Univalent fragments of antibody: A study by electron microscopy

Immunochemistry. Pergamon Press 1965. Vol. 2, pp. 169-176. Printed in Great Britain

UNIVALENT FRAGMENTS OF ANTIBODY: A STUDY BY ELECTRON MICROSCOPY J. D. ALMEIDA,~t B. CINADERand D. NAYLOR Subdivision of Immunochemistry, Division of Biological Research, Ontario Cancer Institute; and Departments of Medical Biophysics and Pathological Chemistry, University of Toronto, Toronto, Ontario, Canada (Received 2 November 1964)

Abstract--Univalent fragments, fractions I and II, of polyoma virus antibody, obtained by papain digestion, followed by chromatography, were examined, after negative staining, in the electron microscope. The mean length of the univalent fragments was 70 A and the estimated maximum length was 90 A. Univalent fragments were often seen attached to the virus in V-formation; the width of fragments so arranged was significantly smaller than that of fragments attached randomly. Divalent antibody can also, though rarely, be seen in V-formation. The width of divalent antibody, so arranged, is also significantly smaller than that of the unselected divalent antibody molecules. INTRODUCTION THE fine structure of antigen-antibody aggregates and the dimensions of antibody molecules have been investigated by electron microscopy of shadowed (1-5) and of negatively stained le) preparations. I~-n) Divalent antibody, combined with antigen, appears as a rod-shaped flexible molecule, which is attached to antigen at the two short ends of this cylindrical molecule. The dimensions of antibody molecules, evaluated from electron micrographs, have been found to be in fairly good agreement with those obtained from hydrodynamic measurements.14, m Details of the fine structure of the antibody molecule have not been resolved, though it appears as if the molecule was not uniform throughout its length and consisted of five beads linked by narrow strands, m~ Definitive information on fine structure will depend on advances in the preparation and staining of specimens, and particularly on improvements of supporting materials for specimens and reduction in the granularity of staining reagents. It may, however, also be possible to gain additional information on fine structure from a study of fragments of antibody from papain digests of divalent antibody. Two types of such univalent antibody fragments have been obtained (12~ and are derived from two distinct families of divalent antibody molecules.(13,14~ The interaction of these univalent antibody molecules with antigen differs in several respects from that of divalent antibody molecules, presumably because univalent antibody molecules cannot participate in the formation of antigen-antibody lattices. This view is supported by the increase in inhibitory capacity if the complex of univalent antibody with ribonuclease is treated with divalent antibody, directed against the univalent fragments.~ls) The nature of the complex of three components: antigen, univalent antibody directed against the antigen, and divalent antibody directed against univalent antibody, is thus of some interest. Though the change * Present Address : Department of Medical Microbiology, St. Thomas's Hospital, London, S.E.1, England. 169

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J.D. ALMEIDA, B. CINADER and D. NAYLOR

from divalency, to univalency, could account for most of the altered properties of univalent antibody-fragments, it is also quite possible that additional configurational changes may affect these properties. The shape of the univalent fragments thus deserves attention. In the following experiments, it is our aim to investigate the properties of antibody further--by evaluating the dimensions of univalent rabbit antibody fragments, examining the attachment of such fragments of rabbit antibody to polyoma virus and the reaction of such complexes with divalent antibody directed against fragments of rabbit y-globulin. MATERIALS AND M E T H O D S

Phosphate buffered saline, pH 7.5, 0.0062 M Na2HPO4, 0.00135 M KH2PO4, 0.15 M NaCI. ~161

