Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/34—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood group antigens
• • 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
  • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/975—Kit

Definitions

• the present invention relates to a method and kit for typing feline blood.
• the present invention relates to a kit having a mixture of two monoclonal antibodies, which recognize feline specific A antigens on the feline erythrocytes.
• feline blood group antigens is important to determine so as to eliminate adverse reactions that can occur after blood transfusions.
• A, B, and AB type A predominates in the more common Domestic shorthair/Domestic longhair (DSH/DLH) cats.
• Blood type B is much more likely to occur in certain breeds, particularly the Persian, Abyssinian, Birman, and related modern breeds, such as the Himalayan and British Shorthair.
• the AB blood type is by far the least common of the three and is characterized by the presence of both A and B antigens on the erythrocyte surface.
• blood type may be hypothesized with a fairly high degree of accuracy based on inherited characteristics, an individual's blood type can only be hypothesized if the blood type of the parents is known. Further, a statistical estimate can only be made if the parents are of different blood types. It is essential to type a subject's blood in order to determine the blood type. This is the only method to accurately determine the blood type.
• feline anti-A antiserum harvested from type B cats is used as the typing reagent for detection of blood type A.
• a population of type B cats must be housed for a long time period.
• the cats must be periodically bled and the serum ' must be separated to use as the typing reagent.
• This process is problematic because the antibody titer, or concentration of antibody varies from one cat to another, introducing a variable that must be monitored between different batches of typing sera. Resultantly, these procedures can be costly.
• the periodic bleeding of cats to harvest serum for blood typing purposes can be criticized or even prohibited by animal rights' groups and governmental agencies in many parts of the world.
• the present invention relates to a method and kit for determining feline blood type.
• the present invention also relates to at least one isolated feline antibody, which binds to one or more receptors on an antigen that characterizes feline type A blood.
• Both the kit and method include the use of two different monoclonal antibodies, which recognize feline blood group specific A- antigens.
• One of the antibodies will recognize glycolipid A antigen (NeuGc) 2 Go 3 .
• the other antibody will recognize a second glycolipid A antigen other than (NeuGc) 2 Go 3 .
• Any monoclonal antibody may be used, as long as feline blood type A can be accurately recognized and the erythrocytes are agglutinated.
• the invention is used to determine whether a blood sample contains feline glycolipid A antigens.
• the kit is formed by selecting a substrate member, such as a card, which facilitates contact between a mixture of the first and second monoclonal antibody with a blood sample.
• any system or device that allows the antibody mixture to be contacted with a blood sample may be used.
• Alternative testing systems include the use of a test tube.
• an agent which agglutinates with blood type B may be included in the kit.
• the antibodies can be used in any of a variety of concentrations, as long as the blood visibly agglutinates. It is also preferred that the antibodies be mixed with other constituents, which inhibit the degradation of proteins. It is further preferred if the moisture in the antibody mixture is eliminated so as to preserve the proteins. One way to preserve the proteins is to lyophilize the mixture.
• the method includes collecting a blood sample from a feline subject and contacting an amount of the sample with the antibody mixture.
• the sample may also be contacted with an agent which agglutinates blood type B.
• the antibody and sample mixture is then observed to see if the blood agglutinates with the anti-A antibody, and, optionally, the anti-B agent.
• Any antibody can be used with the present invention, as long as it has a receptor for binding with feline type A blood.
• a preferred monoclonal antibody will have a receptor site for glycolipid A antigen (NeuGc) G D3 .
• a preferred second monoclonal antibody will have a receptor site for glycolipid A antigen that is similar to (NeuGc)G ⁇ 3 .
• the present invention is advantageous because it provides for a quick and simple method for testing feline blood type. Additionally, the present method and kit alleviate the need to house a population of cats from which serum is harvested for use in blood typing. The present invention is also advantageous because it is more accurate than traditional testing methods. It is also more accurate than relying on known inheritance characteristics. Importantly, the present invention uses two separate monoclonal antibodies for determining blood type A.
• the present invention relates to a method for typing feline blood samples and a kit associated therewith.
• the method and kit use a mixture of first and second monoclonal antibodies, each of which recognizes feline blood group specific A antigens.
• the kit and method can be used to accurately type feline blood.
