fixation ofthe first of complement (c'la)...

Fixation of the First Component of

Complement (C'la) by Human AntibodiesWENDELLF. ROSSEwith the technical assistance of JuDrm PARKER

From the Duke University Medical Center, Department of Medicine,Durham, North Carolina 27706

A B S T R A C T The fixation of the first compo-nent of complement (C'la) by human antibodiesand human cells has been studied by the use of theC'la fixation and transfer test (C'la FT test) ofBorsos and Rapp.

Cold agglutinin antibodies appear to requireno more than one antibody molecule to fix onemolecule of C'1a.

Most warm agglutinin antibodies are IgG inimmunoglobulin type and require at least twomolecules of antibody to fix a molecule of C'la.Donath-Landsteiner antibody has the same re-quirements for C'la fixation. A single example ofa warm agglutinin antibody which appears to re-quire one molecule of antibody for the fixation ofC'la was found.

Antibodies of the Rh system do not fix signifi-cant amounts of C'la in the absence of anti-anti-body when antiserum of a single Rh specificitywas used. However, when three antisera at dif-ferent specificity are present, C'la may be fixed.Under these conditions cells from a patient withparoxysmal nocturnal hemoglobinuria may belysed when fresh serum is added to provide theother components of complement.

The presence of IgG antibodies could be detectedby the use of anti-IgGHu antiserum and a one-to-one relationship between the concentration of anti-serum in the reaction and the amount of C'la fixedcould be established.

The effect of temperature, ionic strength, pa-painization of the red cells, and repeated washingof the red cell-antibody aggregates on the amountof C'la fixed was investigated. Conditions of maxi-

Received for publication 28 December 1967 and in re-vised form 15 July 1968.

mal C'la fixation were established for each classof antibodies.

Globulins present in normal isologous or autolo-gous serum are absorbed in small amounts to nor-mal red cells in a manner analogous to warm ag-glutinin antibody. Their presence is detectable bythe C'la fixation and transfer test only with anti-globulin antiserum.

Within certain limits, the C'la fixation andtransfer test provides a quantitative measure ofthe reaction of human red cells and antibodies toantigens on their surface.

INTRODUCTIONThe fixation by antibody of complement compo-nents to red cells of patients with immune hemo-lytic anemia is, in certain circumstances, an im-portant event leading to the destruction of thosecells. In some instances, complement may damagethe cell by making small "holes" in the cell mem-brane (1). When this occurs, intravascular he-molysis results with consequent hemoglobinemiaand hemoglobinuria. In other instances, the pres-ence of complement components on the surface ofthe circulating red cell appears to render the cellabnormal and to lead to the sequestration and de-struction of the cell by the reticuloendothelial sys-tem (2). Hence, information about the fixationof complement by human antibodies is importantin understanding the events leading to hemolysis inpatients with immune hemolytic anemia.

The first step in the fixation of complement byantibody is the fixation of the first component,C'1 (3) .1 Once this component is attached by

1The following symbols are commonly used in dis-cussing the reactions of complement: E = a red blood

2430 The Journal of Clinical Investigation Volume 47 1968

antibody, the remainder of the components willinteract sequentially if all are available and cationand temperature requirements are met. Borsos andRapp (4) and Ishizaka, Ishizaka, Borsos, andRapp (5) have studied the fixation of C'la by avariety of rabbit antibodies and by human isoim-mune anti-A antibodies. They have shown thatwith these antibodies, a single 1gM antibody mole-cule is sufficient for the fixation of a molecule ofC'la whereas two IgG antibody molecules injuxtaposition are required.

In the present investigation we have studied thefixation of C'la by "autoimmune" and isoimmunehuman antibodies to human red cells, using theC'la fixation and transfer test of Borsos and Rapp(6). By this test, the number of molecules ofC'la fixed by antibody on each red cell may be cal-culated and the relationship of this to the amountof antibody present may be determined. Thistechnique not only provides a method for deter-mining the way in which C'la is fixed but also pro-vides a quantitative measure of the amount ofantibody attached to the red cell.

METHODS

A. Buffers and complement components

Buffer. Veronal-buffered saline (VBS) was made ac-cording to the formula given in reference 3. When buf-fers of reduced ionic strength were required, this bufferwas mixed with appropriate amounts of a buffered iso-tonic sucrose solution made according to the formulagiven in reference 7. VBS-sucrose buffer refers to asolution of 40% VBS-60% isotonic sucrose (,g =0.065)unless otherwise stated.

EDTA solution. A 0.1 M solution of EDTA (ethy-lenediaminetetraacetate, disodium salt) pH 7.4 was madeaccording to the formula given in reference 8. Lower

cell (usually from sheep unless otherwise indicated),A = antibody to that red cell (usually made in rabbitsunless otherwise indicated), and C' =complement takenas a whole. The individual components are denominatedby numbers 1-9 and the activated form of the compo-nent is indicated by the letter "a" (e.g. C'la = the acti-vated form of the first component of complement). Cel-lular intermediates in which some but not all of thecomponents of complement are present at the cell sur-face are denominated by symbols such as EAC' la, 4.The sequence of reactions of complement componentsis thought to be as follows: E+A -*EA + C'1EAC'la + C'4 .-* EAC'la, 4 + C'2 -> EAC'la, 2EAC'la, 4, 2a + C'3 -- EAC'la, 4, 2, 3 + C'5 -> EAC'la,4, 2, 3,5 . . +C'9*EAC'la, 4, 2, 3, 5, 6, 7, 8, 9lysis.

concentrations of EDTA were obtained by diluting thisisotonic solution with appropriate amounts of VBS.

Alsever's solution was made according to the formulagiven in reference 3.

Whole blood was obtained weekly from healthy sheepand was stored at 4°C in an equal volume of Alsever'ssolution.

Complement. Fresh frozen guinea pig serum, ob-tained from Suburban Serum Laboratories, SilverSprings, Md., was absorbed twice with a 1/10 volumeof washed sheep cells, and was stored at -90°C.

C'la. A solution containing a high concentration ofC'la and lacking C'2 and C'4 activity was prepared fromguinea pig serum by the method of Borsos and Rapp (9).This solution contained at least 10 molecules of C'la/ml and was absorbed twice at 370C and twice at 0°Cwith packed human red cells before use.

C'2. A solution containing a high concentration ofC'2 and lacking C'la, C'4, and "C'3" activity was pre-pared from guinea pig serum by the method of Borsos,Rapp, and Cook (10). This solution contained at least5 X 10" molecules of C'2/ml as determined by themethod of Borsos and Rapp (11).

EAC'4 was made according to the method given inreference 3. In later experiments, the revised method ofBorsos and Rapp, using EAC'la and C'-EDTA, wasused for making EAC'4 (11).

B. Humanmaterial

Human red blood cells (El'). Blood was removedaseptically by venipuncture, placed with an equal amountof Alsever's solution, and stored at 4C. At the timeof use, the cells were incubated with a 1/10 volume ofisotonic 0.1 M EDTA for 5 min, washed once in 0.01 MEDTA in VBS and three times in either VBS orVBS-sucrose. One part of packed cells was added to 18parts buffer and this suspension was adjusted so that a 1in 25 dilution in 0.01 M NH4OHhad an optical densityin a spectrophotometer (Beckman Instruments, Inc., Ful-lerton, Calif.) of 0.210 at a wavelength 541 mg in a 1.000cm cuvette. Such a suspension contains about 2.2 X 10'red blood cells/ml.

