DK153521B - Process for the preparation of protein concentrates containing immunological factors from milk - Google Patents

Process for the preparation of protein concentrates containing immunological factors from milk Download PDF

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DK153521B
DK153521B DK131078A DK131078A DK153521B DK 153521 B DK153521 B DK 153521B DK 131078 A DK131078 A DK 131078A DK 131078 A DK131078 A DK 131078A DK 153521 B DK153521 B DK 153521B
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milk
ml
characterized
ig
whey
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DK131078A
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DK131078A (en
DK153521C (en
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Helmut Hilpert
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Nestle Sa
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/04Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from milk
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23JPROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/20Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey
    • A23J1/205Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey from whey, e.g. lactalbumine
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; THEIR TREATMENT, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES
    • A23V2002/00Food compositions, function of food ingredients or processes for food or foodstuffs

Description

DK 153521 B DK 153 521 B

Den foreliggende opfindelse angår en fremgangsmåde til fremstilling af proteinkoncentrater indeholdende immunologiske faktorer fra mælk, specielt aktive ikke-denaturerede immunoglobuliner. The present invention relates to a method for the production of protein concentrates containing immunological factors from milk, in particular active non-denatured immunoglobulins.

Man har allerede foreslået at indføre immunologiske faktorer stammende fra mælk i diætetiske produkter til nyfødte og ammebørn ved i mælken at inkorporere disse faktorer, idet den orale indtagelse af disse produkter vil muliggøre transporten i ammebarnets blod af disse faktorer uden samtidig at se de ledsagende omstændigheder eller følgerne af en sådan generel passiv immunisering. It has already been proposed to introduce immunological factors derived from milk in dietetic products for new born and breast-feeding children by the milk incorporating these factors, the oral intake of these products will enable the transport of breastfeeding the baby's blood of these factors without also see the accompanying circumstances, or the consequences of such a general passive immunization.

I fremlæggelsesskriftet DE 1 810 438 foreslås med henblik på immunisering af kalve at isolere immunlactoglobulinerne fra mælken fra vaccinerede køer ved koagulation af mælk, ved udvindelse af lac- In the presentation document DE 1810438 is proposed for the purpose of immunization of calves to isolate immunlactoglobulinerne from the milk of vaccinated cows by coagulating the milk, recovering the lac

2 DK 153521 B 2 DK B 153 521

toserum og selektiv udfældning af lactoglobulinerne med f.eks. toserum and selective precipitation of lactoglobulinerne, for example. ammoniumsulfat efterfulgt af dialyse overfor rent vand, filtrering og tørring. ammonium sulfate followed by dialysis against pure water, filtration and drying. Undersøgelse af serobeskyttelse hos mus i forbindelse hermed har vist, at parenteral injektion af disse immuniserende principper frembringer en almindelig passiv beskyttelse overfor visse enteropathogene bakterier og vira, dersom mikroorganismerne indføres i dyrene på samme måde. Examination of seroprotection in mice in this connection have shown that parenteral injection of such immunizing principles produces an ordinary passive protection against certain bacteria and viruses enteropathogene if the microorganisms are introduced into the animals in the same manner.

Endvidere kan nævnes DK paténtskrift 87.610, hvori angives en fremstillingsmetode til beskyttelsesstoffer mod antigener. In addition, there may be mentioned paténtskrift DK 87610 which sets a manufacturing method for protecting fabrics against antigens. Man indsprøjter udvalgte antigener i yveret på et kl'ovdyr i dyrets ikke-malkende periode og fortsætter med infusioner i den malkende periode. Injecting selected antigens in the udder of an animal's kl'ovdyr in the non-milking period and continues infusions in the milking period. Mælk eller colostrum indsamles, skummes og casein fjernes ved sur fældning. Milk or colostrum is collected, foamed and casein removed by acid precipitation. Herefter udfældes γ-globulin med kold alkohol, ud-dispergeres i en vandig saltopløsning, genudfældes med alkohol og lyophiliseres. Then precipitated γ-globulin with cold alcohol, out-dispersed in an aqueous salt solution, re-precipitated with alcohol and lyophilized.

I US patent 3.911.108 beskrives lignende fremstillingsmetoder for beskyttelsesstoffer mod antigener. US Patent 3,911,108 discloses similar manufacturing methods for protecting fabrics against antigens. Disse beskyttelsesstoffer skal anvendes på smågrise. These protective substances to be used in piglets.

Ved fremgangsmåden benytter man et kemisk antibakteriemiddel, nemlig β-propiolakton, til sterilisering og fraskiller antistof ved fældning med ammoniumsulfat og påfølgende dialyse. In the process one uses a chemical anti-bacterial agent, namely, β-propiolactone, for sterilization and antibody is separated by precipitation with ammonium sulfate and subsequent dialysis.

Ingen af disse fremgangsmåder er særlig egnet til industriel udnyttelse. None of these approaches is particularly suitable for industrial application.

Man har nu fundet en fremgangsmåde til i industriel målestok at fremstille proteinkoncentrater indeholdende immunoglobuliner uden denaturering af disse ved hjælp af teknologiske processer. It has now been found a process for industrial scale to manufacture protein concentrates containing immunoglobulins without denaturing them by means of technological processes. Dette produkt frembringer en lokal passiv immunisering i tarmen uden resorption og uden bemærkelsesværdig ødelæggelse af aktiviteten i fordøjelseskanalen. This product produces a local passive immunization in the intestine without resorption and without any remarkable deterioration of the activity of the digestive tract.

Fremgangsmåden ifølge opfindelsen er ejendommelig ved, at man a) opsamler fra mælkeproducerende drøvtyggere, især køer, der er hyper immuniserede ved vaccination, idet der successivt administreres antigener paren-teralt og lokalt ca. The process according to the invention is characterized in that a) collecting from milk-producing ruminants, especially cows that are hyper-immunized by vaccination, taking successively antigens administered parenterally and locally ca. 8 til 1-2 uger før kælvningen og peroralt i de ca. 8 to 1-2 weeks before calving and orally in the approximately 2 uger før kælvningen og, eventuelt, en serie successive administreringer af antigen fra ca. 2 weeks prior to calving, and, optionally, a series of successive administrations of antigen at about 2\ måned før det antagne ophør af mælkesekretionen med skift mellem parenteral, lokal og peroral administrering. 2 \ month before the successful suppression of milk with toggle parenteral, local or oral administration. Ovennævnte mælk består af colostrum fra 1 og 2 timer efter kælvningen, overgangsmælk fra 3-8 dage efter kælvningen og, eventuelt, mælk fra de sidste 60 dage af lacta- 3 The above-mentioned milk consists of colostrum of 1 and 2 hours after calving, transitional milk from 3-8 days after calving and, optionally, milk from the last 60 days of lactams 3

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tionsperioden; tion period; b) fraskiller fløden og urenhederne; b) separating the cream and the impurities; c) koagulerer den klarede og skummede mælk; c) coagulating the clarified and skimmed milk; d) fraskiller caseinet, filtrerer./ ultrafiltrerer og steriliserer proteinet fra vallen ved filtrering.; d) separating the casein, filtrerer./ ultrafiltering and sterilizing the protein from the whey by filtration .; : og e) inddamper og tørrer produktet under sådanne betingelser, at man ikke denaturerer immunoglobulinerne og bevarer steriliteten. : And e) evaporating and drying the product under such conditions that do not denature the immunoglobulins and maintains sterility.

Den medfølgende tegning er en skematisk gengivelse af fremgangsmådens forskellige trin. The accompanying drawing is a schematic representation of the process different stages.

Det foretrækkes at behandle en blanding af ovennævnte mælkearter for at få en forhøjet antistofmængde under perioden så længe som muligt. It is preferred to treat a mixture of the above species milk to get an elevated antibody amount during the period as long as possible.

Hyperimmuniseringen af koen udføres ved en kombineret vaccination. Hyperimmuniseringen of the cow is performed by a combined vaccination. F.eks. Eg. ved intracisternal indførsel i .brystkirtlen, ved parenteral injektion (subcutan, intravenøs), ved injektion i det retromammære lymfesystem, ved skarifikation, ved peroral indgift eller ved en kombination af flere af metoderne. by introduction into the .brystkirtlen intracisternal, by parenteral injection (subcutaneous, intravenous), by injection into the lymphatic system retromammære, by scarification, by oral administration, or by a combination of several of the methods.

Det foretrækkes at anvende en kombination af metoderne, idet man følger et omhyggeligt lagt immuniseringsskema for at opnå en tilfredsstillende antistoftiter i hver type anvendt mælk. It is preferred to use a combination of the methods, following a carefully laid immunization schedule in order to achieve a satisfactory antibody titer in each type of milk used. Således er vaccinationen for colostrum og overgangsmælken parenteral og intracisternal i 8-2 uger førend kælvningen og peroral i ugen førend kælvningen. Thus, the vaccination of colostrum and milk release parenteral and intracisternal in 8-2 weeks before calving and oral during the week prior to calving.

Til mælken fra slutningen af lactationen anvender man et vaccinationsskift mellem parenteral, lokal og peroral indgift fra 2.1/2 måned før det antagne ophør af mælkesekretionen. For the milk from the end of the lactate ion used is a toggle parenteral vaccination, local and oral administration of 2.1 / 2 months before the successful suppression of milk.

Den anvendte vaccine er fordelagtig en udvalgt blanding af antigener og et adjuvang på basis af homolog blodserum fra immuniserede køer, idet det således tillader en detoksificering af vaccinen og dannelsen af immunkomplekser. The vaccine used is advantageously a selected mixture of antigens, and a adjuvang on the basis of homologous blood serum of immunized cows, thus allowing a detoxification of the vaccine and the formation of immune complexes.

Koen tjener som produktionsdyr, de i proteinkoncentratet indeholdte Ig-forbindelser er principielt IgG-forbindelser (specielt IgG·^) forskellige fra modermælken, som fremherskende indeholder IgA som Ig-forbindelsen. The cow serves as productive livestock, the protein concentrate contained in the Ig compounds are in principle IgG compounds (particularly IgG · ^) different from human milk, which contains the predominant IgA-like Ig compound.

Indholdet af Ig i proteinkoncentratet, af hvilket proteinerne udgør 70 til 80 vægt-%, er 25 til 35 vægt-%. The content of Ig in the protein concentrate from which the proteins constitute 70 to 80% by weight, is 25 to 35% by weight. Visse fordele er knyttet til den kendsgerning, at Ig-forbindelserne ikke ved processens i- Certain advantages associated with the fact that the compounds Ig does not know the process i-

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4 gangsætning er adskilt fra de øvrige proteiner i lactoserum, som således ligeledes er til stede i det opnåede koncentrat. 4 once phrase is separated from the other proteins in lactoserum, which therefore is also present in the resulting concentrate. Man undgår således de selektive udfældningstrin, f.eks. Thus, to avoid the selective precipitation step, for example. med ammoniumsulfatet og dialysen. with ammonium sulfate and dialysis.

Yderligere genfindes i proteinkoncentratet visse bakteriosta-tiske eller antivirusfaktorer, f.eks. Further the protein concentrate is recovered in certain bacteriostatic synthetic or antiviral factors, for example. lactoferrinet, eller de enzymatiske systemer, som f.eks. lactoferrin, or the enzymatic systems, such as. lactoperoxidase, ribonuclease, som er til stede i lactoserummet. lactoperoxidase, ribonuclease, which is present in lactoserummet.

