271
Clinica Chimica Acta, 78 (1977) 271-284 @ ElsevierlNorth-Holland Biomedical Press
CGA 8623
A SOLID PHASE FLUORESCENT IMMUNOASSAY FOR THE QUANTITATION OF THE C3 COMPONENT OF HUMAN COMPLEMENT
MICHAEL
W. BURGETT
Bio-Rad Laboratories, (Received
January
*, SANDRA
Richmond,
18th,
J. FAIRFIELD
Calif. 94804
and JAMES
F. MONTHONY
(U.S.A.)
1977)
Summary A non-competitive method for the determination of the C3 component of human complement in serum is described. This procedure involves use of a specific antibody covalently attached to derivatized polyacrylamide beads and a fluorescently labeled specific antibody. Reproducible results were achieved for C3 in serum in the range of 20 mg/dl to 195 mg/dl within 2 h. C3 levels as low as 125 ng/ml can also be measured. Fluorescent immunoassay and radial immunodiffusion were used to determine C3 levels in healthy adults. Good agreement was found between the two methods.
Introduction The C3 component of complement in human serum currently can be immunologically assayed by three commercially available methods. These methods, radial immunodiffu,.on (RID) [ 1,2], electroimmunoassay (EIA) [ 31 and nephlometry [4] all suffer from one or more drawbacks. RID lacks sensitivity, requires extended incubation time and is often difficult to interpret. EIA also is lacking in sensitivity and is difficult to interpret. Nephlometry requires the filtration of all reagents and some samples and an expensive apparatus. The purpose of this paper is to describe a solid phase fluorescent immunoassay (FIA) for human C3 which is rapid, sensitive, relatively simple and can be performed with ordinary laboratory equipment and an inexpensive filter fluorometer. In the method described, an excess of a commercially available solid phase antibody is reacted with a diluted serum sample. The antigen solid phase antibody complex thus formed is reacted with fluorescently labeled antibody. * Correspondence should be addressed to: Michael W. Burg&t. Ph.D., Senior Research Chemical Division, Bio-Rad Laboratories. 32nd Griffin Avenue, Richmond, CA 94804,
Biochemist, U.S.A.
278
The solid phase antibody antigen fluorescently labeled antibody sandwich is then separated from unreacted labeled antibody. The fluorescence of the solid phase antibody complex is determined and is directly proportional to the amount of C3 present. This method differs from previously described solid phase fluorescent immunoassays [5,6,7] in that the total fluorescence of a bead suspension is measured rather than the fluorescence of individual beads. Materials and methods Immobilized antibody The immobilized antibody used in this study, Immunobeadt” C3 Reagent (Catalog No. 170-5178, Control Nos. 15088 and 20972), was obtained from Bio-Rad Laboratories, Richmond, Calif. This reagent is a derivatized polyacrylamide bead support to which a highly purified rabbit anti-human C3 (P-l-A, p-1-C globulin) is covalently bound. The small size (l-8 pm diameter) and hydrophilic nature of the bead support allows them to form suspensions which are stable for up to 2 h. The reagent was supplied in a lyophilized form. When rehydrated with phosphate buffered saline (PBS, 0.01 M potassium phosphate buffer, pH 7.2, 0.15 M NaCl, 0.01% NaN,) the reagent was found to be stable for at least 30 days at 4°C. Fluorescent labeling of antibody Rabbit immunoglobulins against human C3 (P-l-A, p-1-C globulin) also were obtained from Bio-Rad Laboratories. The preparations used were shown to be monospecific by immunoelectrophoresis [8] and cross rocket immunoelectrophoresis [9]. These immunoglobulins were labeled with fluorescein isothiocyanate (FITC) by a modification of the method of Rinderknecht [lo]. A 1% immunoglobulin solution was dialyzed extensively against 0.05 M sodium bicarbonate, pH 8.5, containing 0.075 M NaCl. Approximately 1.4 mg of FITC on Celite (Calbiochem, La Jolla, Calif.) was added for each mg of immunoglobulins and the mixture was stirred slowly for 1.5 h. Celite was removed by centrifugation at 1400 X g for 20 min. Unbound FITC was separated from the immunoglobulin by dialysis against PBS. The FITC labeled immunoglobulin could be used without further purification if the fluorescein to protein ratio of the material was between 2 and 4. The FITC labeled immunoglobulins were stable for more than 2 months at 4°C and could be lyophilized for longer storage without loss of activity. Determination of the optimal amount of immobilized antibody Our assay required that the immobilized antibody be maintained in excess so that all the C3 present in the samples to be tested would be bound. To determine the optimal amount of immobilized antibody to be used in the assay, increasing amounts of the immobilized antibody (0.25 mg to 3.5 mg) were added to a dilution of human serum which contained 538 ng of C3. Blank samples containing the immobilized antibody, but no C3, were run concurrently. The final volume of each reaction tube was brought to 1.0 ml with PBS. After 2.5 h at 37°C an additional 2.0 ml of PBS was added to each reaction tube. The samples were mixed and centrifuged at 1000 X g for 8 min.
