Quantitative micro-complement fixation for detection of small differences in protein structur

Levine, L.

Reichlin, M.

Wilson, A. C.

English

NASA Technical Reports Server

i ** 
, 
Use of Quantitative Micro-Complement Fixation for Detection 
of Small Difference8 in Prote in  Struoture  
Morris R e l ~ h l i n , ~  Allan C. Wilson, and Lawrenoe Levine 
Graduate Department of Brandeis University, 
U a l t h a m ,  #aasachuaetts 
https://ntrs.nasa.gov/search.jsp?R=19650020051 2020-03-17T01:59:11+00:00Z
f .  ,/. - 4” * 
Abstract 
T h r e e  techniques (the quantitative prec ip i t in  reaction, tuacro- 
complement f ixat ion and micro-complement f ixat ion)  have been compared 
for  their capacity t o  distinguish chickenfkom turkey H4 l a c t i c  
dehydrogenase, human- chimpanzee serum albumin, and chicken h Q m  
turkey ovalbumin. W i t h  each immune system, greater serological 
differences w e r e  found by micro-complement fixation. It is suggested 
t h a t  the greater sens i t i v i ty  exhibited by micro-complement f ixat ion 
may r e f l e c t  the measurement of a more avid fract ion of the antibody 
population. Hence, even a sl ight ly  different  conformation in a 
heterologous antigen might be expected t o  lead to dissociation of 
the antigen-antibody aggregates that are required for complement 
f ixat ion.  
, -  0 
1 
J 
U s i n g  quantitative nicro-comglement (0') fixation, rabbit  antisera 
t o  human hemoglobin A 
and the mutant hemoglobins S and C (1) which d i f fe r  in only one of 
146 amino aoide in the beta chain (2 ) .  The same rerbbit antisera 
did not discriminate these hemoglobins when measured by the quan- 
titatire preelpi t ln  teohnique of Heidelberger and Ksndall (3) o r  
by the C' fixation technique of Mayer, & a& (4). 
detected differences between hemoglobin A 
between 
A 
To determine whether micro-C' f ixation is always more seneitive 
t o  differences I n  protein structurs, other imraune systems were 
examfned by the same three serological techniquee, The rabbit  
antisera used were anti-chicken & l ac t i c  dehydrogenase, anti-human 
serum albumin and anti-chicken ovalbumin, The proteins thit were  
examined serologioally were ohicken and turkey HJ+ laaticr dehyitro- 
genase, human and chimpanzee serum albumin, aad chicken and turkey 
ovalbumin, 
tian WELEI a m!om dslkate  precsckiie f o r  B s j ~ m e t ~ U t i n g  diifferencee 
in protein struoture, 
With each antiserum, it w a s  found that m i o r o - C '  flxa- 
MATERIdLs MYD HETHODS 
PreDaratlon of Imnuni%in~ Anti~ene. Human serum albumin (HSA) 
was crystall ized three t b e s  as the aeroary Umsr by the method of 
Hughes (5). 
the dlmer against an ex0888 of eyeteine t o  remove the mercuq and, 
f inal ly ,  against several changes of water, Crystalline, ahloken 
heart l ac t i e  dehydrogenaee (LDH) was prepared aaeording t o  Pesee 
Xeraaptoalbumln monomer was prepared by dialysis of 
- e t  -0 a1 ( 6 ) .  The enzyme was electrophoretioally and ultracentri-  
fugally homogeneous and correeponded t o  the H4 form described by 
Cahn -- e t  a l ,  ( a ) .  
obtained from Pentex Corp, 
Chicken ovalbumin, five tiPle8 arystall ized, was 
2 
Preparation of Antisera, Rabbit anti-HSA (No, 669-1) was pre- 
pared by the Intravenous injection o f  increasing doses of alum- 
precipi ta ted antigen on a l te rna te  days f o r  five weeks, A t o t a l  
of 60 mag of protein was administered, 
af ter  the las t  injection, For production o f  anti-chioken LDH 
(175%) and anti-chicken ovalbumin (95B3), 10 m g  of protein in a 
volume of 1,0-1,5 ml mixed w i t h  an equal volume of complete Freund's. 
