Patent Application: US-201113178017-A

Abstract:
the present invention provides the method for prevention and treatment of specific localized intestinal infections and diseases using iga and igm antibodies obtained from the eggs of hens which have been hyperimmunized to the same specific infections and diseases . the invention describes the high functionality of iga and igm antibodies present in the white of an egg from a hyperimmunized chicken as compared to the igy from the same egg . the invention also describes the resistance of iga and igm to low ph environments such as stomach acids . the invention also describes the inhibition of bacterial growth when bacteria are exposed to iga antibodies specific to said bacteria together with lysozyme from egg whites .

Description:
the present invention relates to a new alternative approach , as opposed to antibiotics , for the prevention and treatment of the causes of localized intestinal infection due to bacteria , viruses , protozoa , toxin or fungus . more precisely , the invention relates to iga and igm antibodies from the egg whites of domestic chickens . the iga and igm antibodies may be concentrated , but it is unnecessary to separate the antibodies from the egg white , so that processing and administration are convenient and inexpensive . as used herein , the term “ total antibody functionality ” refers to the ability of a certain amount of polyclonal chicken egg antibody , of a specific class , to bind to a given antigen . as used herein , the term “ antibody prevention ” refers to a process by which antibodies ( or together with lysozyme ) obstruct , delay or destroy intestinal infection or disease causing agents or their products and said antibodies are present in the locale of the intestine prior to the arrival of the infection or disease causing agents . as used herein , the term “ antibody treatment ” refers to a process by which antibodies ( or together with lysozyme ) obstruct , delay or destroy intestinal infection or disease causing agents or their products which are not currently attached to the intestinal mucosa , and said antibodies arrive in the locale of the intestine after the infection or disease causing agents . iga and igm , for the purpose of prevention and treatment of the causes of localized intestinal infection in human and non - human mammals are obtained from eggs of domestic chickens which have been actively immunized and boosted by direct injection or other means against said one or more pathogenic organisms or noxious agents . this invention , i . e . the use of iga and igm for the prevention and treatment of the causes of localized intestinal infection in human and non - human mammals , is not obvious for several reasons explained herein . the invention disclosed herein , includes the discovery that iga and igm are obtained in sufficient functional quantities from egg whites to be useful for the purpose of antibody prevention and antibody treatment of the causes of localized intestinal infection or disease . the inventiveness of the method is explained . 1 . the total antibody functionality of igm and iga together , from an egg white , is the same or greater than the total antibody functionality of igy in the egg yolk of the same , said egg . discussion in the background has illustrated that the total antibody functionality of eggs , specifically in egg yolks , is deemed to be enough for use on a commercial scale in preventing and treating the causes of localized intestinal infection or disease . this said , the amount of iga and igm in eggs from immunized hens are of sufficient functionality and quantity to be of commercial scale use through oral ingestion to prevent and treat the causes of localized intestinal infection or disease . 2 . iga and igm are resistant to mammalian stomach acid , unlike egg yolk antibodies as discussed in the background . this said , iga and igm from immunized hens are not degraded by stomach acid , therefore the egg white antibodies , intact , functional , unprotected by buffering or enteric coating will reach the locale of the intestine after oral ingestion to prevent and treat the causes of localized intestinal infection and disease . 3 . iga from an immunized hen &# 39 ; s egg in conjunction with lysozyme naturally found in egg white will inhibit the growth of bacteria to which the iga antibody is specific through said immunization . this said , iga contained in egg white in conjunction with lysozyme also found in the same egg white are a natural bactericide . the techniques for immunization of hens against selected antigens or immunogens are well known to those practiced in the art . the preferred method of immunization is through intramuscular injection of the desired antigen in conjunction with a suitable adjuvant such as freund &# 39 ; s complete or incomplete adjuvant . booster injections are usually given within a few weeks of the initial immunization . titers of the desired antibody in the immunized hen &# 39 ; s eggs can be confirmed by immunological tests , such as direct elisa , which are well known to those practiced in the art . eggs from immunized hens may be pooled and the antibody extracted by methods explained herein or the egg may be separated and the egg white used in its natural form or dried through spray drying or other means . the following examples are given for the purpose of illustration only and are not intended to limit the scope of the present invention . each of the following three examples uses eggs from chickens immunized using inactivated e . coli 0011 . immunization through injection with adjuvant was performed on 60 hens using techniques well known to those skilled in the art of immunization . a booster injection was given after approximately 30 days and eggs were collected after approximately 45 days . igy was obtained from 57 egg yolks and purified through ammonium sulphate precipitation , dialysis and ion exchange . iga and igm were obtained from the whites of the same 57 eggs using the method described in u . s . patent application ser . no . 13 / 177 , 114 filed jul . 6 , 2011 . both iga and igm were purified through precipitation and ion exchange . the resulting igy , iga and igm titers were in the range of 1 : 250 , 000 to 1 : 500 , 000 as examined by direct elisa . agglutination tests to show functionality of igy , iga and igm antibody binding normalized for in - ovo volume proportions agglutination titer tests are a measure of the functionality of a particular antibody , not just a quantification of how much antibody is present in the sample . functionality requires the antibody to bind to the sample antigen in a useful manner causing matrix formation . agglutination tests were carried out according to the methods set out by a . a . benedict in methods in immunology and immunochemistry vol . 