Polyoma virus. Mouse embryo cells were infected with polyoma virus. Ten days later, cells showing a distinct cytopathogenic effect were frozen and thawed three times. The suspension was centrifuged at low speed for 10 rain and the resulting supernatant was centrifuged for 1 hr at 40,000 g. An aliquot of the pellet, so obtained, was suspended in distilled water and checked in the electron microscope. The remainder was suspended in phosphate-buffered saline. Immunization. Antibodies to polyoma virus were raised in rabbits by repeated intravenous injection. Antibodies to papain fragments of rabbit y-globulin were raised in goats. Preparation of y-globulin. A serum fraction insoluble in 12 per cent Na~SO4 was prepared ~17~and was dialyzed against phosphate-buffered saline. Preparation of univalent fragments of rabbit antibody. Treatment of rabbit 7-globulin with papain and isolation of fragments was carried out by the method of Porter.ll2~ Compounds qf virus and divalent antibody. To assay the concentration of virus, the virus was mixed with a known concentration of 880 A latex particles and the number of virus particles was determined by the method of Pinteric and Taylor. ~ts~ Immune sera were clarified by centrifugation at 2°C and 34,000 g for 2 hr. Constant quantities of virus were mixed with varying quantities of immune sera. The volume of the mixtures was kept constant by the addition of phosphatebuffered saline. The mixtures were kept for 1 hr at 37°C and then at +2°C overnight. They were subsequently centrifuged at 2000 rev/min at +2°C and the precipitates were then washed twice with a volume of phosphate-buffered saline equal to the original volume of the antigen-antibody mixture. Compounds of univalent fragments of antibody and virus. Preparations of Porter's fraction I and II were clarified by centrifugation for 2 hr at 2°C at 34,000 g in a Servall centrifuge. Univalent antibody and virus were mixed, kept for 1 hr at 37°C and then overnight at +2°C. The mixtures were then centrifuged for 2 hr at 17,000 rev/min and 2°C in a Servall centrifuge (34,000 g). The precipitates were washed twice with a volume of phosphate-buffered saline equal to the original volume of the antigen-antibody mixture. Complexes between virus, univalent fragments and divalent antibody directed against the univalent fragments. Compounds between univalent fragments and virus were

Umvalent Fragments of Antibody: A Study by Electron Microscopy

171

prepared and were suspended in phosphate-buffered saline. Divalent goat antibody, directed against univalent fragments of 7-globulin, were added to these suspensiom. The resulting mixtures were kept at room temperature for 10 min and were then centrifuged. The precipitates were washed and then centrifuged at 2000 rev/min and 2°C. Electron microscopyof complexes. Washed pellets of the antigen-antibody complex were suspended in a small quantity, usually 0.1 ml, of distilled water. Immediately afterwards, one drop of this suspension was mixed with an equal amount of 3 per cent phosphotungstic acid adjusted to pH 6 with potassium hydroxide. A drop of the virus-phosphotungstate mixture was placed on a 400 mesh carbon-formvarcoated grid, allowed to settle for a few seconds, and excess fluid was gently withdrawn with filter paper. As soon as this preparation was completed, the grid was placed in the electron microscope. Micrographs were taken at an initial magnification of 40,000 and 94,000, using the double condenser illumination. RESULTS The structure of aggregates between varying quantities of divalent antibody and polyoma virus has been previously described. The region of extreme antibody excess was defined in terms of the absence of antibodies cross-linking antigen molecules, m) Some of the salient features of complexes between divalent antibody and virus will be shown here, for comparison with the complex between univalent antibody and virus. The outline of icosahedral polyoma virus is quite clear and sharp when the virus is not combined with the antibody (Fig. l(a)). This appearance is obscured, however, when the virus has been treated with divalent antibody. When antigen is in excess, or is at equivalence, particles of polyoma virus appear in partially ordered arrays (Fig. l(b)). Individual antibody molecules can be seen stretched between two virus particles. Such stretched molecules occasionally appear as if they were beaded. When divalent antibody was present in extreme excess, stretched molecules of antibody were never seen. All the antibody molecules are kinked and bent and the dense tangle surrounding the antigen rarely allows the course of an individual antibody molecule to be clearly followed (Fig. l(c)). The appearance of complexes of virus with univalent antibody fragments Fig. l(d-f)) is very different from complexes formed between virus and divalent antibody (Fig. l(b-c)). The virus particles appear to be spiked with rods which seem more rigid and are strikingly shorter than the divalent antibody molecules. In contrast to divalent antibody (7 per cent), the univalent antibody molecules can frequently (16-23 per cent) be seen, combined with virus, in V-formations, in which two univalent antibody molecules appear to be attached to one capsomere. Occasionally it appears as if univalent antibody molecules were beaded, and in these cases, two beads seem to compose the antibody molecule. The general appearance of univalent antibody fragment I and fragment II is very similar. The dimensions of univalent fragments of antibody molecules were obtained by direct measurement. Mean width and mean lengths are given in Tables 1 and 2. The standard errors quoted, are the standard errors of the means, themselves, and not those of the individual variations; they are small because each mean is calculated

