SERUM ANTIGENS OF CATTLE. IV. IMMUNOGENETICS OF A SOLUBLE ANTIGENIC DETERMINANT DERIVED FROM

LYSED ERYTHROCYTES1

JAN W A C Z , NANCY KORDA AND W. H. STONE

Laboratory of Genetics, Uniuersity of Wisconsin, Madison, Wisconsin 53706

Manuscript received September 10, 1974

ABSTRACT

This paper describes an allotypic system in cattle called Ec (Erythrocyte cattle). The antigenic determinant is derived from red cells and is detected in lysates by gel precipitation with alloimmune serum. The Ecl specificity is controlled by an autosomal dominant gene and occurs with variable fre- quencies in different cattle breeds. The Ec system is a very useful genetic marker because the homozygous and heterozygous genotypes show a dosage effect and can be distinguished by the size of the precipitin rings in double diffusion gel precipitation tests.

N the first paper of this series (RAPACZ, KORDA and STONE 1968) we described I a n allotypic system, called M c l , which contained an antigenic determinant carried by macroglobulin molecules. Later, BLAKESLEE, BUTLER and STONE (1971) described two immunoglobulin specificities: an AI marker ( B u d ) which was located on the Fc portion of IgG, heavy chains and a B1 marker (B&) presumably on the light chains and found on IgG, IgA and IgM molecules. More recently, BLAKESLEE, RAPACZ and BUTLER (1971) discovered the antitheti- cal allele (Bva,) of the Bval gene.

This paper describes another system which is also detected as an allotype in serum. However, the antigenic determinant in this system is derived from lysed erythrocytes and is not the direct product of the serum. We call this system Ec (Erythrocyte cattle) and the determinant Ecl.

MATERIALS A N D METHODS

Preparation of immunogen and immunizations: The immunogen was prepared from an ammonium sulphate fraction of fresh adult cattle serum, as described previously ( F ~ P A C Z , KORDA and STONE 1968). The preparation contained about 70% protein by dry weight. Starch gel and hunaelectrophoretic analyses showed that the preparation contained high concentra- tion of y and /3 globulins, lesser concentrations of a globulins, and lipoproteins and traces of albumin. The ammonium sulphate fractions of the donor serum had the advantages over whole serum of reduced volume, less albumin, and providing for dry, weighed samples. Later, when it was learned that the antigen was derived from lysed erythrocytes (cells), a lysate of cells was used as the immunogen.

Paper No. 1789, Laboram of Genetics. Supported in part by NIH Grants AI-032M and GM454&2 and AEC Grant COO-1210-87.

Genetics 80: 323-329 June, 1975.

324 J. RAPACZ, N. KORDA A N D W. H. STONE

The immunizations were carried out as already described (RAPACZ, KORDA and STCXE 1968). Briefly, each of six adult Holstein-Friesian cows was immunized with immunogen obtained from different donors of either the Brown Swiss or Ayrshire breeds. Only one of the six cows (306) produced precipitins for the Ec factor, and only after ten intramuscular injections at three- to six-week intervals with immunogen from donor cow 73. Each injection consisted of an equal mixture of complete adjuvant (Difco) and antigen preparation containing approximately one gram of antigen. More recently, alloimmunization using partially purified antigen from lysed erythrocytes yielded strong and specific antisera after only three osr four monthly injectioas. lisually, the best antisera were obtained between one and two weeks after the last injection. The antisera were stored frozen without preservative until used.

Gel precipitation tests: After it was discovered that the antigenic determinant was derived from lysed cells, samples for testing were prepared from lysates either by adding distilled water to packed washed cells in a ratio 1:lO (cel1s:water) o r by repeated freezing and thawing of a 10% suspension oE washed cells in an autologous serum. The method used to prepare the cell lysates did not appear to affect the reactivity. The samples were tested by a double diffusion precipitation test on microscope slides, using a modification of the technique described by HIRSCHFELD (1963). 'The details of the technique have already been described (RAPACZ: KORDA and STONE 1968).

