C03HA16GB 8E28-01 30852801 Purified Phytohaemagglutinin
2001 Remel Inc. / Printed in the UK / Murex Purified Phytohaemagglutinin
PURIFIED PHYTOHAEMAGGLUTININ BIBLIOGRAPHY 1 Börjeson, J. et al. (1964). Purification of the Mitosis-Stimulating Factor from
Phytohaemagglutinin (PHA), derived from extracts of Phaseolus vulgaris
Phaseolus vulgaris. Biochim, biophys. Acta, 82,158.
seeds, has been used for a number of years, on account of its twin
Calne, R.Y., Wheeler, J.R. and Hurn, B.A.L. (1965) Combined
properties of causing erythroagglutination and of stimulating progressive
Immunosuppressive Action of Phytohaemagglutination and Azathioprine (Imuran)
lymphocyte mitosis in cell culture3,5,13,18. Both agglutinating and mitogenic
on dogs with Renal Homotransplants. Brit. Med. J., ii,154.
activities appear to be associated with the protein fractions of crude
Carstairs, K. (1962). The Human small Lymphocyte: Its possible Pluripotential Quality. Lancet, i, 829.
extracts, which are sufficiently alike in their physico-chemical properties
Cooper, E.H., Barkhan, P. and Hale, A.J. (1963). Observations on the
to have caused difficulty in attempts to separate them1,17,19. One other
Proliferation of Human Leucocytes Cultured with Phytohaemagglutinin. Brit. J.
factor complicating fractionation procedures has been the slow and
Haemat., 9, 101.
imprecise method of assay for the mitogenic activity. Fortunately,
Dorset, M. and Henley, R.R. (1916). Production of clear and sterilized Anti-Hog-
relatively crude extracts selected for their ability to yield good mitotic
Cholera Serum. J. agric. Res., 6, i, 333.
pictures (as in reagent grade Phytohaemagglutin) have proved entirely
Elves, M.W. (1966). In Proc. Symp. ‘Biological Effects of Phytohaemagglutinin’.
satisfactory for use in routine lymphocyte culture for chromosome
Robert Jones and Agnes Hunt Orthopaedic Hospital Management Committee,Oswestry, England, 11-26.
studies. PHA has proved of interest in the study of the immune
Gamble, C.N. (1966). The effect of Phytohemagglutinin on the Primary Antibody
response2,6,7 lymphocyte kinetics4,9,14,15,20, and bone marrow
response of mice to rat Erythrocytes and Human Gamma Globulin. Int. Arch.
dynamics8,10,12, and for these purposes it is desirable that a substance
Allergy, 29, 470.
of more closely reproducilble qualities and known potency should be
Hayes, D.M. and Spurr, C.L. (1966). Use of Phytohemagglutinin to stimulate
used. Improvements in the technique of assaying mitogenic activity16
Hematopoiesis in humans. Blood, 27, 78.
have made it possible to quote a value for each batch of Purified
Holm, G. and Perimann, P. (1965). Phytohaemagglutinin-Induced Cytotoxic action of Unsensitized Immunologically competent cells on Allogeneic and
Phytohaemagglutinin in terms of a reference standard preparation.
Xenogeneic tissue culture cells. Nature (Lond.), 207, 818. COMPOSITION Humble, J.G. (1964). The treatment of Aplastic Anaemia with Phytohaemagglutinin. Lancet, i, 1345.
Purified Phytohaemagglutinin is a freeze-dried, highly refined protein
11 Hurn, B.A.L. (1966). In Proc. Symp. ‘Biological Effects of Phytohaemagglutinin’.
fraction of selected Phaseolus spp. seed extract in which the specific
Robert Jones and Agnes Hunt Orthopaedic Hospital Management Committee,
mitogenic activity and the mitogenic/haemagglutinating activity ratio
have been increased by a factor of about 100/1 during purification11.
12 Israel, L., Sors, Ch. and Bernard, Et. (1966). La Polychimiothérapie prolongée
Each bottle contains the stated weight and activity of PHA, dried from
des cancers Broncho-Pulmonaires inopérables. Sem. Hôp. Paris, 29, 1825.
a small volume of buffered saline without preservative. Li, J.G. and Osgood, E.E. (1949). A method for the rapid separation of Leukocytes and Nucleated Erythrocytes from blood or marrow with a PRECAUTIONS
Phytohemagglutinin from red beans (Phaseolus vulgaris). Blood, 4, 670.
14 Ling, N.R. (1968). ‘Lymphocyte Stimulation’. Amsterdam; North Holland Press.
15 McIntyre, O.R. and Ebaugh, F.G. (1962). The effect of Phytohemagglutinin on
Although Purified Phytohaemagglutinin may be employed for routine
Leukocyte cultures as measured by p32 incorporation in the DNA, RNA, and
lymphocyte culture, the conditions necessary for its successful use
Acid Soluble Fractions. Blood. 19, 443.
are much more stringent than those required for less pure reagents. Mosedale, M.B. and Parke, J.A.C. (1966). Assay of the Lymphocyte stimulating factor in Phytohaemagglutinin. In Proc. Symp. ‘Biological Effects of
In particular, the concentration of PHA giving maximal lymphocyte
Phytohaemagglutinin’. Robert Jones and Agnes Hunt Orthopaedic Hospital
stimulation without inhibition or toxicity lies within very narrow limits.
Management Committee, Oswestry, England, 97-103.
The use of Reagent Grade Phytohaemagglutinin is recommended for
17 Nordman, C.T., de la Chapelle, A. and Gräsbeck, R. (1964). The Interrelations
all purposes that do not require precise dosage of mitogen with minimal
of Erythroagglutinating, Leucoagglutinating and Leucocyte-mitogenic Activities
haemagglutinating activity and inactive impurities.
in Phaseolus vulgaris Phytohaemagglutinin. Acta med. scand. Suppl., 412.
18 Nowell, P.C. (1960). Phytohaemagglutinin: An initiator of Mitosis in Cultures of
Normal Human Leukocytes. Cancer Res., 20, 462.
The reagent may be reconstituted to any desired volume in sterile saline
19 Rigas, D.A. and Johnson, E.A. (1964). Studies on the Phytohemagglutinin of
(if buffered, preferably to a pH between 6.5 and 8) or distilled water,
Phaseolus vulgaris and its Mitogenicity. Ann. N.Y. Acad Sci., 113, 800.
20 Sell, S., Rowe, D.S. and Gell, P.G.H. (1965). Studies on rabbit Lymphocytes In
using a sterile disposable hypodermic syringe. The cap of the bottle
Vitro J.exp. Med., 122, 823.
should be sterilized by wiping with ether, the needle should pierce thecentre of the rubber plug and be held in a vertical position duringreconstitution. If desired, the risk of bacterial contamination
of reconstituted Phytohaemagglutinin may be minimized by addition of
a few drops of chloroform to the solution: alternatively, antibioticsor any of the usual preservatives may be added if the proposed use of
Murex Biotech LimitedCentral Road, Temple Hill
LIFE AND STORAGE
The dried material will retain full potency at least until the date shown
on the bottle label when stored at 2 to 8°C. The reconstituted materialmay be stored at –20°C for six months or at 2 to 8°C for two weeks
Available in the U.S.A. from:
without loss of activity (in the absence of bacterial contamination). For Technical Assistance call: Toll Free - 800 255 6730 / 913 888 0939
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