adjuvant w a s  Injected into the toepads and lnt~amuscularly. 
weeks l a t e r  the rabbits boosted w i t h  5 mg of the antigen intravenously, 
After a fur ther  three weeks they were boosted again. 
taken seven days following the l as t  intravenous injection, 
The rabbi t  was bled six days 
!Phree 
w e r e  
A 
Bleeding6 were 
Preparation o f  Antigens ror Serological Analssis. In the LDH 
studies,  c rys ta l l ine  chicken and turkey hear t  LDH's, prepared and 
characterized by the same procedures as described In the preparation 
of immunizing antigens, were used, 3
a33mufr?Irrna\ W I D l e  human s e m  and a chimpanzee s e m  obtained from 
Dr, Morris Goodman were used as souroes of serum albumin, The Slbu- 
m i n  content o f  these sera  wae assumed t o  be 40 m g / m l ,  For serologi- 
ca l  analysis of the ovalbumin systems, b o t h  crystal l ized crhicken 
ovalbumin (used f o r  immunization) and ohtoken and turkey egg white8 
were used. The .ovalbumin content of egg white was assumed to  be 60%. 
SeroloRioal  Hethode, For quantitative prec ip i t in  analyeis, the 
method of Heidelberger and Kendall (3) was used, The twice-washed 
Immune preoipi ta tes  were dissolved i n  2.0 ml of  0.1 l!i NaOH and the 
protein concentration measured a t  287 mp. 
f ixa t ion  using 100 C' %o (macro-C' f ixa t ion) ,  the method of  Mayep 
- e t -0 a1 (4) w a s  used, 
man and h v l n e  (8) was used w i t h  the modifleation they describe In 
For quantitative C' 
For micro-C' f ixation, the procedure of  Wasser- 
3 
footnote 3, 
of mcfiterlony (9). 
Agar diff'ueion analyses were performed by the method 
IiESULTS 
Reaction of Chicken and -key HA LDH with Anti-chfcken H1, LDH, 
When tes ted by double d i f fus ion  In agar, performed In a t r iangular  
pat tern,  both chicken and turkey Q LDH (10 pg) gsve a s i n g l e  band 
of  precipi ta t ion and a reaction o r  ident i ty  w i t h  undiluted ant i -  
chicken LDH, 
turkey LDH did exis t ,  it was n o t  detected by this double diffusion 
technique, 
If any antigenic difference between chicken and 
The resd.ts o r  quantitative precipi t in ,  macro-C' f ixa t ion  and 
micro-C' f ixat ion w i t h  chioken o r  turkey LDH' and anti-chicken 
LDE are shown In  Fig, 1, For the quant i ta t ive pree ip l t ln  analyeee, 
the constant amount o r  antiserum u8ed w a s  1.0 m l  of a 1/3 dilut ion,  
Supernate analyses of both homologous and heterologoue prec ip i t in  
reaction mixtures revealed no overlapping zones of precipi ta t ion,  
If the antibody content is caloulated from the antigen-excese aide 
o f  the equivalence zone af ter  subetraction of the added antigen, 
chicken and turkey LDH precipitated 301 pg and 274 118 antibody I re- 
spectfvely/.33 m l  of undiluted antiasrum, a difremmce of  9%. 
