modified procedure for hansa test serum ii. sero dilution

Indian J. Dairy Science, 35, I, 1982
MODIFIED PROCEDURE FOR HANSA TEST SERUM
II. SERO DILUTION TECHNIQUE FOR BULK PRODUCTION
BY SPECIES SPECIFIC REAGENP
RT. JAIRAM t and P.G. NAIRt
National Dairy Research Institute, Karnal-132001.
INTRODUCTION
The procedures that have been described so
far (Nair et al., 1965, 1970) are suited for producing highly specific Hansa test serum, However, the large increase in demand for the serum
from the Tndustry has made it necessary for
one to think of methods for mass-production
of specific serum. In a previous paper (Jairam
and Nair, 1980) the method for bulk-production
of antiserum using micellar casein as the antigen
have been described. The traditional method
of "purifying" the serum is by absorbing out
the cross-reacting antibodies using cow casein
as tbe absorbant. But this method has the
disadvantage that along with cros~-reactillg
antibodies, many anti buffalo milk antibcdies
a re also removed by the process of antibodyabsorption so that the rCHllting serum reagent
has a titre reduced to 1 or less than the orjginal.
An alternate method, the technique of "Serodilution" along with a method of testing using
diluted substrate is described in this paper.
MATERIALS AND METHODS
Immunizations with micellar casein, production of antiserum and titrations were done as
described by Jairam and Nair (1980). Readings
"NDRI Publication No. 80-7, ;nd a part of M.Sc.
Thesis of the first author submitled to Panjab Univtr.
ail),. Chandigarh.
Present address: t College of Veterinary Science,
Tirupati-S17 502.
iSoutbtmReliooal Station of National Dairy Research
lostitule, R~n galo.e-S60 030.
were recorded within 2 min after mlXlRg the
antigen and antibody. Pooled bulk milk
~ arllples of buffalo and cow diluted with different
proportions of di stilled water were tested against
various dilutions of antiserum with saline for
finding out the optimum dilutions and the
nature of homologous and heterologous antigen .
antibody reactiol1s (Sero-dilution technique).
In order t<l control false positive reactions,
tests were conducted with antiserum diluted
with distilled water, saline (0.9 %), Sorensen's
M i lS phosphate buffer ranging from pH '6.0
8.0, Verona] buffer (pH 8.4) and borate buffer
(pH 7.0) (Jairam , 1967).
RESULTS AND DISCUSSION
The pattern of antigen-antibody reactions
011 antibuffalo m.il: . (micellar casein) serum
diluted with saline in different proportions to
it~ maximum titre (1 /32) and tested against its
homologous milk diluted with distilled water
to various proportions has been given in Fig. I.
Similar pattern of reactions of the antiserum
"g"inst cow milk diluted to several proportions
have been represented in Fig. 2. It was observed that buffalo milk diluted between 29 and 59
parts with water gave strong positive
+ +)
reactions with the homologous antiserum
diluted in various proportions upto a maximum
of 1/32. As observed in Fig. 2, the antiseruin
diluted to 8 times with saline, produced from
negative to strongly positive reactions against
heterologous cow milk . with increase in dilutions. The antiserum diluted 16 times with
saline gave clear negative reactiOn with cow
31
(+
Hansa Tes, Serum
32
Fig. J
Aatlge.-antibod1 readiollS of antl.erulIl diluted and tested
against bOlllol<)gous milk diluted at diffe..,nt proportions.
Note: 0 .5 "egative, ± as doubtful, + a~ weakly positive, + + as fairly positive, and +, I ''" strongly
positive reactions.
Serum dilutions:
as ~. t • • • • • • as 1,
- - - .. I/B,_' - , -asljl6,and-o-oas 1/32.
.....
...
g
....
.,
....I!0
.~
.".
'
a
.2
•
~
~
...."
s
•
ID
,..
.6
••
...
..
II.
, ..
Dilution of buffalo milk
Fig. 2
Antlgen·antibody reactions of ·"nti.eruPl diluted and t.sted
against heterologous (cow) milk dilllted a( different
proportio....
