between -20°C and -80°C if the sample is not to be analysed immediately. Frozen samples are to be thawed before analysis.
  • a buffer e.g. pH neutral isotonic phosphate buffered brine
  • centrifugation e.g. 800 x g
  • the mucous sample Prior to analysis of transferase activity the mucous sample is prepared by adding to the washed mucus sample a detergent solution, e.g. containing "Triton® X-100" and subjecting it to mechanical or physical impact, e.g. ro ⁇ tating knifes and/or ultrasound - followed by centrifu ⁇ gation of the supernatant which contains dissolved en- zymes.
  • a detergent solution e.g. containing "Triton® X-100"
  • mechanical or physical impact e.g. ro ⁇ tating knifes and/or ultrasound - followed by centrifu ⁇ gation of the supernatant which contains dissolved en- zymes.
  • washing of the mucus sample may be omitted, and the detergent solution may be added as the first step of preparation.
  • the amount of protein in solution is quantified, and the activity of the transferases is determined by quantifying the amount of the product which the transferases can pro ⁇ quiz either in that the transferases incorporate labelled sugars in an acceptor, or in that the product which they produce is specifically identified with antibodies or lec ⁇ tins.
  • Several known methods of performing this quantifi ⁇ cation are known.
  • ligand antibody or lec- tin
  • the mucus may be bonded directly to a solid surface and then be analysed; or the mucus may be bonded via a ligand which is attached to a solid surface.
  • This ligand will typically be an antibody or a lectine which binds to mucus or mucus portions, but not to structures which are irrelevant to the analysis, thereby providing a certain degree of purification.
  • mucus which has been dissolved by various proce- dures by means of detergents or cleaving enzymes, e.g. mercaptoethanol and trypsin.
  • This example uses mucus which has been brought into solution by homogenization in a PBS buffer containing about 1% "Triton®X-100" as well as mercaptoethanol (cleavage of disulfide bonds) and protease inhibitors (phenylmethylsulfonylfluoride/PMSF). After this homogenization the mucus solution is ultracentrifuged 30,000 x g), and the supernatant is repeatedly precipi ⁇ tated with 75% ethanol.
  • the precipitate includes gluco- proteins, and these are resuspended in H ⁇ 0, dialysed over- night and freeze-dried. Then the protein content of the sample is measured by the BioRads method, and the sugar content is measured by periodic acid Schiffs reaction. The sample is then applied to either a nitrocellulose or a nylon membrane with a known amount of protein or PAS posi- tive sugar. The membrane is then blocked e.g. with albumin and incubated with lectin or antibody in a suitable dilu ⁇ tion. The optional binding of lectin or antibody is then evaluated using a radioactive isotope, an enzyme, a metal or the like which gives a signal, e.g. a colour, that can be identified.
  • a radioactive isotope an enzyme, a metal or the like which gives a signal, e.g. a colour, that can be identified.
  • the enzyme peroxi- dase is bonded directly to the lectin, while the antibody binding is detected by binding an anti-globulin label with the same enzyme directly to the antibody.
  • the membrane is then washed, and a colour reaction is produced by 4- chloro-1-naphthol which is converted to a blue substance by the enzyme.
  • This colouring matter is particularly suitable because the colour reaction proceeds to an end point within a reasonable period of time (less than 30 minutes) without background colour. This provides a colour product which linearly reflects the peroxidase enzyme concentration.
  • the blue substance is then quantified in a chromatoscanner, which is adjusted for absorption in a maximum wave range for the blue colour (evaluated by spectral analysis) .
  • a method as described has shown the presence of blood group antigen H (Fuc l-2 Gal 01-3/4 Glc NAc-R) in rectal mucus from all humans with intestinal cancer, but not in mucus samples from healthy humans.
  • blood group antigen H Fuc l-2 Gal 01-3/4 Glc NAc-R
  • antibodies and lectins used include:
  • Antibodies Anti-T, Tn, Le , Le x , Le y , A (all subtypes), B, H (all subtypes) as well as antibodies against derivatives of these substituted with other sugars, e.g. sialic acid.