279
The supernatant fluid was discarded from each sample and the pellet was resuspended in 1 ml of PBS. A large excess of FITC labeled antibody (108 pg) was then added to each sample; they were then mixed and incubated 1.5 h at 37°C. The immobilized antibody complex was washed twice by centrifugation and resuspended in PBS to remove unreacted FITC labeled antibody. Borosilicate disposable culture tubes (12 X 75 mm) were used both as reaction tubes and as cuvettes for fluorescence determination. The washed, immobilized antibody complex was resuspended in 2.25 ml PBS and the fluorescence of the sample determined with a Perkin-Elmer Model 204 spectrofluorometer (excitation wavelength 485 nm, emission wavelength 525 nm). A typical experiment is shown in Fig, 1. An estimation of the C3 binding capacity of the immobilized antibody can be made by extrapolating the linear portion of the curve to 100% maximal fluorescence. In the example shown 1.8 mg of immobilized antibody was required to bind the 538 ng C3 added. Therefore, the capacity was approximately 300 ng C3 per mg of immobilized antibody. The two lots of Immunobeadst” C3 reagent used in this study had capacities of approximately 300 ng and 260 ng C3 per mg of immobilized antibody respectively. Determination
of the optimal amount
of FITC
labeled antibody
As with the immobilized antibody, it was necessary that excess labeled antibody be present in the assay system in order to react with all the C3 present in the sample to be tested. To determine the optimal amount of FITC labeled antibody, assay tubes were set up containing 2.0 mg of immobilized antibodies and 538 ng human C3 in a final volume of 1 ml PBS. Blank tubes containing only the immobilized antibody in 1 ml PBS were run concurrently. After incubation at 37°C for 2.5 h, 2 ml of PBS was added to each sample; they were then mixed and centrifuged at 1000 X g for 8 min. Supernatants were .
100
1 80 -
mg of Immobilized rabbit anti-C, reagent. Increasing amounts of rabbit anti-C3 Fig. 1. Fixation of C3 by rabbit antiC Immunobeadstm Immunobead Reagents were added to a constant amount (538 ng) of C3. The reaction was completed and the fluorescence determined as described in Materials and methods.
discarded and each pellet was resuspended in 1 ml PBS. Increasing amounts of FITC labeled antibody (3.5 pug-104 pg) were then added to the samples and they were incubated at 37°C for 1.5 h. The immobilized antibody complex was washed twice by resuspension in PBS and centrifugation. The fluorescence of each sample was determined as described previously. A typical experiment is shown in Fig. 2. To insure that the FITC labeled antibody was in excess, 1.5 times the minimal amount required was used in the assay described below. C3 standards A pool of normal human serum was obtained from Pacific Biological Company, Richmond, Calif., for use as a C3 standard. The pool was standardized against a standard human serum purchased from Behring Diagnostics, Somerville, N.J. (Catalog No. 17-592-006, Lot 975H). Four dilutions of this pool were made using 1% bovine serum albumin (Calbiochem, La Jolla, Calif.) in PBS as the diluent and l-ml aliquots of each dilution were lyophilized. The standards were stable for more than six months dry and for one month after rehydration when stored at 4°C. Typically, standard curves were linear over the range of the four dilutions: 25.8 ng to 258 ng (see Fig. 3). When the standard dilution (1 : 151) for serum was used in the assay described below, the four dilutions could be converted to mg/dl levels of 195 mg/dl, 130 mg/dl, 65 mg/dl and 19.5 mg/dl. Fluorescent
immunoassay
of C3 in human serum
The fluorescent immunoassay of C3 in human serum is outlined in Table I. The human serum samples were assayed on the day they were drawn. When aliquots of these samples were stored at 4°C for 24 h or -20°C for seven days, no change in C3 level was found by the FIA. All human serum samples were
I
1
20
I
I
I
40 60 60 !g of FITC labeled rabbit an&C,
1
100
Fig. 2. Titration of FITC labeled rabbit anti-C3. Increasing amounts of FITC labeled rabbit anti-C3 were added to a constant amount of immobilized antibody-C3 complex. The reaction was completed and the fluorescence determined as described in Materials and methods.