Human red cells were treated with papain as follows.Equal volumes of a standard suspension of EHU in VBSand of a 1% papain solution in VBS were -mixed andincubated for 30 min at 37°C. The cells were washedthree times with VBS and suspended in the originalvolume of VBS or VBS-sucrose.

Antisera. Serum from patients with hemolytic dis-ease due to cold-reacting antibodies was obtained fromblood drawn with a warmed syringe and kept warmuntil the clot had separated. After centrifugation at37°C, the serum was heated to 56°C for 30 min and wasstored at - 20°C until use.

Serum from patients with hemolytic disease due towarm-reacting antibodies was obtained from blood col-lected at room temperature and allowed to 'lot at -roomtemperature. This serum was stored and used as outlinedabove. Eluates were made from the red cells of pa-

Fixation of C'la by Human Antibodies 2431

TABLE IBrief Clinical Summary of Patients with Immune Hemolytic Anemia

DirectCoombs

Lowest testDura- recorded Retic-tion of hemo- ulocyte anti- anti-

Patients Age Sex disease globin count' IgG C' Treatment Other disease

yr yr g/100 %ml

Cold-reacting antibodyAgglutinin

M. G. 63 F 13* 6.3 10.0 - + Steroids,t 6-MP§ Carcinoma of colonT. B. 60 M 8.0 7.2 14.0 - + ?A. D. 58 F 0.5* 5.8 22.0 - + Carcinoma of colonA. P. 60 F 0.4* 4.2 5.0 - + Steroids Carcinoma of lung

Polycythemia rubra veraJ. M. 26 M 0.3* 4.8 2.0 - + Steroids, 6-MP, vin- Acute lymphocytic leukemia

cristineDonath-Landsteiner

J. J. 24 M 0.3 6.0 18.5 :1 Steroids Hypergammaglobulinemia

Warm-reacting antibodyR. G. 66 F 9.0 4.6 56.0 + + Steroids, splenectomy Systemic lupus erythematosusM. P. 36 F 1.8 6.1 22.0 + + " " Systemic nocardiosisA. G. 23 F 5.0 4.8 60.0 + - " " Thalassemia minorJ. S. 72 M 1.0* 5.4 32.0 +A. P. 42 F 1.5 7.6 5.6 + + " Systemic LupusB. B. 36 M 1.4 6.7 24.0 +A. B. 72 F 1.5* 6.8 12.0 + - " Chronic lymphocytic leukemiaE. B. 63 F 1.0 3.8 52.0 + + " Systemic lupusL. W. 56 M 0.7 10.3 9.3 + -C. F. 1.5 F 1.0 6.3 15.0 + - " Multiple infectionsJ. B. 62 M 0.5 5.5 17.0 + - " Chronic lymphocytic leukemia

* Patient subsequently deceased.t Usually prednisone.* 6-mercaptopurine.

tients with warm-reacting antibodies by the method ofRubin (12). A brief summary of the clinical status ofthese patients is given on Table I.

Isoimmune antibodies of the Rh system were obtainedfrom Ortho Laboratories, Raritan, N. J. or from pa-tients sensitized during the course of pregnancy. Theseantibodies were stored at -20'C until use and did notappear to lose their ability to react as antibodies over aperiod of 18 months.

Human complement. Human serum for complementwas obtained from blood collected by venipuncture fromnormal donors and placed immediately into cooled tubeswhich were centrifuged at once for 4 min at 10,000 rpmin a refrigerated centrifuge. The plasma was removedand allowed to clot. The fibrin clot was removed withapplicator sticks and the serum was stored at -900Cuntil use.

C. Antisera to human immunoglobulinsAnti-human globulin. Rabbit antiserum against whole

human serum (anti-WSH") was obtained from OrthoLaboratories. This antiserum was found on immuno-electrophoresis to react with IgG, IgA, and IgM im-munoglobulins, Bic (C'3), and serum albumin. The anti-IgGHU antibodies were found to be IgG in type by chro-matography on Sephadex G-200 column.

Goat anti-human immunoglobulin antisera. Antibodiesagainst human IgG (IgGHU), IgM (IgMHU), and IgA(IgA'u) immunoglobulins were made by injecting indi-vidual goats with single purified immunoglobulin pro-teins according to the method outlined in footnote 2.These antisera were absorbed with both type I andtype II Bence Jones protein as well as mixed normalhuman red cells of all major blood groups. These anti-sera were monospecific as determined by immunoelec-trophoresis and hemagglutination inhibition.

Rabbit anti-human immunoglobulin antisera. Antibodyto IgGHU, IgMHU, and IgA'u were made by injectingsingle purified human immunoglobulins mixed withFreund's complete adjuvant into the foot pad of rabbitstwice weekly for 2 weeks. The 0.5 ml of the immuno-globulin-containing solution was then injected subcu-taneously three times weekly for 6 more weeks. Bloodwas obtained at weekly intervals thereafter. The serumwas removed after clotting, heated to 560C for 30 min,and absorbed successively with mixed human red cellsand Bence Jones proteins. The anti-IgGHu was absorbedwith purified IgMHU and IgAHU protein.

The anti-IgM"u was absorbed with human fetal serum

2 Buckley, E. E., III, and R. Hornsby. Personal com-munication.

2432 W. F. Rosse

and the anti-Ig A" with serum from a patient withmacroglobulinemia whose serum was deficient in IgA.

D. Procedures

Separation of proteins on Sephadex columns was per-formed according to the method given in reference 13.Sephadex G-200 was boiled 5 min, allowed to settleseveral times while the finer granules were removed,and was put on a 100 cm X 1.0 cm column. The effluentwas collected in 3.5-4.0 ml fractions and analyzed forprotein content by determining the optical density at278 m/A using a Beckman DU spectrophotometer.

The C'la fixation and transfer test (C'la FT test) ofBorsos and Rapp was performed as outlined in refer-ence 6 with minor modifications. The first part of thetest involved the fixation of C'la by EHu and antibody,the removal of the excess C'la, and the release of C'lafrom the cell-antibody aggregates. For cold-reacting an-tibodies, the following procedure was used. 0.1 ml of astandard suspension of EHu was mixed in a siliconized testtube at 00C with 0.1-0.4 ml of antiserum and allowedto incubate for 60 min in an ice bath. If a low con-centration of serum was used, 0.3 ml C'la solution wasadded at the onset of the incubation. If a high con-centration of serum was used, the cells and antiserumwere allowed to incubate 1 hr, were washed twice withice-cold VBS-sucrose, and were incubated for a further60 min with 0.3 ml of C'la solution. At the end of thisincubation, the cells were washed once in ice-cold VBS-sucrose, carefully poured over into a second set of sili-conized tubes, and washed three times more with 8 mlof cold VBS-sucrose. At the end of the washing, thecells were suspended in 9 ml of VBS, appropriate dilu-tions were made, and the amount of C'la in solutionwas determined by the method of Borsos and Rapp (9).