1 , p . 229 . a two - fold dilution series was performed on each antibody beginning with a 100 μl sample of each of the three antibody types . samples of suspensions of 10 7 e . coli 2592 and e . coli 0011 bacteria per treatment well were tested independently . agglutination titers were read for each e . coli type and each of the three antibodies for all 57 eggs in the study . volumes of antibody in the whole egg were normalized by multiplying the agglutination titers by the volume of each of the igy , iga and igm containing fractions in in - ovo proportions for each individual egg studied . fig1 shows the results of the study of the agglutination units per egg study . the results are presented as a percentage of total , in - ovo , antibody binding functionality possessed by each of the three egg antibody components against each antigen , i . e . igy , iga and igm against e . coli 2592 and e . coli 0011 . using e . coli 2592 antigen the volume normalized antibody functionality measured is igy : 18 %, iga : 69 % and igm : 13 %. using e . coli 0011 antigen the antibody functionality measured is igy : 31 %, iga : 47 % and igm : 22 %. these results show that the antibody functionality present in in - ovo egg white ( i . e . iga and igm only ) is greater than the antibody functionality present in in - ovo egg yolk ( i . e . igy only ). fig1 . is a histogram and bar graph showing agglutination units measured for igm , igy and iga antibodies to two e . coli antigens expressed as a percentage of total agglutination activity in a single egg . agglutination to e . coli 2592 shows 13 %, 22 %, 69 %, e . coli 0011 shows 22 %, 31 %, 47 % for igm , igy , iga respectively . functionality of iga and igm antibodies after exposure to low ph environments iga and igm antibody agglutination titers to e . coli 0011 were tested at time 0 and after 1 hour of exposure to a ph 2 . 0 environment . the ph of the antibody solution was lowered using 0 . 01m hcl and raised to neutral ph using bicarbonate solution after the prescribed time period . agglutination tests were carried out according to the methods set out by a . a . benedict in methods in immunology and immunochemistry vol . 1 , p . 229 . a two - fold dilution series was performed on each antibody sample beginning with a 100 μl sample of each of the antibodies . a sample suspension of 10 7 e . coli 0011 bacteria was used per treatment well . agglutination titers were read for the two antibodies for all 57 eggs in the study . iga at time 0 had a titer of 1 : 200 , after 1 hour the titer was 1 : 275 igm at time 0 had a titer of 1 : 8 , after 1 hour the titer was 1 : 4 all agglutination titers from these two - fold dilution series have no more than a single dilution step difference in titer indicating that there is no significant difference in titer from time 0 to time 1 hour . these results demonstrate the resistance of both iga and igm to low ph environments . demonstration of the ability of the combination of iga antibodies and lysozyme to inhibit bacterial growth 10 6 e . coli 0011 bacteria were added to 10 ml nutrient broth for each treatment combination . 1 mg / ml samples of each antibody ( igy , iga ) were used for treatment . a 1 mg / ml quantity of dialyzed , lyophilized lysozyme from chicken egg white obtained from sigma - aldrich was used in each appropriate treatment . treatment combinations ( all treatments in nutrient broth ): 1 . 10 6 e . coli 0011 2 . lysozyme + 10 6 e . coli 0011 3 . iga + 10 6 e . coli 0011 4 . igy + 10 6 e . coli 0011 5 . iga + lysozyme + 10 6 e . coli 0011 6 . igy + lysozyme + 10 6 e . coli 0011 e . coli were allowed to enter the log phase for one study and allowed to enter the stationary phase for the second study . counts of colony forming units ( cfu ) were taken for each treatment combination at time 0 and after 3 hours incubation at 37 ° c . for the log phase study and for the stationary phase study . in addition , e . coli samples from the iga + lysozyme treatments were dispersed and re - suspended in new nutrient broth after the initial study in order to test whether the treatment actually affected the nutrient broth and not the bacteria themselves . a second , independent , experiment measured turbidity , indicating bacteria growth . the treatments consisted of untreated 10 5 e . coli 0011 per ml in nutrient broth as the control and 10 5 e . coli 0011 per ml + 1 mg / ml iga + 1 mg / ml lysozyme in nutrient broth for each of the 57 eggs in the study . turbidity was measured in a time step of 15 minutes beginning in the lag phase and through log and stationary phases of growth . fig2 shows the percent survivors during the log and stationary phases of bacteria growth for each of the treatments . during the log phase of growth the iga + lysozyme treatment significantly reduced the number of surviving cfu (& lt ; 20 % surviving ) after incubation as compared to time 0 . all other treatments did not show significant reductions in survivors during the log phase . in the stationary phase the iga + lysozyme treatment had the greatest reduction in surviving cfu ( 70 % surviving ). re - suspension of e . coli samples from the iga + lysozyme treatments in new nutrient broth showed no further increase in colony forming units . fig3 shows the turbidity measured at 650 nm at a time step of 15 minutes . at approximately time step 38 the e . coli samples treated with iga and lysozyme showed markedly less turbidity than the control samples of untreated e . coli . fig2 . is bar graph and histogram showing the survivorship of e . coli colonies in two sets of six treatment groups expressed as a percentage of the untreated e . coli group . the first set of measurements is for treatments beginning in log phase , the second set is for treatments beginning in stationary phase . log phase shows 100 %, 76 %, 80 %, 84 %, 18 %, 97 %, stationary phase shows 100 %, 87 %, 104 %, 107 %, 72 %, 93 % for untreated , lysozyme , iga , igy , iga + lysozyme , igy + lysozyme treatment groups respectively . fig3 . is a line graph showing growth of treated and untreated e . coli , as measured by absorbance at 650 nm in 15 minute time steps from 0 to 86 steps . treated and untreated e . coli growth is equal in both groups from step 0 and absorbance of 0 . 048 until step 34 and absorbance of 0 . 480 . after step 35 the untreated group continues growth to 1 . 178 absorbance . after step 35 the treated group continues to 0 . 691 nm absorbance . results , shown in all studies in example 3 , demonstrate the ability of the combination of iga and lysozyme to inhibit growth of bacteria , especially during the log phase of growth . benedict , a . a . 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