J. D.

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CINADERand D. NAYLOR

from many observations. The mean width of fragments in V-formation was found to be lower than that derived from measurements of antibodies not arranged in V-formation. This held true for both univalent fragments, fraction I and fraction II (Table 1). Any two widths, based on measurements of unselected divalent antibody, or univalent fragments (fraction I and fraction II), were significantly different at the 1 per cent level. Any two mean widths of molecules in V-formation were significantly different at the 5 per cent level. These conclusions are based on Scheffe's multiple comparison procedure. TABLE I . THE WIDTH OF DIVALENT RABBIT ANTIBODY AND OF UNIVALENT FRAGMENTS

Fraction I, in total Fraction I, V-formation Fraction II, in total Fraction II, V-formation Divalent antibody, in total Divalent antibody, V-formation

Mean A

S.E. of mean A

Maximum# A

24.8 21.4 21.9 19.3 27.6 22.5

0.32 0.40 0.40 0.46 0.19 0.75

34.4 31.5 29.3 28.4 41.3 27-5

Difference in Distribution x2 d.f. "~ 66.4

4

l

16-6

3

25.4

4

* The maximum quoted is the largest observation plus a small correction for grouping. TABLE 2. THE LENGTHOF UNIVALENTFRAGMENTSOF RABBITANTIBODY

Fraction Fraction Fraction Fraction

I, in total I, V-formation II, in total II, V-formation

Mean A

S.E. of mean A

Maximum* A

67.5 72.5 70.0 71.2

0.86 1.24 1.09 1.25

97.5 93.8 93.8 92.5

Difference in Distribution xz d.f. 9-22

7

1.45

6

* The maximum quoted is the largest observation plus a small correction for grouping. As already mentioned, divalent antibodies are only rarely (7 per cent) seen to be combined in V-formation. However, a careful search of photographs revealed that such formations can be found, occasionally. A comparison of widths of divalent antibody arranged in V-formation with unselected divalent antibody molecules again showed that antibodies arranged in V-formation had a narrower width than those not so arranged (Table 1). The maximum length of univalent fragments was estimated to lie between 92.5 and 97.5 A. The difference between lengths of univalent fragments, fraction I and fraction II, was not significantly different from zero at the 5 per cent level, either for measurements of unselected antibody molecules or for measurements of molecules in V-formation (Table 2). There was no significant difference between the length of univalent fragments, fraction I, and the length of univalent fragments, fraction II. Since the length of divalent antibody has been previously estimated to be 250-270 A (11) the length of univalent fragments is approximately one-third of the length of divalent antibody.

9

ii.....

e

FIG. |. Electron micrographs, Magrrrfication / 2 8 0 , 0 0 0 .

To face p. I72

see over for legend

(a) Polyoma virus, not treated with antiserum. The outline of the virus is smooth; the capsomeres can be clearly seen. (b) Polyoma virus combined with divalent rabbit antibody. The structure of the virus is obscured. Some antibody molecules can be seen linking two virus particles. The antibody molecules linking two virus particles appear as straight rods. Arrow points to a stretched divalent antibody molecule showing beading. (c) Polyoma virus combined with excess of divalent rabbit antibody. The virus particles are completely surrounded by a halo of antibody molecules. The divalent antibody molecules, anchored on only one antibody molecule, show considerable flexibility in at least one and frequently several places. (d) Group of polyoma virus pamcles combined with univalent rabbit antibody, fraction I t12~ showing short, relatively rigid rods, attached to the wrus particles. Arrow points to antibody in V-formation. (e) Group of polyoma virus particles, combined with univalent rabbit antibody, fraction II a-"~ showing short, relatively rigid rods, attached to the virus particles. Arrow points to antibody in V-formation. (f) Group of polyoma virus particles combined with univalent rabbit antibody, fraction II cte~ showing V-formatmn, presumably due to the attachment to the same capsomere of two univalent fragments. Arrow points to antibody In V-formation. (g) Polyoma virus treated with univalent fragments (fraction I) of rabbit antibody and subsequently with divalent antibody directed against the umvalent fragments. The haloes of antibodies surrounding the virus particles form a ring approximately 300 ~- wide, considerably wider than the haloes surrounding virus particles treated with umvalent antibody, only (d and e). The antibody molecules are more nearly aligned to radial potations than those seen attached to v~rus pamcles treated with divalent rabbit antibody, only (c). Virus particles not treated with univalent antibody and treated with divalent goat antibody directed against univalent fragments of rabbit gamma globuhn show no attached rod-shaped structures, and are not distinguishable from untreated virus pamcles shown in (a).

