Dec., 1942
PRECIPITATION REACTIONS OF POLYHAPTENIC SIMPLESUBSTANCES
the seven monohaptenic substances failed to precipitate. It is pointed out that these results provide strong support of the framework theory of the precipitin reaction.
[CONTRIBUTION FROM
THE
3003
Data on the amounts of precipitate formed are discussed in relation to the structure of the simple antigens. PASADENA, CALIFORNIA
RECEIVED JULY 6, 1942
GATESAND CRELLINLABORATORIES OF CHEMISTRY, CALIFORNIA INSTITUTE OF No. 8861
TECHNOLOGY,
The Serological Properties of Simple Substances. 11. The Effects of Changed Conditions and of Added Haptens on Precipitation Reactions of Polyhaptenic Simple Substances BY LINUSPAULING, DAVIDPRESSMAN, DANH. CAMPBELL, AND CAROL IKEDA During the course of the investigation of precipitation reactions of polyhaptenic simple substances reported in the preceding paper of this series' we found it desirable to carry out a study of the effects of changed conditions of precipitation and washing on the amount of residual precipitate. We also made some experiments on the inhibition of precipitation by added haptens, in order to see how great would be the effects of monohaptenic impurities possibly present in the substances studied. The results obtained are presented and discussed in this paper. Experimental Methods.-The experiments were carried out in the way described in the preceding paper (I). In addition to antisera C and D mentioned in paper I, three antisera, E, F, and G, were used. E and G contained amounts 0.6 and 3.2 mg. per ml., respectively, of antibody precipitable by azo-ovalbumin test antigen ; the strength of F was not determined. '
The borate buffer solutions were made by adding suitable amounts of 0.16 N sodium hydroxide solution to 0.2 M boric acid solution containing 0.9% sodium chloride. The Effect of Changed Conditions of Precipitation and Washing on the Amount of Precipitate.-It is seen from the data reported in Tables I, 11, and 111, obtained with ,two antigens (VI and X) and three antisera (C, E, and F), that the antigen-antibody precipitate is either dissolved slightly or carried away mechanically by the saline or borate buffer solutions with which i t is washed. The loss in this way is, however, small, amounting to about 5 to 15y0for eight or ten extra washings with 10-ml. portions of solution. A few experiments were made (Tables I1 and 111) to test the effects of changing the time and temperature of precipitation. It was found that increasing the precipitation time from one day to two days increases the amount of precipitate by
TABLE I EFFECT OF NUMBER OF WASHINGS ON AMOUNT OF RESIDUAL PRECIPITATE 3 ml. antigen VI, 25 pg./ml. in saline solution, plus 3 ml. antiserum C
Washings
Composition of precipitate Antibodya Antigen0
Washings
Composition of precipitate Antibody0 Antigen"
2
a
2 ml. antigen VI plus 2 ml. antiserum E Composition of precipitate Washings Antibodya Antigen0
1665 8.9 6 1600 8.7 3 1660 8.4 1540 8.7 1660 8.9 1590 8.7 10 3 1600 8.7 7 1590 8.7 15 1600 8.9 1530 8.9 1545 8.7 1510 8.9 4 1545 8.7 8 1510 9.2 1630 8.7 1570 8.9 8.7 1480 8.1 1660 5 1570 8.9 10 1520 9.5 1570 8.9 1510 7.6 1570 9.5 1320 9.5 Amounts of precipitated antibody and antigen in micrograms. pH of all supernates 8.1.
(1) Linus Pauling, David Pressman, Dan H. Campbell, Carol Ikeda, and M. Ikawa. THISJOURNAL, 64, 2994 (1942). Wr shall refer to this as paper 1.
1110 1125 970 930 960
5.7 6.2 5.7 6.0 6.0
about 10%. The amountof precipitate formed seems to increase with increase in temperature of
:100-1
Lrwus PAULING, DAVIDPRESSMAN, D. H. CAMPBELL AND CAROL IKED.A TABLE I1
1':FFILCT OF C H A N C R D C O N D I T I O N S ON A M O U N T O F PRECIPITATE
3 ml. antigen VI, 12.5 pg./rnl., in saline solution, plus 3 ml. antiserum E. ;bH of supernates 8.0 to 8.3. Condition. of precipitation
Room 1 hr., refrigerato] 24 hr.
Washings
3 10
15
Composition of precipitate Antibody, Antigen, pg, Irg.
1600 1610 1190 1310 1140 1140 1750 1590 1590 1640 1610 1690 1080 980
9.2 8.9 8.4 8.1 8.7 9.2 9.2 8.9 8.9 9.2 8.9 8.9 7.0 7.n
1701.