Alle slags antigener af virus eller bakterieoprindelse kan anvendes, og de opnåede antistoffer er afhængige af de antigeners natur, det er administreret til koen. Any kind of antigens of viral or bacterial origin can be used, and the resulting antibodies are dependent on the nature of antigens, it is administered to the cow. Man kan f.eks. One can for example. opnå en beskyttelse mod enteropathogene bakterier og virus, der er ansvarlige for gastroenteriter med samtidig kraftig diarré med dehydrering, ved inkorporering af proteinkoncentrater indeholdende Ig-forbindel-serne i alt tilskud anvendt til peroral administrering. obtaining a enteropathogene protection against bacteria and viruses, which are responsible for gastroenteriter with concomitant severe diarrhea with dehydration, by incorporation of protein concentrates containing compounds Ig-conditions a total of grants used for oral administration. Dette tilskud kan være et vilkårligt hjælpestof eller fortrinsvis et diætetisk produkt som mælk eller mælkepulver til småbørn, en såkaldt"hu-maniseretf' mælk eller tilpasset ammebørn, mælk specielt tilpasset en særlig gruppe nyfødte, som f.eks. mælk til for tidligt fødte børn eller børn med ringe fødselsvægt osv. This grant can be any excipient or preferably a dietary product such as milk or milk powder for infants, called "hu-maniseretf 'milk or adapted breastfeed children, milk specially adapted to a particular group of newborns, such as. Milk for premature babies or children with low birth weight, etc.

Dersom man vil anvende f.eks. If you want to use for example. et koncentrat indeholdende Ig-anti E. coli i profylaktisk dosis med mælk som tilskud, inkorporere] man fra 0,8 til 3 g af koncentratet i 100 g af det tørre materiale og indtil 6 g, når det drejer sig om en terapeutisk dosis. a concentrate containing Ig-anti E. coli in prophylactic dose with milk grant, incorporate] to 0.8 to 3 g of the concentrate in 100 g of the dry material and up to 6 g, in the case of a therapeutic dose.

For at udføre fremgangsmåden er det nødvendigt at sørge for, at afskumningstrinnene og klaringen foregår meget omhyggeligt for at undgå en efterfølgende tilstopning af filtrene og ultrafiltrene. To perform the procedure, it is necessary to make sure that skimming steps and clarification takes place very carefully to avoid a subsequent clogging of filters and ultrafilters.

Afskumningen udføres fortrinsvis i to etaper, først under køling for at fjerne størstedelen af fedtstofferne og urenhederne (f. eks. blod, der ofte findes i colostrum) og til sidst under opvarmninc for fuldstændig at fraskille disse. Defoaming is preferably carried out in two stages, first under cooling to remove the bulk of the fats and the impurities (f. Eg. Blood, often found in colostrum) and finally during opvarmninc for completely separating these. Koagulationen kan udføres ved caseiriets isoelektriske pH i nærværelse af osteløbe med tilsætning af syre, f.eks. The coagulation may be effected by caseiriets isoelectric pH in the presence of rennet with the addition of acid, for example. citronsyre. citric acid.

Det foretrækkes at udføre koaguleringen ved syretilsætning, f.eks. It is preferred to carry out the coagulation by addition of acid, for example. saltsyre, indtil en pH-værdi omkring 4,6 og opvarmning indtil 40°C. hydrochloric acid until a pH about 4.6 and heating up to 40 ° C.

Fraskillelsen af caseinet medfører en klaring af serummet, der muliggør, fraskillelse af udfældningerne. The separation of casein leads to a brightening serum that allows separation of the precipitates.

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5 .......................... : 5 ..........................:

Det er nødvendigt at udføre trinnene, der nødvendiggør en termisk behandling (inddampning og tørring) under styrede forhold for at undgå inaktivering af Ig-forbindeiserne. It is necessary to carry out the steps requiring a thermal treatment (evaporation and drying) under controlled conditions in order to avoid inactivation of the Ig forbindeiserne.

For at formindske tabene aflg, hvoraf en del fjernes under af-skumningen af fløden og en anden del med caseinet, når dette fraskilles, anbefales det at ekstrahere fløden og tykmælken med vand og behandle vaskevandet indeholdende Ig-forbindelserne for at genvinde dissei ; In order to reduce the losses aflg, part of which is removed in de-foaming of the cream and a second portion of the casein when this is separated off, it is recommended to extract the cream and the curds with water and treat the wash water containing the Ig compounds in order to recover dissei; I de følgende eksempler belyses opfindelsen nærmere, idet mængder og dele er angivet i vægtenheder, med mindre andet er anført. In the following examples illustrate the invention in more detail, with amounts and parts are in weight units unless otherwise stated.

Eksempel 1 Køer af forskellig race blev hyperimmuniseret efter nedenfor anførte skema med en vaccine, hvis fremstilling er anført nedenfor, og man opsamlede mælken de to sidste måneder af lactationsperioden og de otte første dage efter kælvningen. Example 1 cows of different races were hyperimmunized by below scheme with a vaccine, the preparation of which is given below, and to the milk collected the last two months of the lactation period and the first eight days after calving.

Fremstilling af vaccine: Preparation of vaccine:

Man anvendte følgende enteropathogene serumtyper af E. coli: 0 18 : K 76 (B 20) 0 111 : K 58 (B 4) 0 20 : K 17 (L) 0 112 : K 68 (B 11) 0 26 : K 60 (B 6) 0 119 : K 69 (B 14) 0 44 : K 74 (L) 0 124 : K 72 (B 17) 0 55 : K 59 (B 5) 0 125 : K 70 (B 15) 0 78 : K 80 (-) 0 126 : K 71 (B 16) 0 86 : K 61 (B 7) 0 127 : K 63 (B 8) 0 128 : K 68 (B 12) But using the following enteropathogene serotypes of E. coli: 0 18: K 76 (B 20) 0111: K 58 (B 4) 0 20: K 17 (L) 0112: K 68 (B 11) 0 26: K 60 (B 6) 0119: K 69 (B 14) 0 44: K 74 (L) 0124: K 72 (B 17) 0 55: K 59 (B 5) 0125: K 70 (B 15) 0 78 : K 80 (-) 0126: K 71 (B 16) 86 0: K 61 (B 7) 0127: K 63 (B 8) 0128: K 68 (B 12)

De forskellige stammer gav gastroenteriter hos hospitalsindlagte børn. The different tribes gave gastroenteriter in hospitalized children. Serotypningen udførtes med antisera, der var multi- og monovalente (Difco). Serotyping was performed with antisera multi- and monovalent (Difco). Bakterierne dyrkedes på flydende minimalsubstrat indeholdende 2% aminosyrer fra casein (casaminosyrer Difco) og 2% glucose i 24 timer ved +37°C uden omrystning af kolberne. The bacteria were cultured on liquid minimal medium containing 2% amino acids from casein (casamino acids, Difco) and 2% glucose for 24 hours at + 37 ° C without shaking the flasks. For at inaktivere bakterierne adskilte man kulturerne ved centrifugering i bundfældede celler og supernatant. In order to inactivate the bacteria to separate the cultures by centrifugation of the pelleted cells and supernatant. Cellerne genopslemmedes i suspension og inkuberedes ved 37°C i 24 timer i nærværelse af 0,5% formaldehyd, medens den supernatante væske inåktiveredes som angivet, men med 0,05% formaldehyd. The cells are reslurried in suspension and incubated at 37 ° C for 24 hours in the presence of 0.5% formaldehyde, whereas the supernatant liquid inåktiveredes as indicated, but with 0.05% formaldehyde. Efter en ny centrifugering og fjernelse af su-pernatanten anbragtes bakterierne i opslemning i den oprindeligt in-aktiverede supernatant. After another centrifugation and removal of supernatant were placed SU-bacteria in the slurry in the originally in-activated supernatant. Denne fremgangsmåde tillader at bevare den supernatante væske indeholdende bakterieexotoksiner til vaccinen. This method allows to preserve the supernatant liquid containing bakterieexotoksiner to the vaccine.

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6 6

U U

Man opnåede en vaccine med 1-2 x 10 bakterie/ml, og man fortyndede herefter blandingen med en opløsning af 2% Al(OH)g og homolog blodserum fra immuniserede køer. This gave a vaccine with 1-2 x 10 bacteria / ml and the mixture then was diluted with a solution of 2% Al (OH) g or homologous blood serum of immunized cows.

Fremgangsmåde ved immuniseringen. Procedure for the immunization.

A. Immuniseringsskema til opnåelse af colostrum og overgangsmælk indeholdende Ig-anti-E.coli-forbindelser: A. immunization for obtaining colostral and transition milk Ig containing anti-E.coli compounds:

Dage for forventet kælvning = X Days of calving = X

Behandlingstidspunkt Vaccine + evt. Treatment Time Vaccine below. .adjuvans Administreringsmåde 7 X - 8 uger 5 ml vaccine (5 x 10 Subcutan injektion bakterier/ml)^ + 5 ml serum 7 X - 7 " 5 ml vaccine (5 x 10 Intravenøs injektior bakterier/ml + 5 ml 2% Α1#(ΌΗ)3 + 5 ml serum 7 X - 6 " 10 ml vaccine (5 x 10 Subcutan injektion bakterier/ml 7 X - 5 1/2 uger 20 ml vaccine (5 x 10 Injektion i det re- bakterier/ml + tromammære lymfesy- 10 ml 2% Al(OH), stem ό 7 X - 5 uger 40 ml vaccine (5 x 10 Infusion på 4 x 15 bakterier/ml) ml i yveret med pat- + 20 ml serum tekanyler Q ££ X - 4 1/2 uger 24 ml vaccine (5 x 10 Subcutan injektion bakterier/ml) + 8 ml serum* 7 X - 4 uger 40 ml vaccine (5 x 10 Infusion på 4 x 15 bakterier/ml) ml i yveret med pat- + 20 ml serum tekanyler .adjuvans of administration X 7 - 8 week 5 ml of vaccine (5 x 10 subcutaneous injection bacteria / ml) ^ + 5 ml of serum-X 7 - 7 '5 ml of vaccine (5 x 10 Intravenous injektior bacteria / ml + 5 ml of 2% Α1 # (ΌΗ) 3 + 5 ml of serum-X 7 - 6 "10 ml of vaccine (5 x 10 subcutaneous injection bacteria / ml 7 x - 5 1/2 weeks 20 ml of vaccine (5 x 10 re-injection into the bacteria / ml + tromammære lymphatic 10 ml 2% Al (OH), Stem ό 7 X - 5 weeks 40 ml of vaccine (5 x 10 Infusion of 4 x 15 bacteria / ml) ml of the udder with mammalian serum + 20 ml tekanyler Q ££ X - 4 1/2 weeks 24 ml of vaccine (5 x 10 subcutaneous injection bacteria / ml) + 8 ml serum-7 * X - 4 weeks 40 ml of vaccine (5 x 10 Infusion of 4 x 15 bacteria / ml) ml of the udder with a teat + 20 ml of serum tekanyler

O ISLAND

X - 3 " 10 ml vaccine (5 x 10 Subcutan injektion bakterier/ml) 8 X - 2 uger 5 ml vaccine (5 x 10 Intravenøs injektion bakterier/ml),+ 5 ml 2% Al (OH) , X - 1 uge 1(50 ml xaccine·3 (1 x 10y bakterier/ml) Peroral indgift under drøvtygningen X - 1/2 uge 100 ml yaccine (1 x 10y bakterier/ml) Peroral indgift under drøvtygningen + Disse serumtilsætninger foretages kun, når man er nødt til det, eller en ko immuniseres for første gang. X - 3 "10 ml of vaccine (5 x 10 subcutaneous injection bacteria / ml) 8 X - 2 Weeks 5 ml of vaccine (5 x 10 Intravenous injection bacteria / ml) + 5 ml 2% Al (OH), X - 1 Week 1 (50 mL xaccine · 3 (1 x 10y bacteria / ml) Oral administration during rumination X - 1/2 week 100 ml yaccine (1 x 10y bacteria / ml) Oral administration during rumination + These serum additions are carried out only when one has to it, or a cow immunized for the first time.