  • Lectins PNA (Peanut Agglutinin), Jacalin, UEA (Ulex Europaeicus Agglutinin), WA (Viscia Villosa Agglutinin), DBA (Dolichos Biflorus Aggluti ⁇ nin) . Determination of g-2-L-fucosyl transferase activity in rectal mucus sample
  • the supernatant which contains the transferases, is incubated in a suitable buffer containing an acceptor suitable for determination of precisely this transferase as well as a radioactively labelled sugar - here fucose.
  • a suitable buffer containing an acceptor suitable for determination of precisely this transferase as well as a radioactively labelled sugar - here fucose.
  • the product is separated from the other substances by chromatography, e.g. on thin layer plates or on paper.
  • the product is now quantified in a scintillation counter and is compared with the starting amounts, it being established how much of the added sugar has been incorporated in the acceptor by means of the transferase, and thus how great an activity the transfe ⁇ rase has.
  • Tests have demonstrated an activity of ⁇ -2-L-fucosyl transferases in mucus from the rectum in cancer patients which is more than 10 times higher than the activity in normal healthy patients. Also a permanently increased ac ⁇ tivity of this transferase has been demonstrated in pa ⁇ tients who have had prestages of cancer in the rectum, such as polypi.
  • the examined humans may also optionally be divided according to their erythrocyte phaenotype.
  • transferases which are known to be relatively stably ex ⁇ pressed in the population may advantageously be analysed, e.g. 01-3N-acetylglucosaminyl transferase which is known to increase its activity in case of cancer in the colon.
  • 96 micro-well titertrays also called immunoplates
  • WGA Wood Germ Agglutinin
  • PNA Pieris Agglu ⁇ tinin
  • DBA Dolichos Biflorus Agglutinin
  • conA Concanavalin A
  • MPA Methylura Pomifera Agglutinin
  • UEA 1 Ulex Europaeus 1 Agglutinin
  • BSA 1 Bovine submaxillary mucin, asialofetuin, asialomu ⁇ in, asialo- glycophorin, D(+)-galactose, D(+)-glucose, o-L-fucose, N- acetyl
  • Bovine serum albumin and "Tween®20" were from Merck, Darmstadt, FRG. Cellulosenitrate, 0.45 am, was from Schleicher and Schuell, Dassel, FRG. Monoclonal anti- bodies against Lewis a, Lewis b, A, and B antigens were from Biotest Serum-Institut, Frankfurt, FRG. Monoclonal antibody against H antigen, rabbit anti-mouse IgG con ⁇ jugated with HRP, and 1,2-phenylenediamine-dihydrochloride (OPD) tablets 2 mg, were from Dakopatts, Glostrup, Den- mark. Gelatine G was from Grindsted Products, Brabrand, Denmark. Galactose 01-0-PAP-HSA, Lacto-N-tetraose-O- phenyl-HSA, and GDP-fucose were from Biocarb AB, Lund, Sweden.
  • Secretion from the distal part of the large bowel was collected by two different procedures.
  • a piece of cloth was inserted into the gut lumen through the re ⁇ toscope during rectoscopic exa- mination and allowed to suck-up secretion. After this, the piece of cloth was taken out and frozen in an appropriate container until further analysis.
  • a special device according to this invention consisting of a rod covered with sucking foam, was used to collect the secretion. In this case no rectoscopic examination was necessary, however digital exploration of the rectum, be ⁇ fore insertion of the device into the gut, was used to assure that no obstruction of the rectum by tumors, or other abnormalities, was present.
  • the rod which may be lubricated on the tip by a lubricating gel, was inserted into the rectum by gently pressing the tip of the rod against the anus.
  • the rod covered by the sucking foam was gently moved in direction of the umbilicus (as when inserting a thermometer) until the foam covered part of the rod had passed the anal sphincter muscles.
  • the foam covered part of the rod was then moved in a circular fashion to allow secretion of mucus from the distal part of the gut to be absorbed or adhered to the foam.
  • a time period (appr. 20 seconds) the rod was pulled out and placed in its con ⁇ tainer, the container was closed and the rod kept frozen until further analysis was taking place.