macro-C' fixation, ueing as the constant quantity o f  antfeerum 1.0 
ml o r  a 1/250 di lu t ion  (3.6 pg antigoas N/reaction m i x t u r e ) ,  the 
heterologoue LDH reacted more erieotlrely than the  homologous LDH, 
58 and 52 0' H50 maximum 0: rixation r e s ~ a t i v e l y ,  
f ixat ion,  however, ueing 1.0 ml o r  a 1/20,000 di lu t ion  o r  m t i e e r u ~ ~  
(0,045 
chicken and turkey LDH wars eaaily deteoted, 
a leo be distinguished by etareh gel deot~phQZwB18 (Wilson, nnpub- 
lashed observation), 
In 
By mioro-C' 
an t i f rob  N/reaetion mixture) the ditference between the 
The two enzymes Ban 
4 
Reaction of Eum and OhhpanZee SA w i t h  mt i -HSA,  In a Study 
of the lnnaunologlcal relationship of primate plasma proteins,  Goodman 
(10) using chiaken anti-HSA did discriminate between CSA and HSA 
as measured by a turbidometric technique, Ihe difference w a s  of 
the order of rSrrr+. 5%. 
which gave a single band of precipitation w i t h  whole human serum and 
whole chlaPpanzee serum (0.1 m l  of a 1/200 d i lu t ion  of each serum 
used as antigen) gave a pattern of ident i ty  when tested by the Ouch- 
terlony procedure, The data obtaf ied when t h i s  rabbit-anti-HSA 
reacted with HSA and CSA by the quantitative precipitin,macro and 
m i c r o - C '  f ixat ion techniques are shown in Fig, 2. Supernate analyeis 
of the HSA and CSA prec ip i t in  reaotlon mixture suggested inuaunologi- 
The rabbi t  anti-HSA used In the present study 
ca l  homo&meity as judged by the absenoe of any overlapping preci- 
p i ta t ion ,  'phe antibody content of this serum was calculated to be 
611 pg antlhody N/ml of undiluted serum. 
antibody N when calculated from a similar region of the prec ip i t fn  
curve (antigen excess side of the equivalence zone), and assuming 
CSA precipi ta ted 540 pg 
that the same amount of CSA was required t o  reach this point of the 
curve, a difference o f  11%. 
antibody N/reaction mixtures, HSA and CSA fixed 59 and 57 C' B50,& 
respectively, &ab, micro-C' f ixa t ion  waa the moat diacrlmlnatlng 
technique, aa shown by the percent C' f ixa t ion  using 0.7 l3g antibody 
BT/reaction mixture, HSA and CSA fixed C' maximally a t  values of 
68 and 32% respectively, a difference of 52$. 
By macro-C' f ixat ion,  using 10 ~ r g  - 
Rsaotion o f  Bhicken and h k e s  Ovalbumins w i t h  Anti-chicken 
O v a l b u r n i n .  A reaction o f  partial ident i ty  w a s  seen when chicken 
egg white, turkey egg white and undiluted anti-chfeken ovalbumin 
was tested by agar diffusion set up ln  a t r iangular  pattern,  Only 
5 
. 
one band of precipi ta t ion w a s  observed w i t h  16 t;rg o r  chicken egg 
white, A second band of precipitation w a s  seen, however, when 200 
pg of chlaken egg white we~z~uaed, 
can a l so  be seen by inepection of t he  quant i ta t ive prec ip i t in  curve 
i n  which the contaminating Immune syetenl l e  reflected by the r i ee  in 
precipi table  B in the antigen excess side of the prec ip i t in  o u r v 8 ,  
both w i t h  chicken and turkey egg w h i t e  (Fig, 3). TheWbrogeneity, 
however, w a s  not  seen by analysis of the prec ip i t in  reaction m i x t u r e  
aupernates. 
valence zone showed tha t  chicken egg white, turkey egg white and 
crys ta l l ine  ovalbumin precipitated 606, 580, and 540 pg antibody N, 
respectively, The Increased antiboay N precipi ta ted by the chicken 
egg white re f lec ts ,  in part, antibody t o  the contahlna t lng  antigen 
which is probably a t  a higher ooncantration in the egg white. 
we compare the antibody rJ values obtained w i t h  the egg wh%te prepara- 
tione, the difference betiweexi “&e chicken iiai& t i i l = i i ; e ~  azitigem iii s a y  
4%. 