64
TABLE IV THEEFFECTOF DILUTIONWITH BUFFER SOLUTION ON AMOUNTOF PRECIPITATED ANTIBODY 2.5 ml. antigen VI, varying volume of borate buffer solution with pH 8.0, 2.5 ml. antiserum C. Volume of huffer solution
0 2.5 5 10
Amount of antigen used (fig.) 15.6 31.3 62.5 Amount of precipitated antibody
(rrs.)
750 600 480 470
1375 1030 845
775 990 525
655
450
well-defined compound. These observations provide further evidence of the heterogeneity of the antibodies in immune sera. 3 The Effect of Hydrogen-ion Concentration on Refrigerator 48 hr. 3 Amount of Precipitate.--The results of experiments to test the effect of change in pH on the Room 24 hr. 3 amount of precipitate are given in Table V. It is seen that for this antigen-antibody system the TABLE I11 optimum pH is about S.1, the amounts of preEFFECTOF CHANGED CONDITIONS ON AMOUNTOF PRECIPIcipitate in this region being greater than for either TATE 5 ml. antigen X, 20 pg./ml. in saline solution, 2.5 ml. more acidic or more basic solutions. Evidence antiserum F, and 7.5 ml. borate buffer of pH 9.0. that the buffering substances do not have a large ComDosition of direct influence on the reaction is given by the pricipitate Conditions of Washing Antibody, Antigen, agreement of the values a t pH 8.1 for added precipitation solution Washings fig, Mg. saline solution and added borate buffer. Room 1 hr., re- Saline 3 520 3.3 520 3.3 frigerator 24 The optimum antigen concentration is seen to 10 525 hr . Saline 3.1 he changed only slightly by change in PH. 530 3.1 Less extensive experiments were also carried Saline, 3 585 3.3 out with the amide antigen XIV (R"-R") in place iced 520 2.9 of the azo antigen; the results obtained, given in Buffer, 3 600 3.3 2.0 Buffer, 3 630 Table VI, are similar. iced 2,9 600 The Effect of Dilution with Normal Serum or 3 2 .9 Saline .5so Room 1 hr., reBuffer Solution.-In order to determine the 3.9 frigerator 48 hr. 593 effect of change in the strength of a serum of :i (ill) 3.1 Room 3 hr., re- Saline fixed antibody composition on the position of the 550 3 .1 frigerator 22 hr 3 675 3.1 Room 24 hr Saline optimum zone, identical experiments were made 605 3.1 with sera obtained by mixing antiserum G and 3 2.9 ;j6 1 hr., re- Saline 560 normal serum. The results are given in Table A55 3 9 frigerator 24 hr. T'II. It is seen that for both antigens (I11 and the tube during the first few hours of the pre- VI) the maximum precipitation occurs a t an cipitation period (Table 111). However, the amount of antigen about midway between 25 and evidence is inconclusive as to whether the final 50 pg. (for 2 nil. of antiserum), and that on twoamount of precipitate is increased or not by iold dilution of the antiserum, decreasing its refrigeration during the later part of the pre- antibody concentration by the divisor 2, the optinium amount of antigen is also decreased by cipitation period. The data reported in Table IV show that the about the divisor 2. The effect of diluting the amount of precipitated antibody is decreased by antiserum is hence to cause the optimum zone the addition of buffer solution to the antigen- to shift in such a way as to keep constant the ratio antibody mixture. The decrease is not propor- of antigen to antibody. The same result is given by experiments on the tional to the volume of buffer added, so that the pheriomenon is not analogous to the solution of a effect of dilution with buffer solution, reported Room 1 lir., refrigerator -18hr. Refrigerator 24 hr.
0
3
Dec., 1942
PRECIPITATION
REACTIONS OF
TABLEV EFFECTOF HYDROGEN-ION CONCENTRATION ON AMOUNT OF PRECIPITATE 3 ml. saline solution VI, 3 ml. antiserum E, and 3 ml. saline or buffer solution; 48 hrs. in refrigerator. precipitate, micrograms. A = antigen in precipitate, micrograms. Amount of antigen (pg.) Added solution p H of supernate
Boric acid
7.6-7.7
Saline
8.1-8.2
Borate buffer, PH 8 . 0 Borate buffer, pH 9 . 0
B
22.2
640 640 890 840 820 790 680 650
8.1 8.8-8.9
Amount of added antigen (pg.) Added pH of solution supernate
Boric acid
7.6
B
4.9 4.6 4.6 4.6 5.7 5.7 5.1 4.6
820 820 1140 1140 1080 1080 780 820
Saline
8.0-8.2
Borate buffer, pH 8.0 Borate buffer, pH 9.0
8.1 8.8-9.0
12.5 25 50 Amount of precipitated antibody ( p g . )
200 250 270 290 270 290 215 280
275 290 305 345 290 375 290 295
260
240 270 265 225 325 240 270
TABLEVI1 THE EFFECTOF DILUTIONWITH NORMAL SERUMON AMOUNT OF PRECIPITATED ANTIBODY 3 ml. of antigen I11 or VI plus 2 ml. of mixture of normal serum and antiserum G. Amount of antigen used ( p g . ) Normal Antiserum serum
12.5 26 50 100 Amount of precipitated antibody (pg.)