  • mice Twenty mice were sacrificed and the small bowel was extirpated, cut open, and the mucosa was gently scraped off with an object glass.
  • the pooled mucosas were homo- genized with an ultraturrax homogenizer in the presence of ammonium acetate containing 1% "Triton®X-100".
  • the homo ⁇ genate was centrifuged at 30 000 x g and the supernatent dialyzed for 48 hours against distilled water.
  • the dialysate was then freeze dried and stored frozen in small aliquots.
  • Latex beads 500 ⁇ l of Latex beads were centrifuged for a few seconds at 800 x g, the supernatant was discarded, and the pellet consisting of the Latex particles was suspended in a 1 mg/ml solution of Ligand (PNA, DBA, anti-Le etc. ) in carbonate buffer, pH 9.6. After incubation for 2 hours at 25 °C, with occasional shaking, the solution was centri- fuged at 800 x g and the supernatant discarded. The pelleted Latex particles coated with ligand were then washed three times in PBS, and finally diluted appro ⁇ priately (1:5 - 1:20) in PBS.
  • Ligand PNA, DBA, anti-Le etc.
  • Coating buffer 0.1 mol/1 sodiumhydrogencarbonate, pH 9.8. Washing solution, and solution for dilution of samples, ligands and blocking reagents: PBS (0.5 M NaCl, 0.0075 M Na 2 HP0 4 , 2 H 2 0, 0.0025 M NaH 2 P0 4 , H 2 0), pH 7.2 containing 0.1% "Tween®20".
  • Enzyme substrate 8 mg OPD in 12 ml 0.1 M citric acid/phosphate buffer pH 5.0. 5 ⁇ l 30% H 2 0 2 were added 5 minutes before use. Scanning: All wells were read in a Tim 200 immunoreader (Teknunc, Roskilde, Denmark), at 492 nm.
  • Blocking with blocking buffer 200 ⁇ l of the following solutions were tested: 2% BSA in washing buffer, or 2% Gelatine G in washing buffer, for 10 min at 20 °C 4. Washing once in washing buffer.
  • DBA-HRP, conA-HRP, and UEA 1-HRP in concentration ranges between 0.1 ⁇ g/ml and 1 ⁇ g/ml, and monoclonal antibodies against Lea, Leb, A, B, and H antigens diluted 1:100 to
  • the ligands were diluted in washing buffer containing Mn ions, and the pH of the buffer was varied between 6.0 and 10.0 to assure optimal specific binding.
  • the incubation time was 60 min.
  • WGA coated latex beads were used as a label which could be read semiquantitatively by the naked eye, or by use of a magnifying glass.
  • this step in ⁇ cluded washing in washing buffer.
  • this step included incubation with Rabbit anti-Mouse conjugated with HRP, diluted in washing buffer 1:500 to 1:4000 for 60 min, followed by washing once in washing buffer.
  • the following assay conditions were kept constant: The nitrocellulose membrane was mounted in a 96 well dot blot applicator (BioRad) and 250 ⁇ l PBS were added to each well.
  • Sample buffer PBS. Washing buffer: 0.1% "Tween®20" in PBS. Blocking buffer: 5% BSA in washing buffer. Color de ⁇ veloping reagent: 4-chloro-l-naphtol. Buffer for diluting lectins and antibodies: 0.1% BSA in PBS.
  • washing buffer composition was tested with BSA 0.5% to 5% (w/v)
  • the blocking buffer was tested with BSA from 0.5 to 5% (w/v)
  • "Tween®20" from 0.01% to 2%.
  • the HRP-labelled lectins tested were: PNA, WGA, WA, Jacalin, UEA 1.
  • Monoclonal antibodies tested were anti- Le , -Le , -A,-B, -Le , and anti-H, as well as anti-CEA (Roche) and Ca 19-9 (Centrocor).
  • the following example shows an assay for ⁇ l-2-L-fucosyl- transferase activity in rectal secretions.
  • the nucleotide sugar used was GDP-fucose
  • the detecting ligand was UEA 1 lectin which detects ⁇ l-2 linked fucoses.
