Академический Документы
Профессиональный Документы
Культура Документы
Protected by copyright.
J. clin. Path. (1963), 16, 70
SYNOPSIS An elution technique is described which gives eluates at least as potent as previously
described methods. Elution can be completed in only 15 minutes. The method, a modification of
that of Vos and Kelsall, is simple to carry out and requires the minimum of equipment. Studies
have shown that the coating antibody is almost, but not completely, eluted.
Antibody protein adherent to the red cell surface can method requires about three hours but with some
be detected in several ways. However, in order to minor modifications (Jensen, 1959) it can be accom-
determine their serological specificity it is necessary plished in about one and a half hours without
to elute them so that they can then be tested with red reducing the antibody content significantly. Kom-
cells of known antigenic content. A number of ninos and Rosenthal (1953) suspended red-cell
methods have been devised to elute the antibodies stroma in 8 % hypertonic sodium chloride at room
but each method suffers from one of two faults; temperature and obtained good eluates. An addi-
either weak eluates are obtained or potent eluates are tional 48 hours was required, however, to bring the
obtained only after several hours' or days' work. A eluate to isotonicity by dialysis before it could be
rapid simple procedure yielding high-titred etuates tested.
would be of considerable practical value. Greenwalt (1956) precipitated stroma in cold
By far the simplest procedure for elution is the 0-25% formalin and then separated the stroma on
original method of Landsteiner and Miller (1925). glass wool. Elution is accomplished at 56'C. This
Although requiring only 10 to 15 minutes to carry takes somewhat more than two hours to perform.
out, this method, unfortunately, gives eluates which Vos and Kelsall (1956) treated acid-precipitated
are low in antibody activity. Other and later methods stroma with diethyl ether and obtained very good
are more elaborate and time-consuming, entailing eluates in about one and a half to two hours.
first separating red blood cell stroma from haemo- The purpose of the present study was to compare
globin as completely as possible which adds signi- some of the less time-consuming methods of anti-
ficantly to the time involved in the procedure, and body elution and if possible to improve on them. It
then eluting the antibody from the stroma. Kidd was found that the methods of Weiner and Vos and
(1949) eluted antibodies by exposing stroma to a of Kelsall gave the strongest eluates and by modify-
buffer at pH 3-2 to 3-4 at room temperature but this ing the latter method eluates have been obtained as
method is complicated. Selwyn (1952) prepared strong as those obtained by the original method
stroma in the same way but then eluted the antibody although the working time has been reduced to 15
by heating the stroma to 60°C. at a pH of 6-6 to 6-8. minutes. The modified method is described. In addi-
Mitchell (1956) attempted to define the optimum tion some observations on the sensitized cells of
conditions of temperature and pH at which anti- patients with auto-immune haemolytic anaemia are
bodies are best eluted and found that for different reported.
types of antibodies, different optima applied. He also
stated that the texture of the initial stromal precipi- METHODS
tate affected the yield of antibody. His method as
finally evolved requires two dialysis steps and 48 GENERAL The general techniques applied in this study
hours before the final eluate was obtained. were those previously described by Dacie (1956). Com-
parisons between methods were always begun on the
Weiner (1957) precipitated stroma and antibody in same day. When sensitizing cells with antibody, the same
cold 50% ethanol and then eluted at 37°C. The source of fresh red cells or cells stored in acid-citrate-
'Trainee, National Institute of Arthritis and Metabolic Diseases, dextrose solution for not more than one week was used.
National Institutes of Health, Bethesda, Maryland. In the initial studies an incomplete warm type of anti-D
Received for publication 11 July 1962. was used as a prototype antibody.
70
J Clin Pathol: first published as 10.1136/jcp.16.1.70 on 1 January 1963. Downloaded from http://jcp.bmj.com/ on March 17, 2020 by guest. Protected by copyright.
Antibody elution from red blood cells 71
TECHNIQUE OF ELUTION An equal volume of 0-85 % saline enzyme-treated cells and it is irrelevant when using the
is added to one volume of washed and packed red cells. indirect antiglobin technique.