When warm-reacting antibodies were used, the 0.1 mlof a standard suspension of EHu was mixed with 0.1-0.4 mlof antiserum diluted appropriately and the mixture wasincubated for 30 min at 30'C. The cells were washed-twice with 8 ml of VBS-sucrose and 0.1-0.6 ml of anti-globulin antiserum was added if necessary for theexperiment; the mixture was then incubated for 30 minat 30'C. and the cells again washed with 8 ml VBS-sucrose. 0.3 ml of C'la solution was added and the mix-ture incubated for 30 min at 300C. The cell-antibody-C'laaggregates were treated as in the test for cold-reactingantibodies.

The second part of the C'la fixation and transfer testis the determination of the C'la content in solution, us-ing sensitized sheep red cells and guinea pig complementcomponents. After appropriate dilution of the C'la-con-taining solution (to achieve partial lysis of the sheepcells at the end of the experiment), 0.5 ml was mixedwith 0.75 X 108 sheep EAC'4 contained in 0.5 ml of VBS.After allowing the reaction to proceed at 30'C for 15min, 0.5 ml of C'2 solution containing at least 10'° mole-cules of C'2/ml in isotonic sucrose solution was addedand allowed to react for 10 minutes. Following this, 5ml of a 1/50 dilution of guinea pig serum in ice-cold

0.015 M EDTA was added and the cells placed in a370C water bath for 60 min. The remaining cells werethen removed and optical density of the supernatantfluid at a wavelength of 412 m/A was determined in aspectrophotometer (Gilford Instrument Company, Ober-lin, Ohio). Controls for these experiments consisted of:(a) a "cell blank" consisting of 0.5 ml of EAC'4+6.0ml of VBS-sucrose, (b) a C' color blank consisting of1.5 ml VBS-sucrose + 5.0 ml C'-EDTA, (c) a "completeblank" consisting of 0.5 ml EAC'4, 0.5 ml VBS-sucrose,0.5 ml C'2, and 5.0 ml C'-EDTA, and (d) "a completelysis control" consisting of 0.5 ml EAC'4+6.0 ml 0.01MNH40H.

The net optical density of each sample (optical den-sity less the optical density of the cell control and ofthe complement color control) was divided by the netcomplete lysis control (the optical density of the com-plete lysis control less the optical density of the cellcontrol). This fraction was subtracted from 1.0, andthe negative natural logarithm of the result was found(z). The value of z for the complete blank was sub-tracted and corrected value of z was multiplied by thedilution of the original sample. This yields the numberof C'la molecules fixed per EHU, assuming 80%o efficiencyin transfer and detection of C'la molecules (6).

In all experiments, controls in which EHu were incu-bated with C'la in the absence of antibody and (or)anti-globulin antibody were performed. The number ofmolecules of C'la fixed per EHu was usually small (10-50) and this number was subtracted from all experi-mental values. All values represent the mean of two ormore determinations of the C'la content of the solutionderived at the end of the first part of the C'la FT test.

RESULTS

Characteristics of the antibodies

The cold-reacting antibodies. To characterizethe molecular species of the- antibodies containedin the serum of patients with cold agglutinin he-molytic anemia, 2-ml aliquots of serum were chro-matographed on Sephadex G-200 and the effluentwas analyzed for protein content and for antibodycontent as judged by ability to fix C'la to redcells. In each case, the antibody activity was foundin the first (excluded) peak of protein which con-tained macroglobulin proteins. The antibody inantiserum T. Bu. was further characterized bypurification by ultracentrifugation as outlined inreference 1. The antibody activity was found to bein the macroglobulin fraction by this method aswell.

Serum from patient J. J. containing a Donath-Landsteiner antibody was chromatographed onSephadex G-200; the antibody activity was de-


tected using the C'la FT test by reacting 0.2 mlof effluent with E'u, followed by 0.2 ml of anti-WSHUantibody. The antibody activity was foundprimarily in the second peak which contained IgGprotein.

The warm-reacting antibodies. The antibodiesin serum from patients with warm-reacting anti-body hemolytic disease were characterized in twoways. (a) The immunoglobulin type of the anti-bodies was determined by affixing the antibody toEHU, reacting the EHu-antibody aggregates withspecific anti-IgGHu, anti-IgMHu or anti-IgAHuantisera, and determining the amount of C'lafixed by the EHu-antibody-antiantibody aggre-gates by the C'la fixation and transfer test; theresults are shown in Table II.

(b) Certain of the antisera were passed throughSephadex G-200 columns and the antibody ac-tivity of the effluents determined by sensitizing0.1 ml of standard EHu with 0.2 ml of effluent,reacting the EHu-antibody aggregates with anti-WSHU, and determining the amount of C'la fixedto the cells by this combination. With the exceptionof antisera from patients M. W. and M. P., theantibody activity was found in the second peakwhich contains the IgG immunoglobulins.

Immunological specificity of the antibodies

Cold-reacting antibodies. Cold agglutinins areusually classified by their reaction with so-called"I" red cells, i.e., red cells from newborn infantsand red cells from rare adults said to be lackingthe "I" antigen (14). The ability of the cold ag-glutinins used in these studies to fix C'la to nor-mal group 0 red cells was compared to their fixa-tion to group 0 cord cells. The results are re-

TABLE 1 I

Fixation of C'Ia by HumanAntibodies in Combination withHeterologous Anti-IgGHu, Anti-IgMHu, and Anti-IgAHu

Antibodies

Anti-antibody

Anti- Anti- Anti-Antibody Dilution IgGHu IgMHu IgAH~i

C'la molecules fixed/EHfCold-reacting

AgglutininT.Bu. 1/100 0 1238 16

Warm-reactingAutoimmune

J. B. 1/1 155 0 0E. B. 380 0 0A. G. 230 6 2J. S. 390 0 0R. F. 370 0 12M. P. 1355 20 100

Isoimmuneanti-e 180 3 0anti-c 170 3 30anti-D 196 13 102anti-C 95 13 21

Normal SerumJ. P. 1/1 112 30 100

ported in detail elsewhere 3 but are summarizedin Table III. For each antibody, the variation inthe amount of C'la fixed by red cells from dif-ferent donors was great in both groups but themean value was lower in each case for the "Inegative" cells, except for antiserum T. B.

Warm-reacting antibodies. Certain warm-reac-

3 Rosse, W. F., and J. B. Sherwood. Cold agglutininantibodies: differences in C'la fixation by "I+" and "I"red cells. Manuscript in preparation.

TABLE IIIFixation of C'Ia by Cold Agglutinin Antibodies to Red Cells front Adult and Newborn Donors

EHu-adult EHunewborn

Cla molecules fixed/ Cia molecules fixed/EHu EHu

No. of donors No. of donorsAntiserum Reciprocal of dilution tested Mean SD tested Mean SD

V. L. 1600 14 6920 1930 9 3555 1115M. G. 10 24 1470 1410 12 550 520T. B. 100 16 1110 330 8 1340 1570A. D. 100 6 3000 950 10 1620 930A. P. 10 10 1320 485 14 190 80

2434 W. F. Rosse

ting antibodies occurring in "autoimmune" hemo-lytic anemia have been found to be specific forantigens of the Rh system (15), whereas othershave been found to act with all cells possessing anyantigens of this system but not with cells lackingall these antigens, the so-called Rhnull cells (16).The immune hemolytic antibodies used in thepresent experiments were tested with cells ofknown Rh phenotype. Only antibody of patientA. B. was found to have specificity for a definedRh antigen, e (hr").