  • Other assays tested - but not shown - include tests for ⁇ l-4-L- fucosyltransferase activity with anti-Lewis antibodies as detecting ligands, and tests for ol-3-L-fucosyltransferase activity using anti-Le antibodies as detectors.
  • the following reaction mixture was then added to each well: 7 ⁇ l GDP-fucose, (tested at concentrations from 0.16 nmol/ ⁇ l to 5 nmol/ ⁇ l), 5 ⁇ l ATP 0.1 M, 10 ⁇ l MnCl 2 0.2 M, 50 ⁇ l Tris/HCl.
  • the enzyme reaction was initiated by adding 20 ⁇ l of the rectal secretion sample to the reac- tion mixture, and incubating the titer-tray for 60 - 120 min at 37 °C.
  • the pH of the buffer containing WGA-HRP was varied from pH 6-10 as was the NaCl concentration in the buffer from 0.9% (w/v) to
  • the carbohydrate specificity of the PNA binding was tested by preincubating the PNA-HRP label with D-galactose, D- glucose, L-fucose, and N-acetylglucosamine (Fig. 6). As expected, the PNA binding was inhibited by D-galactose even at very low concentrations of the monosaccharide. D- glucose showed some inhibition whereas the other sugars, whose conformations deviate more from galactose, were unreactive (Fig. 6) .
  • this assay has a very high specificity and sensitivity for detecting cancer in the colorectal area - and cancer in other or- gans. If one excludes individuals with severe colitis symptoms, who would be diagnosed anyway, the specificity and sensitivity in this small material are both 100%.
  • the WGA - WGA- HRP assay could be used as a reference value for the con- tent of WGA binding structures in the secretion sample, thus making protein determination unnecessary.
  • This has the advantage compared to protein determination, that only structures able to be caught by the WGA catching ligand, and, thus, only the structures able to react with the PNA- HRP label are measured, irrelevant proteins are neglected by this assay.
  • the con- tamination with irrelevant proteins is important, as the WGA - WGA-HRP assay correlated very well with the protein content.
  • the WGA - WGA-HRP values could be used to make a ratio between these values and those obtained from the WGA - PNA-HRP assay, thus, making protein determinations un ⁇ necessary.
  • the DBA ligand thus, seems to be complemen ⁇ tary to the PNA ligand, and could be used alone as a marker of carcinoma in the large bowel and other organs, or as a marker of severe colitis.
  • a reference value for normal individuals would be a DBA value >0.8 ⁇ g/ml (calibrated against asialomucin) - meaning that lower values indicate the presence of car ⁇ cinoma or severe inflammatory bowel disease.
  • the DBA measurement could be used for screening patients or selected populations to get diag ⁇ nostic information.
  • the disease colitis ulcerosa is usual ⁇ ly diagnosed based on its symptoms (frequent diarrhoeas, abdominal pain), however the DBA measurement could be of importance as a prognostic marker (colitis patients have an increased risk of colorectal carcinomas), or as a marker of disease progression, as discussed above con ⁇ cerning the WGA - PNA-HRP assay.
  • DBA assay A last possibility for the DBA assay was to measure the ratio between DBA binding and WGA binding to samples caught by the immobilized WGA. As discussed above for the PNA assay, such a procedure could make protein determi ⁇ nations unnecessary, and be a check for dilutional errors.
  • a standard was prepared from mouse small intestine. This standard contained crude intestinal material compared to the purified glycoproteins used as standard reagents for calibration. However, it is possible to use this standard instead of the glycoproteins, and to express the lectin binding in arbitrary units.
  • a titer-tray microwell was used as the solid phase for binding of the catching ligand. How ⁇ ever, any solid material able to bind the ligand could be used, and either the catcher could be bound to the con- tainer (like a tube or well) containing the sample, or the solid support carrying the catcher could be placed in a container housing the sample solution.
  • results from this assay are shown in table 4.
  • the results are in some ways comparable to those obtained in the titer-tray assay.
  • this assay suffers from the drawback compared to the titer-tray assay, that the re- suits cannot be quantitated, and the presence of irrele ⁇ vant proteins in large amounts in the solution e.g. in the case of intraluminal bleeding (a very common finding in colorectal cancer) can block the membrane, and thus lead to false negative results.
