Note: Descriptions are shown in the official language in which they were submitted.
107061Z
This invention relates to a method for the
immunofluorescent assay of antigens (or haptens) and their
antibodies. More particularly, it relates to the use of
an immune reactant related to the antibody or antigen
5 (or hapten) to be determined which is covalently bonded -~
or coupled to polymeric particles whose size permit direct
measurement of a labeled immunological reagent's fluorescence
in an aqueous suspension thereof.
The covalent coupling of antigens (or haptens)
and antibodies to water insoluble polymers is well documented.
Typical reports on this topic are:
Campbell, D.H., Leusher, E., and Lerman, L.S.
Proc. Nat. Acad. U.S. 37, 575 (1951)
Weliky, N. and Weetall, H.H., Immunochemistry
2, 293 (1965)
Campbell, D.H., and Weliky, N., Methods in
Immunology and Immunochemistry, Editors:
Williams C.A. and Chase, M.W. Vol. 1,
Academic Press, N.Y. (1967~
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The technique has been used to detect various con-
stituents and is the subject of several patents. In U.S.
Patent No. 3,555,143 issued January 12, 1971 to R.E.A.V.
Axen, et al., the patentees covalently bound antibodies
S to water insoluble polymers, mixed them with radioactive
protein and unlabeled protein in a competitive binding
technique.
In U.S. Patent No. 3,867,517 issued February 18, 1975
the patentee Chung-Mei Ling coated a test apparatus
with hepatitis associated antibody or antigen, then con-
tacted the apparatus with a solution containing the antigen
or antibody. Thereafter this was exposed to hepatitis
antigen or antibody labeled with radioactive isotope I125
to give a sandwich. After washing, the amount of isotope
lS attached could be quantitated.
~07061Z
1 Molday, et al (Molday, R.S., Dreyer, W.J., Rembaum, A.
2 and Yen, S.P.S., Nature 249, May 3, 1974) and (The Journal
3 of Cell Biology, Vol. 64, 75-88, 1975) report on the
4 synthesis of latex spheres equal to or less than 1300 A in
diameter, the fluorescent or radioactive ta~ging of the
6 spheres, and the covalent bonding of antibodies to the
7 spheres. These were subsequently used to locate antigens on
8 cell surfaces.
9 In~an & Dintzis (Inman, J.K. and Dintzis, H.~.,
Biochemistr~ 8 (10~ 4074-4082, 1969) described the chemical
11 modification of polyacrylamide beads to introduce a wide
12 variety of functional groups. The chemically reactive bead
13 derivatives were used to covaIently link antibodies or
14 enzymes.
Ohno and Stahmann (Ohno, Y. and Stahmann, M.A.,
16 Immunochemistry 9, 1087-1093, 1972) subsequently determined
17 that beads of about 10 microns in size, to which penicillin
18 had been attached, gave better agglutination reactions than
19 red blood cells when used in detecting antibodies to
penicillin.
21 Immunochemical labeling techniques have been reviewed
22 in Methods in Immunology and Immunochemistry, Editors:
23 Williams, C.A. and Chase, M.W., Vol. 1, Acad. Press, N.Y.
24 (1967) and Vol. III (1971).
The use of fluorescently Iabeled antibodies has been
26 reviewed by Coons (Coons, A.H., Fluorescent Antibody
Methods, J.F. Danielli (Editors) General Cytochemical
28 Methods, Vol. 1, Academic Press, N.Y. 195~) and has found
29 widespread use in the detection of microbial and tissue
30 antigens by fluorescent microscopy.
31
32
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Coons, et al (Weller, T.H. and Coons, A.H.) Proc. Soc.
Exptl. Biol. Med. 86, 789 (1954) also described a
method of determining cell bound antigen using a specific
antibody plus a fluorescent anti-gamma globulin antibody.
The same authors (Coons, A.H., Leduc, E.H. and Connolly, J.M.,
J. Exptl. Med. 102, 49, 1955) described a technique for
determining cell bound antibody by using specific antigen
plus specific fluorescent antibody.
Capel (Capel, P.J.A., J. of Immunological Methods 5,
165-178, 1974) coupled antibodies or antigens to the surface
of agarose beads of a size ranging from 40-190 u. In this
work he had to adjust conditions to prevent the antigen
from penetrating the pores of the bead or the ensuing anti-
gen-antibody reaction would be hindered. In his work he
attached human IgG to agarose beads, reacted them with
rabbit anti-human IgG antibody and then fluorescein iso-
thiocyanate (FITC) labeled horse anti-rabbit Ig serum.