++ Dénne subcutane injektion gentages i det lymphatiske system, som følger: DK 153521 7 2 x 4 ml i de pre-scapulære lymfesystemer 2 x 4 ml i depost-scapulære lymfesystemer 2 x 4 ml i lyskelymfesystemerne 2 x 4 ml i de poplitiske lymfesystemer. This ++ subcutaneous injection is repeated in the lymphatic system, as follows: DK 153,521 7 2 x 4 ml in the pre-scapular lymph systems 2 x 4 ml in depost-scapular lymph systems 2 x 4 mL lyskelymfesystemerne in 2 x 4 ml in the lymph systems poplitiske .

B. Immuniseringsskema til opnåelse af mælken fra lactationens slutning (to sidste måneder af lactationen) indeholdende Ig-anti- E.coli-forbindelser. B. immunization for obtaining the milk from lactationens end (last two months of the lactate ion) containing Ig anti-E.coli compounds.

Denne immunisering foretages kun på køer, der allerede er blevet immuniseret under den colostrale periode og overgangsperioden. This immunization shall be carried out only on the queues have already been immunized during the period colostral and transition period.

Antal dage for ophør af mælkesekretionen = X Behandlingstidspunkt Vaccine + øvt. Number of days for suppression of milk = X Treatment Time Vaccine + øvt. adjuvans. adjuvant. Administreringsmåde 7 X - 70 dage 5 ml vaccine (5 x 10 Intravenøs injektion bakterier/ml) + „ „ 5 ml 2% Al (OH)-, a X - 65 20 ml vaccine ^5 x 10 Injektion i det retro- bakterier/ml mammære lymfesystem X - 60 " 24 ml vaccine (5 x 10^ Subcutan injektion** bakterier/ml) (som under A) X - 55 " 100 ml vaccine (1 x 10^ Peroral indgift under bakterier/ml) drøvtygning 7 X - 50 " 40 ml vaccine (5 x 10 Infusion i yveret som Of administration X 7 - 70 days 5 ml of vaccine (5 x 10 Intravenous injection bacteria / ml) + "" 5 ml 2% Al (OH) -, X a - 65 20 ml of vaccine 5 x 10 ^ injection into the retro-bacteria / ml mammary lymphatic system X - 60 "24 mL of vaccine (5 x 10 ^ subcutaneous injection ** bacteria / ml) (as in A) X - 55" 100 ml of vaccine (1 x 10 ^ Oral administration the bacteria / ml) rumination 7 X - 50 "40 mL of vaccine (5 x 10 Infusion into the udder as

bakterier/ml + 20 ml i A bacteria / ml + 20 ml of A

serum 9 X - 40 lOO ml vaccine (1 x 10 Peroral indgift under bakterier/ml) drøvtygning Serum X 9 - 40 loo ml of vaccine (1 x 10 Oral administration the bacteria / ml) rumination

O ISLAND

X - 30 " 20 ml vaccine (5 x 10 Injektion i det retro- bakterier/ml) mammære lymfesystem 7 X ~ 25 " 40 ml vaccine (5 x 10 Infusion i yveret som X - 30 "20 mL of vaccine (5 x 10 injection into the retro-bacteria / ml) mammary lymphatic system ~ 7 X 25" 40 mL of vaccine (5 x 10 Infusion into the udder as

bakterier/ml) + 20 ml i A bacteria / ml) + 20 ml of A

„ serum g X - 10 100 ml vaccine (1 x 10 Indgift under drøvtyg- bakterier/ml) ningen. "Serum-X g - 10 100 ml of vaccine (1 x 10 Administration during ruminant bacteria / ml) scheme.

Eksempel 2 Mælk fra de otte første dage efter kælvningen opsamledes fra vaccinerede køer som beskrevet i eksempel 1 og behandledes på følgende måde: Example 2 Milk from the first eight days after calving was collected from vaccinated cows as described in Example 1 and treated in the following manner:

Afskumningen udførtes i to trin: trin A koldt og trin B varmt. Defoaming was carried out in two steps: Step A Step B cold and hot. A. 1100 kg mælk sattes koldt til en afskumningscentrifuge og ledtes til en tillukket beholder på 1500 liter, hvorefter den pumpedes i en slamcentrifuge ved forhøjede accelerationer (ca. 11000 g) for til sidst at fjerne blod og urenheder (snavs). A. 1100 kg cold milk was added to a skimming centrifuge and passed to a sealed container of 1500 liters, after which it was pumped to a sludge centrifuge at elevated accelerations (about 11000 g) and finally to remove blood and impurities (dirt).

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B. Den slamc entrif ugerede kolde mælk opbevaredes i en beholder på 2500 liter og pumpedes gennem en tempereret opvarmningsanordning på 40°C ud i afskumningscentrifugen, som allerede havde været anvendt forud, hvorved der fraskiltes 110 kg fløde, som man opsamlede, og 5 kg snavs, som man bortkastede. B. The slamc entrif ugerede cold milk was stored in a tank of 2500 liters and was pumped through a temperature-heating device at 40 ° C, is prepared in afskumningscentrifugen, which had already been used before, thereby separated 110 kg cream, which were collected, and 5 kg dirt, as one cast away. Som en variant kan man anvende en selvudtømmende slamafskummer til det kolde og varme afskumningstrin og en slamcentrifuge mellem operationerne og således nedskære afskumningens varighed. As a variant, one can use a selvudtømmende slamafskummer to the cold and hot skimming step and a sludge centrifuge between operations and thus reduce the duration of scum.

Den afskummede og afslammede varme mælk (985 kg) anbragtes herefter i fire firkantede koaguleringskar på hver 750 liter udstyret med et rør'eværk for at koagulere. The scum shaped and afslammede warm milk (985 kg) was then placed in four square koaguleringskar on each 750 liters equipped with a rør'eværk to clot.

Man tilsætter en opløsning af saltsyre på 1 N ved 37°C, indtil pH stabiliseres omkring 4,6, og man opretholder temperaturen i 20 minutter under omrøring. Adding a solution of hydrochloric acid of 1 N at 37 ° C until the pH stabilized around 4.6, and maintaining the temperature for 20 minutes under stirring. Derefter afkøler man til ca. Then is cooled to about 15°C. 15 ° C.

Det efterfølgende trin er adskillelsen af caseinet. The subsequent step is the separation of the casein. Den består af to centrifugeringer efter hinanden af vallen efter afskumning af mælk, koagulering og skæring. It consists of two spins succession of whey after skimming of milk coagulation and cutting. Den første centrifugering f rå skiller størstedelen af det udfældede casein ved hjælp af en klarings-autoslamcen-trifuge, idet der fraskilles 150 kg casein, og serummet går over i en tillukket beholder. The first centrifugation f crude separates the majority of the precipitated casein by means of a autoslamcen The clearing-centrifuge, while separating 150 kg of casein and the serum merges into a sealed container. Den anden centrifugering fjerner alle spor af partikler, som kan forstyrre den efterfølgende filtrering og ultrafiltrering, f.eks. The second centrifugation will remove all traces of particles which can interfere with subsequent filtration and ultra filtration, for example. ved hjælp af slamanordningen anvendt i operation A til afskumning. by the slurry used in the device operation A for defoaming. Man opnår 854,7 kg klaret serum, som pumpes over i en beholder på 2500 liter. To give 854.7 kg clarified serum, which is pumped into a tank of 2500 liters.

Herefter fortsætter man med filtrering efterfulgt af ultrafiltrering af serummet. Then continue with the filtration followed by ultrafiltration of the serum. Filtreringen skal være meget omhyggelig for at lette ultrafiltreringen, idet man fraskiller de meget fine partikler af casein. The filtration has to be very careful in order to facilitate the ultrafiltration, taking the separating very fine particles of casein. Man anvender "Seitz Supra" 100 filter i stykker på 20 x 20 cm. Using "Seitz Supra" filter 100 into pieces of 20 x 20 cm. Filtratet oplagres i et reservoir på 3000 liter og pumpes derefter til uitrafiltret. The filtrate is stored in a reservoir of 3000 liters and is then pumped to uitrafiltret. Ultrafiltreringen sker i to eller tre etaper med recyclisering af det tilbageholdte materiale (for nemheds skyld i det følgende kaldet retentatet) til reservoiret. The ultrafiltration is done in two or three stages with recyclisering of the retained material (for convenience, hereinafter referred to as the retentate) to the reservoir. To centrifugepumper i serie sikrer strømningen mod et indgangstryk i ultrafiltrets indgangsafdeling på ca. Two centrifugal pumps in series ensures the flow at an inlet pressure of ultra filter input area of ​​approximately 7 bar, idet udgangstrykket holdes på ca. 7 bar, the outlet pressure is maintained at about 4,5 bar. 4.5 bar. For at undgå genopvarmning af retentatet ved den energi, som kommer fra pumperne, afkøles dette til 10-12°C. In order to avoid re-heating of the retentate by the energy that comes from the pumps, this is cooled to 10-12 ° C.

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Det første trin eller selve ultrafiltreringen muliggør adskillelse af de opløste stoffer med høj molekylvægt, proteinerne, der er tilbageholdt på membranen, og som findes i retentatet, medens en del af lactosen, vitaminerne, mineralsaltene og vandet overføres til per- meatet. The first step or the ultrafiltration allows separation of the solutes with high molecular weight, the proteins retained on the membrane, and which is found in the retentate, while a portion of the lactose, the vitamins, the mineral salts and the water is transferred to the per- meatet. Dersom man anvender en model DDS med membran 800 (overflade-2 membran = 7 m ), vil forholdet mellem retentat/permeat være ca. If one uses a model with DDS 800 membrane (surface-membrane 2 = 7 m), the ratio of retentate / permeate will be about 1 til 3,1, idet man således opnår 580 kg permaet I og 274,7 kg re- 2 tentat med en strømningsmængde på 14,17 kg permeat/m time. 1 to 3.1, as one thus obtains 580 kg permaet I and 2 re- 274.7 kg retentate at a flow rate of 14.17 kg permeate / m hour.

Det andet trin erN en diafiltrering, i løbet af hvilken man kontinuert til retentatet sætter 825 kg vand, og man cirkulerer opløsningen under samme betingelser, som forefindes i det første trin. The second step is N diafiltration, during which it continuously to the retentate sets 825 kg water, and circulating the solution in the same conditions which are present in the first step. Diafiltreringen standses, når den elektriske ledningsevne af permea-tet opnår den ønskede værdi, idet forholdet mellem retentat/tilføjet vand vil være ca. The diafiltration is stopped when the electrical conductivity of the permeation-tet achieves the desired value, the ratio of retentate / water added would be approximately 1 til 3. På denne måde fremstiller man 825 kg per- meat II og 274,7 kg retentat med en middelstrømning på 11,77 kg per-2 meat/m time. 1 to 3. In this manner one prepares 825 kg permeate II and 274.7 kg of retentate with an average flow of 11.77 kg per meat-2 / m hour. Det tredje trin er en koncentrering, hvor man, dersom det ønskes, recirkulerer retentatet på membranen indtil et indhold af tørstof på ca. The third step is a concentration, where, if desired, recirculating the retentate on the membrane until a dry matter content of about 10%, idet man fjerner 82,5 kg permeat III. 10%, by removing 82.5 kg permeate III. Man opnår således 192,2 kg prekoncentreret opløsning. This gives 192.2 kg prekoncentreret solution. Som alternativ kan man undlade dette tredje trin, og gå direkte til sterilfiltrering. Alternatively, you can omit this third step and go directly to sterile filtration.