• the present invention also relates to at least two monoclonal antibodies which can be used to identify feline blood type A.
• the antibodies included in the kit recognize feline A antigen (NeuGc) 2 G D and at least one other feline A antigen.
• the kit is made by first forming monoclonal antibodies which recognize feline blood group specific A antigens. It is preferred to form a hybridoma cell line that produces the monoclonal antibodies.
• the monoclonal antibodies can be formed, however, according to any other procedure, which will result in the production of the desired antibodies.
• the preferred method for forming the monoclonal antibodies and the hybridoma cell line is specifically disclosed in Example 1.
• the preferred method starts with isolating blood from a type A cat.
• the erythrocytes are then separated from the serum followed by extracting the lipids from the erythrocyte membranes. Using thin layer chromotography it is possible to isolate the glycolipids from the remainder of the lipids.
• Liposomes which incorporate the isolated erythrocyte membrane glycolipids, are next prepared. Mice or other host-organisms are then given an intraperitoneal injection of the liposome suspension. This will facilitate production of IgM antibodies. It is hypothesized, however, that other serum proteins having the same feline antigen A receptor site could be used. Lymphocytes are then harvested from the mice and fused with mouse myeloma cells. The resultant hybridoma culture cells are then screened for antibodies that recognize the feline specific A antigens. These cells, which produce the desired antibody, are further grown to expand the population.
• two hybridoma cell lines which produce two different monoclonal antibodies for detecting feline specific A antigens.
• one antibody recognizes glycolipid A antigen (NeuGc) G D3 , the major glycolipid antigen of type A feline blood.
• the other antibody recognizes a second glycolipid antigen, which may resemble (NeuGc)G ⁇ 3 .
• the antibodies are specifically different because they have different antigen receptors. The inclusion of two different antibodies is important because the antibodies do not always recognize blood type A when used alone.
• blood type AB has at least one A antigen, which can be either (NeuGc) 2 GD 3 , (NeuGc)G ⁇ 3 , or an A antigen similar to (NeuGc)G ⁇ 3 .
• the glycolipid detected by one of the antibodies is likely (NeuGc)G ⁇ 3 , or another (NeuGc) containing ganglioside. Such gangliosides are found on type A red blooe cells. If two different antibodies directed at two different antigens are used correct identification of blood type AB is more likely to occur. It is necessary to use two different antibodies because blood type AB cats are heterogeneous. In order to accurately type such cats, two different antibodies that recognize a distinct A antigen are required. The preferred monoclonal antibodies produced by the hybridoma cell lines are labeled
• 13G3 and 4E10 are murine IgM antibodies.
• 4E10 corresponds to the antibody, which recognizes A antigen (NeuGc) 2 Go .
• the 13G3 antibody recognizes the A antigen, which corresponds to the A antigen (NeuGc)G ⁇ 3 or a related antigen.
• These two antibodies can be used in combination to type feline blood. Any of a variety of different substrate kits or testing procedures can be used so long as these two antibodies or homologous antibodies thereof can be used to type the blood sample.
• Antibodies having receptor sites homologous to the glycolipid A antigen receptor sites discussed above can be used in the alternative to the antibodies labeled 13G3 and 4E10. Regardless, the antibody must accurately type feline blood group A samples.
• the two antibodies could be used in any application that currently allows for the use of antibodies for typing blood. Additionally, the two antibodies can be used in future applications, including diagnostic use, research use, and therapeutic use. Such applications include, but are not limited to, red cell agglutination, microsphere agglutination, enzyme linked immunoassay, fluorescent antibody applications, immunoprecipitation, agar gel irnmunodiffusion. immunohistochemical applications, antibody-based antigen purification from tissues or fluids, and others. The antibodies could also have as-yet realized therapeutic applications.
• the antibodies can be used alone or can be included as part of a kit.
• a kit To prepare the kit it is preferred to obtain a card member having at least two wells. More preferably, the card member will contain enough wells for two controls and a sample to be tested. The additional wells are preferred to insure the accuracy of the results.
• Any of a variety of commercially available cards may be used so long as a monoclonal antibody mix can be readily placed on the card.
• An example of a preferred card is a PathoDx ® card, made by Diagnostic Products Corp., Los Angeles, California.