N, the macro-C’ fixation was performed with 2.7 I J ~  antibody lV/reac- 
t ion  mixture and the mloro-C’ fixationwfth 0.07 Pg antibody N/reac- 
t ion mixture, In the macro- and mioro-C’ f ixa t ion  procedure8, the 
contaminating Immune system was not  eeen. 
di lu ted  out o r  It ha8 l e s s  e f f ic ien t  C’ firing propertlee than the 
major  antibody, anti-ovalbumin. When measured by macro-C’ f ixat ion,  
turkey ovalbumin reacted 89% as effect ively as chicken ovalbumin. 
By micro-C’ f ixa t ion  no reaction w i t h  the heterologous antigen was 
observed a t  the antiserum dilution employed to denonstrate the 
honologoue reaction, 
This immunochemical heterogeneity 
Calculation from the antigen excess s ide  of the equi- 
If 
Using the antibody content of anti-ovalbumin as 540 pg antibody 
Probably it has e i the r  been 
- .  
6 
DISCUSSIOH 
rphe percent of cross reaction a8 measured by quantitative preci- 
p i t in ,  macro- and m i c r o - C '  f ixation is summarized in 'Fable 1. 
~n order t o  explain the greater aens i t iv i ty  of nicro-C' fixa- 
tion, the following experimental findings must be considered. 
Wallace - e t  a. (11) have shown that rabbit ant isera  t o  bovine s e m  
albumin, when compared a t  equal levels  of  antibody 18, -led in C' 
fixing capacity from serum t o  serum. This variation i n  C' f ix ing  
capacity w a s  large in antisera after one course of immunization, 
By micro C' fixation, we aleo find a variation in C' fixing capacity. 
Equal C' fixation w a s  obtained w i t h  0.045 Pg anti-chicken H4 LDH I, 
0.07 pg anti-ohicken ovalbumin 8,  and 0.7 pg anti-HSA EJ (the anti- 
HSA is a first course antidlenun; the other immune a em are third 
course ant isera) .  
HI11 and Osler (12) t h a t  C' fix- capacity varies even among ant i -  
bodies i n  a single Immune serum. i n  rnicr0-C' z'ixatisl-i, thci tsxiaentra- 
t ions of antigen and antibody are each 2-3 orders of magnitude below 
those used f o r  the quantitative prec ip i t in  reaction and 1-2 orders 
of magnitude lower than those w e d  by Hill and Osler (macro-C' 
f ixat ion) .  
antibody, only  a fratstion of th6 antibody population is being 
measured. 
s i t e s  of maximum complementarity t o  each antigenic determinant. 
Since C' f ixat lon measures antigen-antibody aggregates (12,13) and 
since the rabbit-Immune p rec ip i t a t e  can be appreciably soluble (14), 
even a slight ohange in confornation of an antigenic determinant 
m y  render imperfect the f i t  between avid antibody and i t a  de- 
terminant and lead t o  a greater dissooiation of the aggregate. 
More pertinent, perhaps, is the observation by 
Thus, it is possible that a t  very low concentrations of 
This avid fract ion would be expected t o  have reeeptor 
7 
Horeomr, a slight change In eonformation of one antigenic deter- 
minant may infiuenoe the binding o f  neighboring antigenic determi- 
nants t o  their antibodies and thus lead t o  a diseoclation of the 
antigen-antibody aggregate into smaller complexes inaapable of 
fixing C', 
SUMMARY 
1, The antigen-antibody Interaction of chioken and turkey H 4 
LDH w i t h  rabbit anti-chieken H4 LDH as measured by quantitative 
precipit in,  macro- and micro-C' fixation gave 91$, 112% and 32% 
cross reaction values respeatively. 
2, The antigen-antibody interaction o f  human and chimpanzee 
serum albmnin w i t h  rabbit anti-HSA aEa measured by qusntitative pm- 
c ip i t in ,  mcm- anU miero-Ut fixation gave 89$, 97% and 46% cro88. 
reaction values reepecrtively. 