Sterilfiltreringen udgør et vigtigt trin i hele fremgangsmåden, fordi varmebehandlinger ikke er anvendelige til sterilisering, da · de vil føre til denaturering af Ig-forbindelserne. Sterile filtration is an important step in the entire process, because the heat treatment is not useful for sterilization since they · will lead to denaturation of the Ig compounds. Sterilfiltreringen fjerner mikroorganismer fra retentatet, som endnu måtte findes dér (størstedelen af disse er allerede fjernet under fraskil-lelsen af fløden, caseinet og urenhederne). Sterile filtration removes microorganisms from the retentate, which still had to be found there (the majority of these have already removed under Separate-feeling of the cream, the casein and impurities). For at undgå tilstopning af sterilfiltrene er en fjernelse af urenheder ved centrifugering og en tredje filtrering af retentatet nødvendig. In order to avoid clogging of the sterile filters is the removal of impurities by centrifugation, and a third filtration of the retentate necessary. Efter fjernelse af u-renheder opbevarer retentatet i reservoiret, og ledes ad en filtreringslinie, der omfatter en prefiltrering på tre filtre "Seitz Supra" 100,ΕΚ, EKS anbragt i række, derefter sterilfiltrering på filtrene "Seitz Supra" 20 og "Millipore" HA 0,45μ anbragt i række og fortrinsvis steriliseret med overopvarmet vand. After removal of the U-purities keeping the retentate in the reservoir, and passed through a filter line, comprising a prefiltration of three filters "Seitz Supra" 100, ΕΚ plus arranged in series, then sterile filtration on filters "Seitz Supra" 20 and "Millipore" HA 0,45μ arranged in series and preferably sterilized with superheated water. Det steriliserede retentat opbevares i et sterilt reservoir på 500 liter. The sterilized retentate stored in a sterile reservoir of 500 liters.

Alle de øvrige operationer sker under sterile forhold. All other operations are conducted under sterile conditions. Ved By

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10 hjælp af komprimeret nitrogen og sterilt filter kan man f.eks. 10 using the compressed nitrogen and the sterile filter can, for example. overføre til den efterfølgende inddampning. transfer to the subsequent evaporation.

Inddampningen udføres med en filminddamper (med en strøm, der 2 falder over en overflade på 1 m ) ved en opvarmningstemperatur på 75°C, idet produktets temperatur vil være på ca. The evaporation is performed with a wiped-film evaporator (with a current which 2 drops of a surface of 1 m) at a heating temperature of 75 ° C, the temperature of the product will be approximately 30°C indtil et indhold af tørstof på mindst 20%. 30 ° C until a dry matter content of at least 20%. Por at undgå enhver infektion udføres overførelsen med en peristaltisk pumpe fra retentatreservoiret til koncentratreservoiret, hvilket er forbundet i en ring omfattende inddamperen, idet koncentreringen foregår i et lukket kredsløb. Por to avoid any infection is carried out with the transfer of a peristaltic pump from retentatreservoiret to koncentratreservoiret, which are connected in a ring including the evaporator, the concentration takes place in a closed circuit. Denne operation påvirker ikke aktiviteten af Ig-forbindelserne. This operation does not affect the activity of the compounds Ig.

Man fortsætter herefter med tørring af koncentratet (96 kg) ved almindelige metoder, f.eks. One then proceeds with the drying of the concentrate (96 kg) by conventional methods, for example. ved frysning efterfulgt af lyofilise-ring. by freezing followed by lyophilization. Frysningen kan udføres på plader på -40°C. The freezing can be carried out on plates at -40 ° C. Ig.forbindelserne kan opbevares ved -30°C uden at tabe aktiviteten i ca. Ig.forbindelserne can be stored at -30 ° C without losing activity of the ca. 45 dage. 45 days. Produktet ved -30°C lyofiliseres på plader i en ovn ved 0,5 torr ved en kondensationstemperatur på -50°C og en opvarmningstemperatur på 30-35°C. The product at -30 ° C lyophilized on plates in an oven at 0.5 Torr at a condensation temperature of -50 ° C and a heating temperature of 30-35 ° C. Tørringsmetoden påvirker ikke aktiviteten af Ig-forbindelserne. The drying method does not affect the activity of the compounds Ig. Man opnår på denne måde 19,1 kg af et tørt produkt indeholdende 25-35% Ig-forbindelser, som man konditionerer sterilt. Are obtained in this manner 19.1 kg of a dry product containing 25-35% Ig compounds that can condition the sterile.

Eksempel 3 example 3

Man går frem som i eksempel 1, idet man behandler mælken fra de første otte dage efter kælvningen og mælken, der er opsamlet i løbet af de sidste fire uger af lactationsperioden, hvorved man opnår et proteinkoncentrat med gennemsnitssammensætningen: The procedure is as in Example 1 by treating the milk from the first eight days after calving and the milk collected during the last four weeks of lactation period to yield a protein concentrate with average composition:

Proteiner 75-5% 40 -5 % immunogluboliner 15-5 % α-lactalbuminer 35-5 3-lactoglobuliner 5 -2 % serumalbuminer 5 -2 % andre mindre proteiner rest fugtighed 4-0,5% lactose 10-2 % mineralsalte 5-2 % nitrogenforbindelser, + ikke-proteiner. Proteins 75-5% 40 -5% immunoglobulins 15-5% α-lactalbumins 35-5 3-lactoglubulins 5% serum albumins -2 -2 5% of other minor proteins residual moisture from 4 to 0.5% lactose 10-2 5% of mineral salts -2% nitrogen compounds, + non-proteins. 5-2 % 11 5-2 11%

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Eksempel 4 example 4

Man går frem således som beskrevet i eksempel 2, men med den forskel, at fløden og caseinet ekstraheres med vand for at genudvinde en del af de tabte Ig-forbindelser, idet vaskevandet recirkuleres på fabrikationslinien. The procedure is as described in Example 2, but with the difference that the cream and the casein is extracted with water to recover the part of the lost compounds Ig, since the washing water is recirculated at the manufacturing line.

Den kolde afskumning (etape A) udføres med en linie parallel med vandekstraktionen af proteinfraktionen, der indeholder Ig-forbindelserne fjernet med fløden. The cold defoaming (Stage A) is carried out with a line parallel to the water extraction of the protein fraction containing the Ig compounds removed with the cream. Den fløde, der fremkommer ved den første centrifugering (115 kg), opbevares i et reservoir på 1000 liter med omrøring og blandes med 150 kg lunkent vand ved 40°C, hvorefter det hele blandes i 30 minutter og centrifugeres i en mejericentrifuge. The cream that is obtained by the first centrifugation (115 kg), stored in a reservoir of 1000 liters with stirring and mixed with 150 kg of lukewarm water at 40 ° C, after which the whole is mixed for 30 minutes and centrifuged in a milk centrifuge.

Man fraskiller således 115 kg fløde og 150 kg opløsning, som forenes med den skummede og klarede mælk. It separates thus 115 kg cream and 150 kg of solution, which is combined with foamed milk and clarified. På denne måde opnår man 130 kg væske, som føres til varmskumning (etape B) og koagulering. In this way one obtains 130 kg liquid which is fed to the hot foaming (Stage B), and coagulation.

Fraskillelsen af caseinet giver ud fra 1145 kg skummetmælk, der er tilsat løbe og syrnet, 991 kg serum og 154 kg casein. The separation of the casein provides from 1145 kg of skimmed milk was added and acidified run, 991 kg and 154 kg serum casein. En parallellinie tillader at ekstrahere proteinfraktionen indeholdende Ig-forbindelserne, der er koaguleret i vand. A parallel line allows to extract the protein fraction containing the Ig compounds that are coagulated in water. Koagulatet opbevares ved udgangen af klarings-autoslamcentrifugen i et reservoir udstyret med en omrører og omrøres med 300 kg lunkent vand ved 40°C i 30 minutter, hvorefter man leder det til en kolloidmølle. The coagulate is stored at the end of The clearing autoslamcentrifugen in a reservoir equipped with a stirrer and stirred with 300 kg of lukewarm water at 40 ° C for 30 minutes, then passing it into a colloid mill. Suspensionen af det formalede koagel adskilles herefter i en klarings-autoslam-centrifuge. The suspension of the milled curd is separated then in a auto The clearing sludge centrifuge. Man udvinder 154 kg vasket casein og 300 kg vaskevæske, som man slår sammen med serummet fra de øvrige operationer. Are recovered 154 kg washed casein and 300 kg wash bandied together with the serum from the other operations. 1291 kg serum filtreres og ultrafiltreres, hvilket giver 2010 kg permeat (I, II og III) og 216 kg prekoncentrat. 1291 kg serum is filtered and ultra-filtered to obtain 2010 kg of permeate (I, II and III) and 216 kg prekoncentrat. Inddampning giver 108 kg koncentrat, som giver 21,6 kg tørt produkt ved tørring. Evaporation yielded 108 kg of concentrate, which give 21.6 kg of dry product by drying. Man får således ca. One gets so about 14% Ig-forbindelser mere end ved fremgangsmåden fra eksempel 2. 14% Ig compounds over the method of Example 2.

Eksempel 5 1 kg pulver fremstillet som i eksempel 2 eller 4, henholdsvis 2 kg pulver fremstillet som i eksempel 3, blandes med 100 kg mælkepulver på ensartet måde, og denne konditioneres sterilt til at udgøre et produkt, der indeholder én profylaktisk dosis ig til en næringsmiddeldosis svarende til 140 cal/kg/dag svarende til 250 mg koncentrat Ig/kg/dag fremstillet som i eksemplerne 2 eller 4 henholdsvis til 500 mg koncentrat Ig/kg/dag fremstillet som i eksempel 3. Example 5 1 kg powder prepared as in Example 2 or 4, respectively 2 kg of powder prepared as in Example 3 were mixed with 100 kg of milk powder in a uniform manner, and this conditioned sterile to make up a product, containing a prophylactic dose in grams to a nutritional dose equal to 140 cal / kg / day, equivalent to 250 mg concentrate in g / kg / day prepared as in examples 2 or 4, respectively, concentrate to 500 mg Ig / kg / day prepared as in example 3.

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12 ' * 12 '*

Eksempel 6 example 6

Forskellige forsøg in vitro og in vivo er blevet udført med proteinkoncentratet fremstillet som i eksempel 2 eller 4 og i det efterfølgende kaldt "koncentrat Ig" for at vise: at Ig-mælkeforbindelserne har en antistofspecifitet, som man normalt finder hos de menneskelige Ig-forbindelser, og som bevares under fremstillingsprocessen, at de specifikke Ig-mælkeforbindelser udviser en beskyttende aktivitet, idet de interfererer i de pathogene mekanismer fra entero-pathogene mikroorganismer. Various tests in vitro and in vivo have been performed with the protein concentrate prepared as in Example 2 or 4 and hereinafter referred to as "concentrate Ig" to display: the Ig-lactic compounds have a antistofspecifitet, as normally found in the human Ig compounds and which is maintained during the manufacturing process, that the specific Ig-lactic compounds exhibits a protective activity, in that they interfere in the pathogenic mechanisms of entero-pathogenic microorganisms.

Passiv hæmagglutination. Passive hemagglutination.

