вход по аккаунту


The detection of anti-globulin factors utilizing pre-coated latex particles.

код для вставкиСкачать
The Detection of Anti-Globulin Factors Utilizing
Pre-Coated Latex Particles
A modified serological procedure for the
detection of anti-gamma globulin factors
has been developed. This test employs
polystyrene latex particles with a diameter of 0.25 microns. The particles are
coated with gamma globulin and the excess protein in solution is removed by
repeated washing. A solution of these
gamma globulin coated particles is stable
for at least one year, and yields more
reproducible titers than those originally
described for the latex agglutination test.
Esseva disveloppate un modificate procedimento pro le detection de factores
anti globulina gamma. Le test utilisa
particulas de latex polystyrenic con un
diametro de 0,25 micron. Le particulas
es revestite de globulina gamma e le
excess0 de proteina es eliminate per
repetite ablutiones. Un solution de tal
particulas revestite de globulina gamma
es stabile durante a1 minus un anno. Le
titros obtenite con ill0 es plus reproducible que illos describite pro le test original de agglutination a latex.
KIFORM SIZE LATEX PARTICLES 8000 A in diameter (Dow Chemical) have become widely used as particulate carriers of antigens for the
detection of many antibodies or antibody-like substances.l The technic as
originally applied to the serological measurement of rheumatoid, or antigamma globulin factors is known as the latex fixation or latex agglutination
reaction (FII-LP) .*
Bozsoky” recently used the FII-LP reaction as a prototype to emphasize the
hazards in the reporting of results using a “standard test in different laboratories. Using standard lyophilized dried sera he asked 30 laboratories in 14
countries to perform FII-LP and sensitized sheep-cell (SSC) tests on a blindfold basis. Great variations in results were obtained and these proved to be
due to small changes in technic. These changes included the use of latex
particles from different commercial sources, variation in the size and concentration of the particles, differing concentrations of coating gamma globulin
(ranging from 0.025 per cent to 5 per cent), inactilration of the sera and
gamma globulin in some laboratories (in contradiction to standard technic),
incubation periods ranging from none to 2 hours, and reading times from
immediately to 19 hours later. It therefore was not surprising that a standard
positive serum gave results from 1:32 to 1:20,000.
This I ariability is particularly troublesome in research investigaticns and
epidemiologic studies. It gives rise to obvious difficulties in the comparison
of results obtained among various institutions at different times, if there has
been a change in personnel. As a consequence, a modification of the latex
fixation test has been developed utilizing latex particles 2300 A 2 200 A in
From the Laboratory Division, Department of Microbiology, Mont4ore Hospital,
Bronx, New York and the Department of Medicine, State Uniuwsity of New York, Downstate Medical Center, Brooklyn, N e w York.
Aided in part b y grants No. AM 06052-01-02 from the National Institute of Arthritis
and Atetaholic Diseases, National Institutes of Health untl by the N e w York Chapter of
the Arthritis Foundation.
diameter, ( Lytron Monsanto Chemical). These particles are stable and are
most effectively pre-coated with protein material.
1. Gamma Globulin preparations #I812 and #2015 were obtained from E. R. Squibb
& Sons through the courtesy of the American Red Gross, Washington, D. C.
2. Fraction V Human Albumin was obtained from E. R. Squibb and Sons.
3. (3ycine-snline buffer at p H 9.6, ionic strength 0.24 (25 C.) was prepared liy adding 100 ml. of 1 N NaOH to 116 nil. of 2M glycine and bringing the volume to 1,000
ml. with distilled water, p H was adjusted to 9.6 and then 9 grams of sodium chloride
were added to each 1,000 ml. of buffer.
4. Polystyrene Lytron particles were obtained through the courtesy of the Monsanto
Chemical Company, Springfield, Mass. Lytron 601 (1700 A diameter) and Lytron 61518-1076 (serial #RF-2146) (2500 A diameter) were used.
5. Polystyrene Latex particles were prepared by Dr. John W. Vanderhoff of the Dow
Chemical Co., Midland, Mich., who kindly provided the physico-chemical characteristics
of each batch. Dow LS-040-A, LS-052-A, LS-463-E, LS-061-A, LS-063-A, LS-449-E, L6053-33-1, L-5309-19-3 and L-4849-44-2, varying in size from 500 A to 280.000 A, were
6. Merthiolate solution ( 1:10,000 in glycine-saline buffer) was added to prevent bacterial contamination.
7. Stabilaty i 5 defined as a uniform colloidal suspension with no visible agglutination or
aggregation and with turbidity corresponding to the light transmission in a Coleman, Jr.