He measured the amount of fluorescence attached by visualiza-
tion with fluorescent microscopy. If antigen was to be
measured, the anti-human IgG antibody was attached to the
agarose beads, reacted with human IgG and FITC labeled
horse anti-human Ig serum added and the fluorescence deter-
mined by fluorescent microscopy.
By way of summary, in the application of covalently
coupled immune reactants with insoluble polymers to assays,
immune reactants labeled with either radioactive tracers
or fluorescent compounds have been employed. Radioactive
tracers have the disadvantages of limited life and special
handling requirements, as well as requiring
~"`' ` 107061Z
1 expensive detection instrumentation. With respect to the
2 prior use of fluorescent tracers, applications that might be
3 considered an assay have been limited to indirect or
4 relatively tedious and time consuming procedures such as
measuring fluorescence of individual particles by
6 visualization with fluocescent microscopy.
7 The method of the present invention permits measurement
8 of fluorescently labeled part1cles by direct optical
9 spectroscopy. The key to the method is in the selection of
polymeric particle sizes which provide a substantially
11 homogeneous suspension during execution of the assay. It
12 has been discovered that such a condition exists upon which
13 direct fluorometric measurements can be made where the .
14 polymeric particles have a size of about .l-lO microns and
preferably where the particles have a size distribution
16 within this range centered about 5 microns.
17 Utilizing such particles, an appropriate immune
18 reactant immunologically related to unknown antigen (or
hapten) or antibody to be determined is covalently bonded
thereto. The particles, unknown immune reactant, and
21 appropriate fluorescently labelled immune reactant are mixed
22 under conditions so that a quantity of the labelled immune
23 reactant proportional to the concentration of the unknown
24 immune reactant is immunologically bound, directly or
indirectly, to the particles. The particles can then be
26 readily physically separated and their fluorescence directly
27 r,leasured by fluorometry.
28
29
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As used herein, "immunologically related" means that
, .
the immune reactant is either the same as the immune reac-
tant being referred to or its homolog. An antibody is the
"immunological homolog" of an antigen which produced it and
vice versa. In the methods of this invention antigens
and haptens perform entirely analogous functions. This
will be indicated by referring to them as alternatives
throughout.
More particularly, the present invention provides
an improved immunofluorencence assay comprising providing
a plurality of water insoluble hydrophilic polymeric
particles of about .l-10 microns in size and having
covalently bonded thereto an immune reactant immunologically
related to an unknown immune reactant to be determined,
providing an appropriate amount of fluorescently labeled
immune reactant immunologically related to said unkno-~n,
immunologically binding said particles in aqueous solution
with a quantity of said labeled immune reactant proportional
to the concentration of saidunknown, separating said particles
from said aqueous solution, and measuring the fluorescence
of an aqueous suspension of said aqueous particles by
fluorometry to obtain information from which the concen-
tration of said unknown immune reactant can be determined.
The immune reactant is preferably an immunological homolog
for the antigen (or hapten) or antibody to be determined.
-- 6 --
107061~ ~
1 The method is especially suited ~or deter~lning
2 antigens (or haptens) selected from protein~i and
3 polypeptides utilizing antibodies a~ainst the pLo~ein or
4 polypeptide. These antibodies are covalent1y bound to
suitable water insoluble polymeric particles. Thus, in a
6 preferred embodiment the improved assay comprises providing
7 a plurality of water insoluble hydrophylic polymeric
8 particles of about .l-lO microns in size and having
9 covalently bonded thereto-antibody for unknown antigen (or
hapten) to be determined. Unknown antigen (or hapten) is
11 immunologically bound to said particles in aqueous solution.
12 In addition, fluorescently labeled immune reactant
13 is combined to immunologically bind a portion thereof either
14 directly or indirectly (through bound antigen or hapten) to
said particles so that the bound labels are separable with
16 said particles. The particles are separated from unbound
17 immune reactant and the fluorescence of a fluid suspension
18 is measured in a fluorometer to obtain information for the
19 assay of unknown antigen (or hapten).