Røde blodlegemer fra får sensibiliseredes med en urinstofekstrakt af specifikke serotype E. coli. Sheep red blood cells were sensitized with a urea extract of E. coli serotype specific. Dette ekstrakt fremstilledes som beskrevet af Brodhage (Brodhage H., (1961) J. Hyg., 1961, 148, 94). This extract was prepared as described by Hage Brod (Brod Chin H., (1961) J. Hyg., 1961, 148, 94). Bakterierne dyrkedes på næringsagar ("Difco") ved 37°C i 30 timer i en Roux-kolbe. The bacteria were grown on nutrient agar ( "Difco") at 37 ° C for 30 hours in a Roux flask. Man høstede kulturen med 10 ml af en saltvandsopløsning buf ret med phosphat (8,8 g NaCl + 1,86 g · 2H20 + 0,43 g KH2P04/1000 ml H20) indtil pH 7,2. One culture harvested with 10 ml of a saline solution with phosphate buf right (8.8 g of NaCl + 1.86 g · + 2h20 KH2P04 0.43 g / 1000 ml H20) up to pH 7.2. Efter tilsætning af 10 g urinstof ("Merk”) henstod opslemningen i 90 timer ved 37°C under let omrøring. Efter centrifugering dialyseredes supernatanten fuldstændig over for en saltvandsopløsning bufret med phosphat til pH 7,2, fortyndedes til et slutrumfang på 50 ml med en saltvandsopløsning bufret med phosphat til pH 7,2, hvorefter man nedfrøs i små portioner ved -20°C. Sensibilisering af røde blodlegemer fra får med dette antigen udførtes ved en kombination af metoderne beskrevet af Avrameas et coll. (Avrameas S., Taudou B., Chuilon S., Immunochemistry, 1969, j5, 67) og Otto et coll. (Otto H., Takamiya H., Vogt A., J. Immul. Methods, 1973, 3^ 1937). Fårets røde blodlegemer var forudbehandlet med glutaraldehyd (slutkoncentrationen af røde blodlegemer fra får 5% og 0,5% glutaraldehyd), hvorefter der vaskedes og opslemmedes med 20% af en saltvandsopløsning bufret med phosphat til pH 7,2, og blanding af det samme ekstraktvolumen med urinstof. Efter 16 timers inku-beri After adding 10 g of urea ( "Merk") was allowed to stand, the slurry for 90 hours at 37 ° C with gentle stirring. After centrifugation, the dialyzed supernatant completely against a saline solution buffered with phosphate to pH 7.2, was diluted to a final volume of 50 ml with a saline solution buffered with phosphate to pH 7.2, then froze in small portions at -20 ° C. Sensitization of sheep red blood cells with this antigen was performed by a combination of the methods described by Avrameas and coll. (S. Avrameas, Taudou B., Chuilon S., Immunochemistry, 1969, J5, 67) and Otto and coll. (Otto H., Takamiya H., A. Vogt, J. Immul. Methods, 1973, 3 ^ 1937). sheep's red blood cells were pre-treated with glutaraldehyde (final concentration of sheep red blood cells 5% and 0.5% glutaraldehyde) and then washed and slurried with 20% of a saline solution buffered with phosphate to pH 7.2, and mixing the same extract volume with urea. After 16 hours of incubation-beri ng ved 20°C under let omrøring vaskedes de sensibiliserede røde fåreblodlegemer adskillige gange og opslemmedes indtil 1% til umiddelbar brug. ng at 20 ° C with gentle stirring was washed sensitized sheep red blood cells several times and was slurried up to 1% for immediate use. De røde fåreblodlegemer kan opbevares i en suspension på 10% ved 4°C i adskillige uger. The sheep red blood cells can be stored in a suspension of 10% at 4 ° C for several weeks.

Før filtrering bør hver prøve, der skal undersøges, være absorberet på røde fåreblodlegemer forbehandlet med glutaraldehyd (0,1 ml røde fåreblodlegemer udfældet pr. 1 ml Ig-koncentrat, 37°C, 60 mi- 13 Prior to filtration, each sample to be tested, be absorbed onto sheep red blood cells pre-treated with glutaraldehyde (0.1 ml sheep red blood cells per precipitated. 1 ml of Ig-concentrate, 37 ° C, 60 micro 13

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nutter). Nutter).

Titreringerne udførtes med mikrotitersystemet (Sever JL, J. Immul., 1962, 8j3, 320 og Conrath TB, Handbook of Microtiter Procedures, 1972), idet man anvendte polystyrenplader med udhulinger i form af V. Man udførte en række fortyndinger på 50μ liter hver i normalt kaninserum fortyndet til 1/200, og man tilføjede 25μ liter sensibiliserede røde fåreblodlegemer på 1% til hver fortynding. The titrations were carried out with mikrotitersystemet (Sever JL, J. Immul., 1962, 8j3, 320 and TB Conrath, Handbook of Microtiter Procedures, 1972), using polystyrene plates with cavities in the form of V. We performed a series of dilutions of each 50μ liter of normal rabbit serum diluted at 1/200, and added to 25μ liter of sensitized sheep red blood cells at 1% for each dilution. Man fremstillede en række med røde fåreblodlegemer, der ikke var sensibiliseret, som ikke-specif ik agglutinationskontrol. Was prepared with a number of sheep red blood cells that were not sensitized, and non-specif ik agglutinationskontrol. Man af læste result-taterne efter 2 timer, idet hæmagglutinationstiteren angaves ved den sidste fortynding, der førte til en klar positiv reaktion. One result of the read-The keys after 2 hours, the hæmagglutinationstiteren was said by the last dilution which resulted in a clear positive reaction.

Resultaterne opnået for 5 serotyper E.coli er angivet i tabellen nedenfor. The results obtained for 5 E. coli serotypes are indicated in the table below.

Serotype E.coli_Agglutinationstiter 0 55 1/256 0 111 1/ 64 0 119 1/128 0 127 1/128 0 128 1/ 64 kontrol - Serotype E.coli_Agglutinationstiter 0 55 1/256 0111 1/64 1/128 0119 0127 1/128 0128 1/64 Control -

Bakteriostatisk aktivitet in vitro. Bacteriostatic activity in vitro.

Man undersøgte den bakteriostatiske virkning på væksten af forskellige serotype E.coli ved tilsætning af Ig-koncentrat midt under dyrkningen. The study of the bacteriostatic effect on the growth of different serotype E. coli by addition of the Ig-concentrate in the middle of the cultivation. Man anvendte mikrotitersystemet tilpasset til dyrkning, hvilket muliggjorde at anvende minimale prøvemængder og samtidig undersøge et maksimum antal prøver- But using mikrotitersystemet adapted to the culture, allowing the use of minimal quantities of sample and analyzed at the same time a maximum number of the samples

Hver kultur havde et slutvolumen på 0,25 ml dels på et substrat indeholdende mindst 1% glucose, dels på et rigt dyrkningssubstrat. Each culture had a final volume of 0.25 ml partly on a substrate containing at least 1% of glucose, partly on a rich culture medium. For hver undersøgt dyrkningstid udførte man to prøveudtagninger for at formindske fejlen på grund af variationer i rumfanget. For each studied cultivation was carried out two sampling to minimize error due to variations in volume. Vurderingen udførtes ved dobbelttælling af mængder på 10μ liter på ' plader af næringsagar. The assessment was carried out by double-counting the quantities of 10μ liter of the 'plates of nutrient agar. Bakterieinoculum bestod af en fortynding af 3· en 16 timers kultur fra et miljø, der mindst indeholdt 1 til 2 x 10 bakterier/ml. Bakterieinoculum consisted of a dilution of a 16 · 3 hour culture from an environment containing at least 1 to 2 x 10 bacteria / ml. Inkuberingen udførtes i en fugtig atmosfære ved 37°C. The incubation was carried out in a humid atmosphere at 37 ° C.

Man tilsatte Ig-koncentrater på slutværdier på ^g/ml, 10μg/ml, 3 100μg/ml og 1 mg/ml til dyrkningssubstratet førend podning med 2 x 10 E.coli/ml. Was added Ig concentrates on the end values ​​of ^ g / ml, 10 □ g / ml, 3 100μg / ml and 1 mg / ml to the culture medium prior to inoculation with 2 x 10 E. coli / ml. Man konstaterede en ren væksthæmning af E.coli 0 111 : B4 i løbet af 4 timers inkubering med en Ig-koncentration på 10μg/ml. It was found a clean growth inhibition of E. coli 0111: B4 during 4 hours incubation with an Ig concentration of 10 □ g / ml.

Ved sammenligning med kontrollen, der bestod af dyrkningssubstratet By comparison with the control which consisted of the culture medium

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14 alene, fandt man en bedre vækst i nærværelsen af 1 mg/ml protein fra skummetmælk fra ikke immuniserede køer. 14 alone, exhibited a better growth in the presence of 1 mg / ml of protein from the milk of non-immunized cows.

Clearance af E.coli 0 111:B4 hos mus. The clearance of E. coli 0111: B4 in mice.

Til hvert forsøg anvendte man 8 mus (hvide svejtsiske hanmus på 20 til 24 g), 2 som kontroller og 6 til 3 forsøg hver udført to gange. For each experiment, use was made 8 mice (Swiss white mice of 20 to 24 g), 2 as controls and 6-3 experiments each performed in duplicate. Alle musene modtog en subcutan injektion af 0,1 ml heparin (500 UI/ml). All of the mice scored subcutaneous injection of 0.1 ml of heparin (500 IU / ml). En 16 timers dyrkningsbouillon af E.coli 0 111 : B4 fortyndedes til 1/10 med en steril saltvandsopløsning, hvorefter man fortyndede til 1/100 ved tilsætning af foetal kalveserum ("Difco") fortyndet 1/10 som kontroller og indeholdende tre udvalgte kon centrationer af Ig-koncentrat. A 16-hour culture broth of E. coli 0111: B4 was diluted to 1/10 with a sterile saline solution and then diluted to 1/100 by the addition of fetal calf serum ( "Difco") diluted 1/10 as controls and three selected containing the Kon concentrations of Ig-concentrate. Man injicerede 0,2 ml af den opnåede opløsning intravenøst i den kaudale vene, idet hver mus (2 mus som One injected 0.2 ml of the obtained solution intravenously into the caudal vein, with each mouse (mice 2

C C

kontroller og 2 x 3 som forsøg) fik ca. controls and as 2 x 3 trials) were given approximately 2 x 10 bakterier, idet den 2 x 10 bacteria, the

Q Q

oprindelige bakterieopslemning indeholdt 10 bakterier/ml. original bakterieopslemning contained 10 bacteria / ml. Umiddelbart efter injektionen (til tiden tO) og efter 20, 40 og 60 minutters forløb udtog man en blodprøve på 50y liter fra det ophtalmiske ve-neplexus ved hjælp af kalibrerede hepariniserede kapillarrør, og man overførte straks prøverne til 5 ml sterilsaltvandsopløsning (blod- -2 -3 -4 fortynding 10 ), og man fortyndede herefter til 10 og 10 med saltvandsopløsning. Immediately after the injection (at time TO) and after 20, 40 and 60 minutes, were removed, a blood sample of 50y liters from the ophthalmic RE neplexus by means of calibrated heparinized capillary tube, and was transferred immediately samples to 5 ml of sterile saline solution (blood - 2 -3 -4 10 dilution), and then was diluted to 10 and 10 with saline. Man udførte tællingen på agarplader ("Difco") med 1 ml af hver fortynding og bestemte phagocytoseindekset K efter The count was performed on agar plates ( "Difco") with 1 ml of each dilution and for certain phagocytoseindekset K

Biozzi et coll. Biozzi et coll. (Biozzi G., Stiffel C., Halpern BN, Le Minor L., (Biozzi G., C. Stiffel, Halpern BN, Le Minor L.,

Mauton D., J. Immul., 1961, 87_, 296): log C-, - log C9 K = ---— T2 - ^ C·^ og C2 svarende til tællinger ved tiden og T2. Mauton D., J. Immul., 1961, 87_, 296): log C-, - C 9 log K = ---- T 2 - · ^ C ^ and C2 corresponding to the counts at the time and T2.

