J. Rejnek

Impermeability of pig placenta for antibodies

After passive immunization of pregnant swine 24 and 48 hours before delivery with isolated fraction of 7S gamma globulin or with the whole serum of a high titer of antibodies to diphtheria and tetanus toxoid no traces of antibodies were detected in newborn piglets by passive hemagglutination even if the globulin fraction of their sera was many times concentrated.

Study of the effect of antibodies in the intestinal tract of germ-free baby pigs

The effect of antibodies in the intestinal tract was studied in germ-free baby pigs whose intestinal barrier was closed to macromolecules by the peroral administration of modified cows milk for the first 72 hours after birth. They were then all contaminated with the pathogenic strainEscherichia coli 055 in amounts of 109 bacterial cells per animal. The controls, which were not given any antibodies, all died within 24 hours. All the experimental animals given 12.5–50ml immune colostrum or serum survived, while of those given 50ml normal serum or colostrum containing natural antibodies reacting with theEscherichia coli test strain, 50% survived. No circulating antibodies were found in the serum of the experimental animals after the administration of serum or colostrum. The antibodies present in colostrum thus appear to protect the newborn organism directly in the intestinal tract, which is the first site of bacterial invasion, as well as after infiltration into the blood stream.

Isolation and characterisation of immunoglobulins in the serum of precolostral piglets

The presence of actively synthetized immunoglobulin in the serum of newborn precolostral germfree piglets was confirmed. This immunoglobulin, being of IgG antigenic type, carries determinants of typical IgG heavy and light chains and has a sedimentation constant of 4S. The first antibodies formed in germfree piglets after immunization with sheep red blood cells are of macroglobulinemic (IgM) nature. They are followed by formation of more slowly sedimenting antibodies of the IgG type. No fast sedimenting antibodies of the IgG type were detected.

Two structurally different types of rabbit light chains.

Disulphide bonds of rabbit γ-G-globulin and the antibody of the γ-G-globulin type against the 2,4-dinitrophenyl group were split both by the oxidative sulphitolysis at pH 8.6 and by the reduction with 2-mercaptoethanol followed by carboxymethylation. The fractionation was carried out in 0.05 m formic acid containing 6m urea, in 1m propionic acid or in 6m guanidine hydrochloride. Both heavy (H) and light) (L) chains are released from the I+J fraction preceding on an elution diagram H chains when rechromatographed in a stronger desaggregation medium. A small amount of the L chains is also released on rechromatography of the H chains (isolated from 1m propionic acid) in 6m guanidine hydrochloride. The separation of the degraded γ-G-globulin in 0.05m formic acid containing 6m urea or in 6m guanidine hydrochloride showed a separation of the L chains to two fractions differing by electrophoretic properties, peptide maps and N-terminal amino acids. However, these chains exhibit a similar molecular weight, immunoelectrophoretic behaviour and similar properties on reactivation of the antibody H chain.

Studies on the immunoglobulin spectrum of porcine serum and colostrum

An analysis of the immunoglobulin region of porcine blood serum proved the presence of a number of protein fractions which is higher than in other animal species. The presence of antibody activity to tetanus anatoxin examined by radioimmunoelectrophoresis was found in three γ G-globulin fractions, which could be distinguished from each other, and also in two additional fractions. One of the latter was the γ-macroglobulin fraction and the other one might correspond to γ A-globulin even though its mobility was higher than the mobility of γ A-globulin of human serum. A comparison of the immunoglobulin spectrum of porcine serum, colostrum, and of the suckling pig serum after ingestion of the colostrum showed that the main component of porcine colostrum constitutes the γ G-immunoglobulin, even though the colostrum seems to contain other components of the latter which have a higher electrophoretic mobility. The antibody activity of colostrum was found only in fractions γ G and γ M. The fraction of colostrum which might correspond to γ A did not display any activity at all. The activity in suckling pig serum was also concentrated in fractions γ G and γ M.

Use of zinc ions for fractionation of pig γ-G-globulin and its structural subunits

The different behaviour of the two fractions of pig γ-G-globulin prepared by interaction with zinc ions during oxidative sulphitolysis is described. The γ-G-globulin fraction which is not precipitated by zinc ions is dissociated more readily, as seen from the finding that, unlike the other fraction, it contains practically no incompletely dissociated molecules. Fractions of the light chains, with different molecular weights, were also isolated from this fraction. A technique was elaborated for separation of the component with H antigenic specificity which is present in the light chain preparation. Detailed study of this component showed that it is probably part of the heavy chain. The origin and formation of the component is discussed.