Any suitable water insoluble polymeric particle may be
21 utilized. Generally the particle will be in spherical or
22 bead form and will be selected from polymers which can be
23 derivatized to give a terminal primary amine, terminal
24 carboxyl, or hydrazide group. The antibody or antigen (or
hapten) is then immobilized on the~particle under
26 conventional reaction conditions to produce a covalent
27
28
29
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107061Z
bond therebetween. PreEerred polymeric particles are formed
21 from cross-linked polyacrylami~es. Immobilization of immune
31 reactants on such preferred substrates are reported by Inman
I ~ Dintzis as cited above. Other suitable polymeric
51 particles include those reported in u.s. Patent No.
61 3,555,143 in particular, as-well as the other references
7 ¦ above cited.
8 ¦ In carrying out the present assay method, a number
9 ¦ of alternative techniques are available. The choice will
10 ¦ usually be made depending upon the nature of the particular
11 ¦ antigen (or hapten) or antibody to be determined and their
12 ¦ availability. In general one of the following sequences
13 ¦ will provide the most satisfactory option:
14 ¦ Sandwich Technique
15 j The immobilized antibody (covalently attached to the
16 ¦ polymeric particle, preferably in spherical or bead form) is
17 ¦ reacted in an appropriate solution with specific bivalent or
18 ¦ multivalent antigen (or hapten) in such concentrations that
19 I there is always an excess of antibody. After the reaction
has gone to completion, fluorescently labeled antibody
21 specific to the antigen (or hapten) is added in slight
22 excess. Since the antigen (or hapten) has two or more sites
23 for reaction and only one is occupied, the second labeled
24 antibody will react with the unoccupied antigen (or hapten)
site(s). The antibody beads, combined with the antigen (or
26 hapten) and labeled antibody, are separated and measured in
27 a fluorometer. The concentration of the antigen (or hapten)
28 is directly related to the amount of fluorescence attached
to the beads.
31
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~,
Sequential Saturation
Another approach is to react an excess of the immob-
ilized antibody with the antigen (or hapten) in question.
After the reaction has occurred, labeled antigen (or hapten)
can be added which will occupy the available sites remaining
on the antibody. The immobilized antibody-antigen (or
hapten) complex can be separated and the label measured.
The amount of labeled antigen (or hapten) immobilized will
be inversely related to the amount of antigen (or hapten)
in the sample. This approach may be necessary in the case
of monovalent antigens (or haptens). ~ -
Antigen Excess
When antigen (or hapten) is in excess a competitive
binding technique may be employed. Antibody specific for
an antigen (or hapten) is attached to the particles. The
amount of bound or solid phase antibody added to the system
is sufficient to bind a limited amount of antigen (or hap-
ten). The specific antigen (or hapten) in question and
homologous labeled antigen (or hapten) are added to the
antibody. Since the number of binding sites on the immob-
" ilized antibody is limited, the labeled and unlabeled anti-
gens (or haptens) will compete for the sites. The amount
of labeled antigen (or hapten) bound will be inversely
related to the concentration of unlabeled antigen (or
hapten) in the system and can be used as a means of quanti-
tation of the unlabeled antigen (or hapten) in the system.
This technique can be combined with a second antibody
technique. By the correct selection of an antibody
fraction, the solid phase antibody can be used as a second
antibody in the analyses, for example, of haptens that
have only one combining site with antibodies. In this procedure,
107061Z
1¦ antibodies to a hapten, such as thyroxine, dinitroE)henol or
21 a steroid are prepared by well known procedures. The hapten
31 is conjugated with the protein of one species and injected
4¦ into a noncompatible species. That is, the hapt~n can be
51 conjugated to human serum albumin and injected into rabbits.
6 ¦ The rabbit will produce antibodies against the protein-
7 ¦ hapten conjugate.
8 ¦ In this procedure, a second antibody is required
9 1 and second antibodies are produced against, for example, a
10¦ fracti-on of rabbit globulins by injecting them into a goat.
11 ¦ This produces goat anti-rabbit antibodies. These can be
12 ¦ conjugated to small polyacrylamide beads, for example, and
13 ¦ used as a second antibody for any system in which the first
14 ¦ antibody was produced in rabbits. ~ny series of animals may
15 1 be used as long as antibodies against the first species are
16 ¦ produced in a noncompatible second species.