Man observerede således en normal nedgang i antallet af bakterier ved eliminering gennem det reticulo-endotheliale system i nærværelse af foetal kalveserum, medens tilsætningen af 10yg Ig-koncentrat i injektionsopløsningen fremkaldte en 99,6% forsvinden af bakterierne fra blodstrømmen i løbet af 20 minutter. It was observed by the following a normal decrease in the number of bacteria by elimination by the reticulo-endothelial system in the presence of fetal calf serum, while the addition of 10yg Ig-concentrate in the injection solution induced a 99.6% disappearance of the bacteria from the blood stream over 20 minutes.

Resultaterne er opgjort i tabellen nedenfor: The results are compiled in the table below:

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15 15

Tiden Bakterier i % af det oprindelige antal tilgti- (minutter efter den 0 efter intravenøs injektion af 2 x 10° indsprøjtning E.coli 0 111:B4 af E.coli)_ Bacteria time in% of the initial number of tilgti- (after 0 minutes after intravenous injection of 2 x 10 ° injection E. coli 0111: B4 E. coli) _

Kontrol 1:10 Prøver Ig-koncentrat foetal kalve- 1000 ug 100 ug 10 ug serum 0 100 100 100 100 20 27,8 0,14 0,22 0,4 40 ------------- 15,8 -----------0,02 ......0,02·--" ------- 0,12 ' 60 10,0 0,02 0,02 0,06 Control Samples 1:10 Ig-concentrate fetal calf 1 000 ug 100 ug 10 ug serum 0 100 100 100 100 20 27.8 0.14 0.22 0.4 40 ------------- 15.8 ----------- 0.02 ...... 0.02 · - "------- 0.12 '60 10.0 0.02 0, 02 0.06

Man kontrollerede opsoniseringens specificitet ved fuldstændig absorption af Ig-koncentratet med serotype 0 111:B4 E.coli, og man konstaterede, at denne absorption nssten tilintetgjorde hele aktivitetsforøgelsen af phagocytosen selv med en mængde på 1 mg Ig- koncentrat . Opsoniseringens specificity was monitored by complete absorption of Ig-concentrate with serotype 0111: B4 E. coli, and it was found that this absorption nssten destroyed throughout the activity increase of phagocytosis even with an amount of 1 mg Ig concentrate.

Tabellen herunder giver phagocytose K-værdier opnået i løbet af 20 minutter. The table below gives phagocytosis K values ​​obtained in the course of 20 minutes.

Kontrol 1000 g Ig- 1000 g Ig- 1:10 foetal koncentrat koncentrat kalveserum ikke-absorberet absorberet K 0,015 0,128 0,039 Control 1000g 1000g Ig Ig concentrate Concentrate 1:10 fetal calf serum unabsorbed absorbed K 0.015 0.128 0.039

Forsøg med beskyttelse hos mus. Experiments with protection in mice.

Man fremstillede logaritmiske fortyndinger (log^g) af en kultur af E.coli 0 111:B4 i næringsmiddelbouillon ("Difco") inde- 9 —4 -5 -6 holdende 10 bakterier/ml til fortyndinger på 10 ,10 , 10 og _7 10 i mucin på 5% (granulær mucin, type 1701-W til potentiering af virulensen af bakterier fra Wilson Laboratories, Chicago, Illinois). Dilutions were prepared logarithmic (log g g) of a culture of E. coli 0111: B4 in nutrient broth ( "Difco") containing 9 -4 -5 -6-holding 10 bacteria / ml to dilutions of 10, 10, 10 and _7 10 in 5% mucin (granular mucin, type 1701-W for potentiation of the virulence of bacteria from Wilson Laboratories, Chicago, Illinois).

Man blandede derefter 3 ml af hver fortynding med 0,6 ml forsøgsopløsning, henholdsvis med saltvandsopløsning, Ig-koncentrat, proteiner fra almindelig valle (ikke immuniserede køer). Was mixed then 3 ml of each dilution with 0.6 ml of test solution, respectively, with brine, Ig-concentrate, whey proteins from normal (non-immunized cows). Idet man anvendte 5 mus til hver fortynding, indsprøjtede man 0,6 ml af den opnåede blanding i hver mus intraperitonealt. But using 5 mice for each dilution, injected 0.6 ml of the obtained mixture into each mouse intraperitoneally. Forsøgsresultaterne i form af døde mus pr. The test results in terms of dead mice per. 5 behandlede mus efter 48 timer er angivet i tabellen nedenfor. 5 treated mice after 48 hours, are indicated in the table below.

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Forsøgsmateriale Antallet af døde mus/5 be handlede "inus. Test Material The number of dead mice / 5 be traded "inus.

Koncentration af bakterier ved behandlingen 5 x 104 5 x 103 5 x 102 5 x 101 Concentration of bacteria in the treatment 5 x 104 5 x 103 5 x 102 5 x 101

Saltvandsopløsning 5555 1°μ * Saline 5555 ° 1 μ *

Ig-koncentrat 5553 25μ * Ig-concentrate 25μ 5553 *

Ig-koncentrat 5400 100μ Ig-concentrate 5400 100μ

Ig-koncentrat 0000 10μ proteiner fra almindelig valle (ikke immuniserede køer) 5555 25μ proteiner fra almindelig valle 5 5 5 5 100μ proteiner fra almindelig valle 5 5 5 5 udregnet som totalproteiner indeholdende 35-45% Ig-forbindelser fra mælk. Ig-concentrate 0000 10μ whey proteins from normal (non-immunized cows) 5555 25μ whey proteins from normal 5 5 5 5 100μ whey proteins from normal 5 5 5 5 calculated as total proteins containing 35-45% Ig compounds from milk.

Man så, at 100 ]ig Ig-koncentrat gav fuldstændig beskyttelse over for alle koncentrationer af bakterieprøver, medens 100 ng valleproteinér isoleret fra ikke-immuniserede køer ikke gav nogen beskyttelse. One so that 100] ig Ig-concentrate gave complete protection against all concentrations of bacterial samples, while 100 ng valleproteinér isolated from non-immunized cows did not give any protection.

Eksempel 7 I. En første række kliniske forsøg viste, at Ig-forbindelser fra mælk modstod proteolytisk nedbrydning til inaktive dele i tarmkanalen. I. Example 7 A first series of clinical trials showed that the Ig compounds from milk resisted proteolytic degradation into inactive components in the intestinal tract.

11 børn (9 drenge, 2 piger) hospitalsindlagte af forskellige grunde, men ikke lidende af gastroenteriter, og hvis alder var fra 2 uger til 1 år, madedes med mælk indeholdende et Ig-koncentrat fremstillet som i eksempel 2 eller 4 i kaloriedoser svarende til 2 g/kg legemsvægt fordelt ensartet over 24 timer. 11 children (nine boys, two girls) hospitalized for various reasons, but not suffering from gastroenteriter, and whose age was from 2 weeks to 1 year, fed the milk containing an Ig-concentrate prepared as in Example 2 or 4 in calorie doses equivalent to 2 g / kg body weight evenly distributed over 24 hours. De første og sidste måltider, hvor Ig-koncentrat var inkorporeret, mærkedes med 0,2 g car-minrødt. The first and last meal, wherein the Ig-concentrate was incorporated, were labeled with 0.2 g of carbon minrødt. Mindst én afføringsprøve o'psamledes førend administreringen af Ig og systematisk alle de følgende afføringer i løbet af 72 timer, idet afføringen opbevaredes ved -20°C. At least one o'psamledes stool sample prior to the administration of Ig and systematic all of the following bowel movements within 72 hours, with the faeces were stored at -20 ° C.

Man fremstillede ekstrakter af afføringerne ved tilsætning af 2 dele afføring til 1 del saltvandsopløsning bufret med phosphat til C tr- λλλ f -»- 17 Extracts were prepared by the stools by the addition of 2 parts of stool to 1 part saline solution buffered with phosphate to C TR λλλ f - '- 17

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pH 7,2 og dispergerede afføringerne ved 0°C med en glasspatel. pH 7.2 and dispersed stools at 0 ° C with a glass rod. Man omrørte herefter suspensionen i 20 minutter med en miniomrører (400 omdrejninger/minut) ved stuetemperatur. Then the suspension was stirred for 20 minutes with a miniomrører (400 rotations / min) at room temperature. Herefter afkøledes suspensionen hurtigt til 0°C, og man centrifugerede ved denne temperatur ved 3500 xgi 15 minutter. Next, the suspension was cooled rapidly to 0 ° C and centrifuged at this temperature at 3500 xg for 15 minutes. Til sidst fjernede man omhyggeligt su-pernatanten. Finally removed carefully su supernatant.

Tilstedeværelsen af aktive Ig-forbindelser i afføringsekstrakten bekræftedes ved dobbelt immunodiffusion (Ouchterlony's forsøg) og ved immunoelektroforese. The presence of active compounds in the Ig afføringsekstrakten was confirmed by double immunodiffusion (Ouchterlony's test) and by immunoelectrophoresis. Antisera anvendt til disse forsøg fremstilledes ved subcutan og intravenøs injektion gentaget flere gange af 1 mg mælkeantistof eller 0,1 mg Ig G ^ på kaniner. Antisera used for these experiments were prepared by subcutaneous and intravenous injection repeated several times of 1 mg antibody milk or 0.1 mg Ig G ^ in rabbits. Til Ouchter-lony's forsøg dækkedes glasplader (94 x 84 mm) med et lag af en 1% agargel i en saltvandsopløsning bufret med phosphat til pH 7,2, i hvilken gel man har udskåret hulrum med 4 mm's afstand på 9,5 mm med hullemaskinen ("LKB-Produkteur AB"), idet man anvendte et anti-proteinantiserum fra colostrum valle fra -køer og et monovalent antiserum specifikt anti-Ig fra komælk. For Ouchter-Lony's test was covered glass plates (94 x 84 mm) with a layer of a 1% agar gel in a saline solution buffered with phosphate to pH 7.2, in which the gel has been cut out cavity of 4 mm apart of 9.5 mm and paper puncher ( "LKB Produkteur AB"), using an anti-protein antiserum from colostrum whey from the queues and a monovalent antiserum specific anti-Ig of cow's milk.

Til immunoelektroforesen dækkedes glaspladerne (100 x 85 mm) med 12 ml 1% agargel i 0,05Ή barbiturat bufret til pH 8,3 (10,3 g natriumbarbiturat + 0,5 g citronsyre + 0,5 g oxalsyre i 1 liter vand), og den elektroforetiske adskillelse udførtes ved stuetemperatur i løbet af 90 minutter ved 4 volt/cm. For immunoelektroforesen blanketed glass plates (100 x 85 mm) with 12 ml of 1% agar gel in 0,05Ή barbiturate buffered to pH 8.3 (10.3 g natriumbarbiturat + 0.5 g of citric acid + 0.5 g of oxalic acid in 1 liter of water) and the electrophoretic separation was carried out at room temperature over 90 minutes at 4 volts / cm. Efter diffusion af antisera (24 timer) vaskedes pladen med en saltvandsopløsning, tørredes og fremkaldtes med azocarmin B. After diffusion of the antisera (24 hours), the plate was washed with a saline solution, dried, and was developed with Azocarmine B.