The ability of autoimmune and isoimmune anti-bodies to fix C'la to Rhnull cells in the presence ofanti-IgGHu antibody was tested (Table IV). Allautoimmune antibodies fixed nearly the sameamount of Cfa to Rhull cells as to normal cellswhen cell-antibody aggregates were reacted withanti-WSHu antiserum. Isoimmune antibodies of

TABLE IVFixation of C'la by Autoimmune and Isoimmune. Warm

Reacting Antibodies to Rh-Containing and Rhk,1(---/---) CeUs in combination with Anti-WSHu

Antiserum

Molecules Cla fixed/EHu

Test cellshaving

Test cells RhuniAntiserum or having Rh (------.)

eluate Phenotype antigens phenotype

AutoimmuneR.F. CCDee 3710 3150M. P. 640 1070A. G. 3240 3020L. S. 2340 1800A. P. 755 730B. B. 425 550A. B. CCD ee 920 18

Cc D Ee 510Isoimmune

Anti-c cc d ee 860 4Cc D ee 1290

Anti-C CCd ee 1110 22Cc D ee 270

Anti-D CCD ee* 580 0Cc D eel 970

Anti-E cc d EE 4020 79ccdEd 2200

Anti-e cc d ee 810 0cc d Ee 430

* Presumed genotype CDe/CDe from family studies.t Daughter of above; presumed genotype CDe/cde fromfamily studies.

Antibody

Cold agglutininV. L.T. Bu.M. G.J.M.A. D.A. P.

Donath-LandsteinerJ.J.

Warmagglutinin*R. F.M. P. eluateJ. S. serum

M. W. eluateNormal serum*

V. P.

Dilution O'C 170C 300C 370C

C'la molecules fixed/lEu

1/16001/1001/101/101/1001/10

930057704830

23029502220

25601410254

5101520

0

0

0

1570

0

1/5 4140 - 137*390 - 18

1/41/11/11/8

40203160

7601090

20602330

850910

160 180

B. Temperature changed during first stage of C'la FT test.

Incubation temperatureMolecules of

1st 2nd Cla fixed/Antibody incubation incubation EHU

M. G. 00 00 109000 370 0

370 370 0

* EHu-antibody aggregates incubated with anti-IgGHubefore C'la was added.

the Rh system did not fix C'la to these cells un-der the same experimental conditions.

The effect of alteration of the test conditionson the fixation of G'la by human antibodies

Temperature. The fixation of Cila by humanantibodies and EHU at different temperatures was

tested. The results are shown in Table V, part A.Cold-reacting antibodies showed a marked de-crease in the amount of C'la fixed at higher tem-peratures whereas the warm-reacting antibodiesshowed a less marked difference or no differenceat all.

To determine whether C'la fixed at 00C bycold agglutinin antibody remained attached to thecell when warmed to 370C, we prepared threetubes containing 0.1 ml of a standard suspension


TABLE V

Effect of the Temperature of Reaction of EHu andAntibody of the Fixation of C'la

A. Tempeiature constant during first stage of Cla FT test.

Temperature

of EHu-adult 0.1 ml of a 1/50 dilution of antiserumM. G. containing cold agglutinin antibody, and 0.3ml of C'la solution. The first tube was incubated at0C for 30 min., the second at 0C for 30 min andthen at 370C for 30 min, and the third was incu-bated at 370C for 60 min. The amount of C'lafixed per EHu was determined at the end of the in-cubations by the C'la fixation and transfer test.The results are shown on Table V, part B. C'lawas fixed by the antibody in the cold but was re-leased during incubation at 370C.

Ionic strength. The effect of ionic strength onthe reaction of antibody and EHUwas tested by re-acting red cells and antibody in either VBS (pu =0.15) or VBS-sucrose (pm = 0.065), washing theantibody-cell aggregates in the respective buffer,and determining the amount of C'la fixed by theC'la FT test. Whenwarm-reacting antibodies wereused, anti-IgGHu in VBS or VBS-sucrose wasadded to the cell-antibody aggregates and the cellswere washed twice with the same buffer beforethe addition of C'la. The results are shown inTable VI; in each instance, the amount of antibodyfixed was increased by the reduction of the ionicstrength by isotonic sucrose.

Repeated washing of the antibody-EH" aggre-gates. The effect of repeated washing of the anti-body-red cell aggregates was assessed by mixing0.1 ml of EHu and 0.1 ml of antiserum or eluate ina series of tubes. These were incubated at 300C

TABLE VIThe Effect of Ionic Strength on the Fixation of

C'la by Antibody

Molecules of C'a fixed/EHu

Ionic strength of reaction mixture. p =0.15 p =0.065

Antibody

Cold agglutininT. B. 1/100 580 715M. G. 1/10 130 210

Warmagglutinin*R. F. 1/1 630 1040

Normal serum*1/1 170 150

Control:Cold 50 57Warm 60 73

* Anti-WSHu antiserum added.t No antiserum added.

TABLE VI IEffect of Repeated Washing of EH.-A ntibody Aggregates

on the Amount of C'la Fixed

No. of times washed

Antibody Dilution 2 3 4 6

C'la molecutes/EHVCold'agglutinin

T. Bu. 1/100 660 520 620 640M. G.* 1/10 155 93 21 -

WarmagglutininR. G.$ 1/4 1570 1700 1700 1700

Normal serumJ. P4: 1/1 150 120 60 50

* No Cla present during incubation of Efu and antibody(see Reference 27).$Anti-WSHu antiserum added to washed EHu-antibodyaggregates.

for 30 min and the cell-antibody aggregates werewashed two, three, four, and six times, respectively.C'la fixation was estimated by the C'la FT test.The results are shown on Table VII.

Papainization of the EHU before reaction withantibody. To assess the effect of papainizationof the red cell on the fixation of antibody, we per-formed parallel C'la fixation and transfer testsusing either unpapainized cells or papainized cellsand several human antibodies. The results areshown on Table VIII.

The fixation of C'la in the presence and in theabsence of antibody to human immunoglobu-lins.

The effect of the addition of heterologous anti-bodies to the human immunoglobulins on theamount of C'la fixed by various human antibodiesto human red cells was determined by C'la fixa-tion and transfer tests. In each two tubes, 0.1 mlof a standard suspension of EHU was mixed withantiserum or eluate and incubated at either 0C or300C. The cell-antibody aggregates were washedand 0.1 or 0.2 ml of anti-WS"u was added to oneof the cell pellets. After incubation for 30 min thecell-antibody-antiantibody aggregates were washedand C'la was added to both tubes. The amount ofC'la fixed was determined by the C'la fixation andtransfer test. The results are shown in Table IX.

The four groups of antibodies differ in theirfixation of C'la in the absence of anti-globulinantiserum. The cold agglutinin antibodies fix largeamounts of C'la even at high dilutions of antiserum

2436 W. F. Rosse

TABLE VI I IEffect of Papainization of the Test Red Cells on the Fixation of C'la by HumanAntibodies

Molecules of Cla fixed/EHuRatio of papainized

Antibody Dilution Non-papainized Papainized nonpapainized

Cold-reactive agglutininV. L. 1/1600 980 1915 1.95T. B. 1/100 490 1720 3.51M. G. 1/10 67 560 8.3A. D. 1/100 100 250 2.5

Donath-LandsteinerJ. J.* 1/8 410 980 2.4

Warm-reactive autoimmune*C. F. 1/1 885 840 0.95R. F. 2070 2250 1.1A. G. 260 2304 8.9M. P. 3240 2440 0.75J. S. 1840 6230 3.4M. W. 1160 1560 1.35

Normal serum*71 149 2.1

* Anti-IgGfu used in tests involving those antibodies.