17 ¦ In this procedure, the antibody-hapten or antigen
18 ¦ combination does not differ significantly from unreacted
19 ¦ antibody to allow convenient separation or precipitation. A
20 ¦ second particle bound antibody, to the globulin fraction of
21 ¦ the animal used to produce the first antibody, is utilized
22 ¦ to cause precipitation to occur. In this case, the
¦ initially competitively bound antigen-first antibody
24 ¦ combination~may be considered the immunological homolog of
the particle bound second antibody.
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~1070612
In another technique, hapten protein conjugates
are used in a manner analogous to that described above
for preparing antibodies to haptens. The antibodies to
the hapten are covalently linked to the polyacrylamide
beads to be used as a reagent for the determination of a
hapten. Then the same hapten protein conjugate is prepared ~ -
and the protein fluorescently labelled. This is used
in a competitive binding assay or sequential saturation
assay with the native hapten to be assayed. The technique
provides an amplified molar fluorescent response.
Another variation of the technique is to covalently
bind the antigen to the bead. The antigen bound to the bead
is placed in competition with native antigen for limited
amount of homologous antibody. The beads are separated
and reacted with an excess of a fluorescently labelled
second antibody directed against the antigen-antibody
complex. The beads can be separated and the fluorescence
measured. The amount of fluorescence is inversely related
to the serum concentration of antigen to be measured.
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The present invention contemplates the use of any
suitable fluorescent compound in combination with antigens
(or haptens) or antibodies as a label. The following are
typical examples of suitable compounds, together with
references pertaining to their use as labels.
1. Fluorescein-isothiocyanate
The, ~. H. and Feltkamp, T.E.W., Immunology,
18,865 (1970)
2. Rhodamine B isothiocyanate
Chen, R.F. Arch. Biochem. Biophys. 133, 263 (1969)
3. DNS chloride (5-dimethylamino-1-naphthalene
sulfonylchloride) Weber, G., Biochem J., 51,155
(1952)
4. NBD chloride (7-chloro-4-nitro-benzo-2-oxa-1,
3,-diazole)
Ghosh, P.B. and Whitehouse, M.W., Biochem. J.,
108, 155 (1968)
5. MDPF (2-methoxy-2,4-diphenyl-3(2H)-furanone)
Weigele, M., DeBernardo, S., Leimgruber, W.,
Cleeland, R. and Grunberg, E., Biochem. Biophys.
Res. Comm. 54,899 (1973)
6. Fluorescamine (Flu~am -Roche Diagnostics)
Bohlen, P., Stein, S., Dairman, W. and Udenfriend,
S., Arch. Biochem. Biophys. 155, 213 (1973)
7. O-Phthalaldehyde
Benson, J.R. and Hare, P.E., Proc. Nat. Acad.
; Sci. (USA) 72, 619 (1975)
8. ANS (9-anilinonaphthalene-1-sulfonate)
Hartman, B.K. and Udenfriend, S., Anal. Biochem.
30, 391 (1969).
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: 10~061Z
1 TYPICAL GENERAL PROCE:D~RES
2 I. P~PA~TION OF ANTIBODY L3E~I)S
3 A. ~erivatized polyacrylamide beads, havin(3 a
4 functional capacity of 0.25 meq/g to 6 meq/g, are
used for antibody attachment. (These may be
6 obtained from Bio-Rad Laboratories of Richmond,
7 California, as Affi-Gel 701" 702, 703 or
8 derivative thereof.)
9 In addition, polyacrylamide beads, with an
exclusion limit of 6-7,000 daltons, may be
11 hydrolyzed by treatment with 2 M NaOH for 18 hours
12 at 40C. The beads are neutralized with I~Cl and
13 washed with deionized water. The carboxyl
14 capacity of the beads is measured by direct
titration and preferably should be about 6 meq/g
16 dry weight.
17 B. Carboxylate beads are suspended in 0. 003 M
18 phosphate buffer, pH 6.3, to a final concentration
19 of 10 mg beads/ml.
C. A globulin fraction of an antiserum, specific
21 for the antigen under test, is added to the beads
22 at a concentration of 12 ug antibody/mg of beads.
23 The reaction mixture is adjusted to p~ 6. 3.
24 D. A water soluble carbodiimide such as l-ethyl-3(3-
dimethylaminopropyl) carbodiimide (EDAC) is added
26 at a concentration of 0.25 meq EDAC/meq of
27 functional capacity of the bead. The reaction is
28 maintained at pH 6. 3 for one hour by the addition
29 of dilute acid and/or base. After the first hour,
the pH usually remains constant and the reaction
31 is allowed to proceed over night at 4C.