Spectrophotometer of. a barium sulfate standard prepared by adding 3 ml. of 0.1 N barium
chloride to 3 ml. of % N sulphuric acid.
Unstable is defined as the presence of visible agglutination or aggregation and a light
transmission of 100 p a cent.
Partial Instability is defined as visible agglutination or aggregation and a light transmission betwem that of the barium sulfate standard and 100 per cent.
Pre-Coating is defined as adsorption of gamma globulin on latex particles with subsequent washing until no more gamma globulin remains in the supernatant.
The general procedure was to add one ml. of latex suspension one ml. of solution of
gamma globulin and to bring the total volume to 10 ml. with glycine-saline buffer at p H
9.6. The tubes were incubated for 1 hour at room temperature (approximately 23 C.) and
centrifuged for 1 hour at 12,000 r.p.m. in a Servall refrigerated centrifuge. The particles
cannot be sedimented readily at lower speeds. The supernatant was discarded and the
sediment gently and gr'idually ( t o avoid flocculation and clumping) resuspended in one
to two ml. of buffer, and the suspension brought to a final volume of 10 ml. This washing
was repeated one more time. After the second washing the resultant stock solution was
diluted with buffer to a 5 per cent transmission in a Coleman, Jr. Spectrophotometer,
Model 11, at 650 mp. Each batch of coated latex particles was tested for agglutination activity using a standard rheumatoid arthritis serum with a 1:10,000 positive titer. The tubes
were incubated in a water bath at 56 C. for 1%hours, refrigerated overnight at 4 C. and
then centrifuged at 2,500 r.p.m. for 5 minutes. The resultant visible agglutination ranging from 1+ to 4+ was read with the naked eye. Agglutination titers were recorded as
the reciprocal of the serum dilution.
Selection of Particle Size. Thirteen batches of uniform-size latex particles ranging from
500 to 280,000 A in diameter were tested for stability in glycine-saline buffer at p H 9.6.
Stability was determined utilizing uncoated and coated particles. Each batch was adjusted to the same surface area of 35 x 1018 A2 per ml. of latex suspension. Three con-
Table 1.-A Comparison of the Stability of 0.25 Micron Lytron Particles Coated
with Various Amounts of Gamma Globulin and Their Reactivity
with a Rheumatoid Arthritis Serum
Concentration of
Gamma Globulin Added
in mg./ml.
Ratio of Protein Added in
mg. per em2of Particles
Stability Particle
4.3 x 10-3
3.0 x 10-3
1.3 10-3
4.3 10-4
1.7 10-4
4.3x 10-5
2.2 x 10-5
1.1x 10-5
5.5 x 10-0
Lytron #615 RF-2146-1.5 mg./ml.
Gamma Globulin Squibb #2015.
Glycine Saline Buffer p H 9.6, p = 0.24.
Total Volume = 10 ml.
centrations of gamma globulin 0.6 mg./ml., 1.5 mg./ml., 4.5 mg./ml., were added to each
batch and tubes were processed according to the general method described. Only two
suspensions of latex particles were stable when uncoited and yielded reprodncible titers
when coated. These particles were Lytron-601 and Lytron-615 diameter 2500 A, and
these particles were selected for further testing.
Efect of p H . Detailed investigations of p H and ionic strength of the 0.25, Lytron
particles have been previously presented.4,5 Stability of human gamma globulin coated
latex particles was studied at pH’s varying from p H 3 to p H 11, ionic strength 0.24. The
particles were stable only below p H 5.0 and above p H 8.0. Glycine saline buffer, p H
9.6 was utilized for the test procedure since there was slight instability of the coated
particles at p H 8.2.
Concmtrution of Added Gammu Globulin. Varying amounts of gamma globulin (Squibb
#2015) were added to Lytron 615 Iatex particles (concentration 1.5 mg./mI.) a t a constant volume of 10 ml. (table 1).
The concentration of added gamma globulin varied from 0.0187 mg./ml. to 15 mg./ml.