TABLE IXFixation of C'la by "Autoimmune" Antibodies in the Presence and in the Absence

of Anti-Globulin Antibodies

Neat serum or eluate Diluted serum or eluate

No anti- With anti- No anti- With anti-Antiserum WSHU WSHu Dilution WSHU WSHu

molecules of C'la molecules of C'lafixed/EHW fixed/Emu

Cold-reactiveagglutinin

V. L. 1/1600 380 1150

Donath-LandsteinerJ. J. serum 1/5 540 7970

Warm-reactiveAutoimmune

R. F. serum 380 1230 1/8 21 1260eluate 175 4940

J. S. serumeluate 41 3490 1/4 10 2000

M. W. serum 23 1410eluate 70 2475 1/8 6 1270

E. B. serum 160 370eluate 77 1370

A. B. serum 14 845

M. P. serum 890 2100eluate 250 850 1/2 108 270

Normal serum 21 150


Phenotype- of EHUo

.8 _

. o~~~~~~~~ccddee

.6

4 - ,,--~cc ddEe4

,,,,--cc d

FIGURE 1 (a) The fixation of anti-E (anti-rh") by red cells homozygous, heterozygous,

/ 00,,-' and lacking the E (rh") antigen. The relative2 _ /;#' antibody concentration is plotted on the abscissa,

the number of molecules of C'la fixed in thepresence of anti-globulin antiserum is plotted

cc dd EE on the ordinate; (b) The same as a, using theanti-e (anti-hr") antiserum.

Relative Anti-e Antibody Concentration27

but only somewhat more is fixed in the presence ofanti-antibody (cf. Tables II, III, and IX). TheDonath-Landsteiner antibody fixes a considerableamount of C'la but this is markedly increased bythe addition of anti-human globulin antibodies.Most autoimmune warm agglutinin antibodies fixlittle or no C'la in the absence of anti-human glob-ulin antibodies but fix large amounts in theirpresence.

Antibodies of the Rh system fix insignificantamounts of C'la in the absence of anti-globulinlu.In the presence of anti-antibody, C'la is fixed andthe amount varies, depending upon the antibodyused and the antigenic composition of the cell withwhich it is reacted (see Table IV). In the case

of the antibodies anti-E and anti-e, about 1/2-2/3

as much C'la is fixed at maximal antibody concen-

tration by cells homozygous for the specific antigenas by cell heterozygous for that antigen (see Fig.1). However, no such simple relationship is ap-parent for other antibodies of the Rh system.

The effect of the presence of more than one spe-cific antibody of the Rh system on the red cell at a

time on the ability to fix C'la was tested. Red cellswere incubated successively with one, two, or threeantibodies of the Rh system and the amount ofC'la fixed in the presence and absence of anti-anti-body was measured. Little or no C'la was fixed bysingle antibodies or combination of 2 antibodieswhereas a large amount of C'la was fixed whenall three antibodies are present on the red cellsimultaneously. (See Table X.)

2438 W. F. Rosse

0.

0.

0.'

0

x

wI

0

04-

0

O.

b3

TABLE X

Fixation of C'la and the Lysis of PNHRed Cells (Phenotype Cc D ee) by Rh AntibodiesAlone and in Combination

Molecules of Cla fixed/E %Lysis by C' (whole fresh serum)

Antisera No anti-IgGHu With anti-IgGHu No anti-IgGHu With anti-IgGHa

Anti-c 0 2220 0 30.3Anti-D 27 2550 0 37.9Anti-e 21 1220 0 12.9

Anti-c + Anti-D 105 4770 0 33.0Anti-c + Anti-e 55 3440 0 23.4Anti-D + Anti-e 45 3890 0 39.0

Anti-c + Anti-D + Anti-e 2810 4090 41.3* 41.6

* 42%of the cells were complement-sensitive (see reference 17).

Whencells from a patient with paroxysmal noc-turnal hemoglobinuria were used in such experi-ments, the ability of such cells to be lysed by C'was tested by adding fresh human serum to thecells after sensitization with 1, 2, or 3 Rh anti-bodies with and without anti-antibody. The re-sults are shown in the table. Lysis of about 40%of the cells (all the complement-sensitive cellspresent (17) occurred only in the presence of anti-antibody or, in the absence of anti-antibody onlyif all three Rh antibodies were present on the cell.These studies indicate that Rh antibodies are in-trinsically capable of fixing C'la. The ability tofix C'la and amount of C'la fixed varies with dif-ferent cells and different antibodies. Studies on thecombinations of antigens and antibodies necessaryfor the fixation of C'la are in progress.

The relationship between the amount of anti-body present and the amount of G'la fixedin the absence of anti-antibody.

Cold agglutinins. C'la FT tests were performedin which the amount of antibody reacting withEzu was reduced by twofold serial dilution. Inthe range of limited antibody concentration, thenumber of molecules of C'la fixed is directly pro-portional to the amount of cold agglutinin-contain-ing antiserum in the reaction mixture (Fig. 2).If the logarithm of the relative antibody concentra-tion is plotted against the logarithm of the numberof molecules of C'la fixed, a straight line results,the slope of which is about 1.0 for all cold agglu-tinin-containing antisera tested (see Fig. 3, TableII). This indicates that no more than one anti-

body molecule is required for the fixation of aC'la molecule.4 The number of C'la moleculesfixed by cold agglutinin antibody is therefore a di-rect measure of the amount of antibody present onthe cell.

Warmagglutinin and Donath-Landsteiner anti-bodies. When the amount of warm agglutinin orDonath-Landsteiner antibody reacting with EHU isreduced serially, the amount of Cla fixed is notdirectly related to the relative concentration of theantibody (Fig. 4). The logarithmic plot of therelative concentration of antibody against the num-ber of molecules of C'la fixed per cell is a straightline, the slope of which is 1.7-2.1 (see Fig. 5, Ta-ble II). This indicates that more than one anti-body molecule is required for the fixation of amolecule of C'la.

4 The importance of the slope of the logarithmic plotof relative antibody concentration against the number ofmolecules of C'la-fixed EHU is derived from the follow-ing considerations: The relationship between the con-centration of reactant and product is given by theequation

[product] = K[reactant]Rwhere n = the number of molecules of a reactant which,reacting together, produce the product and K is a pro-portionality constant. If the logarithm of both sides ofthe proportionality are taken,

log [product] = nlog [reactant] + log Kwhich is the formula of a straight line of slope n. Hence,plotting the logarithm of the reactant concentrationagainst the logarithm of the product concentration yieldsa straight line of slope n. The numerical value of it givesthe mean number of reactant molecules yielding a mole-cule of the product, i.e., the number of antibody mole-cules required to fix a molecule of C'la.


00

0.3

IC

U.

( 0.2/0

0

0

0.1_

0 I1 2 4 8

Relative Antibody Concentration

FIGURE 2 Titration of IgM (autoimmune cold aggluti-nin) antibody with C'la. The relationship between theamount of cold agglutinin antiserum (V. L.) in the reac-tion mixture and the amount of C'la fixed as determinedby the C'la FT test.