Note: 0 as negative, ± .. doubtful, + as weakly posi·
tive, + + as fairly posilive, and + + + as ."ongly
positive reaclions
Serum dilutjons:
as L T'
as i,- as lIB, - I - , -- a. 1/16. and - 0 - 0 - as 1/32.
+ •••
••
•
•
••
...
.,
,
• ••
,
,,
~
...
---~
,-
..
I·
DilUlion of cow milk
)0
milk diluted uptn 30 times with water, and the
<lntiserum diluted 32 times gave negative reactions with the heterologous milk diluted 40
time~ and beyond.
According to Landsteiner
(1945), in allied species, the protein differences
are often too minule to be detected by direct
precipitation tests. The scro-absorption technique was not successful with antimicellar casein
serum (Jairam and N~ir, 1980). A useful
property of precipitating systems is that precipitation or flocculation occurs first at, or near,
the equivalence point ,md the ratio of antigen
dilution to antiserum, where the first flocculation
occurred is referred to as the optimum proportion point (Cushing and Campbell, 1957) .
In the present investigation, a zone of optimum
proportions of antigen and antibody was observed revealing high inten~ity of reactions with the
antiserum diluted in all proportions upto 1/32
and tested against homologous milk diluted
in the proportions ranging between 11'30 and
1/60. On the other hand, the specificity of
reactions was possible at higher dilutions of
nnliscrum and tested against heterologous milk
at slightly excess antigen 'lOne (Fig. 2). It was
therefore concluded that when the maximum
titre w,IS 1/32. (he nn(iscrum diluted 16 times
could :;epecijically id<:ntify the presence of
homologous milk upto a dilution 1/30 and the
antiserum diluted 32 times could specifically
distinguish the presence of homologom milk
diluted 40 times. The zone of specificity of
antigen-anlibody reactions by the present modilied method would not only facilitate bulk production of antiserum but aho aid in the supply
of the reagent at lower cost .
...
The specificity of antigen-antibody reactions
were ,tudied further at different pH (Table I)
lIsing different diTlltors (Table 2). Highly specilic reaction~ were observed at pH ranging bocween 7.0 and 8.0 with phosphate buffer when
the antiserum ww; diluted to 1/8 with buffer
and tested against buffalo and cow milk both
being diluted to 1 nO. Kabat and Mayer (1961)
suggested that the pH of the 'lntigenic solutions
B.T. Jairam and P.C. Nair
33
TABLE 1
Reactions of .nti·OOII'alo (micellar casein) .. ilk _UID dnuted 8 times with phosphate buft'er mixtures at
different pH
Hydrogen Ion concentration (pH)
Milk diluted with nine p;!r1s
of water
6.0
+ ++
Buffalo milk
Cow milk
-
7.0
7.5
8.0
++ +
1++
+ ++
6.5
+ ++
::I:
NeBative reaction.
+++
± Doubtful reaction.
Strona posilive reaction.
TABLE 1
Rl!lIctlons of Rhti-bulfai" (mkellar caseio) uulk serum dll.led 8 limes wil' ... ter, salin (0.9%).
aid bolfen
Anti-buffalo mille serum diluted. eight times with
Milk diluted Wllh nine
parts of water
Buffalo milk
Cow milk
-
Nosalive.
±
.- - ... - -
DistjIJed
Saline
(0.9%)
+++
++ ~
::I:
±
Doubtful,
+++
-----
Phosphate buffer
pH 7.0
Veronal buffer
pH 8.4
Borate buffer
pH 7.0
+++
+-'-+
+++
Strongly positive reaClion.
TABLE 3
a
Readions of anli-balT.lo milk SNIIID, dil.tcd
ti ...... lrith buffer, against QDe part of buft'alo and
mind In dirrerent proportions and diluted with 4 part. DC waler
cow milk
Mixtures of I part of buffalo milk with different proportions of cow milk
I :1
1:3
1:7
1 , 14
I : 31
1 : 63
+++
+ ++
+++
-t-'-+
++
±
Ncptive.