The ratio of added gamma globulin varied from 5.5 x 10-6 mg./cm.z to 4.3 x 10-3
mgJcm.2 of particles. The solutions were standardized to match a 5 per cent light transmission in a Coleman Jr. Spectrophotometer. Each solution was tested with a standard
rheumatoid arthritis serum and with a control of glycine-saline buffer p H 9.6. Table 1
demonstrates that coating with small amounts of gamma globulin (0.0187 mg./ml. to
0.075 mg./ml.) results in an unstable solution in both controls and test tubes. With a
higher concentration of gamma globulin the particles were stable and yielded increasing agglutinating titers up to a concentration of 1.5 mg,/ml. Above this concentration
(which may represent the saturation point of the particles for gamma globulin) there
is no increase in titer. For this reason, the concentration of Squibb #2015 gamma globulin
used to coat Lytron #615 latex particles (2500 A in diameter, 1.5 mg./ml.) in glycinesaline buffer yH 9.6 ionic strength 0.1, 25 C., was 1.5 mg./ml. brought to a total volume
of 10 ml.
Increase of Stability by the Addition of Albumin. Since some of the gamma globulin
coated latex suspensions, even when prepared under the described optimal conditions,
tended to become unstable after standing (even under refrigeration) for a period of time,
albumin was added to the stock suspension as a stabilizing agent. Y Albumin 5 pg.
N/ml. of stock solution was utilized to stabilize the solution since this amount did
not interfere with the titer or strength of agglutination. The suspension with stabilizing
albumin incorporated has proved to be stable under refrigeration for at least one year.
Detailed Final Method
Before preparing a large stock solution of Lytron gamma globulin suspension, the
optimal amount of gamma globulin to be added must be determined. One 1171. of a
suspension of Lytron particles #615 (serial RF-2146) containing 48 to 50 per cent
solids is diluted to 1.5 per cent of solids with distilled water. A 3 per cent solution of FII
gamma globulin in glycine-saline buffer p H 9.6 is prepared. A preliminary titration is
then performed. One ml. of Lytron containing 15 mg. of solid particles is placed in each
of five tubes and 7 ml. of glycine-saline buffer is added to cach tube. Two ml. of gamma
globulin containing 0.75 mg., 1.5 mg., 6 mg., 15 mg., or 45 mg., are added to the Lytron
buffer mixture respectively. Tubes are carefully shaken to assure mixing and incubated
for one hour at room temperature. The tubes are then centrifuged under refrigeration a t
4 C. for an additional hour at 12,000 r.p.m. Supernatants are discarded and the sediments
washed twice with 10 ml. of glycine-saline buffer. The sediments are carefully resuspended
by stirring the particles with an applicator stick. Following the second washing the
sediment is resuspended in 1 ml. of glycine buffer. Of this stock solution 0.1 to 0.5 ml.
is added to 10 ml. of glycine buffer until a 5 per cent transmission is reached at 650
rnp in a Coleman Jr. Model 11, Colorimeter in a 13 x 100 nim. square cuvette. The five
working solutions are then added to five rows of a serially diluted rheumatoid serum (1: 10
to 1:10,240) and a control of glycine buffer. Latex gamma globulin 0.5 ml. is added to
0.5 ml. of serially diluted serum. The tubes are then incubated for 1 hour in a water
bath at 56 C., refrigerated overnight at 4 C. and centrifuged the next day at 3,0008 r.p.m.
for 5 minutes. The resulting visible agglutination (1+ to 4 + ) is read with the naked
eye. The most suitable coated particles are those which show no agglutination in the
control tubes and produce the highest agglutination titer with the standard serum. The
least amount of gamma globulin consistent with the maximum agglutination titer should be
Once this preliminary selection of the proper amount of gamma globulin to be added
to the Lytron suspension has been made, a large batch of particles may be prepared
using the same proportions of material. The only differences in preparation of this large
batch are that following the second washing, a glycine-saline buffer p H 9.6 is used which
contains merthiolate ( 1:10,000). Also, 5 y pg. nitrogen of albumin per ml. of stock
solution is addcd to preserve stability under refrigeration for at least twelve months.
The stock solution is diluted as previously described to make the working solution.
The test itself is performed by a technique similar to that of the standard latex test.2
Serum should not be inactivated. The wosrking pre-coated gamma globulin latex suspension
is prepxed so that a 5 per cent light transmission is reached at 650 mp in a Coleman Jr.,
Model 11 Colorimeter in a 13 x 100 nun. square cuvette. One half nil. of this solution is
added to 0.5 ml. of the serially diluted serum to be tested, utilizing glycine-saline buffer
at pH 9.6.