  • this assay could therefore expect this assay to have a lower specificity and sensitivity com ⁇ pared to the assay principle where a catcher, in our case the WGA lectin is used to immobilize the relevant struc ⁇ tures and therefore to partly purify the samples before labelling these with PNA and DBA.
  • the figures show a seemingly higher sensitivity, and the problems of a lower specificity that often follows a higher sensitivity.
  • Some normal individuals are positive with PNA as ligand, which was not the case in the titer-tray assay. Whether these normal individuals are completely normal or carry a disease - maybe even a cancer disease - which has not reached a level where it gives symptoms can only be elu ⁇ cidated by prospective clinical studies.
  • WGA binds to all samples as does anti-Le after neuraminidase treatment.
  • the latter finding is important as it shows that an antibody against neuraminic acid (or sialic acid) substituted Le structures might be used as a catcher instead of WGA in the titer-tray-assay.
  • the anti ⁇ bodies against ABH blood group antigens are not very specific in the detection of carcinomas compared to anti- bodies against Leb antigens, and Lev * * antigens. Both anti- bodies react very little with normal secretion, but to some extent with malignant secretion. They could be sup ⁇ plementary to PNA and DBA as tumor markers.
  • the HRP- labelled lectins and antibodies were substituted with identical ligands coupled to large Latex beads. These beads can be seen by the naked eye, if necessary assisted by a magnifying glass.
  • the assay thus, consisted of a WGA - Lectin or Antibody-Latex sandwich, with the sample structure caught between these ligands. The procedure was exactly like the one used with the HRP-conjugated ligands except that the reaction sequence ended with a wash after incubation with the ligand coated latex particles.
  • the sample container used to place the sample collection rod inside could be used as one large reaction "well”.
  • Exactly the same reactions that take place in the titer-tray wells can be performed in the container - if this is made of a material, or coated with a material, capable of being coated with the ligands used - preferably WGA.
  • other combinations could be used e.g.
  • the container could be coated with two different lectins at the same time, thus permitting simultaneous measurement of binding of tumor associated ligand, and a control ligand (WGA or antibody against sialosyl-Le ) for eliminating false nega ⁇ tive results.
  • the micro-well enzyme assay could be performed in other containers than micro-well. Especially the container used for housing the sample rod, would be a relevant site for performing the test.
  • the container could be coated with the sugar acceptor, Galactose-R, and the entire reaction mixture could be present in the container so that just the secretion sample having the enzyme activity, if from car- cinoma patients, would be needed to start the reaction.
  • the next step would then be washing out the mixture after an incubation time, and labelling the product, if any, with UEA 1 coupled to enzyme (color reaction), isotope (counting) or solid particle (e.g. Latex particles, seen by the naked eye or with a magnifying glass).
  • enzyme color reaction
  • isotope counting
  • solid particle e.g. Latex particles, seen by the naked eye or with a magnifying glass.
  • kits can be tailored for different uses of the assay according to the invention.
  • One kit may be designed for use at home, which implies that it should be simple and must not require extra equipment.
  • Another kit could be intended for general practitioners, small clinics and out-patient's clinics. Here greater demands may be made as to complexity of the procedures and possibly minor equipment.
  • a third kit could be intended for laboratories with all the possibilities implied.
  • a minimum kit for structural assays is characterized by comprising a container which on at least part of its in- terior surface is coated with first or second ligand, a buffer solution containing a detergent, such as "Triton®X- 100", for solubilizing the body fluid, a washing buffer, and a labelled second or first ligand.
  • a detergent such as "Triton®X- 100"
  • the kit comprises a sample-taking device ac ⁇ cording to the invention. Then the container in which the absorbing material is placed after the sample-taking may be used for carrying out the assay, the container being coated at the bottom, so that the foamed plastic does not scrape off the catcher ligand.
  • the bottom of the container could be coated in a particular symbolic pattern.