32 -13-
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1¦ ~. The coupled beads are washed twice ~ith PBS
2 ¦ (physiologically bufEered saline, 0.15 M NaCl -
31 0.01 M phosphate bufEer, pH 7.2), three times with
4 ¦ 5M guanidine HCl in 0.05 ~I phosphate buffer, pH
¦ 7.5, two more times with PBS and finally twice
6 ¦ with 0.005 M phosp'nate buffer, pH 7.5. The volume
7 ¦ of the washes was about 50 mljlOO mg beads. The
8 I washes are carried out at 4C for maximal antibody
9 ¦ activity.
10 ¦ F. The beads may be stored in 0.005 M phosphate
11 ¦ buffer containing 0.01~ sodium azide at 4C.
12 ¦ II. ANTIGEN EXCESS ASSAY (Competitive Binding Assay)
13 ¦ A. Aliquots of 200 ul of a 10 mg/ml antibody bead
14 ¦ suspension (~2 mg antibody beads) are added to a
15 ¦ 13 x 100 mm borosilicate test tube containing 1.1
16 I ml of PBS. The beads are pelleted by
17 ¦ centrifugation ~V8,000g for~vl min.)
18 ¦ B. The reaction is started by the addition of 10 ul
19 ¦ labeled antigen (containing 10 mg antigen/ml), a
20 ¦ sample of serum (10 ul of whole serum should fall
21 ¦ in the range of the assay for lgG) and enough PBS
22 ¦ to bring the assay mix to 1.5 ml. The mixture is
23 ¦ shaken with a vortex mixer and incubated for 30
24 ~ minutes at room temperature.
26
27
310 ~
32 -14-
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C. At 30 minutes, 4 ml of PBS is added to the
-~ assay mix. The sample is mixed and then centri-
fuged as above. The supernatant fluid is
carèfully discarded and the pellet is resus-
pended in 5 ml PBS. After about 10 minutes the
suspension is recentrifuged and the supernatant
fluid is again discarded.
D. The amount of labeled antigen on the beads is
directly determined in a fluorometer. The
quantity of labeled antigen picked up by the
test sample is divided by the quantity pic~ed ,
up by a control sample tantibody beads plus
labeled antigen with no serum) and plotted as
a~function of the concentration of antigen in
mg/ml on log logit paper.
III. SANDWICH ASSAY
A. Aliquotes of 200 ul of a 10 mg/ml antibody bead
suspension (~2 mg antibody beads) are added to
a 13 X 100 mm borosilicate test tube containing
1.2 ml of PBS. The beads are pelleted by
, centrifugation (~8,000 g for~ 1 min.)
B. A dilution of the serum is made (~1:1000 for IgG,
100 for IgM and IgA). A 100 ul aliquot
of the dilution is added to the tube from (A).
The sample is mixed with a vortex mixer and allowed
to incubate at room temperature for 3 hours.
.
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107061Z
C. A lO0 ul aliquot of labeled antibody which
should contain 20-50 ug antibody is added to
the tube from (B) and the mixture is incubated
an additional 30 minutes.
D. At 30 minutes, 4 ml of PBS is added to the
assay mix. The sample is mixed and then centri-
fuged as above. The supernatant fluid is care-
fully discarded and the pellet is resuspended
in 5 ml PBS. After about 10 min. the suspension
is recentrifuged and the supernatant fluid is
again discarded.
E. The relative fluorescence of labeled antibody
on the bead is determined and this valve is
plotted versus the concentration of antigen in
ug on log-log paper.
The following examples of specific embodiments will
illustrate the invention in connection with rabbit anti-
human IgG (Ro~HIgG) coupled to cross-linked polyacrylamide
beads. In the drawings referred to in the Examples:
Figures l and 2 both relate relative fluorescence
to antigen concentration.
- 16 -
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- 107061Z
1 l EXAMPLE 1
21 A gram sample of terpolymer microbeads (less than 10
3 microns in diameter) was hydrolyzed by treatment with 2M
41 NaOH for 18 hours at 40C. The beads were neutralized with
I HCl and washed six times with DI (de-ionized) H2O.