Tests with the original antiservm showed that this antigen-antibody system was extremely sensitive to variations in the amounts of reactants. Using a 1:l ratio of antigen to undiluted antiserum, precipitation was observed only when the concentration of erythrocytes in hemo- lysates was between 5% to 20%. The optimum reactions were regularly obtained using a 10% lysate. The antisera obtained later by alloimmunization with lysates were also very sensitive to the concentration of reactants. Therefore, each antiserum was titrated with varying amounts of lysates to determine the concentrations and amounts of each that gave optimum reactions. These tests were performed using a Hamilton Syringe (The Hamilton Co. Inc., Whittier, CA) which permitted accurate pipetting of microliter volumes. With the stronger antiserum, the optimum reactions were obtained using 2 ,pl of antiserum and 6 pl of 10-15% lysate. However, as will be described later, lysates from homozygous cells showed a dosage effect and had to be appropriately diluted for testing. The tests were read after 18 and 36 hrs incubat: ion at room temperature (22"-24") in a sealed moisture chamber, The reactions were fully developed at 18 hrs and easy to read with a small hand lens (1.5X) and blue-filtered light from a microscopic lamp with a small aperture.

Immunoelectrophoresis: A modification of the immunoelectrophoretic procedure described by HIRSCHFELD (1960) was used as detailed previously (RAPACZ, KORDA and STONE 1968). The main precaution was to use 40-50% suspension of cell lysate as antigen; otherwise the reactions were weak and difficult to read.

Radial diffusiosn tests: A modification of the technique described by MANCINI, CARBONARA and HEREMANS (1965) was used as a quantitative test to distinguish between samples from homozy- gous and heterozygous individuals. The details of this single diffusion technique were described by BLAKESLEE, BUTLER and STONE (1971). Briefly, 5 ml of the antiserum-agar mixture are pipetted onto 3 x 4 inch glass slides. After cooling, 2.5 mm wells are punched and filled with the samples to be assayed. As many as 48 samples can be tested with the antiserum on a single slide. The size of the precipitation ring around the antigen well is diagnostic of zygosity.

RESULTS

Serologic studies: One alloimmune serum produced in a Holstein-Freisian cow (306) against the ammonium-sulfate fraction of serum from an Ayreshire cow (73) was used throughout most of these studies. The Ec specificity was first observed using this antiserum in a routine test for lipoprotein allotypes (unpub- lished results). A test serum gave a strong reaction in Ouchterlony gels. The pre- cipitin band stained for protein but not for lipoprotein. Later, another sample

SERUM ANTIGENS OF CATTLE 325

of serum obtained from the same animal did not react with this antiserum. For- tuitously, it was observed that the original serum was heavily hemolyzed, where- as the newer serum was not. A hemolysate of fresh, washed cells from the same animal gave a reaction of identity with the reaction observed in the hemolyzed serum. As shown in Figure I, the specificity detected by antiserum 306 behaved as a single serologic factor, giving reactions of identity with the three adjacent samples of cell lysates. This observation was confirmed by absorption tests in which each positively reacting hemolysate removed the reactions from the anti- serum for all other reactive samples.

In a study of 17 offspring, from as many matings involving only parents who were both positive for the Ecl factor, 7 gave weak diffuse reaction bands close to the antibody well, which disappeared after two days of incubation. This sug- gested that these atypical reactions were the result of excess antigen produced by a double dose of the causative genes. When these 7 samples were diluted 1/2, they gave normal reactions, thus supporting this hypothesis. Confirmation that there was a dosage effect was obtained by using the radial diffusion technique. AS shown in Figure 2, the precipitation ring of a homozygous sample was con- spicuously larger than the ring produced by a heterozygous sample. In addition, the ring produced by the homozygous sample, diulted 50% with a negative sample, gave a precipitin ring equivalent to a heterozygous sample. Similarly, when the heterozygous sample was diluted equally with a negative sample, the diameter of the precipitin ring was reduced proportionately. .

FIGURE l.-Alloimmune antiserum 306 (Ab) detecting the Ecl specificity i s in center well. Wells no. I, !2, 3 and 5 contain hemolysates from different positive cows; wells no. 4 and 5 contain hemolysates fnrm different negative cows. Note the reactions of identits among the hemolysates in wells I, 2 and 3.