The tubes are then incubated, refrigerated and centrifuged as described in the preliminary titration and the agglutination read with the naked eye. Any titer obtained using
this precoated particle system is considered positive.
The sera of 128 patients with rheumatoid arthritis, 343 with other arthritic
and non-arthritic diseases and 250 controls were tested by both the standard
FII LP and modified pre-coated FII LP tests. Results of the two tests closely
paralleled each other, but the pre-coated FII LP yielded titers which were one
Table 2.-Results of Precoated Gamma Globulin Latex Test in Sera
with Rheumatoid Arthritis and Other Conditions
Rheumatoid arthritis
a. Adult
b. Juvenile
Systemic lupus erythematosus
Rheumatic fever
Cirrhosis of the liver
Neoplastic disease
a. Apparently healthy
Hospital population
b. Healthy individuals
Per Cent
Total Sera
to two dilutions lower. Table 2 shows the results using the pre-coated FII LP
68.3 per cent of adult rheumatoid arthritis patients were positive as contrasted with only 20 per cent of the juvenile rheumatoid arthritis patients.
Thirty patients with systemic lupus erythematosus yielded 30 per cent positive results, a figure in agreement with that generally accepted for the standard
latex test. Twenty per cent of sera from patients with scleroderma were
positive but sera from patients wih rheumatic fever, osteoarthritis and gout
gave, as expected, a very low incidence of positive results. An incidence of
25 per cent positive tests for anti-gamma globulin factors in sera from chronic
liver disease was encountered. Sera from four of forty patients with syphilis
and positive Wassermann reactions had positive titers. Only 0.8 per cent of a
group of sera from 125 apparently healthy non-hospitalized individuals was
positive. 1.6 per cent of an apparently healthy population (physicians, nurses,
technicians) were positive, a figure in agreement with those results reported
utilizing standard technics. Twenty sera from patients with rheumatoid arthritis and a positive titer of 1:2560 were tested monthly for a period of six to
twelve months utilizing the stock suspension of pre-coated particles. There was
no more than one tube variation during this time.
The FII-LP test has been widely m0dified.l Certain modifications, however, carry with them fundamental changes which may lead to great variability in results. The standard latex test utilizes only particles 0.8 micron in
diameter.2 Different batches of alcohol-fractionated gamma gIobulin vary in
degree of- denaturation, amount of aggregation, stability at an alkaline pH
and degree of contamination by other plasma protein^.^,^ Gamma globulin
prepared by sodium or ammonium sulfate fractionation may react differently
due to the presence of other plasma proteins which act as stabilizers of the
I 99
colloidal latex gamma globulin suspension. Pure 7s gamma globulin used for
coating of the particles will be more sensitive, but less stable, than aggregated
gamma g l o b u h 6
For these reasons, the present test was developed in an effort to eliminate
many of the factors which lead to variability in the reporting of result^.^^^
The stability of 0.25 micron diameter latex particles (Lytron) at various pH
and ionic strengths has previously been r e p ~ r t e d . The
~ , ~ amount of gamma
globulin used to coat the particles was determined as described.1° If 15 mg. of
2300 A Lytron particles (2.3 x
particles with a total surface of 4.1 square
feet are mixed with 15 mg. of gamma globulin in a total volume of 10 ml.
then interpolation in an experimentally established bonding isotherm shows
that one is well within the area where the isotherm curve tends to flatten out
out horizontally, that is, within the area where the amount of bound gamma
globulin approaches saturation. This proportion and concentration were
chosen SQ that even a decrease of up to 50 per cent in gamma globulin concentration will not appreciably diminish the amount of gamma globulin
bound per unit surface of particle. The use of concentration of gamma globulin solution 10 per cent or more dilute than the recommended standard may
lead to instability of the resultant latex gamma globulin suspension. In order
to increase the stability of the Lytron gamma globulin suspension so that a
standard batch may be kept for a year or more, small amounts of albumin
( 5 Y g.nitrogedml.) were added because of its protective capacity in
colloidal suspensions. This albumin had no effect on the tests for anti-gamma
globulin factors.
The presently described test, using the stated precautions, is much less
variable than the standard FII LP test. The technic is somewhat more difficult
and this suggests that the standard latex fixation test should still be used for
routine screening purposes. The new test may be performed in positive and
doubtful cases and in epidemiologic and other research.