The dose response curve of antibody M. P. wasconsistently different than that of the other warmagglutinin antibodies when this antiserum was re-acted with EHu in the absence of anti-antibody. In

IXw

N.

U-

4-

00

0so H

Q2 0.6 OB. 1.0 2.0Relative Antibody Concentration

FIGURE 4 Titration of IgG (autoimmune warm agglu-tinin) antibody with C'la. The relationship between theamount of warm agglutinin (R. F.) present in the reac-tion mixture and the amount of C'la fixed per red cellin the absence of anti-globulin antibodies.

the range of limited antibody, the plot of the log-arithm of the relative concentration of antibodyagainst the logarithm of the number of moleculesof C'la fixed per cell was a straight line, the slope

6.0O

-4.0o

0

Iwi 2.000

0i.

,. 1.0

IV 0.8

z0.6

0.41Slope = 1.0

I1 2 4


- Slope = 2.08

/ 1 1 10.4 0.6 0.8 1.0 2.0


FIGURE 3 The data shown in Fig. 2 regraphed as the FIGURE 5 The data shown in Fig. 4 regraphed as thelogarithm of the relative concentration of antiserum logarithm of the relative concentration of antiserumagainst the logarithm of the number of molecules of C'la against the logarithm of the number of molecules of Clafixed per cell. fixed per red cell.

2440 W. F. Rosse

0.61

0.41

0.2-

o

0

xw

L._0

U-a0

0

0

.0

0.1

0.08

0.06

I I

of which was about 1. At high concentrations ofantiserum, the slope approached 2.

The relationship between the amount of anti-body present and the amount of C'la fixed inthe presence of anti-antibody.

Warm agglutinin antisera and eluates were di-luted in twofold falling dilutions and the amountof C'la fixed by 0.1 ml of a standard suspension ofEHu and equal volume of graded dilutions of warmagglutinin antisera or eluates in the presence of aconstant amount of anti-human globulin was de-termined by the C'la fixation and transfer tests.The amount of C'la fixed was proportional to theamount of autoimmune or isoimmune antibodypresent (in the range of limited antibody). Theplot of the logarithm of the relative concentration

TABLE XISlope of Logarithmic Graph of Relative Antibody Concen-

tration vs. Molecules of C'la-Fixed/EHu for AutoimmuneAntibodies in the Presence and Absence of

Anti-Immunoglobulin Antibodies

Amount of anti-antibody

Antiserum 0 ml 0.1-0.2 ml

A. Cold-reactingAgglutinin

V. L. 1.06M. G. 0.95 -T. B. 1.1 -D. M. 1.0A. P. 1.05J. M. 0.98

Donath-LandsteinerJ. J. 1.7 1.0

B. Warm-reactingAutoimmune

R. F. 1.9 0.95M. P. 0.9-2.0* 1.2A. G. 1.1M. W. 1.0E. S. 2.1E. B. 1.8 1.0

IsoimmuneAnti-D - 1.1Anti-c - 1.1Anti-C - 1.2Anti-e 0.9Anti-E - 1.0

Normal serumJ. P. - 1.0

* See text.

of autoimmune or isoimmune antibody against thelogarithm of the number of molecules of C'lafixed yields a straight line, the slope of which isabout 1 (Table II). The effect of variation in theamount of anti-antibody on the amount of C'lafixed and the slope of the dose-response curve isdiscussed elsewhere.5

The absorption of globulin in normal serumto normal EHU

Globulins present in normal serum appear tobe adsorbed to normal red cells in a manneranalogous in some respects to the adsorptionof antibody. This adsorption is demonstrable inthe reaction of red cells and serum from the donorof the cells or from different normal donors. Theadsorption of this material is increased by reduc-tion of ionic strength of the reaction mixture andby papainization of the red cells prior to reaction,and is reduced by repeated washing of the cell-se-rum aggregates. The material reacts with anti-IgGHU and to a lesser extent with anti-IgMHu andanti-IgAHu. About 80-100 molecules of C'la arefixed per EHU by 0.2 ml of normal serum in thepresence of anti'WSHu antiserum, whereas muchless C'la fixation occurs in the absence of anti-antibody. The amount of material on the cells de-tected by anti-WSHu antiserum is directly pro-portional to the amount of normal human serumpresent.

DISCUSSION

The fixation of complement by antibodies from pa-tients with autoimmune hemolytic anemia is inpart determined by the immunoglobulin type ofthe antibody. Borsos and Rapp (4) and Ishizakaet al. (5) have shown that only one molecule ofIgM antibody appears to be necessary for the fixa-tion of a molecule of C'la whereas at least twomolecules of IgG antibody in a "doublet" are re-quired. In the present studies, the cold agglutininantibodies were found to be IgM in immunoglobu-lin type, as has been previously described (18, 19);these antibodies were found to require no morethan one molecule of antibody to fix a molecule ofC'la. The Donath-Landsteiner antibody and most

5 Rosse, W. F. The fixation of the first component ofcomplement (C'la) by heterologous anti-human im-munoglobulin antibodies in combination with human anti-bodies to erythrocyte antigens. Manuscript in preparation.


of the warm agglutinin antibodies appeared to beIgG in type and to require at least two moleculesof antibody to fix a molecule of C'1.

In a previous publication, we have proposed anhypothesis to explain this difference in molecularrequirement for C'la fixation deduced from knownproperties of the structure of IgG and IgM mole-cules and the study of inactivation of the hemolyticactivity of IgG and IgM rabbit anti-Forssmanantibodies by ionizing radiation (20). The IgMmolecule consists of at least five subunits, eachabout the size of an IgG molecule (21). WhenIgG antibodies were irradiated, only one radiosen-sitive target was found per molecule whereas amean of 3.2 targets was found in IgM antibodies.Using this data, we have suggested that in theIgM molecule, two adjacent subunits are able toact together to fix C'la in the manner of a doubletof IgG molecules.

This difference among different antibodies inthe requirements for C' binding is important indetermining differences in the manifestations ofthe hemolytic disease. The antibodies in cold ag-glutinin disease probably have only a transitoryattachment to the red cell as the blood passes to thecooler skin and extremities during circulation.Therefore, the alteration on the surface leading todestruction is not due primarily to antibody butto the complement components fixed to the surface,some of which are detected in the "non-y" Coombstest or as /.ic and 81E. The requirement of onlyone molecule of antibody to initiate a complementsequence means that such sequences can be initiatedefficiently during the passage through the cooledextremity. The present data and the data of Boyer(22) suggest that as the cold reacting antibodydissociate, C'la leaves the cell as well. However,the human C' system is probably analogous to theclassic guinea pig C' system, and in that system,once the sequence has proceeded through the addi-tion of C'4 to the cell surface and the activationof C'2 at the site of reaction, the remainder of thereactions can proceed in the absence of C'1 (3).

Complement rarely causes direct intravascularlysis of the cell but brings about sequestration ofthe cell in the reticuloendothelial system. Thework of Mollison (2) suggests that cells coatedwith C' components are removed from the circu-lation by all organs of the reticuloendothelial sys-tem, including both liver and spleen, and that the

number of cells removed by the liver is greater dueto its greater content of reticuloendothelial elements.Hence, splenectomy would be expected to be oflittle value in treating cold agglutinin disease andseldom is (23).