  • the interior bottom surface of the con- tainer may be coated with a stripe of WGA and a stripe of PNA the two stripes being essentially at right angles to each other, and the labelled ligand provided with the kit be WGA. Then, if the sample material contains altered saccharide structures, it will bind to both stripes, and when the bound sample material is visualized by labelled WGA a + sign will mark that the assay is positive. If no altered saccharide structures bind to the PNA, a - sign will mark that the assay is negative. If the sample has been taken erroneously and for example only contains water, no symbol will appear at all.
  • a clinical kit would contain either the device for taking samples of secretion or a number of loose coated tubes in which to carry out the assay, so that it is easier to carry out many at the time.
  • the kit could then be extended by e.g. a magnifying glass for reading the latex reactions through the bottom of the tubes or a sort of mini-scanner for measuring enzyme-developed color.
  • a titer tray micro-well kit intended for laboratories may be used for precise quantitation of the binding of ligands. It should comprise one or more micro-well plates and a ligand coating liquid or pre-coated micro-well plates; a concentrated solution of a standard glyco- compound or a series of solutions diluted to different known concentrations or a standard series pre-bound to catcher ligand in the tray; and labelled ligands. It may also comprise a high and a low control and, if appro ⁇ priate, enzyme substrate for a possible color reaction. Usually the kit will also comprise a set of directions for use.
  • Kits for enzymatic assays are, in principle, designed like the above kits for structural assay, only supplying the reaction mixture as an extra component.
  • a minimum kit for enzymatic assays is characterized by comprising a container which on at least part of its in ⁇ terior surface is coated with an acceptor substrate, a buffer solution containing a detergent for solubilizing the body fluid, and enzyme reagent buffer containing a donor substrate, a washing buffer and a labelled ligand.
  • the kit may comprise a sample-taking device according to the invention, the assay being conducted in the container of this device; or it may comprise titer tray micro-wells as explained above.
  • Benign colorectal diseases included colitis ulcerosa, whereas small adenomas were included in the group of normals.
  • Le +Nase Staining with antibody against Le following neuraminidase treatment.

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Abstract

On détecte et on quantifie des structures de saccharide modifiées sur des glycoprotéines, des glycolipides, des polysaccharides et des oligosaccharides par mise en contact du liquide biologique avec un premier ligand se liant à toutes les structures de saccharide, et avec un second ligand se liant soit à une partie terminale spécifiquement modifiée ds structures de saccharide soit uniquement à la partie terminale anormale, un desdits ligands étant immobilisé sur une phase solide, l'autre étant marqué. On détecte et on quantifie une enzyme de glycosyltransférase produisant de telles structures de saccharide dans un liquide biologique ou un homogénat cellulaire ou tisssulaire par l'emploi similaire d'une chaîne de sucre immobilisée constituant un substrat accepteur pour l'enzyme et un nucléotide-sucre constituant un substrat donneur pour ladite enzyme. Un dispositif de prélèvement d'un échantillon de sécrétion via un orifice du corps, de stockage et de traitement de l'échantillon comprend un récipient (14) ainsi qu'une tige (2) dont une partie terminale est dotée d'une matière absorbante (8), et dont l'autre partie terminale s'étend de manière coulissante dans un couvercle (12) dudit récipient (14). L'invention concerne également divers kits utilisés dans les analyses précitées.
PCT/DK1989/000265 1988-11-10 1989-11-10 Analyses structurelles et enzymatiques sur des glycocomposes, leur emploi dans le diagnostic du cancer, kits utilises dans les analyses et dispositif de prelevement d'un echantillon de secretion via un orifice du corps WO1990005304A1 (fr)

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DK626488A DK626488D0 (da) 1988-11-10 1988-11-10 Fremgangsmaade til udtagning af en sekretproeve via en legemsaabning og til praeparation af proeven med henblik paa kvantitativ bestemmelse af molekylaere strukturer og enzymaktivitet i proeven samt proeveudtagningsindretning til brug derved
DK6264/88 1988-11-10

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Cited By (10)

* Cited by examiner, † Cited by third party
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US5180674A (en) * 1990-04-16 1993-01-19 The Trustees Of The University Of Pennsylvania Saccharide compositions, methods and apparatus for their synthesis
US5403717A (en) * 1987-08-11 1995-04-04 Pacific Northwest Research Diagnosis of premalignant or malignant conditions of human secretory epithelia
EP0640835A3 (fr) * 1993-08-23 1996-04-03 Takara Shuzo Co Méthode pour la détermination de la structure d'une chaîne de sucre.