Umvalent Fragments of Antibody: A Study by Electron Microscopy

173

When divalent goat antibody, directed against univalent rabbit fragments, was allowed to combine with a complex between univalent fragments and the virus, complexes were seen which differed from the initial compound between univalent antibody and virus, and from virus particles treated with divalent virus antibody. The halo of antibody molecules surrounding the virus (Fig. l(g)) was much wider than the halo, surrounding viruses, treated with univalent fragments only (Fig. 1(d-f)). The antibody molecules in the three component complex seemed to be in a more orderly arrangement and the periphery of the antibody molecules was much closer to a circle, than in the complexes between divalent virus antibody and the virus (Fig. 1(c)). The distance between the edge of the virus particles and the periphery of the antibody halo was approximately 300 A. V-formations could be seen much more frequently than in the complex between divalent virus antibody and the virus. When virus particles were treated with divalent goat antibody molecules, directed against monovalent fragments of rabbit 7-globulins, no change in the appearance of the virus particles was noted and they were undistinguishable from untreated virus particles as shown in Fig. l(a). DISCUSSION To identify the rods, seen in electron micrographs, with antibody, their specificity must be demonstrated. We have done this, previously, with divalent antibodies, by showing that a mixture of two different icosahedral viruses, distinguishable by their size, and treated with antibody to one of the two viruses, contains the rod-shaped appendages only on the virus against which the antibody was raised, m~ In the present study, proof of specificity is obtained from a different kind of experiment in which a goat antibody, directed against rabbit y-globulin fragments, was employed. If virus preparations were treated with this divalent antibody, the electron microscopic appearance of treated particles showed no difference from untreated particles. If, however, the complex between virus and univalent antibody was treated with the divalent antibody, the appearance of the resulting complex was quite distinct from that of the complex between virus and univalent antibody, alone. The periphery of the halo that surrounded such virus particles was about 300 n wide, and since the filaments in this halo were rarely arranged along straight radial lines their length was clearly in excess of 300 A, and hence much greater than the previously estimated length (250-270 A) of goat antibody. The combination of divalent antibody (250-270 A in length) with univalent antibody 90 A in length, would account for the width of the halo. This experiment, therefore, provides evidence that the short rods seen attached to virus, which has been treated with univalent antibody, constitute fragments of rabbit ~-globulin. The difference between estimates of width for randomly chosen antibody and for antibody in V-formation may be connected with overlap between antibodies, not in V-formation, or with asymetry of the antibody molecule and a special orientation of those seen in V-formation. The first of these alternatives is based on the assumption that univalent antibody fragments have a circular cross section and are always arranged in V-formation so

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B. CINAI)ERand D. NAYLOR

that those not seen in V-formation are oriented at an angle to the photographic plane. In the latter case, the two molecules constituting the V-formation, may not be exactly superimposed and measurements of width may consequently include the overlapping part of a second molecule. Overlap would rarely occur when molecules are observed in V-formation and measurements, confined to molecules seen in this formation, would include a relatively large proportion of values close to the true width. The circular cross section of the molecule would be obtained from the mean of measurements of molecules in V-formation. The second of the above alternatives to be considered, is based on the assumption that antibody molecules have an elliptical cross section and that those seen in V-formation are all uniformly aligned, possibly because they are directed against the same determinant of the virus-capsomere. Antibody molecules, as seen on equatorial capsomeres, would all be oriented along one particular axis of the elliptical bases. Antibody molecules, not seen in V-formation, would be directed to a variety of determinants or combined with the single capsomere determinants, not on 'equatorial' capsomeres, so that a variety of aspects of the width of the molecule would be measured when unselected antibody molecules are considered. The dimensions of the two axes of the elliptical cross section may be approximated from the mean estimate of width based on measurements of the antibodies in V-formation and from the maximum width-estimate for the randomly chosen molecules. T A B L E 3. A COMPARISON OF THE PERCENTAGE VOLUME OF UNIVALENT FRAGMENTS BASED ON THE ASSUMPTION OF CYLINDRICAL SHAPE W I T H EITHER CYLINDRICAL OR ELLIPTICAL CROSS SECTION AND OF PERCENTAGE MOLECULAR WEIGHTS BASED ON HYDRODYNAMIC DATA