I11
230 495 495 265 215 555 230 305 340 200 105 305 280 215 55 225 170 105 10 230 170 105 0 395 1150 1070 170 400 1100 1240 170 495 455 195 65 425 510 205 70 290 120 40 0 330 200 45 0 8 . 1 8.1 8.1 8.1 8.2 8.3 8.4 8 . 5
Antigen
VI
pH of supernate
2
0
1
1
0.5
1.5
2
0
1
1
0.5
1.5
I
antigen I11 antigen VI
33.3
A
TABLEVI EFFECTOF HYDROGEN-ION CONCENTRATION ON AHOUNT OF PRECIPITATED ANTIBODY 1 ml. antigen XIV, 1 ml. antiserum G, 2 ml. saline or buffer solution.
in Table I V .
In these experiments, in which the amount of antibody is kept constant, the position of the optimum zone does not change significantly. These conclusions agree with those reached by many earlier investigators with protein antigens. The Inhibition of Precipitation by Hapten.I t was discovered by Landsteiner2 that a hapten (2) K. Landsteiner, Biochcm. Z.,95, 117 (1919); 104, 280 (1920); K. Landsteiner and J.v an der Scheer, J . ExPtb M c d . , 48, 315 (1928); SO 407 (1929); 64, 298 (1931); 611, 781 (1932).
3OOti
POLYHAPTENIC SIMPLE SUBSTANCES
50
A
B
5.9 5.7 6.5 6.5 8.4 7.3 5.9 6.5
820 820 1020
940 920 640 720
B = antibody in 75
A
B
5.7 5.7 6.2 6.2 7.0 6.0 6.2 6.2
610 620 610 550 650 590 520 520
A
4.1 5.4 3.0 3.8 4.9 5.4 5.4 4.3
such as arsanilic acid present in reasonable concentration in a mixture containing an azoprotein (made from this hapten) and the hapten-homologous antiserum decreases the amount or inhibits the formation of the antigen-antibody precipitate. It has also been founda that this phenomenon occurs with polyhaptenic simple substances replacing the azoprotein. Quantitative results were obtained by us with antigens VI, X, and XX, containing the groups R, R', and R", respectively, and haptens XXI, XXII, XXIII, and XXVII. These are given in Tables VI11 to XI and Figs. 1 and 6. It is seen (Table VIII) that addition of hapten decreases the amount of precipitate without noticeable shift in the equivalence zone, and (Tables X, XI) that haptens differ in their inhibiting power. TABLEVI11 THEINHIBITION OF PRECIPITATION BY HAPTEN 3 ml. antigen VI, 1 ml. hapten XXI, 3 ml. antiserum E. PH of supernates 8.2. Amount of antigen (pg.) 15 22.2 31.3 60 Amount of hapten fpg.) Amount of precipitated antibody (pg.)
0 12.5 25 50 100 200 400
540 520 520 340 330 290 200
790 740 580 560 360 340 270
1280 910 630 610 430 290
1020 850 780 640 550 440
75
560 540 530 530 450 360 270
TABLEI X INHIBITION BY HAPTEN 1 ml. antigen VI (12.5 pg.), 0.5 ml. hapten XXIII, 1 ml. antiserum D. PH of supernates 8.1. Amount of hapten, pg.
0
3.13 6.25 12.5 25 50
Amount of precipitated antibody, fig.
505 470 505 480 355 220 70
(3) K. Landsteiner and J. van der Seheu, ibid.. 66, 399 (1932).
.
3006
LINUSPAULING., DAVIDPRESSMAN, D. H. CAMPBELL AND CAROL IKEDA TABLE X INHIBITION BY
system antigen-antibody-hapten when the order
HAPTEN
of combining the reactants is changed.
0.5 ml. antigen X (25 pg.), 0.2 ml. hapten X X I I or XXIII, 0.5 ml. antiserum D. Amount of precipitated antibody, pg Hapten XXII XXIIl
Amount of hapten, Pg.
U 1.25
570 565 505 495
2.51,
5 10 20 40
420
465 295 2 10 8.5
pH of supernates
565