One of the purposes of introduction of the new test is to produce titers
which not only are reproducible from one laboratory to another but which
may provide information concerning variation in titer of rheumatoid factor in
the same patient at various stages of disease. Inherent in serologic testing is a
small titer variation which in general should not exceed two dilution tubes.
Various rheumatoid sera were tested by the new technic using a single batch
of pre-coated particles over a period of 1 ear.^,^ Only one tube dilution variation was noted. The new test is therefore well within the normal limits of
variability. Reproducible titers are important in studies which reflect possible
changes in rheumatoid factor according to the stage and seventy of disease,
to determine whether there is a change in reactivity of patient serum with
various methods of treatment, to determine whether there is a relationship between titer and prognosis and to reflect possible increases in titer during such
complications of rheumatoid arthritis as vasculitis.
Epidemiologic studies, both of a static and progressive nature, will benefit
by the use of a test which yields reproducible titers. Since the presence of any
anti-gamma globulin factors in serum is abnormal, any positive titer utilizing
the relatively stable technic here described should be considered as a positive
The present test utilizes pre-coated Lytron which has so far proved to be
stable under refrigeration for at least one year. It may possibly prove to be
stable much longer. Laboratories may thus prepare their suspensions at less
frequent intervals and some central agency might prepare the pre-coated
particles and distribute them to various laboratories, further reducing the
possibility of variability of results due to changes in technic.
In studies of the presence of anti-gamma globulin factors, both in rheumatoid arthritis and in other diseases, standardized results are essential. This is
true both of the present te3t and of the usual latex fixation and sensitized sheep
cell test. Particular care should be used in the selection and preparation of
particles, gamma globulin and buffer, and they should be tested against
standard rheumatoid sera which also should be made available by some
central agency. A kit might be provided containing all of the materials for the
test, to serve as reference for the materials utilized by each laboratory.
It has been demonstrated that when an animal is immunized with denatured
homologous gamma globulin the resultant antiserum is reactive with the
-gamma globulin of other species.11-13In the FII-LP test adsorption of 7s
human gamma globulin on the surface of the latex particles produces denaturation or at least distortion of the native human gamma globulin molecules.
This surface denaturation uncovers reactive sites for human antibodies to
rabbit and other animal gamma globulins. The sensitized sheep cell test, on
the other hand, detects antibodies to rabbit gamma globulin. Various studies
have shown that anti-gamma globulin factors exhibit 3 serologic patterns of
reactivity with human and rabbit gamma g l o b ~ l i n . l These
~ - ~ ~ patterns are:
(1) reactive with human gamma globulin; ( 2 ) reactive with rabbit gamma
globulin; ( 3 ) reactive with both human and rabbit gamma globulin. The
great majority of rheumatoid sera react with both human and rabbit gamma
globulin. However, occasional sera are reactive only with rabbit gamma
globulin in the SSC test. Sera from patients with acute and chronic inflammatory states not related to connective tissue disease frequently follow the pattern of being reactive only with human gamma globulin, i.e. positive FII LP
and negative SSC. However, some sera from this disease group are reactive
with both FII LP and SSC, and very few with SSC only. It is also known
that in this group of patients FII LP titers tend to fall as the patient imp r o v e ~For
. ~ this
~ ~reason
~ ~ the development of a test which yields reproducible
results may result in the accumulation of more consistent and therefore more
reliable data.
1. A modified latex agglutination reaction utilizing pre-coated latex particles
2300 A t 200 A in diameter has been described. Optimal conditions for coating particles with gamma globulin are delineated.
2 . This modification yields reproducible titers and eliminates doubtful results.
3. The latex-gamma globulin suspension is stable under refrigeration for at
least 1year.
I. Singer, J . M.: The latex fixation test in
rheuniatoid clisease. Am. J. Med. 31:
766, 1961.
2. -, Plotz, C.: The latex fixation test.
I. Application to the serologic diagncsis of rheumatoid arthritis. Am. J.
Med. 21:888, 1956.
3. Hozsoky. S.: The problem of standardization in rheumatoid arthritis serology. Arth. & Rheumat. 6:641, 1963.
4. Halbcrstam, D., Singer, J. M., Allen,
E. G. and Plotz, C. M.: Stability
characteristics of 0.2 micron diameter
polystyrene latex particles. Atti. del
X Congresro dela lega Internazionale
contro il rheumatismo., 2:854, 1961
hfinerva hfedica.