326 J. RAPACZ, N. KORDA AND W. H. STONE

FIGURE 2.-A portion of a radial diffusion (double diffusion) test using elloimmune anti- serum (306) with hemolysates from homozygous and heterozygous cows. Well no. 1 contains a homozygous sample (1 724)). Well no. 2 contains the same sample diluted 50% with a negative sample (1 702). Well no. 3 contains a heterozygous sample (1 712). Well no. 4 is the same sample diluted 50% with a negative sample (1702). Note the proportionate sizes of the precipitation rings.

Genetic Studies: Data from 561 matings established that the Ecl factor be- haved as a unit in inheritance. As shown in Table l, the data are consistent with the hypothesis that the Ecl factor is controlled by a dominant gene, E d . As ex- pocted, matings of positive parents did not produce significantly less than 75% positive offspring and matings between positive and negative parents produced at least 50% positive offspring. Three positive offspring were obtained from 345 matings between negative parents. One of these offspring represented a disputed parentage case in which an exclusion was possible using blood types. The parents of the remaining two exceptional offspring could not be blood-typed to determine if the parentage data were correct.

TABLE 1

Phenotypes of parents and ofspring in matings involving ihe &f factor

Tvw of m a t h

Reactioa~ ..i+ mti-Ed Ofispnag

No. + - + X + 55 39 16 + X - 161 a2 79 - x - 345 3* 3a Totals 561 134 437

One of these^ was excluded a the basis of blood types; the other two could not be blood-typed or retested (see text).

SERUM ANTIGENS OF CATTLE

TABLE 2

Segregation data for E d factor from matings of parents with known genotypes

327

offspring

Mating' Genotypes No. of m a w s Ec'/Ecl EC'/ECO E@/@ P

1: Ecl/EcO x Ec'/EcO 8 3 4 1 >.8 2: Ecl/EcO x Ecl/EcO 19 3 13 3 > .4

Totals 27 6 17 4

* 1: Genotypes ascertained by progeny test. 2: Genotypes predicted by radial diffusion test.

As shown in Table 2, the eight specific matings involving two heterozygous (Ec'/Eco) sires whose genotypes were ascertained by pedigree analysis (EcO designates the absence of a detectable allelic product), and eight heterozygous dams whose genotypes were ascertained by radial diffusion tests produced three offspring who tested as homozygotes, four as heterozygotes and one as negative. These results are not significantly different from a 1:2: 1 ratio (p > 0.8).

We were able to study 19 additional matings (Table 2) in which the parents and all offspring were diagnosed as to their zygosity by the radial diffusion tech- nique. The segregation ratio from these matings was very close to the expected 1 : 2: 1 (P > 0.4). Since both homozygous and heterozygous males occur, sex link- age is ruled out. Taken together, these data are consistent with the hypothesis that the Ecl factor is controlled by a dominant allele at an autosomal locus.

Breed distribution: The frequency of the Ecl factor among 1,470 cattle of 9 different breeds is listed in Table 3. The highest frequency was found in the Guernsey breed (63.7%) and the lowest in Red Angus (0.4%). The factor was not found in Black Angus and Jerseys. It shows a wide range of frequencies among the different breeds and clearly is not characteristic of either the dairy or the beef breeds.

Developmental studies: Among 273 calves tested at birth for the Ecl factor, 28 showed strong and specific reactions typical of adults. The reactions of these 28 calves remained stable for up to three years, whereas none of the remaining

TABLE 3

Frequency in descending order of Eci factor in various cattle breeds

Breed No. tested

Guernsey Charolais Brown Swiss Hereford Ayrshire Holstein Red A n g u s Black Angus Jersey

22 214 2.5 2% 24

594 240 99

7

Percent positive

63.7 48.1 36.0 33.4 16.6 8.1 0.4 0.0 0.0

328 J. RAPACZ, N. KORDA A N D W. H. STONE

245 calves became positive. Thus, we conclude that the Ecl factor is fully developed at birth. Tests of samples obtained from 22 fetuses between the ages of 80 and 250 days established that the Ecl factor was detectable, although at reduced levels, as early as 85 days of gestation.