On the other hand, the antibodies in warm ag-glutinin disease are attached very efficiently atbody temperature whereas complement fixation, be-cause of the requirement for spatially aligned anti-body doublets, is very much less efficient. Hence,the major cause of alteration of the red cell sur-face leading to destruction of the cell is probablydue to the presence of antibody. Cells coated withIgG antibody are preferentially sequestered in thespleen (3), probably by attachment to macro-phages there (24). Hence, splenectomy would beexpected to be of value in treatment of this dis-ease, and often is (23).

The Donath-Landsteiner antibody is anomalous.Although it is cold-reacting and apparently IgGin immunoglobulin type and hence requires doub-let formation for C'la fixation, it is a potent he-molytic antibody in vivo in causing hemolysis bythe direct lytic action of C'. Certainly part of thispotency is due to the very large amount of antibodyusually present in the serum but other factors un-doubtedly play a role. In other studies, we havecompared th amount of lysis of normal or PNHred cells induced by limited concentration of com-plement in the presence of Donath-Landsteinerantibody, "Anti-I," anti-P, or anti-shigella anti-body and adsorbed shigella antigen (24). In thistest ("the complement-lysis sensitivity test") onlythe Donath-Landsteiner antibody induced an in-creased sensitivity of normal cells to lysis by C',suggesting that this antibody was able to usesmall amounts of C' more efficiently in bringingabout the lysis of the normal cell than the otherantibodies tested. The reasons for this differenceare currently under investigation.

Antiserum M. P. exhibited several peculiarproperties. The antibody appeared to be warm-reacting but, unlike most warm-reacting antibodies,considerable amounts of C'la were fixed in the ab-sence of anti-antibody. The logarithmic dose re-sponse curve showed a slope of 1 at low concentra-tions of antibody but approached 2 at higher con-centrations. Reactions with the antisera specificfor immunoglobulin types showed IgG antibodies tobe present. However, on Sephadex gel filtration,

2442 W. F. Rosse

antibody activity was found in both the excludedpeak and in the peak containing 7S proteins. Thoseantibodies in the excluded peak readily fix C'lawithout anti-antibody and are probably responsiblefor the slope of 1 on the logarithmic dose-re-sponse curve at low concentrations of antiserum,whereas those in the 7S peak do not fix C'lareadily except at high concentrations of antiserumwhere they are probably responsible for the slopeof 2. Studies are in progress to determine the na-ture of the antibody in the excluded peak.

Immunoglobulin type is not the only factor de-termining the efficiency of fixation of C'la. TheIgG isoimmune (or autoimmune) antibodies ofthe Rh system do not appear to fix C'la even whenpresent in maximal amounts on the cell. This isnot due to the lack of a C'-fixing site on the anti-body molecule since in combinations of several Rhantibodies, C'la is fixed. The inability of antibod-ies of a single Rh specificity to fix C'la may be dueto sparse distribution of antigen sites or to peculiaralignment of the antigen sites. Since the Rh anti-bodies used here are IgG in immunoglobulin type,two molecules in juxtaposition are needed to fixa molecule of C'la. If the antigen sites of a givenspecificity were situated at a distance greater thanthe critical "juxtaposition" distance, C'la could notbe fixed. If antigen sites of other specificity werenear enough, then the simultaneous presence of an-tibody molecules of different specificities could leadto the fixation of C'la. These possibilities are beinginvestigated.

Since the cold agglutinin antibodies requireonly one molecule of antibody for the fixation ofC'la, the C'la FT test provides a direct relativemeasure of antibody concentration on the cellsurface. Whether it provides an absolute measureof antibody concentration is less certain. If morethan one C'la molecule were fixed per antibodymolecule, then the C'la FT test would overesti-mate the number of antibody molecules. However,the findings of Borsos and Rapp with IgM anti-Forssman antibody suggest that only one mole-cule of C'la is fixed per molecule of antibody (25).On the other hand, the number of antibody mole-cules on the cell would be underestimated if allantibody molecules were not capable of fixing C'laeither because of "steric hindrance" or structuraldifferences among molecules. Preliminary reportsby Hoyer, Borsos, Rapp, and Vannier suggest that

such heterogeneity may be present in some IgMIantibody preparations (26) but is probably notpresent in others (25).

The relationship between the number of antibodymolecules on the cell and the number in solutionwill depend in large part upon the dissociability ofthe antibody-antigen complex. Since the cold ag-glutinin antibodies tend to be easily dissociated(some, such as in antiserum M. G. (27), muchmore than others) the C'la FT as performed inthese studies will tend to underestimate the num-ber of antibody molecules in solution.

The dissociation of cold agglutinin antibodiesmay be increased or decreased by several altera-tions in the conditions of the test. Probably themost important factor increasing the association ofantibody and erythrocyte is decreased tempera-ture. Because of this, all procedures designed toestimate the maximal fixation of cold-reactive anti-body are performed at the lowest practical tem-perature.

In previous studies (27) we have shown that thefixation of C'la by erythrocyte-antibody complexesenhances the fixation of the antibody to the redcell. Thus, with highly dissociable antibodies (anti-serum M. G.) very much less antibody is fixed ifC'la is absent during the reaction of cells andantiserum than if it is present. The amount of anti-body fixing to red cells is also increased by theprior treatment of the red cells by papain; this isprobably due to an alteration in the equilibriumconstant of the reaction between antigen and anti-body rather than an "uncovering" of antigen sites(28). Finally the association of antibody and anti-gen can be increased by reduction of the ionicstrength of the reaction mixtures.

Using these facts, the conditions under whichC'la is fixed can be designed so that the leastamount of dissociation of antibody occurs. At anyevent, the amount of the underestimation due todissociation will be the same fraction of the totalantibody concentration in solution on each deter-mination, provided the conditions of the test arethe same. Hence, the C'la FT test provides a di-rect estimate of the number of cold agglutininantibody molecules which are capable of fixingC'la at the red cell surface and a relative estimateof the number of molecules of antibody in solution.

The problems in quantitating the amount of IgGwarm agglutinin on the cell surface or in solution


are different from those encountered with cold-reacting IgM antibodies. These antibodies are gen-erally less dissociable than the cold agglutinin anti-bodies but require a "doublet" of molecules forthe fixation of C'la. Many single antibody mole-cules will be undetected by the C'la FT test whenanti-antibody is not present. However, if heter-ologous antibodies directed against the primaryantibody are added, C'la is fixed. By proper ad-justment of the amount of anti-antibody, a directproportionality between the amount of antibody inthe reaction mixture and the amount of C'la fixedcan be established. The constraints upon thismethod are discussed elsewhere.5

As with the cold agglutinin antibodies, themethod probably underestimates the number of an-tibody molecules in a solution. This underesti-mation due to dissociability of the antigen-antibodycomplex is probably less than with cold reactiveantibodies since these antibodies are, in general,less dissociable. Papainization of the red cells anddecrease in the ionic strength of the reaction mix-ture will increase the amount of these antibodiesattached to the cells. Again, the proportion of anti-body molecules either not attached to the red cellor washed from it during the testing procedurewill be constant regardless of antibody concentra-tion.