Contribution to the structural characterization of gamma globulin from pig colostrum

From the results obtained in the present work it is concluded that gamma globulin of the 7 S type (γG) which represents the main immunoglobulin component of pig colostrum, differs from serum γG globulin by the presence of another type of polypeptide chain, designed L2; the latter was detected after S-sulfonation in starch gel electrophoresis as the fastest moving component and its immunoelectrophoretic pattern shows the presence of two precipitating components.Preparation of soluble heavy and light chains enabled us to study them imunochemically. The heavy chain is represented by two zones; the fainter one was not detected in comparative analysis of the heavy chain of serum γG globulin. The L1 chain of colostral gamma globulin and the light chain of serum gamma globulin seem to contain three precipitating components, one of which appears due to aging of the chain solution in the presence of glycine. The material from colostrum seems to contain a larger amount of this component than serum. Further it was shown that the component arising in this way is antigenically closely related to the heavy chain.AbstractНа основании полученных в настоящей работе результатов можно сделать вывод, что гаммаглобулин типа 7 S, представляющий собой главный иммуноглобулиновый компонент свиного молозива, отличается, как было установлено после S-сульфонации, от сывороточного гамма-глобулина G благодаря присутствию еще одного типа полипептидной цепочки, называемой L2, которая при электрофорезе на крахмальном геле является наиболее быстро движущимся компонентом и которая при иммуноэлектрофорезе характеризуется присутствием двух преципитирующих компонентов.Приготовление растворимых цепочек типа Н и Л сделало возможным их иммунохимическое изучение. Цепочка Н представлена двумя зонами, более узкая из которых не была обнаружена при сравнительном анализе цепочки Н сывороточного гаммаглобулина G. B цепочке L1 гаммаглобулина молозива и в цепочке L сывороточного гаммаглобулина были обнаружены З преципитирующих компонента, один из которых возникает в результате старения раствора этой цепочки в присутствии глицина. На основе данных произведенных до сих пор анализов кажется вероятным, что в материале молозива этого компонента имеется больше, чем в сыворотке.—Далее, было показано, что возникающий таким образом компонент отличается близким антигенным родством с цепочкой Н.

The effect of rivanol on the immune globulins of human colostrum

The behavior of immune globulins of colostrum was investigated during fractionation using rivanol and ethanol. It was found that, in contrast with serum immune globulins, a part of these proteins present in colostrum is precipitable with rivanol. It was also observed that the fraction of colostrum immune globulins which yields a heavy precipitation line in the cathode region of the immunoelectrophoreogram precipitated with normal antihuman serum, represents a heterogeneous mixture of proteins, from the point of view both of their electrophoretic mobility and of interaction with rivanol and solubility in dilute ethanol. We are dealing here with at least three types of protein molecules which all display identical antigen relationship to serum γ-and β2A-globulins. No differences were found between serum β2M-globulin and the colostrum protein of similar properties.AbstractИсследовалось поведение иммунных глобулинов молозива при фракционировании с применением риванола и этанола. Было установлено, что—в отличие от иммунных глобулинов сыворотки —часть этих белков, содержащихся в молозиве, сворачивается под действием риванола. Далее, было обнаружено, что фракция иммунных голбулинов молозива, которая при преципитации нормальной антигуманной сывороткой дает на иммуноэлектрофореограмме выраженную линию преципитации в катодной области, представляет собой смесь разнородных белков—как с точки зрения электрофоретической подвижности, так и с точки зрения взаимодействия с риванолом и растворимости в растворе этанола. Это касается не менее чем З видов белковых молекул, и все они обнаруживают одинаковое антигенное родство с гамма- и бета2A-глобулинами сыворотки. Между бета2M-глобулином сыворотки и белком, отличающимся этими свойствами и присутствующим в молозиве, не было обнаружено разницы.

A component of the fraction of porcine light chains structurally related to heavy chains

In the previous paper (Rejneket al., 1967) we described the fractionation of light chains (L) by Zn ions resulting in an accumulation of antigenic determinants of the heavy chain (H) in the Zn precipitate. Peptide maps of the obtained fractions of the L chains differ considerably from each other. Peptides of the L chains, the position of which corresponds within the experimental error to peptides of the H chain may be detected by comparing them with the peptide map of the H chains. The number of such peptides increases with qualitatively assayed accumulation of the component precipitated with anti-H serum during fractionation. The concentration of N-terminal glutamic acid, characteristic for the H chains increases at the same time.

Presence of allotypic determinants on polypeptide subunits of rabbit gamma globulin

Inhibition of passive haemagglutination showed the presence of the allotypic specificities Aa1 Aa2, Aa3, Ab4, Ab5 and Ab6 on polypeptide subunits of rabbit IgG belonging to the phenotypes Aa1-3/Ab4-4 Aa2-2/Ab4-4, Aa3-3/Ab4-4, Aa3-3/Ab4-5 and Aa3-3/Ab4-6, prepared by oxidative sulphitolysis followed by isolation on Sephadex G-100 in 6m urea and 0.05m formic acid. The determinants Aa1, Aa2 and Aa3 were found only on H chains and Ab4, Ab5 and Ab6 only on L chains. Of the latter, Ab4 and Ab5 were found in both fractions, i.e. L1 and L2, of the light chains, while Ab6 was found only in fraction L1.