DISCUSSION

The membrane structures of the red cells contain many immunogenic com- ponents, as evidenced by the large number of antigenic polymorphisms identified as blood groups. In contrast, polymorphic systems of intracellular or even sub- surface antigens are exceedingly rare, perhaps because they are difficult to detect by standard serologic technics. IANNELLI, BETTINI and ORESTE (1973) described a sub-surface antigen of the red cells of water buffaloes; and SPOONER (1969) reported a transient IgM precipitin in cattle serum produced against hemoglobin presumably by autoimmunization. In addition, a number of polymorphic enzyme systems can be identified by precipitation in gels using red cell hemolysates.

The workers in cattle blood groups have long known that hemolysins against red cells can often be detected in alloimmune cattle serum produced against plasma presumably free of intact red cells. In retrospect, it is not sur- prising to find that serum contains soluble antigenic substances derived from the red cells. However, it is curious that the Ec factor has not been discovered previously since numerous immunizations with large quantities of red cells have been carried out in cattle for many years. Perhaps it is because antiserum pro- duced against red cells would not normally be tested in a gel precipitation system. Even if such tests were performed, the relative concentrations of reactants would have to be just right to detect a precipitation reaction.

Altzmpts to detect the molecule that carries the Ecl determinant have been underway in our laboratory for some time; the results will be reported elsewhere. As previously indicated. the precipitin band stained as a protein. It was located consistently near the antibody well and jn several other respects behaved like a relatively small molecule with a molecular weight under 100,000.

Immunoelectrophoresis of hemolysates at pH 8.4 showed a single precipitin arc spreading from immediately opposite the antigen well toward the anode. At this pH. the Ecl molecule clearly separated from the hemoglobin. Furthermore, immunoelectrophoresis of hemolysates fractionated on carboxymethyl Sephadex columns (kindly performed by DR. R. NIECE) showed that the Ecl antigen was a non-heme protein.

We have not searched for the Ecl factor in other tissues except to show by absorption that it was probably not present in the red cell stromata. But, since it can originate from red cells at least, it is natural t o speculate that it is carried by an isozyme of the erythrocytes. Tests for correlations with the cattle carbonic anhydrase system (CA) were kindly peri-ormed by Ms. YOSHIKO SUZUKI of the Serology Laboratory, University of California, Davis. There was no obvious cor- relation between Ecl and CA among 45 hemolysates tested. Preliminary tests in our own laboratory did not exclude the possibility that the Ecl determinant was carried by an esterase enzyme.

SERUM ANTIGENS O F CATTLE 329

It would be worthwhile exploring whether or not E d is linked to the locus controlling erythrocyte esterases. Linkage between a serum esterase and a blood group locus has been described in rats by GASSER et al. (1973) and in mice by FOSTER, PETRAS and GASSER (1 974).

We believe that the Ecl specificity, irrespective of its chemical nature, is a useful genetic marker in cattle serum especially because the homozygous and heterozygous genotypes can be distinguished easily.

We wish to thank MR. KENT MCLAUGHLIN for help with the immunizations and for obtain- ing the blood samples. We also thank Ms. JUDY RAPACZ for performing many of the tests involving the families. We gratefully acknowledge the cooperation of our University farms in making the animals available for this study.

LITERATURE CITED

BLAKESLEE, D., J. E. BUTLER and W. H. STONE, 1971 Serum antigens of cattle. 11. Immuno- genetics of two immunoglobulin allotypes. J. Immunocl. 107: 227-235.

BLAKESLEE, D., J. RAPACZ and J. E. BUTLER, 1971 Bovine immunoglobulin allotypes. J. Dairy Sci. 54: 1319-1320.

FOSTER, M., M. L. PEW and D. L. GASSER, 1974 The EA-I blood group locus of the house mouse: inheritance, linkage, polymorphism and control of antibody synthesis. Proc. XI1 Intern. Congr. Genetics Vol. I: 245.

GASSER, D. L., W. K. SILVERS, H. M. REYNOLDS, JR., G. BLACK and J. PALM, 1973 Serum esterase genetics in rats: Two new alleles at Es-2, a new esterase regulated by hormonal factors, and linkage of these loci to the Ag-C blood group locus. Biochem. Genet. 10: 207- 217.

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Serum antigens of cattle. I. Immunogenetics Of a macroglobulin allotype. Genetics 58: 387-398.

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