.5. Oreskes, I., and Singer, J. M.: Influence of pH, ionic strength and protein concentration on stability of polystyrene latex siispensions. Proc. Soc.
Biol. & Med. 115:753, 1964.
6. Singer, J. M., Altmann, G., Goldenberg, A. and Plotz, C. M.: The
mechanism of particulate carrier reaction with rheumatoid sera. 11. Sensitizing capacity of various human
gamma globulin for latex particles.
Arth. & Rheumat. 3:515, 1960.
7. -, -, Oreskes, I. and -: The mechanism of particulate carrier reaction
111. The stabilizing effect of serum
protein. Am. J. Med. 30:772, 1961.
8. -, Plotz, C. M. and Eason, E.: The
latex fixation test IV. The serological
app1ic:ition of 0.2 micron diameter
latex particles coated with gamma
globulin. Atti. del X Congress0 della
lega Internazionale contro il Rheumatismo. 1:304, 1961.
9. -, - and Goldberg, R.: A technique of
prccoating polystyrene lytron particles with gamma globulin for the detection of macroglobulins which react with human gamma globulin.
Third Pan-American Congress on
Rheumatic Disease Santiago, Chile,
October, 1963.
10. -, Oreskcs, J., Hutterer, F. and Emst,
J.: The mechanism of particulate
carrier rractions V. Adsorption of
human gamma globulin to 0.2 micron
diameter latex particles and their
agglutination by rheumatoid factor.
Ann. Rheum. Dis. 22:434, 1963.
11. Milgrom, F., and Witebsky, E.: Stndies on the rheumatoid and related
serum factors. J. A. M. A. 174:56,
12. Abnlzzo, J. L., and Christian, C. L.:
Induction of rheumatoid factor-like
substance in rabbits. Arth. & Rheumat. 4:103, 1961.
13, klcCluskey, T. R,, Miller, F. and Benacerraf, B.: Sensitization to denatured autologous gamma globulin. J.
Exper. Med. 115:253, 1962.
14. Vaughan, J. H.: Behavior of the rheumatoid arthritis agglutinating factor
with immune precipitates. J. Immun01. 77:181, 1956.
1.5. LoSpalluto, J. and Ziff, M.: Chromatographic studics of the rheumatoid
factor. J. Exper. Med. 110:169, 1959.
16. Heimer, R., Schwartz, E. R. and FrVberg, R. H.: Different rheumatoid
factors in the serum of one patient
with rheumatoid arthritis. J. Lab. &
Clin. Med. 57:16, 1962.
17. Milgrom, F., Witebsky, E., Goldstein,
R. and Loza, U.: Studies on the rheumatoid and related serum factors. 11.
Relation of anti-human and anti- rabbit gamma globulin factors in rheumatoid arthritis serums. J. A. M. A.
181:106, 1962.
18. Williams, R. C., Jr., and Kunkel, H.
G.: Separation of rheumatoid factors of different specificities using
columns conjugated with gamma
globulin. Arth. & Rheumat. 6:665,
19. Dressner, E . and Trombly, P.: The
latex fixation reaction in non-rheumatic disease. New England J. Med.
261:981, 1959.
20. Williams, R. C., and Kunkel, H. G.:
Rheumatoid factor, complement, and
conglutinin aberrations in patients
with subacute bacterial endocarditis.
J. Clin. Invest. 41:666, 1962.
Jacques M . Singer, M.D., Head, Department of Microbiology,
Division of Laboratories, Montefiore Hospital, Bronx, N e w
York; Clinical Associate Professor of Medicine, State University
of N e w York, Downstate Medical Center, Brooklyn, N e w York.
Charles hl. Plotz, M.D., LMed. Sc.D., Clinical Associate Profesof Medicine, Director Training Program in Rheumatic
Diseuse. State University of N e w York, Downstate Medical
Center, Brooklyn, N e w York.
Rosalie Coldberg, B.A., Research Assistant, Department of
Microbiology, Division of Laboyatories, Montefiore Hospital,
Bronx, N e w York.
Без категории
Размер файла
596 Кб
factors, globulin, detection, pre, coates, latex, anti, utilizing, particles
Пожаловаться на содержимое документа