Hydrodynamic data ~12,t9~

Data from electron micrographs Molecular species

Fracuon I Fraction II Divalent antibody

Cylinder with circular cross section Volume in A3

Per cent of divalent antibody

34,400 27,300 107,400

32 25 100

Cyhnder with elliptxcalcross section Volume in A8 55,300 41,400 197,200

Per cent of Per cent of divalent Molecular dwalent antibody weight antibody 29 21 100

50,000 53,000 188,000

27 28 100

The alternative interpretations of differences between width measured on randomly chosen and on antibody in V-formation would thus lead to different estimates of the cross section and hence the volume of fragments and intact antibody molecules. From these different estimates of the dimension of the base of the cylinder, and assuming cylindrical shape and equal density of the molecules, ratios of molecular weight can be determined and compared with similar ratios based on hydrodynamic data. As can be seen from Table 3, the two models differ considerably in absolute volumes but little in volume-ratio between univalent fragments and divalent antibody. The volume ratios are in reasonable agreement with those based on the molecular weights reported by Porter~12,20~ and Charlwood,tin though the molecular weight of ~,-globulin (188,000), reported by these

Univalent Fragments of Antibody: A Study by Electron Mteroscopy

175

workers is greater than the usually accepted value of 150,000. It is of some interest, in this connection, that the dimensions of the divalent model with elliptical cross section (250 A × 4 1 A×22.5 A; v - - 2 . 0 x l 0 5 h 3) is in fair agreement with that obtained by X-ray scattering experiments (240 A × 5 0 A × 2 2 A; v = 2.0 × 105 Aa). (21~On the basis of a specific volume of v20 ~ 0.745 (Schultze, quoted from 22) the molecular weight of a molecule of the shape of a cylinder with a circular base would be approximately 87,000, that of the elliptical cylinder would be approximately 159,500. The latter value is in good agreement with well-established molecular weights determined by independent methods.~23, 24) Next, we would like to consider and to dismiss the possibility that the observed V-formations of univalent antibody represent a single fragment, with the combining site at the apex. If this assumption were made, the values for the volume of univalent fragments, quoted in Table 3, would have to be doubled and the relative contribution of each univalent fragment to total volumes of divalent antibody would be near or above 50 per cent and thus would be unreasonably high. After the fragments have been split off, the combining site is still at the extremities of the rod-shaped molecule. The basic structure of the univalent fragment seems to be little altered by being split from the remaining portion of the molecule though some contraction of the cross-sectional area may have occurred. In most cases, divalent and univalent antibodies appear as smooth rods. Where detail can be seen, it appears as if five beads constituted the divalent antibody molecule and two beads constituted the univalent antibody molecule. It is difficult to interpret this observation. If the latter is true, two possibilities may be envisaged; several subunits may be linked by narrow chains; alternatively, a cylindrical molecule may be kinked in several places. In the latter case, the phosphotungstate may fill the spaces of the molecules and thus produce the beaded appearance. It is not known whether beading is artifact or reality. The appearance of the univalent antibody fragment is such, that it may be considered one part of a divalent antibody molecule (25)which has undergone minor conformational changes, after its separation from the remaining parts of the divalent molecule. The fine structure of some proteins of low molecular weight appear to have been reasonably well-resolved in the electron microscope and some evidence of the shape of polypeptide chains has been discerned.(26-29) We have no comparable information on the antibody molecule or on the fragments obtained from it. It has been pointed out that the detail obtained with negatively stained preparations may be less for molecules which have open structures and internal hydration; the contrast obtained in negatively stained preparations may thus depend to a greater extent on the structure of proteins under examination and on the consequent penetration of the staining reagent than it does on the resolving power of the microscope or the supporting material for the specimens. (29~

Acknowledgements--Thanks are due to D r M. Cohn for the gift of goat antibody directed against fraction I of rabbit v-globulin; to Dr Rose Sheinin for the lysates of polyomamfected cells and to Dr Ralph Wormleighton for stattstical advice and computations. This work was supported by the Me&cal Research Council of Canada (Grants MT-832 and MA-1589); the National Cancer Institute of Canada; and the National Institutes of Health (Grants 5 T G M 506-03 and CA-04964).

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J . D . ALMEIDA,B. CINADERand D. NAYLOR REFERENCES

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