Man konstaterede tilstedeværelsen af mælke Ig-forbindelser i afføringerne, skønt betydelige forskelle i, hvornår de viste sig, og i varigheden af udskillelsen af Ig, ligesom en god overensstemmelse mellem, at den røde carminfarve og Ig-forbindelsen kom til syne og forsvandt. It was noted the presence of lactic Ig connections in the stools, although significant differences in when they were found, and in the duration of the secretion of Ig, like a good agreement between the red carminfarve and Ig connection appeared and disappeared.

For at bekræfte aktiviteten af Ig-mælkeforbindelserne udførte man en prøve in vivo på serobeskyttelse hos mus (som beskrevet i eksempel 6), idet man anvendte 6 afføringsekstrakter i hver række, og hvis resultater er anført i følgende tabel: 18 To confirm the activity of the compounds Ig milk was carried out a test in vivo on seroprotection in mice (as described in Example 6), using 6 stool extracts in each row, and the results are given in the following table: 18

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Ekstrakt af Intensitet af Ig Antal døde mus/ afføringer i afføringseks- 5 behandlede mus trakt efter im- Antal bakterier anvendt munoelektrofore- ved behanalingen tisk undersøgelse . The extracts of the intensity of the Ig Number of dead mice / stools in afføringseks- 5 treated mice extract by imports Number of bacteria used munoelektrofore- by behanalingen schematically study. , ~ ·, 5x10 5x10 5x10^ 5xlOx , ~ ·, 5x10 5x10 5x10 ^ 5xlOx

Barn MD I 5 5 5 5 (2 uger) II 5 5 5 5 IV + + + + 2 1 0 0 V + + + + 0 0 0 0 VI + + + 1 1 0 0 IX -;- 5 5 3 4 Barn MD I 5 5 5 5 (2 weeks) II 5 5 5 5 IV + + + + 2 1 0 0 V + + + + 0 0 0 0 VI + + + 1 1 0 0 IX -; - 5 5 3 4

Barn SG I -:- 5 5 5 5 (1 måned) III 4 5 5 5 IV + + + + 0 0 0 0 VI + + + + 0 0 0 0 X + + 3 0 10 XI + 5 2 0 0 Child SG I -: - 5 5 5 5 (1 month) III 5 4 5 5 IV + + + + 0 0 0 0 VI + + + + 0 0 0 0 X + 0 + 3 + 10 XI 5 0 2 0

Man konstaterede klart en korrelation mellem de positive resultater ved immunoelektroforese og beskyttelsesevnen fra det materiale, der var indeholdt i ekstrakterne fra afføringen. It was noted clearly a correlation between the positive results of immunoelectrophoresis and protective ability of the material contained in the extracts from the stool. Specielt bestemte man koncentrationen af Ig-mælkeforbindelser fra ekstrakterne V (barn MD) og IV og VI (barn S G.) svarende til mindst 1 mg Ig-koncentrat. In particular, specific to the concentration of Ig-lactic compounds from the extracts V (infant MD), IV, VI (G. child S) of at least 1 mg Ig-concentrate.

II. II. I en anden.rakke kliniske forsøg undersøgte man de terapeutiske og profylaktiske egenskaber af Ig-koncentrater fremstillet som beskrevet i eksempel 2 eller 4. In a anden.rakke clinical trial examined the therapeutic and prophylactic properties of the Ig concentrates, prepared as described in Example 2 or the fourth

Terapeutiske egenskaber. Therapeutic properties.

Denne undersøgelse udførtes med børn på indtil 5 måneder, der led af godartede til heftige gastroenteriter fremkaldt af E.coli. This study was conducted with children up to five months, suffering from benign to violent gastroenteriter caused by E. coli. I forskellige forsøgsrækker administrerede man i alt til 152 patienter dels 2 g koncentrat Ig/kg/dag i 5 dage, dels 1 g/kg/dag i løbet af 10 dage til deres mad. In various test series administered to a total of 152 patients in part 2 g of concentrate in g / kg / day for 5 days and the other 1 g / kg / day over 10 days to their food. Patienterne modtog ingen medikamenter som sulfamider eller antibiotika hverken oralt eller parenteralt. The patients received no medication as antibiotics or sulfamides either orally or parenterally.

Man foretog dyrkning fra fæces dagen efter administreringen, og den sidste mellem 36 og 48 timer efter den sidste administrering af Ig-koncentrat. Cultivation was performed from the faeces the day after the administration, and the last between 36 and 48 hours after the last administration of Ig-concentrate. 43 Patienter behandledes på samme måde, men idet man erstattede Ig-koncentratet med proteiner fra valle, der kom fra ikke immuniserede køer. 43 patients were treated in the same manner, but with the replacement of the Ig-concentrate with the proteins from whey, which came from non-immunized cows.

Man opnåede negative dyrkninger fra fæces for 90 børn (57,7%) under behandlingen.I kontrolrækken viste kun 14 børn (27,7%) en spon- This gave negative cultures from faeces of 90 children (57,7%) under the row behandlingen.I control showed only 14 children (27.7%) have a spontaneous

SΛΛΛ f. IS SΛΛΛ f. IS

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19 tan forsvinden af E.coli fra dyrkningen af facesprøverne. 19 tan disappearance of E.coli face from the culture of the samples. Man må hu~ ske, at naturlige proteiner,' der stammer fra valle af ikke immuniserede køer, in= deholder en ringe maigde Ig anti-E.coli. It is to be hu ~ happen that the natural proteins, 'derived from whey of non-immunized cows, in = maigde rings containing an Ig anti-E.coli. Man observerede ligeledes, at udeblivelse af heldicrt udfald -af behandlingen sås oftere i de tilfælde, hvor man anvendte høje doser i løbet af en meget kort tid. It also observed that the absence of heldicrt outcome -of treatment was seen more frequently in cases where they used high doses over a very short time.

Man undersøgte konsistensen af fæces i hvert tilfælde. The study of the consistency of faeces in each case. Man konstaterede, at diarréen forsvandt og en følelig klinisk forbedring i et stort antal tilfælde også selv om dyrkning fra fæcesprøver forblev positive. It was found that the diarrhea disappeared and a tactile clinical improvement in a large number of cases, even if growing from stool samples remained positive. I det tilfælde, hvor dyrkningen fra fæcesprøverne blev negative, forbedredes konsistensen fra afføringen sig hurtigere, dersom Ig-koncentratet administreredes i et mælkepulver fattig på lactose. In the case where the culture from faeces samples were negative, improved consistency of the stools more quickly if the concentrate Ig was administered in a milk powder low in lactose.

Profylaktiske egenskaber. Prophylactic properties.

De· for tidligt fødte børn udgør en gruppe, der er ekstremt følsomme over for gastroenteriter af E.coli,og derfor valgtes denne gruppe til et klinisk profylakseforsøg. They · premature babies form a group that is extremely sensitive to gastroenteriter of E.coli and therefore chosen this group to a clinical profylakseforsøg.

Undersøgelsen udførtes i løbet af 6 måneder på 2 stuer med 8 kuvøser på hver stue. The study was conducted over 6 months at 2 to 8 incubators living rooms of each living room. Man foretog fordeling af forsøg og kontroller på hver stue, således at alle patienter var udsat for samme betingelser, idet 4 kuvøser tjente som forsøg og 4 som kontrol. Man undertook distribution of tests and controls in each room so that all patients were exposed to the same conditions, with four incubators served as trial and 4 as a control. Hvert tilfælde varede i gennemsnit 41 dage, idet et barn fjernedes efter helbredelse og erstattedes af et andet afhængig af tilhørsforholdet (forsøg eller kontrol). Each event lasted an average of 41 days as a child were removed after healing and replaced by another, depending on the affiliation (experimental or control). Man fulgte 70 tilfælde, 36 som kontrol og 34 som forsøgsgruppe. Following the 70 cases, 36 as the control and 34 experimental group.

Forsøgsgruppen modtog Ig-koncentrat ved hver madning med mælk fordelt over 24 timer med 0,25 g/kg/dag. The experimental group received Ig-concentrate at each feeding with milk distributed over 24 hours with 0.25 g / kg / day. Man undersøgte fæcesprøver fra begyndelsen af forsøget og undersøgte fscesprøver ved dyrkning hver femte dag. We examined stool samples from the beginning of the experiment and examined fscesprøver by growing every fifth day. Af i alt 666 dyrkede fæcesprøver udgjorde 339 kontroller og 327 forsøgsprøver. Of a total of 666 stool specimens cultivated totaled 339 controls and 327 test samples. Blandt de sidstnævnte udviste 33 tilstedeværelsen af E.coli (10,1%), medens 96 ud af 339 kontrolkulturer var positive (28,3%). Among the latter, 33 showed the presence of E.coli (10.1%), while 96 out of the 339 control cultures were positive (28.3%). Dersom man kun undersøger serotype E.coli 0 119:B14 finder man den ofte forbundet med kraftige .former af gastroenteriter, og frekvensen af positive fæcesdyrkninger var 5,5% i forsøgsgruppen imod 23,3% i kontrolgruppen. If one examines only serotype E. coli 0119: B14 feature often associated with strong .former of gastroenteriter, and the frequency of positive fæcesdyrkninger was 5.5% in the test group against 23.3% in the control group.

Man kan konkludere, at inkorporeringen af proteinkoncentrater indeholdende mælkespecifikke anti-E.coli Ig.til· mælken til for tidligt fødte formindsker infektionsgraden kraftigt med E.coli hos denne særligt følsomme gruppe nyfødte. One can conclude that the incorporation of protein concentrates containing milk-specific anti-E.coli Ig.til · milk for premature reduces infection rate strongly with E. coli in this particularly vulnerable group of newborns.

