Latex Antistreptolysin O Test as a

Tube Dilution Procedure

GERHARD L. BACH, M.D., RONALD CADOTTE, B.S., ROBERT A. WIATR, B.S.,
MARUTI BHORADE, M.D., AND TRUMAN O. ANDERSON, M.D., PH.D.
Department of Clinical Immunology and Rheumatology, Cook County Hospital, Chicago, Illinois;
University of Loyola Stritch School of Medicine, Maywood, Illinois; Abraham Lincoln
School of Medicine, University of Illinois College of Medicine, Chicago, Illinois

ABSTRACT
Bach, Gerhard L., Cadotte, Ronald, Wiatr, Robert A., Bhorade, Maruti, and
Anderson, Truman O.: Latex antistreptolysin O test as a tube dilution pro-
cedure. Amer. J. Clin. Path. 57: 209-211, 1972. Recently, several investigators
reported on the usefulness of a latex antistreptolysin O (ASO) test as a screen-
ing procedure for the detection of elevated ASO titers. The present report
deals with applications of a variant of this technic to the quantitation of ASO
titers in serum. One hundred fourteen serum specimens were analyzed for
ASO activity by this tube dilution technic and by two standard hemolytic
methods, the Todd method and the International Unit procedure. T h e results
with all three assays were comparable.

RECENTLY, a latex antistreptolysin O (ASO) of antibody show a negative reaction in the

test has been developed and reported as a
screening procedure for the detection of
significantly elevated ASO titers.1- "•5-7' 9 >"
This simple, rapid technic was shown to
be of value in reducing the cost and effort
needed in the performance of standard
hemolytic assays for ASO by identifying
and eliminating serum specimens with ti-
ters of less than 200 International Units
(I.U.) from further consideration. The pres-
ent report deals with application of the
latex ASO reaction to the actual measure-
ment of antibody titers. Specifically, the
test procedure involves preliminary addi-
tion of sufficient streptolysin antigen to each
serum dilution to react with 100 units of
ASO antibody. This step achieves two ob-
jectives: (1) all sera with less than 100 units
Received January 25, 1971; received revised man-
uscript March 15, 1971; accepted for publication
March 29, 1971.
Address reprint requests to: G. L. Bach, M.D.,
6718 Gruenstadt, Beim Bergtor 12, West Germany.
209
first tube dilution, and (2) the incremental
difference between each serum dilution and
the next must be at least 100 units of ASO
antibody.
For purposes of comparison and valida-
tion, ASO titers were determined in paral-
lel on 114 serum specimens by three pro-
cedures: the latex ASO reaction under
study, and two standard hemolytic tech-
nics—the Rantz and Randall modification 8
of the Todd procedure, 10 and the method
described by Storiko 0 which employs the
International Unit system.
Materials and Methods
The latex ASO tube dilution procedure
uses the following solutions: (A) latex
ASO reagent,* an aqueous suspension of
polystyrene latex particles coated with
streptolysin O antigen, a purified protein
derived from culture of /{-hemolytic group
' Behring Diagnostics, Woodbury, N. Y.

Downloaded from https://academic.oup.com/ajcp/article-abstract/57/2/209/1764703

by guest
on 11 March 2018
 210 BACH ET AL. A.J.C.P.—Vol. 57

Talk 1. Comparison of the Latex ASO Tube Dilution Technic with

Two Conventional Hemolytic Assays

Latex ASO (I.U.) vs.

Rantz and Randall
Latex ASO (I.U.) vs. (T.U.)
Behring Hemolytic Procedure
(I.U.) Approx. No.
International the Sera
Units (I.U.) Same ± 1 tube >±1 same* ± 1 tube Tested

<100 5 — — 5 — 5
100 13 2 — 12 3 IS
200 IS 9 — 18 6 24
300 26 6 — 30 2 32
400 6 2 1 5 4 9
600 8 4 — 10 2 12
800 9 1 1 11 — 11
1,200 6 — — 5 1 6

TOTAL £ 88 24 2 96 18 114
(77%) (84%)

* Since the conventional Rantz and Randall8 modification of the Todd procedure10 employs a dilution series
different from that used in the International Unit system,9 we interpreted the titers obtained by the Todd method
as "approximately the same" when the endpoint was either just above or just below the International Unit end-
point. This was always within one tube dilution.

C streptococci, and (B) the streptolysin O
in solution at a concentration such that
0.3 ml. contained sufficient antigen to bind
100 I.U. of ASO antibody. Although the
latter reagent was always prepared fresh
prior to its use by adding sterile saline so-
lution to the lyophilized enzyme, prelimi-
nary studies suggest that the reconstituted
enzyme retains its activity as antigen in
the latex reaction for at least 10 days at
4C.
Using saline solution, serial dilutions of
sera were employed as follows: undiluted,
1:2, 1:3, 1:4, 1:6, 1:8, and 1:12. T o each
0.1 ml. of these dilutions 0.3 ml. strepto-
lysin O reagent (containing enough strep-
tolysin O antigen to neutralize 100 I.U. of
antistreptolysin antibody) was added. The
tubes were stoppered and the mixture thor-
oughly agitated by shaking 10 to 15 times.
The mixture was then incubated at room
temperature for 15 min. After incubation,
one drop of each dilution and one drop of
latex ASO reagent were mixed on a glass
slide. The reaction was read at 3 min. and
recorded as positive or negative with re-
spect to particle agglutination. Agglutina-
tion reactions occurring after 3 min. were
not considered, since the intensity of the
agglutination reaction was in some measure
time dependent. The 114 serum specimens
studied in the present report represented
a wide range of antistreptolysin titers. They
were obtained from the serology laboratory
of the University Hospital. Those speci-
mens which were not tested within 24 hr.
were frozen and stored at — 20 C. for sub-
sequent use.
Parallel measurements of ASO activity
were done on all serum specimens by the
Todd 1 0 procedure as described by Rantz
and Randall 8 and the technic suggested
by Storiko 9 using commercial reagents.t
fDifco Laboratories, Detroit, Michigan; Behring
Diagnostics, Woodbury, N. Y.