The finding that the incubation of human redcells with autologous serum results in the attach-ment of material which can be detected by its reac-tion with antiserum against the proteins of humanserum is not particularly surprising. Gammaglob-ulins are loosely attached to the membrane surfaceof human red cells and are very difficult to washaway (29). Either these proteins do not act asantibodies or their concentration is insufficient forthe body to declare the cell abnormal since thereis no evidence that the process of random de-struction (destruction without regard to cell age)seen with antibody-coated cells occurs under nor-mal circumstances (30). The finding that undi-luted autologous serum and rabbit anti-whole hu-man serum antiserum can bring about the fixationof a small amount of C'la indicates that results ofserum assays yielding low titers of C'la fixationand transfer must be interpreted with caution withregard to content of specific antibody.

If these constraints upon the estimation of anti-body number on the cell and in the solution are

taken into consideration, the C'la fixation andtransfer test provides a quantitative measure ofimmunologic reactions occurring on human redcells. Its use in the quantitation of the severityof immune hemolytic disease and in the responseof the disease to therapy is the subject of furtherstudy.

ACKNOWLEDGMENTSI wish gratefully to acknowledge the helpful advice ofDr. Tibor Borsos and Herbert Rapp, ImmunochemistrySection, Immunology Branch, National Cancer Institute,the supply of cells with particular antigenic structure byDr. Paul Schmidt, Director of the Blood Bank, ClinicalCenter, National Institutes of Health, and the unflaggingsupport of Dr. R. Wayne Rundles, Division of Hema-tology, Department of Medicine, Duke University MedicalCenter.

This work was supported by Grant 1 R01 CA10267-01AIB from the National Cancer Institute and the Insti-tutional Grant IN-6IG from the American Cancer Society.

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phrey. 1966. Immune lysis of normal human andparoxysmal nocturnal hemoglobinuria (PNHI) redblood cells III. The membrane defects caused bycomplement lysis. J. Ezpt. Med. 123: 969.

2. Mollison, P. L. 1965. The role of complement inhaemolytic processes in eivo. Ciba Found. Symp.Complement. 323

3. Mayer, M. M. 1962. Complement and ComplementFixation in Experimental Immunochemistry. E. A.Kabat and M. M. Mayer, editors. Charles C Thomas,Springfield, Ill. 2nd edition. 133.

4. Borsos, T., and H. J. Rapp. 1965. Complement fixa-tion on cell surfaces by 19S and 7S antibodies.Science. 150: 505.

5. Ishizaka, T., K. Ishizaka, T. Borsos, and H. J. Rapp.1966. C'la Fixation by human isoagglutinins: Fixa-tion of C'1 by yG and -yM but not by yA. J. Immunol.97: 716.

6. Borsos, T., and H. J. Rapp. 1965. Hemolysin titrationbased on the fixation of the activated first componentof complement: evidence that one molecule of hemoly-sin suffices to sensitize an erythrocyte. J. Immunol.95: 559.

7. Borsos, T., and H. J. Rapp. 1963. Effects of low ionicstrength on immune hemolysis. J. Immunol. 91: 826.

8. Frank, M. M., H. J. Rapp, and T. Borsos. 1964.Studies on the terminal steps of immune hemolysis.I. Inhibition by trisodium ethylenediaminetetraacetate(EDTA). J. Immunol. 93: 409.

9. Borsos, T., and H. J. Rapp. 1963. Chromatographicseparation of the first component of complement andits assay on a molecular basis. J. Immunol. 91: 851.

10. Borsos, T., H. J. Rapp, and C. T. Cook. 1961.Studies on the second component of complement III.

2444 W. F. Rosse

Separation of the second component from guinea pigserum by chromatography on cellulose derivatives.J. Immunol. 87: 330.

11. Borsos, T., and H. J. Rapp. Immune hemolysis:a simplified method for the preparation of EAC'4 withguinea pig or with human complement. J. Immunol.99: 263.

12. Rubin, H. 1963. Antibody elution from red bloodcells. J. Clin. Pathol. 16: 70.

13. Borsos, T., and H. J. Rapp. 1965. Estimation ofmolecular size of complement components by Sepha-dex chromatography. J. Immunol. 94: 510.

14. Jenkins, W. J., W. L. Marsh, J. Noades, P. Tippett,R. Sanger, and R. Race. 1960. The I antigen andantibody. Vox Sanguinis. 5: 97.

15. Dacie, J. V., and M. Cutbush. 1954. Specificity ofauto-antibodies in acquired haemolytic anaemia. J.Clin. Pathol. 7: 18.

16. Weiner, W., and G. H. Vox. 1963. Serology ofacquired hemolytic anemias. Blood. 22: 606.

17. Rosse, W. F., and J. V. Dacie. 1966. Immune lysisof normal human and paroxysmal nocturnal hemo-globinuria (PNH) red blood cells. I. The sensitivityof PNH red cells to lysis by complement and spe-cific antibody. J. Clin. Invest. 45: 736.

18. Gordon, R. S., Jr. 1953. The preparation and prop-erties of cold hemagglutinin. J. Immunol. 71: 220.

19. Fudenberg, H. H., and H. G. Kunkel. 1957. Physicalproperties of red cell agglutinins in acquired hemolyticanemia. J. Exptl. Med. 106: 689.

20. Rosse, W. F., H. J. Rapp, and T. Borsos. 1967.Structural characteristics of hemolytic antibodies asdetermined by the effects of ionizing radiation. J.Immunol. 98: 1190.

21. Miller, F., and H. Metzger. 1965. Characterization ofa human macroglobulin 1. The molecular weight ofits subunit. J. Biol. Chem. 240: 3325.

22. Boyer, J. T. 1965. Stability of the antigen-antibodycomples. Bibliotheca Haematol. 23: 19.

23. Dacie, J. V. 1962. The Haemolytic Anaemias Con-genital and Acquired Part II-The AutoimmuneHaemolytic Anaemias. Grune and Stratton, NewYork. 2nd edition.

24. Lo Buglio, A. F., R. S. Cotran, J. H. Jandl. 1967.Red cells coated with immunoglobulin G: Bindingand sphering by mononuclear cells in man. Science.158: 1582.

25. Rapp, H. J., and T. Borsos. 1966. Forssman antigenand antibody: preparation of water soluble antigen andmeasurement of antibody concentration by precipitinanalysis, by C'la fixation and by hemolytic activity.J. Immunol. 96: 913.

26. Hoyer, L. W., T. Borsos, H. J. Rapp, and W. E.Vannier. 1967. Complement fixation by purified rab-bit IgM antibody. Clin. Res. 15: 295. (Abstr.)

27. Rosse, W. F., T. Borsos, and H. J. Rapp. 1968. Cold-reacting antibodies: the enhancement of antibody fixa-tion by the first component of complement (C'la).J. Immunol. 100: 259.

28. Hughes-Jones, N. C., B. Gardner, and R. Telford.1964. The effect of ficin on the reaction betweenanti-D and red cells. Vox Sanguinis. 9: 175.

29. Grob, P. J., D. Frommel, H. C. Isliker, and S. P.Masouredis. 1967. Interaction of IgG and its frag-ments with red cells. Immunology. 13: 489.

30. Berlin, N. I., T. A. Waldmann, and S. M. Weissman.1959. Life span of red blood cell. Physiol. Rev. 39:577.


fixation ofthe first of complement (c'la)...

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