US5874261A (en) * 1988-09-02 1999-02-23 The Trustees Of The University Of Pennsylvania Method for the purification of glycosyltransferases
WO1999039209A1 (fr) * 1998-02-02 1999-08-05 Biogenes Gmbh Dosage immunologique et trousse d'essai pour la determination d'une proteine fucosylee dans un echantillon biologique
US6132982A (en) * 1995-09-22 2000-10-17 Novo Nordisk A/S Oligosaccharide amino alditols and assay method
US6518051B1 (en) 1991-04-11 2003-02-11 The Trustees Of The University Of Pennsylvania Saccharide compositions, methods and apparatus for their synthesis
US6569649B2 (en) 1990-04-16 2003-05-27 The Trustees Of The University Of Pennsylvania Compositions and methods for saccharide synthesis
US6576429B1 (en) * 1999-10-26 2003-06-10 Alimenta Diagnostics Ab Apparatus for intestinal sampling and use thereof
WO2013043644A1 (fr) * 2011-09-21 2013-03-28 The University Of North Carolina At Chapel Hill Procédés utilisant des biomarqueurs de maladies hépatiques

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EP0033437A1 (fr) * 1980-02-02 1981-08-12 MERCK PATENT GmbH Procédé et réactif pour la détermination d'un composant d'un groupe constitué par des récepteurs à liaison spécifique et des substances qui peuvent être liées spécifiquement par ces récepteurs
EP0043359A2 (fr) * 1980-06-30 1982-01-06 Pharmacia Diagnostics Ab Détermination des groupes terminaux de saccharides dans des glycoprotéines
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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5403717A (en) * 1987-08-11 1995-04-04 Pacific Northwest Research Diagnosis of premalignant or malignant conditions of human secretory epithelia
US5874261A (en) * 1988-09-02 1999-02-23 The Trustees Of The University Of Pennsylvania Method for the purification of glycosyltransferases
US5288637A (en) * 1990-04-16 1994-02-22 The Trustees Of The University Of Pennsylvania Apparatus for the synthesis of saccharide compositions
US5180674A (en) * 1990-04-16 1993-01-19 The Trustees Of The University Of Pennsylvania Saccharide compositions, methods and apparatus for their synthesis
US6569649B2 (en) 1990-04-16 2003-05-27 The Trustees Of The University Of Pennsylvania Compositions and methods for saccharide synthesis
US6544778B2 (en) 1990-04-16 2003-04-08 The Trustees Of The University Of Pennsylvania Apparatus for glycosyltransferase-catalyzed saccharide synthesis
US6331418B1 (en) 1990-04-16 2001-12-18 Neose Technologies, Inc. Saccharide compositions, methods and apparatus for their synthesis
US6518051B1 (en) 1991-04-11 2003-02-11 The Trustees Of The University Of Pennsylvania Saccharide compositions, methods and apparatus for their synthesis
EP0640835A3 (fr) * 1993-08-23 1996-04-03 Takara Shuzo Co Méthode pour la détermination de la structure d'une chaîne de sucre.
US6132982A (en) * 1995-09-22 2000-10-17 Novo Nordisk A/S Oligosaccharide amino alditols and assay method
WO1999039209A1 (fr) * 1998-02-02 1999-08-05 Biogenes Gmbh Dosage immunologique et trousse d'essai pour la determination d'une proteine fucosylee dans un echantillon biologique
US6576429B1 (en) * 1999-10-26 2003-06-10 Alimenta Diagnostics Ab Apparatus for intestinal sampling and use thereof
WO2013043644A1 (fr) * 2011-09-21 2013-03-28 The University Of North Carolina At Chapel Hill Procédés utilisant des biomarqueurs de maladies hépatiques

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