61 A 500 mg sample of the above hydrolyzed beads was
71 suspended in 100 ml 0.003 M phosphate buffer, pH 6.3. A 2
8 ¦ ml aliquot of an IgG fraction of rabbit anti-human IgG serum
9¦ (Miles, Lot 14, Code 64-155) contained 2.9 mg/ml antibody in
10¦ a 1% protein solution. The pH of the reaction mixture was
11¦ adjusted to 6.3. An aliquot of 130 mg of EDAC (Bio-Rad) was
12¦ added and the pH of the mixture was maintained at 6.3 with
~3¦ the addition of dilute acid and base for one hour. The
14¦ reaction was allowed to proceed overnight with stirring at
~5¦ 4C. The beads were washed twice with ~100 ml PBS, three
16¦ times with 100 ml of 5 M guanidine HCl containing 0.01 M
'71 phosphate buffer, pH 7.5, and twice more with 100 ml PBS.
18¦ After three hours at 4C the beads were washed twice with
19¦ 100 ml of 0.005 M phosphate buffer, pH 7.5, and then
20¦ suspended and stored in 50 ml of the last buffer containing
22 0.01% sodium azide (final concentration, 10 mg beads/ml).
I, 231 .
24 ~
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32 -17-
107061Z
1 1 EX~MPLE 2
2 ¦ Antigen ~xcess Assay
3 ¦ A 200 ul (2 mg) aliquot of R~ HIgG beads from ~xample l
4 ¦ was added to 1200 ul of PBS in a series of l.5 ml Eppendorf
5 ¦ centrifuge tubes. The beads were pelleted in an Eppendorf
¦ centrifuge ;~odel 3200/30 by centrifugation at maximum speed
7 ¦ for l minute (~12,000 g). A lO ul aliquot of FITC labeled
8 ¦ Human IgG (Cappel), various dilutions of ~1ormal Human serum
9 ¦ and enough PBS to bring the assay mix to l.5 ml was added to
10 ¦ each sample. The reaction was initiated by resuspending the
11 ¦ beads with a vortex mixer. After 30 minutes the beads were
12 ¦ centrifuged as above and the supernatant fluid was decanted.
13 ¦ The beads were washed by resuspension in 1.5 ml PBS followed
14 ¦ by centrifugation as above. The supernatant fluid was again
15 ¦ poured off. This process was repeated once and the beads
16 ¦ were resuspended in 5 ml 0.005 M Tris HCl, p~ 8.5. The
17 ¦ fluorescence of the beads was determined using a Turner
18 ¦ Filter Fluorometer with filter 47B for the excitation light
19 ¦ and filter 2Al2 for the emission light.
20 1 The fluorescence of a tube containing a ~2 mg
21 ¦ suspension of untreated beads is subtracted from the
22 ¦ fluorescence of each test sample. The fluorescence of the
23 ¦ test sample is then divided by the fluorescence of the
24 ¦ control beads (Ab beads + fluorescent antigen with no serum
25 ¦ added) and plotted versus concentration of antigen in mg on
27 log-logit paper as shown in Figure l.
28
29
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` 1070612
1 EXAMPLE 3
2 Sandwich Assay
3 The following components were mixed in a ~ppendorf
4 centrifuge tube and incubated ~or 18 hours at room
temperature. 200 ul of R~ HIgG beads from Example 1, 1,200
ul of PBS containing 1~ BSA and 100 ul of a normal human
7 serum dilution. At 18 hours the beads were centrifuged down
8 in an Eppendorf Model 3200/30 at maximu~ speed for one
9 minute (~12,000 g) and the supernatant fluid was decanted
off. The beads were washed by resuspension in 1.5 ml PBS
11 and centrifuged as above. The supernatant fluid was
12 decanted off and the beads were resuspended in 1 ml of PBS
13 containing 1~ BSA. A 10 ul aliquot of FITC conjugated R~
14 HIgG (Miles Lot 19, Code 64-169) was added to the beads and
they were allowed to react at room temperature. After 30
16 minutes the beads were centrifuged and washed twice with P8S
17 as above. The beads were resuspended in 5 ml 0.005 M Tris
18 HCl, pH 8.5. The Eluorescence of the beads was determined
19 with a Turner filter fluorometer using a ~7B filter for the
excitation light and a 2A12 filter for the emission light.
21 The fluorescence of the test sample less the fluorescence of
22 a blank sample (a sample containing bead which had not been
23 exposed to serum but was reacted with fluorescent antibody)
24 was plotted versus IgG concentration in ug on log-log paper
as shown in Flgure 2.
27
28
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