• the above-discussed monoclonal antibodies can be optionally mixed into solution.
• the solution is preferably comprised of 0.02M phosphate-buffered saline with 2% bovine serum albumin (BSA).
• BSA bovine serum albumin
• Stabilicoat ® may be added at this stage, or can later be contacted with the mixture, as discussed below.
• the Stabilicoat ® is added to the antibody mixture to prevent degradation.
• Other solutions instead of Stabilicoat ® may be used, as long as degradation of the antibodies is inhibited.
• different amounts of PBS and BSA may be used.
• the 13G3 monoclonal antibody is added to solution in an amount ranging
• 13G3 antibody is added in an amount equal to about 68 ⁇ g/ml.
• the 4E10 is added to the
• the 4E10 is added in an amount equal to about 128 ⁇ g/ml. Other amounts can be used;
• the antibody mixture which is optionally in solution, is preferably mixed with an equal volume of plasma Stabilicoat ® .
• the antibody mixture in solution with the Stabilicoat ® is then added to at least one well and more preferably three of the wells on the card member.
• the antibody and Stabilicoat ® mixture is spread out over an entire defined area with a paintbrush.
• an anti-B reagent such as Triticum vulgaris, is added.
• Other anti-B reagents can be used in the alternative. Both the anti-B and anti-A reagents, after the antibodies have been mixed in solution with the Stabilicoat ® , are preferably added in an anti-B reagent, such as Triticum vulgaris, is added.
• Triticum vulgaris Triticum vulgaris
• the 100 ⁇ l amount is selected because it is an
• the cards are preferably then frozen at -20° C for one hour followed by lyophilizing over night at -10° C.
• Alternative methods that allow for long-term storage of a protein mixture may be used.
• the resultant cards can be stored at room temperature, but, they have an extended shelf life of greater than one year if they are stored at 4° C.
• test tube or similar member that can house an antibody mixture may be used, for example. Any substrate device or system can be used that facilitates contact between the antibody mixture and the blood sample. Typically, the antibody mixture will be used in the same amount, regardless of the substrate.
• a blood sample to be tested is taken from a feline subject.
• the blood sample can be comprised only of erythrocytes, or can contain plasma, serum, and other blood constituents.
• An anticoagulent such as ethylene diaminetetracetic acid (EDTA), is added thereto. If a
• lyophilized card is used, 50 ⁇ l of PBS is added to each well to reconstitute the solution. Between 50 ⁇ l and 100 ⁇ l of the cat EDTA whole blood sample is added to an anti-A well and a
• the present kit and method result in accurate identification of blood type in cats 99.9% of the time. Of course, correct procedures must be followed in order to insure accuracy. Also, alternatives to the card kit may be used. For instance, a test tube could be used instead of the card kit. Any type of kit variation may be used so long as the above listed antibodies are included.
• Feline blood samples were obtained from submissions to the Clinical Pathology Laboratory at Kansas State University and from a commercial veterinary laboratory (Colorado Veterinary Laboratories, Broomfield, CO). Blood used for screening of hybridomas and preparation of erythrocyte membranes was obtained from blood-typed A, B, and AB cats and six sheep maintained at the Kansas State University Animal Resources Facility. Sheep erythrocytes were used as a source of known membrane glycolipid profiles. Blood from the cats had been typed by a commercial laboratory (Stormont Laboratories, Woodland, CA) and by blood-typing cards in the laboratory. Samples were obtained by jugular venepuncture and submitted in EDTA anti-coagulant.
• Each blood sample was typed by tube agglutination or by using blood-typing cardboard substrate cards (PathoDx ® cards, Diagnostic Products Corp., Los Angeles, CA). For typing on
• Hemoglobin-free erythrocyte membrane ghosts were prepared within 24 hours of collection by hypotonic lysis (Dodge et al. 1962). If stored overnight, EDTA-anticoagulated
• Liposomes incorporating the isolated erythrocyte membrane glycolipids were prepared
• dipalmitoyl phosphatidylchoiine 50 ⁇ mol/ml C:M 1 : 1), 20 ⁇ l Salmonella minnesota
• lipopolysacchari.de (5 mg/ml C:M 1:1), and 20 ⁇ l of the isolated erythrocyte membrane
• lipids were completely dry. Lipids were resuspended in 5 ml of sterile PBS, heated at 50° C in a
• mice Female Balb/c mice were given a single intraperitoneal injection of 0.5 ml of liposome suspension in order to facilitate production of IgM antibodies for use in agglutination assays (Watarai et al. 1987).