± Doubtful,
.+ +
Fairly strong positive,
+ + -j-
l 00 % cow milk
Strongly positive reactio ....
Hansa Test Serum
34
should be near neutrality and the salt concentration should be near 0.15 M. Otherwise nonspecific precipitation or inhibition might occur.
Jennes and Patton (19W) stated that casein partieles were sensitive to changes in the divalent
cation content of the medium , soch as calcium
and llUlgnesium ion.", which were strongly
bound by casein and served to cause aggregation
of the particles as was seen in the present work.
Phosphates and citrates ordinarily exerted action
opposite to that of calcium and magnesium ions.
The formulation of such complex ions had the
effect of reducing the concentration of calcium
and magnesium ions in the solutions and of
taking care of apparent discrepancy between
cations lind ion s. Soluble rea.gents which thus
tied up a particular ion were called sequestering agents . The use of phosphate buffer was,
therefore, justified fo r maintaining the stability
of cation and the milk proteins. Hipp et af.
(1952) while fractionating caseins studied the
disaggregation property of 0.15 % sodium
chloride which, when added increased the dis.
aggregation of caseins. The use of buffer
in place of saline was beneficiaL It could reduce
the intensity of cross reactions or non-specific
reactions as compared with the serum diluted
with non-buffering sohltions. Use of phosphate
buffer (pH 7.0) or veronal buffer (pH 8.4),
(Table 2), as dilutors of the antiserum was preferable for preparing highly specific reagent.
The pattern of reactions of the antiserum as
tested against the mixture$ of different proportions of buffalo milk and cow milk, diluted with
four parts of water has been given in Table 3.
The presence of 0.3 % buffalo milk in cow milk
could be detected by this test.
SUMMARY
Sero-dilution technique in place of seraabsorption technique was reported to produce
bulk production of highly specific antiserum.
Optimum dilutions of buffalo milk for all dilutions of antiserum W AS found to be when 1 part
of mil k was diluted be tween 39 and 59 parts
with water. S~cific reactions could occur when
the high titred anti·micellar casein serum was
diluted with 15 and 31 parts of saline respectively.
It could specifically identify the presence of
buffalo milk in the admixture with milk of other
species and increase the production of antiserum
adequately. Buffers like phosphate buffer (pH
7.0) would be preferable to saline dilutions for
avoiding false positive reactions.
ACKNOWLEDGEMENTS
The authors wish to ack nowledge the kind
help and sllggestion~ made by Dr. S.N. Ray,
Ex-Director, Dr. N.C. Ganguli. former Head,
Dairy Chemistry, Dr. M.N. Razdan , former
Dairy Physiologist and Sri K.G.S. Nair, Senior
Technician, National Dairy Research Institute,
Kamal , during the period of research.
REFERENCES
CUShitlg, J.E. and Campbell, D.H. (1957) Pri'lcipies of
[mmunology. McGraw Hill, New Yorlc.
Hipp, N.J., Groves, M.L., Custer, H.H. and McMeekin,
T.L. (1952) J. Dairy Sci., 35,272.
Jairam, B.T. (1967) M.Sc. Thesis, Pal\iab University.
Chandigaril.
Jairam, B.T. and Nair, P.G. (1980) 1m/ian J. Dairy Sd.,
33, 51.
Jennes, R. and Patton (1959) Prirv;iples of Dairy ChemiJtry,
John Wiley & Sons, New York.
Kabat, E.A. and Mayer. M.M. (1961) Experim."IQ/
Imml/wokemis"y, II Ed., Charles C. Thomas Springfield, Illinois.
Landsteiner, K. (1945) 1"116 JIHcifiCilY 0/ serololicai
rtoclioll!J, Dover Publications rnc., New York.
Nair, N.O., Nair, K.G.S. and lairarn, B.T. (1970) InditJlf
J. Dairy Sci .• 28, 168.
Nair. P.G., Slldershan Sinah, M . and
(1965) Indian Doi,yma~, 17, 19M.
Mchar Singh.

Download Report