Downloaded from https://academic.oup.com/ajcp/article-abstract/57/2/209/1764703

by guest
on 11 March 2018
 February 1972 LATEX ANTISTREPTOLYSIN O TEST 211

Results in the hemolytic tests. This discrepancy

A comparison of ASO titers obtained by may be a result of the fact that Klein and
the three different test methods is presented colleagues employed a micromethod, where-
in Table 1. The two hemolytic assay meth- as our study used the conventional macro-
ods, the Todd procedure and the Inter- technic.
national Unit measurement, showed titers The results of the present investigation,
equivalent to those found by the latex par- which employed two overlapping doubling
ticle test in 77% and 84% of instances, dilution series of the test sera, suggest that
respectively. Of the remaining 32 serum the latex ASO tube dilution test is as re-
specimens, 30 differed from the latex titer liable and as sensitive as the more time
by ± one tube dilution on hemolytic assay, consuming hemolytic procedures and that
and two differed by more than two tube it has certain technical advantages. The
dilutions. technical performance of the latex ASO test
is simple and reproducible. T h e reagents
Comments are stable and can be stored for many
There have been several other reports in months, thus eliminating the need of ob-
recent literature involving attempts to de- taining and processing erythrocytes.
scribe the quantitative aspects of the latex
ASO reaction system. Farthofer 4 focused on References
a possible relationship between the inten- 1. Bach GL, Wiatr RA, Anderson T O , et al.:
T h e latex ASO test: A rapid screening pro-
sity of the agglutination and the titer of cedure for the detection of elevated antistrep-
the serum, but concluded that correlation tolysin O titers. Amer J Clin Path 52:126-
128, 1969
was not sufficiently consistent to be useful. 2. Baenkler HW, Scheiffarth F, Legler F: Eine
This lack of correlation might be explained Modifikation der Antistreptolysin-Latex-Tech-
nik zur Betimmung eines Titers. Z Rheuma-
in part by our observation that the inten- forsch 28:208-211, 1969
sity of the agglutination reaction is time 3. Eckhardt R, Mai K: ASL-Latex-Test und Anti-
streptolysingehalt menschlicher Seren. Klin
dependent. Farthofer 4 used a three-step di- Wschr 48:295-297, 1970
lution series which permitted a closer fol- 4. Farthofer F: Ober eine abgestufte Latex-Anti-
low-up of the ASO titer in the course of Streptolysin-O-Reaktion lm O b j e k t t r S g e r -
Schnelltest. Z Rheumaforsch 27:424-427, 1968
disease. In the study reported by Baenkler 5. Klein GC, Baker CN, Moody, MD: Comparison
and associates,2 quantitation was achieved of antistreptolysin O latex screening test with
the antistreptolysin O hemolytic test. Appl
either by (a) holding the serum constant Microbiol 19:60-61, 1970
and incrementally increasing the amount 6. Mathies H, Gaedicke H : Die Latex-ASL-Reak-
tion zur groben Differenzierung normaler und
of antigen (streptolysin O) added, or (b) pathologischer Antistreptolysintiter. Med Klin
holding the antigen constant and incre- 63:165-167, 1968
7. Meigel W, Deindl G: Der Latex-ASL-Test.
mentally increasing the amount of serum Munchen Med Wschr 111:198-202, 1969
added. These workers concluded that such 8. Rantz LA, and Randall E: A modification of
an approach was sufficiently accurate to the technic for determination of the anti-
streptolysin titer. Proc Soc Exp Biol Med 59:
provide clinically useful data. 22-25, 1945
One other study deserves comment. Klein 9. Storiko K: Serologische Reaktionen zur Diag-
8 nose rheumatischer Erkrankungen. Labora-
and co-workers reported data showing a toriumsblatter (Behringwerke) 2:1-46, 1967
lack of agreement between the hemolytic 10. Todd EW: Antigenic streptococcal hemolysin.
J Exp Med 55:267-280, 1932
ASO tests conducted using reagents from 11. Winterhoff D: Latex-Antistreptolysin-Reaktion
Behringwerke and reagents from Difco zur quantitativen Bestimmung von Strepto-
kokken-Antikorpern. Z Immunitaetsforsch 137:
Laboratories. We found no such differences 417-420, 1969

Downloaded from https://academic.oup.com/ajcp/article-abstract/57/2/209/1764703

by guest
on 11 March 2018