3, The antigen-antibody interaotioncd chiaken and turkey oai- 
bumin with rabbit  anti-chicken ovalbumin as measured by quantita- 
t ive  preaipit in,  maom- and mlcm-C' fixation gave 96$, 89% and 3% 
croas reaction values reEs6,Otl*ly. 
4, A poselble explanation for the greater senei t ivi ty  of 
mioro-C* rimtioa to dliierenoee in protein etruoture la 
diecaeesd, 
1. 
2. 
3. 
40 
Reichlin, M., Hay, M.,  and Levhe, Lo, Immunochaaistrp, . 
I 1, 21 (1964). 
Ingram, V.H., Natum, 180, 326 (1957). 
Heidelberger, M., and Kendall, F.E., J. lb 0 Medo, 62, 697 (1935). 
Xaysr, W.H., Osler, A.G., Bier, O.G., and Heldelberge~, H., 
Cahn, &Do, Kaplan, N.O.,  Levine, Lo, and Z w i l l i n g ,  E., Science, 
143, 962 (1962). 
Wassexman, E. and Levhe, Lo, J. I u u n ~ o  10 5, 290 (1961). 
-LE 1, Comparison of  Sensitivity of Zxununologloal Methods 
Cross Reaction" 
Quantitative MsCrO- Mlero- 
Heterolwous PmCigitbl complement complenen t 
Bntlslenmr. antigen rdaction fixation f lxa ti on 
A n t i -  
human 
hemoglobin A 
Eumm 
hemoglobin S 
100- 86** 
albumin 
A n t i -  
C'aicken 
ovalbumin 
A n t i -  
c3lctken 
mAIrlpamzee 
serum 
albumin 
Turkey 
ovalbumin 
Turkey 
H4 LDH 
89 
96 
91 
97 
89 
112 
41** 
46 
3 
32 
rea0 tlon. 
. 
c 
LE23i3lDS FOR FIGURES 
Fig. 1. Serological ac t iv i ty  ae measured by the quantitative precipi t in  
reaotion with a l : 3  dilution of antiserum ( lef t ) ,  macro-C' 
fixation with a ii250 dilution of antiserum (eenter), and a 
mltalpQ-C' fixation with a 1:20,000 dilution of mtieerum (right). 
Antiserum: rabbit-anti-chicken H4 LDH, Antieen8: pure chiaken 
H4 LDH (0 -0)  md pure tWkOy Q LDH (4---0). 
Pig. 2. Serological ae t lv i ty  88 meaeured by the quantitative preeipi t in  
reaction with undiluted antisemm ( l e f t ) ,  macro-c' f ixation 
with a 1:60 dilat ion of antleerum (center) , and micro-C' fixa- 
tion w i t h  a 1:g00 dilution or antimerum (right). htiserum: 
rabbit-anti-buman ~erum albumin, &ntigena: human serum ( 0 - 0 )  
and ahlmpanzee serum (o-=-o). 
Pig. 3. Serological ac t iv i ty  a8 measured by tho quantitative preclpi t ln  
reaotion with a 1:2 dilution of antleerum ( l e f t ) ,  macro-C' 
f ixst ion w i t h  a 13200 dilution (aentsr) ,  and miom-C'  f ixation 
with a 1:8,000 dilution o f  antiserum (right). Antiserum: 
rabbit-anti-ohloken ovalbamln. ht igena :  crystal l ine ohlokon 
ovalbumia (0-o), chitaken egg white ( ), and turkey 
egg Whih (*--e). 
c 
Postdoctoral trainee, Public ~ m l t h  service Training Grant 
(5Tla-212.04) 
Publication No. 000. Supported in part by grants from the Ha- 
tiOMl InStitUtt!~8 O f  H-1- (AI-01940), the A m a i c U  C - C a  Society 
( B - 2 2 X )  and the Hational Aeronautics and Space Administration 
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Use of quantitative micro-complement fixation for detection of small differences in protein structure

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