To this mixture is then added two volumes of diethyl ether
A.R. The container is stoppered and mixed by repeated SENSITIZATION INDEX An attempt was made to relate the
inversions for one minute. Mixing for longer periods does antibody content of the final eluates with the degree of
not influence the strength of the final eluate (Table I). The sensitization of the original red cells in order to determine
mixture is then centrifuged for 10 minutes at 3,000 r.p.m. to what extent elution was quantitative. The same rabbit
After centrifugation three clearly defined layers are antihuman globulin reagent (Universal antihuman
visible: an upper layer of ether, a middle layer of de- globulin) prepared in this laboratory was used through-
natured stroma, and a bottom layer of haemoglobin- out. The degree of sensitization of the red cells has been
stained fluid. The two top layers are removed by vacuum scored by taking into account the intensity of agglutina-
suction and the haemoglobin-stained fluid remaining is tion (0 to 4+) and the dilution of the antiglobulin serum
the eluate. This can be tested immediately or stored frozen producing agglutination. The agglutination score was ob-
at - 20'C. In the latter instance, the eluate should first tained by adding together the number of plus signs at
be placed in a 37°C. water-bath for 15 minutes to each dilution of antiglobulin serum and multiplying by
evaporate any residual ether. Eluates prepared in this the number of dilutions in which agglutination occurred.
way have been stored frozen at - 20°C. for three months The time at which agglutination occurs after adding the
without any loss of antibody activity. Vos and Kelsall reagent is also a reflection of the amount of antibody
reported that their eluates kept at - 20°C. for two years coating the cell. An arbitrary index of sensitization was
without any significant change in antibody titre. They, therefore calculated as follows:
however, employed type AB serum instead of saline as an
eluting media and this may influence the potency of the agglutination score
Sensitization index - time
eluates after storage. of agglutination (in seconds)
The method can be applied successfully to fresh red
cells or to cells stored frozen after washing and packing The dilution of the antiglobulin reagent at which the
without the addition of any preservative solutions. agglutination was timed was one which gave strong
The haemoglobin in the eluate, although aesthetically agglutination in all instances, 1 in 64. The average of two
unpleasing, does not interfere with the reading of readings was used and the time was reproducible within
agglutination when its antibody content is titrated with one or two seconds (naked-eye reading).
TABLE I
COMPARISON BETWEEN SHAKING TIME AND STRENGTH OF ELUATES
FOR BOTH WEAKLY AND STRONGLY SENSMZED CELLS
Shaking Time in Minutes Dilution of Eluates
I in I I in 2 1 in 4 1 in 8 1 in 16 1 in 32 Saline Control
Weakly sensitized cells
I + 0 0 0 0 0 0
5 + 0 0 0 0 0 0
15 + 0 0 0 0 0
Strongly sensitized cells
1 ++ + (±) 0 0
5 ++ ++ + (±) 0 0
15 ++ ++ + + ) 0 0
TABLE II
COMPARISON OF ELUTION METHODS
Method of Dilution of Eluates Saline Agglu-
Elution Control tination
I in I I in2 I in 4 1 in 8 1 in 16 1 in 32 1 in 64 1 in 128 Score
Titration of eluates of anti-D with trypsinized cells at 370C. 0
Greenwalt ++ +(±) + (±) 0 0 39
Landsteiner +++ +++ + +() ++ (i) 0 0 91
Weiner +++(±) +++ + +--+ +-r(±) -1+ (±) 0 128
VosandKelsall ++++ ++++±+++ + ++(±) + (±) 0 176
Presentmethod ++++ ++++ ++++ ±+ + ++ +(± + +C4 ) (i) 0 188
Titration of eluates.of anti-D by indirect antiglobulin method
Greenwalt + +(±) ++ + (±) o 0 0 0 0 24
Landsteiner +++ +++ +++ ++ ++ + O 0 0 84
Weiner +±+± ++(±) ±+++ +±+ + ±(±) ++ 0 0 133
VosandKelsall + + + ++++(±) +++ +++ + +-(i ++ + 0 168
Presentmethod +++ ++-(±) +++-+ + ++ +(± -)_
0 152
8
J Clin Pathol: first published as 10.1136/jcp.16.1.70 on 1 January 1963. Downloaded from http://jcp.bmj.com/ on March 17, 2020 by guest. Protected by copyright.
72 Harry Rubin
RESULTS
TAB]LE III
RELATIONSHIP OF SENSITIZATION INDEX TO AGGLUTINATION SCORE OF ELUATES OF ANTI-D
Dilution of Anti-D Serionit Sensitization Index Agglutination Score of Eluate bY Agglutination Score of Eluate with
Indirect Antiglobulin Method Trypsinized Cells at 37°C.
TABLE IV
RESULTS OF ELUTION WITH SENSITIZED CELLS FROM PATIENTS WITH AUTO-IMMUNE HAEMOLYTIC ANAEMIA
Patient Diagnosis Tvpe Antibody, State of Red Blood Cells Eluate