• mice were euthanized 3 days after liposome immunization by inhalant anesthesia followed by cervical dislocation. Splenic lymphocytes were harvested, washed, and fused with AG8U.1 mouse myeloma cells (ATCC, Rockville, MD) using 50% polyethylene glycol.
• Hybridomas were selected in 96-well plates containing Dulbecco's MEM supplemented with 12.5% heat-inactivated fetal calf serum L-glutamine, MEM non-essential amino acids, MEM vitamin solution, and gentamicin sulphate, with added hypoxanthine, aminopterin, and
• Hybridoma culture supernatants were then screened for antibodies to feline type A or B erythrocytes. Erythrocytes from a blood type A cat or a blood type B cat were added to the 100
• Hybridomas exhibiting specific agglutination of type A or B feline erythrocytes were cloned by limiting dilution and expanded in non-selective media.
• Monoclonal antibody was produced in quantity by intraperitoneal injection of selected cloned hybridomas producing the desired antibody into pristane-primed Balb/c mice (Hoogenraad et al. 1983).
• MoAb Monoclonal antibodies
• each strip was incubated for 1 hour with isotype specific reagents, either rabbit anti-mouse IgGl or rabbit anti-mouse IgM (1:15 dilution in PBS-BSA-Tween 20), followed by a 1-hour incubation with goat anti-rabbit IgG horseradish peroxidase conjugate (1 :4000 dilution in PBS-Tween 20).
• substrate solution consisting of 400 ⁇ g/ml OPD in 80 mmol/I citrate-phosphate
• Monoclonal antibodies were produced which could be tested for antigen specificity.
• Example 1 The monoclonal antibodies of Example 1 were tested against a number of feline blood
• MoAbs 13G3 and 4G2 each agglutinated the same 7 of 10 type AB samples.
• MoAbs 23G5 and 4E10 each agglutinated the same 4 of 10 type AB samples, which constituted a subset of the seven samples detected by MoAbs 13G3 and 4G2.
• the remaining three type AB samples were not agglutinated by any of the anti-A MoAbs, although they had agglutination scores of 3-4+ with feline blood-typing reagents.
• Each anti-A MoAb was also tested by rube agglutination with sheep erythrocytes.
• MoAbs 13G3 and 4G2 showed 2-3+ agglutination of erythrocytes from all sheep; MoAbs 4E10 and 23G5 did not agglutinate any sheep blood samples (data not shown). Erythrocyte membranes from one of these sheep were used for subsequent TLC immunostaining.
• the anti-B MoAbs, 9D10 and 17G7 each detected 54 of 55 blood type B samples and 3 of 1428 type A samples.
• the one type B sample not detected by these MoAbs was different for each antibody.
• MoAb 17G7 strongly agglutinated the type B blood sample which did not agglutinate with 9D10, whereas MoAb 9D10 showed 1+ agglutination with the type B sample not detected by 17G7.
• Two of the three type A samples erroneously detected were the same for both anti-B MoAbs.
• Example 3 The following example was performed to show antigen specificity of the monoclonal antibodies of the present invention.
• ganglioside standard (NeuAc) 2 Go 3 was prepared by reconstitution in 1 ml C:M 1:1, and 6-8 ⁇ l was applied per lane. Plates were developed in C:M:NH 4 OH solvent (60:40:9 by volume) as
• the binding specificity of the anti-A MoAbs was evaluated by TLC immunostaining of erythrocyte membrane glycolipids from a type A cat, a type B cat or a commercial (NeuAc) 2 G ⁇ 3 standard, and a sheep, in comparison with human MoAb 32-27 (anti-(NeuGc) 2 G D3 ).
• MoAbs 23 G5 and 4E10 each stained a common band in type A samples which comigrated with (NeuGc) 2 Go 3 .
• MoAbs 13G3 and 4G2 also detected an identical single band in type A glycolipids which migrated slightly behind (NeuGc) 2 Go 3 .