Claims (9)

1. Fremgangsmåde til fremstilling af proteinkoncentrater indeholdende immunologiske faktorer fra mælk, specielt aktive ikke-denaturerede immunoglobuliner, kendetegnet ved, at man: a) opsamler nrallcen fra mælkeproducerende drøvtyggere, især køer, der er hyperimnuniserede ved vaccination, idet der successivt administreres antigener parenteralt og lokalt ca. 1. A process for the production of protein concentrates containing immunological factors from milk, in particular active non-denatured immunoglobulins, characterized in that: a) collecting nrallcen from milk-producing ruminants, in particular cows hyperimnuniserede by vaccination, with sequentially administered antigens parenterally and locally about 8 til 1-2 uger før kælvningen og peroralt i de ca. 8 to 1-2 weeks before calving and orally in the approximately 2 uger før kælvningen og, eventuelt, en serie successive administreringer af antigen fra ca. 2 weeks prior to calving, and, optionally, a series of successive administrations of antigen at about 2\ måed før det antagne ophør af mælkesekretionen med skift mellem parenteral, lokal og peroral administrering; 2 \ måed before the successful suppression of milk with toggle parenteral, local and oral administration; ovennævnte mælk består af colostrum fra 1 og 2 timer efter kælvningen, overgangsmælk fra 3-8 dage efter kælvningen og, eventuelt, mælk fra de sidste 60 dage af lactationsperioden; the above-mentioned milk consists of colostrum of 1 and 2 hours after calving, transitional milk from 3-8 days after calving and, optionally, milk from the last 60 days of the lactation; b) fraskiller fløden og urenhederne; b) separating the cream and the impurities; c) koagulerer den-klarede og skummede mælk; c) coagulates-managed and skimmed milk; d) fraskiller caseinet, filtrerer, ultrafiltrerer og steriliserer proteinet fra vallen ved filtrering; d) separating the casein, filtering, ultrafiltering and sterilizing the protein from the whey by filtration; og e) inddamper og tørrer produktet under sådanne betingelser, at man ikke denaturerer immunoglobulinerne og bevarer steriliteten.. and e) evaporating and drying the product under such conditions that do not denature the immunoglobulins and preserves the sterility ..
2. Fremgangsmåde ifølge krav 3, kendetegnet ved, at vaccinen er på basis af Escherichia coli antigener, der forårsager gastroenteriter hos nyfødte. 2. A method according to claim 3, characterized in that the vaccine is based on the Escherichia coli antigens that cause gastroenteriter in newborns.
3. Fremgangsmåde ifølge krav 1, kendetegnet ved, at koaguleringen udføres ved tilsætning af saltsyre indtil pH ca. 3. The method of claim 1, characterized in that the coagulation is carried out by adding hydrochloric acid to pH ca 4,6 og opvarmning indtil 40°C. 4.6 and heating up to 40 ° C.
4. Fremgangsmåde ifølge krav 1, kendetegnet ved, at man, for at undgå følgende tilstopning af filtre og ultrafiltre, udfører skumningen i to etaper først koldt og derefter varmt, og at skumningen og adskillelsen af caseinet følges af en tvungen klaring. 4. The method of claim 1, characterized in that, in order to avoid the following plugging of filters and ultrafiltration, carries out foaming in two stages until cold and then hot, and that the foaming and the separation of the casein is followed by a forced clarifier.
5. Fremgangsmåde ifølge krav 1, kendetegnet ved, at ultrafiltreringen af vallen udføres med vask af retentatet og re-cyclisering af det vaskede retentat, således at dettes indhold af proteiner forøges. 5. The method of claim 1, characterized in that ultrafiltration of the whey is carried out by washing the retentate and re-cyclization of the washed retentate, such that its protein content is increased.
6. Fremgangsmåde ifølge krav 1, kendetegnet ved, at den prekoncentrerede valle underkastes en prefiltrering efterfulgt af sterilfiltrering. 6. The method of claim 1, characterized in that the prekoncentrerede whey is subjected to a prefiltration followed by filtered sterilization. DK 153521 B DK 153 521 B
7. Fremgangsmåde ifølge krav 1, kendetegnet ved, at inddampningen af den prekoncentrerede og steriliserede valle finder sted i et lukket system under sterile betingelser. 7. The method of claim 1, characterized in that the evaporation of the prekoncentrerede and sterilized whey will take place in a closed system under sterile conditions.
8. Fremgangsmåde ifølge krav 1, kendetegnet ved, at det koncentrerede produkt tørres ved frysning efterfulgt af lyo-filisering og steril konditionering. 8. A method according to claim 1, characterized in that the concentrated product is dried by freezing followed by lyophilization and sterile conditioning.
9. Fremgangsmåde ifølge krav 1, kendetegnet ved, at man for at genvinde de immunoglobuliner, der er fjernet under trin b) og d), ekstraherer fløden og tykmælken med vand og forener vaskevandet med henholdsvis den skummede mælk og vallen. 9. A method according to claim 1, characterized in that, in order to recover the immunoglobulins that are removed during step b) and d), the curds and extracting the cream with water and combine the wash water respectively with the skimmed milk and whey.
DK131078A 1977-04-15 1978-03-22 Process for the preparation of protein concentrates containing immunological factors from milk DK153521C (en)

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Families Citing this family (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE448344B (en) * 1978-02-06 1987-02-16 Stolle Res & Dev The antibody for the treatment of rheumatoid arthritis and seen to be stella this
FR2460135B1 (en) * 1979-07-02 1982-11-19 Liotet Serge
DE3172807D1 (en) * 1981-04-28 1985-12-12 Stolle Res & Dev Method of obtaining an anti-inflammatory bovine milk
JP2561234B2 (en) * 1981-05-12 1996-12-04 スト−ル・リサ−チ・アンド・デイベロップメント・コ−ポレ−ション Anti-inflammatory agents
JPH0552815B2 (en) * 1982-03-09 1993-08-06 Sendai Biseibutsu Kenkyusho
NZ205392A (en) * 1982-09-02 1987-03-06 Unilever Plc Preparation of immunoglobulins against e.coli pili
JPS6112629A (en) * 1984-06-28 1986-01-21 Lion Corp Composition for oral cavity application
DE3432718C1 (en) * 1984-09-06 1986-05-22 Biotest Pharma Gmbh A process for preparing a solution of lactic and / or Kolostralimmunglobulinen
US4816252A (en) * 1985-04-15 1989-03-28 Protein Technology, Inc. Product and process for transferring passive immunity to newborn domestic animals using ultrafiltered whey containing immunoglobulins
US4834974A (en) * 1986-01-13 1989-05-30 Protein Technologies, Inc. Immunologically active whey fraction and recovery process
GB8729031D0 (en) * 1987-12-11 1988-01-27 Express Foods Group Ltd Isolation of immunoglobulin rich fraction from whey
IE76730B1 (en) * 1990-07-30 1997-11-05 Abbott Laboraties Method and product for the treatment of gastric disease
US5258178A (en) * 1990-07-30 1993-11-02 Abbott Laboratories Method and product for the treatment of gastric disease
DE4026365C2 (en) * 1990-08-21 1993-04-22 Biotest Pharma Gmbh, 6072 Dreieich, De
JPH04169539A (en) * 1990-11-01 1992-06-17 Imuno Japan:Kk Preventive and therapeutic agent for alimetary disease and production thereof
US5645834A (en) * 1992-08-28 1997-07-08 Immuno-Dynamics, Inc. Method and product for treating failure of passive transfer and improving milk production in bovine species
AT234855T (en) * 1993-09-20 2003-04-15 Anadis Ltd compositions process for the production of immunoglobulins from colostrum and their use in pharmaceutical
FI97269B (en) * 1993-10-12 1996-08-15 Viable Bioproducts Ltd Process for the preparation of the nutritional beverage
ZA9409789B (en) * 1993-12-30 1995-10-25 Immunotec Res Corp Ltd Process for making undenatured whey protein concentrate
US5670196A (en) * 1995-04-12 1997-09-23 Galagen Inc. Method for microfiltration of milk or colostral whey
US5707678A (en) * 1995-04-12 1998-01-13 Galagen Inc. Method for microfiltration of milk or colostral whey
CA2165937A1 (en) * 1995-05-09 1996-11-10 Immunotec Research Corporation Ltd. Process for producing an undenatured whey protein concentrate
AUPN642795A0 (en) * 1995-11-08 1995-11-30 Northfield Laboratories Pty Ltd Dairy compositions and methods of preparing same
AT325817T (en) 1997-05-29 2006-06-15 Agres Ltd A process for the production of immunoglobulin A in milk
IE970541A1 (en) 1997-07-25 1999-01-27 Michael Anthony Folan Maternal immune secretions and their use in the treatment and/or prophylaxis of the buccal cavity
FI110752B (en) 1999-05-25 2003-03-31 Novatreat Oy A method for treating colostrum
SE0003045D0 (en) 2000-08-29 2000-08-29 Probi Ab New Method
CN100344234C (en) 2001-09-17 2007-10-24 伊莱利利公司 Pesticidal formulation
DE10158009A1 (en) * 2001-11-21 2003-05-28 Begerow E Gmbh & Co Reducing total germination index in aqueous dispersions e.g. animal milk, involves carrying out sterile filtration using deep filtration units
EP1629850B2 (en) 2004-08-24 2013-05-22 N.V. Nutricia Nutritional composition comprising indigestible transgalactooligosaccharides and digestible galactose saccharides
NL1033696C2 (en) * 2007-04-16 2008-10-20 Friesland Brands Bv To milk-derived antigen-specific antibodies, methods for preparing and using them.
US8475789B2 (en) 2008-01-22 2013-07-02 Multimerics Aps Products and methods to prevent infections
BRPI0911994A2 (en) * 2008-05-15 2015-10-20 Health L P W Process for the production of a milk secretory IgA enriched fraction, product, and composition.
EP2424890B1 (en) 2009-04-27 2018-08-01 Immuron Limited Anti-lps enriched immunoglobulin preparation for use in treatment and/or prophylaxis of non alcoholic steatohepatitis
CA2808361A1 (en) 2010-08-17 2012-02-23 Immuron Ltd. Anti-lps enriched immunoglobulin preparation for use in treatment and/or prophylaxis of a pathologic disorder
US20140072649A1 (en) * 2012-09-11 2014-03-13 Al-Urdonia Lemudaddat Al-Ajsam Co. Immunized Camel Milk-Based Composition for the Treatment or Prevention of Gastrointestinal Infections

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK87610C (en) * 1955-04-07 1959-07-27 Berry Campbell A process for the preparation of specific substances protection against antigenic substances.
DE1810438A1 (en) * 1967-11-23 1969-07-10 Twyford Lab Ltd Prophylactic or therapeutic preparation
US3911108A (en) * 1973-02-14 1975-10-07 Diamond Shamrock Corp Process of producing bovine milk products containing specific antibodies
US3930039A (en) * 1971-07-30 1975-12-30 Molkerei J A Meggle Milchindus Method of preparing a protein concentrate from whey

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BE546837A (en) *
DE1022356B (en) * 1955-04-07 1958-01-09 William E Petersen A process for the production of a specific protective material against an antigen
FR1599671A (en) * 1966-06-27 1970-07-20
NL7011786A (en) * 1969-09-24 1971-03-26
BE788120A (en) * 1971-09-01 1973-02-28 Coca Cola Co Processing whey protein

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK87610C (en) * 1955-04-07 1959-07-27 Berry Campbell A process for the preparation of specific substances protection against antigenic substances.
DE1810438A1 (en) * 1967-11-23 1969-07-10 Twyford Lab Ltd Prophylactic or therapeutic preparation
US3930039A (en) * 1971-07-30 1975-12-30 Molkerei J A Meggle Milchindus Method of preparing a protein concentrate from whey
US3911108A (en) * 1973-02-14 1975-10-07 Diamond Shamrock Corp Process of producing bovine milk products containing specific antibodies

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OA5938A (en) 1981-06-30
DE2813984C2 (en) 1987-11-26
CA1101333A (en) 1981-05-19
MX5007E (en) 1983-02-14
FR2387039A1 (en) 1978-11-10
DE2813984A1 (en) 1978-10-26
SE448062B (en) 1987-01-19
IT1156193B (en) 1987-01-28
PH17782A (en) 1984-12-11
IT7848896D0 (en) 1978-04-14
NL7804015A (en) 1978-10-17
CH627079A5 (en) 1981-12-31
JPS6340771B2 (en) 1988-08-12
ZA7801607B (en) 1979-03-28
DK131078A (en) 1978-10-16
GB1573995A (en) 1980-09-03
PH14031A (en) 1980-12-12
AU519091B2 (en) 1981-11-05
JPS53130411A (en) 1978-11-14
NL187516C (en) 1991-11-01
AR218482A1 (en) 1980-06-13
DK153521C (en) 1988-12-05
CA1101333A1 (en)
NL187516B (en) 1991-06-03
GR64433B (en) 1980-03-21
SE7804190A (en) 1978-10-16
AU3490378A (en) 1979-10-18
ES468808A1 (en) 1978-12-01
FR2387039B1 (en) 1981-07-24
MY8100294A (en) 1981-12-31

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