the present invention utilizes an active urease antigen such as jack bean or bacterial urease , which is chemically combined with glutaraldehyde . the method for treatment utilizing the material composition of the present invention involves innoculation with the material composition . in test animals , it has been found that the material composition of the present invention is parenterally non - toxic in quantities representing a dosage increase factor of 100 , 000 over that known in the prior art with unmodified purified natural urease . the period of immunization and the effectiveness of the immunization with glutaraldehyde modified enzyme favorably influences the course in liver disease . moreover , it has been found that the material composition of the present invention offers a long shelf life in lyophilized powder form at room temperature . thus , the material is available for treatment of patients without the need for expensive storage systems . the best mode and preferred embodiment of the present invention is set forth in the following detailed description thereof , taken in conjunction with the accompanying examples . according to the method of the present invention , an active urease , such as crystalline jack bean urease , is diluted and a solution of glutaraldehyde is prepared . the urease solution and the glutaraldehyde solution are then mixed , and a reaction ensues . in the reaction , urease molecules are covalently bonded to glutaraldehyde molecules , producing a molecule with a molecular weight in the range from 250 , 000 to 2 , 000 , 000 . when the reaction has been completed , the excess glutaraldehyde is neutralized and the resulting solution is subjected to lyophilization after being dialized . the resulting powder may be stored at room temperature over an extended period . the method of treatment of the present invention comprises preparation of an injectable dosage by dissolving an appropriate quantity of the powder material in a sterile non - toxic solution , and injecting the dosage into the subcutaneous tissue or muscular tissue of the patient . the material itself is non - toxic and has been shown by vitro tests and animal tests to be totally non - enzymatic , therefore avoiding the ammonia intoxication characterizing the prior art . thus , dosages can be increased by a factor of 100 , 000 over those known in the prior art . subsequent testing of the animals showed that injections of active urease were neutralized , as were the effects of ureaseproducing bacteria injected into various bacteria . the following examples are offered to further explain and exemplify the present invention : while bacterial urease can be used , the readily available jack bean urease has proven satisfactory . commercially available crystaline jack bean urease is dissolved in water or saline solution to give a concentration equivalent to 50 sumner units per milliliter . sumner units are an expression of the activity of the urease and are determined as described below in example 2 . to the solution containing 50 sumner units of urease , glutaraldehyde is added to give a final concentration of 0 . 01 % ( w / v ) glutaraldehyde . the reaction mixture is kept at 37 degrees centigrade for a period not greater than 48 hours . the interval of 48 hours has been previously determined by testing the enzymatic activity of the reaction mixture during the course of the reaction . at this time , approximately 95 % of the urease is found to be inactivated . the reaction is allowed to proceed until all of the urease activity has been neutralized in the mixture . this may require an additional three to six hours . the reaction is then stopped by the addition of sodium bisulfite to neutralize unreacted glutaraldehyde or simply by dialyzing the reaction mixtue . following dialysis , the material is lyophilized and the powder put aside for storage and antibody testing . no enzymatic activity is present in the lyophilized powder which is suspended in saline solution prior to immunization . a substrate is prepared from 11 / 2 grams of urea and 25 milligrams of crystaline bovine serum albumin in 50 milliliters in 0 . 75 phosphate buffered to ph 7 . a known sample of urease enzyme with 0 . 02 molar po 4 buffer at ph 7 to 1 . 0 milliliter . the procedure utilizing the above reagents is carried out immediately as follows : with reagents at room temperature ( approximately 20 degrees centigrade ) two drops of bromcresal green - methyl green kjeldahl indicator are added to one milliliter of substrate and one milliliter of 0 . 02 molar buffer and the enzyme solution titrated with 0 . 1 normal hcl to first purple endpoint . the milliliter of 0 . 1 normal hydrochloric acid needed to reach the endpoint is recorded as ( a ). to assay an unknown concentration of enzyme placed on one milliliter of substrate , 2 drops of indicator is added to one milliliter diluted enzyme at 0 time which is allowed to stand for exactly five minutes at room temperature . ( 20 degrees centigrade ). after 5 minutes , 0 . 1 normal hcl is added to reach the original endpoint and the titration is continued beyond to the first purple color . the total milliliters of the 0 . 1 normal hcl needed to reach the endpoint ( b ) are recorded . a calculation is carried out using the formula ( b - a × 0 . 10 × 14 = the numbers of sumner units of urease per milliliter in the solution to be assayed . rabbits and mice were injected with the lyophilized material from example 1 and were bled at the end of one week and after a second injection at the end of two weeks for determination of antienzyme in the circulating blood . titres of greater than 100 , 000 were usually achieved with a tanned , red cell agglutination test . the serum was tested against unmodified , purified urease . all showed good antibody arcs on an ouchterlony plate when an unmodified crystalline urease enzyme was placed in the trough . this test indicates that the modified urease antigen is capable of producing antibodies in the immunized animal which will neutralize purified , unmodified urease . the serum of the animals when added to urease in solution and mixed with urea inhibits ammonia formation . the lethal dose urease which killed 99 % of the animals ( ld99 ) was 60 to 70 sumner units per kilogram of body weight in nonimmunized rats . however , the ld99 for urease immunized rats was more than 10 times greater . more than 750 sumner units per kilogram weight was required . additionally , humans were immunized with 10 milligrams of the inactive complex in weekly injections over a period of two weeks or three weeks . good enzymes titres were found in patients at the end of fourteen days . isotope studies injecting c 14 labeled urea into immunized humans disclosed that internal urea turnover ( in the large bowel ) was completely prevented . all of the labelled urea was excretion in the urine in contrast to normal humans who cleave some of the radioactive compound and expire c 14 labelled co 2 . no untoward systematic response was encountered and there was no undue inflammatory response at the injection site . purified , crystalized urease is a protein of the globulin type . the molecular weight of the native material is approcimately four hundred eighty thousand with an isoelectric point at a ph of 5 . 0 to 5 . 1 . the glutaraldehyde modified urease has an average molecular weight of approximately two million . while general embodiments of the present invention has been described , it will be apparent to those of ordinary skill in the arts that various alternative configurations and embodiments can readily be adapted to the present invention and are considered to fall within the scope thereof as set forth in the following claims .
the present invention relates to a non - enzymatic and non - toxic urease antigen and a method for its production . administration of the urease antigen in living organisms serves to immunize the same from certain types of ammonia production without causing ammonia toxicity when injected , thereby promoting several advantageous biological activities including the promotion of wound healing , and general immunity from ammonia toxicity and the related effects thereof , and the cessation of toxic ammonia production in infections caused by urea splitting organisms .