• MoAb 32-27 strongly stained three bands in the type A sample, the largest of which comigrated with (NeuGc) Go 3 and another of which comigrated with the band detected by MoAbs 13G3 and 4G2.
• the bands detected in the sheep glycolipids by MoAb 32-27 were different from those detected in the type A sample. None of the four anti-A MoAbs detected bands from either type B glycolipids or a commercial (NeuAc) 2 GD 3 standard. Similarly, MoAb 32-27 did not react with type B glycolipids.
• TLC immunostaining of erythrocyte membrane glycolipids from 38 additional type A cats was done with MoAbs 4E10 and 13G3. In all 38 cats, 4E10 detected a band.
• the TLC immunostaining characterization of the anti-B MoAbs was done with erythrocyte membrane glycolipids from the same blood type A and B cats. Both MoAbs 9D10 and 17G7 recognized the same band in blood type B glycolipids which comigrated with the commercial (NeuAc) 2 Go 3 standard as visualized by orcinol staining.
• the murine MoAb R-24 (anti-(NeuAc) 2 Go 3 ) detected a band at the same position in type B samples. Neither anti-B MoAb nor MoAb R-24 detected bands from type A glycolipids.
• Erythrocyte membrane proteins were separated on polyacrylamide slab gels (7.5%>) containing SDS Sodium dodecyl Sulphate Polyacrylaminde Gel Electrophoresis (SDS- PAGE)/Immunoblotting) using a discontinuous buffer system. Biotinylated high molecular- weight standards were used for reference. Separated proteins were transferred electrophoretically to a PVDF membrane, which was then washed for 20 minutes with PBS.
• biotinylated goat anti-mouse IgG or IgM (1 :200 dilution in PBS-BSA-Tween 20). Blots were washed three times in PBS-Tween 20 and incubated for 1 hour with ABC reagent. After final washes in PBS-Tween 20 and PBS, blots were developed with 4-CN-DAB (4-chloronaphthol- diaminobenzidine (Pierce Chemical Co., Rockford, IL)) substrate according to the manufacturer's directions.
• 4-CN-DAB 4-chloronaphthol- diaminobenzidine
• the following test was conducted to determine the correct amount of each antibody to be used with the test.
• a card agglutination test was performed. Whole blood was used. The specific antibodies and concentrations tested are listed in Table 2, below. To test agglutination, the antibodies were diluted in PBS with 2% of BSA to the desired dilution listed below. The
• Example 5 The procedures of Example 5 were performed using different amounts of individual and combined antibodies.
• Table 4 shows that the 13G3 antibody can be significantly diluted before agglutination is
• a feline blood typing card was prepared as follows:
• PBS Phosphate buffered saline
• bovine serum albumin 0.02 M Phosphate buffered saline
• the concentration of murine IGM monoclonal antibodies 13G3 and 4E10 were,
• Stabilizer (SurModics, Inc. 9924 West 74 th Street, Eden Prairie, MN 55344-3523) was added to
• the blood typing card had eight wells. 100 ⁇ l of the
• monoclonal antibody mixture was placed inside each of four wells and spread over the wells with a #5 paintbrush.
• the "Anti-B reagent” was a lectin from Triticum vulgaris (wheat germ), with 1%> DMSO, and 0.0036g/ml glucose.
• the lectin (6mg) was dissolved in 100 ml of phosphate
• the card was frozen at -20° C for one hour, and then lyophilized at -10° C.
• the card was frozen at -20° C for one hour, and then lyophilized at -10° C.
• Example 7 The procedures of Example 7 were followed with the test cards of the present Example.
• the test cards had an amount of antibody equal to:
• the 4E10 antibody alone, did not result in agglutination with the AB blood type.
• the mixture of 4E10 and 13G3 did result in clear identification of the AB blood type.
• Blood typing cards using the antibodies of Example 1 and the procedure of Example 7 were tested to evaluate for accuracy in comparison to the standard methods described above. Again, a mixture of the two monoclonal antibodies were used to detect blood type A. The typing cards correctly identified 225 of 225 type A samples, did not detect any type B samples (0 of 16 tested), and identified 5 of 5 type AB samples.
• Irwin CC Irwin LN (1979) A simple rapid method for ganglioside isolation from small amounts of tissue.

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