2001 Remel Inc. / Printed in the UK / Murex Purified Phytohaemagglutinin PURIFIED PHYTOHAEMAGGLUTININ
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.
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
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 Manufactured for:
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 Manufactured by:
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

Source: http://e-skola.biol.pmf.unizg.hr/odgovori/PHA.pdf

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