Biomedical Engineering Reference
In-Depth Information
of adjuvant based on mineral oil for a single injection dose of one
rabbit (one-third of these volumes for injection of mice). Prepare
an emulsion by homogenization with a syringe and a thin needle
until a stable white cream is obtained. This water-in-oil emulsion
is stable for several hours.
An immunization scheme for rabbits is given in Table 4.5. Do
not forget to take some milliliters of blood for pre-immun serum
immediately before or after the first immunization.
Table 4.5.
Protocol of rabbit immunization
Day
Amount of
Collected
Remarks
antigen
blood (ml)
µ
(
g/animal)
0 (Preimmun serum)
5
1 (First immunization)
50 - 200
14 (First boost)
50 - 100
5
First test
28 (Second boost)
50 - 100
5
Second test
35 (Third boost)
50 - 100
5 - 50
Third test and/or
first antiserum
43 - 44
25 - 100
Second antiserum
References
Cooper HM, Paterson Y (2000) Preparation of polyclonal antisera. Current
protocols in molecular biology. Wiley, Unit 11.12
Green JA, Manson MM (1992) Production of polyclonal antisera. In: Man-
son MM (ed.) Immunochemical protocols. Methods in molecular bi-
ology, vol. 10. Humana Press, Totowa, N.J., p 1
Harlow E, Lane D (1988) Antibodies. A laboratory manual. Cold Spring
Harbor Laboratory, p 92
Peters JH, Baumgarten H (eds.) (1992) Monoclonal antibodies. Springer,
Berlin, p 39
4.3 Ammonium Sulfate Fractionation
of Immunoglobulins
Stir freshly collected blood with a glass rod to bind fibrin and allow
clotting first at RT for 1 - 2 h and then at 4
◦
C overnight. Pour the
serum into a centrifuge tube and centrifuge at 100−300
g for
10 min. If the serum is not used within the next day, aliquot the
serum, freeze rapidly and store below −20
◦
C.
×
A1M Tr i s
·
HCl, pH 8.0
Solutions/Reagents
B
saturated ammonium sulfate in ddH
2
O (stir about 100 g
(NH
4
)
2
SO
4
in 100 ml ddH
2
O for several hours, then allow to