281
1
4
200 mgldl (265 ng/aliquot)
$, C, Concentration
Fig. 3. Standard curve for the fluorescent immunoassay performed as described in Materials and methods. The corrected relative fluorescence is the fluorescent reading of the sample minus the fluorescent reading of a blank. The C3 concentration is expressed both as ng per aliquot and mg/dl of a normal dilution. Each point is the mean of 15 determinations with the vertical bars representing r2 SD. of the mean.
diluted 1 : 151 before being assayed, 20 (~1 of serum added to 3.0 ml PBS. A 20-/J aliquot of the diluted serum sample was added to the immobilized antibody which was suspended in 0.5 ml PBS. Preliminary experiments showed that the amount of C3 bound by the immobilized antibody increased very rapidly for the first 30 min of the reaction, but remained relatively constant thereafter. Therefore, a 40-min incubation was used in the assay for the reaction of the immobilized antibody with C3. After 40 min at 37”C, the required amount of FITC labeled antibody was added and the reaction mixture was vortexed. Preliminary experiments showed that the amount of FITC labeled antibody bound by the immobilized antibody-C3 complex increased very rapidly for the first 15 min of the reaction, but remained relatively constant thereafter. Thus a 20-min incubation was used in the assay for the reaction of the FITC labeled antibody with the immobilized antibody-C3 complex. After 20 min at 37”C, the immobilized antibody complex was washed twice by centrifugation at 1000 X g for 8 min and resuspended in 3 ml PBS. A standard TABLE
I
OUTLINE Dilute Sample Add Incubate Add Incubate Wash
Measure
OF THE C3 FLUORESCENT
IMMUNOASSAY
PROCEDURE
Mix 20 ~1 serum sample with 3 ml PBS. Place a 20-~1 allquot of dilute serum sample in an assay tube. 0.5 ml Immunobead C3 reagent. 40 mln at 31°C. FITC labeled antibody. 20 min at 37OC. Add 2 ml PBS, vortex, centrifuge at ca. 1000 X B for 8 min. Decant supernatant. resuspend ln 2.5 ml PBS. Vortex. Centrifuge at ca. 1000 X g for 8 mm. Decant supematant. Resuspend sample in 3 ml PBS. Determine fluorescence.
282
curve was constructed by assaying 20 ~1 of each of the prediluted standards described above. The fluorescence of each sample, each standard, and a blank containing no added serum was determined as described or by using one of the followingfilter fluorometers: Turner Model 111 (excitation filter, Corning 5-60; emission filter, Corning 3-69) or Gilson Spectra/g10 (excitation filter 480-3; emission filter, Corning 3-69). The fluorescence of each sample was corrected by subtracting the fluorescence of the blank sample. The C3 level of each sample was determined from the standard curve. Results and discussion Precision
and sensitivity
The method described was reproducible within the normal lo-fold operating range (19.5 mg/dl to 195 mg/dl). Variation within a single run was less than 5.5% (Table II) while day-to-day variation was less than 9.5% (Table III). The operating range of the FIA method can easily be extended by changing the dilution of serum used. If undiluted serum is assayed, C3 levels as low as 125 ng/ml can be measured. However, an undiluted serum sample would add a much higher concentration of protein and other serum components to the reaction mixture than the normal dilution. To see what effect undiluted serum would have on the assay, a diluted serum sample was asssayed in the presence and absence of a 200+1 aliquot of rabbit serum (Table IV). The addition of 1500 times more rabbit serum protein than normally assayed had no effect on the determination of the C3 level in the human serum sample. This suggests that 200 1.11of undiluted human serum could easily be assayed. Since the low standard contains about 25 ng of C3 it also indicates that as little as 125 ng/ml C3 can be assayed by this method. The level of C3 was determined in four specimens of cerebrospinal fluid (CSF). A 20+1 aliquot of unconcentrated CSF was required to perform the assay. The C3 level as measured by the assay of the four samples varied from 1.6 pg/ml to 5.7 pg/ml. The coefficient of variation for 10 replicates of one of the CSF samples was 3.6%. Comparison
of the values for C3 obtained
Samples of serum from 96 healthy bank. These sera were assayed for C3 from 50.1 mg/dl to 122.3 mg/dl (mean a range for normal subjects reported TABLE
II
SAME
DAY
ASSAYED
PRECISION BY
Samples
Low H&h
THE
STUDIES
FLUORESCENT Mean
normal
Middle
by FIA and RID
adults were obtained from a local blood by the FIA method. The C3 level varied 79.9 mg/dl+ 13.7) which was similar to for C3 by Kohler and Muller-Eberhard
normal normal
(mg/dl)
OF
10
REPETITIVE
IMMUNOASSAY
FOR
DETERMINATIONS
S.D.
Coefficient
(mg/dI)
variation
60.0
3.3
5.5
97.6
5.0
5.1
6.5
4.1
157.9
OF
C3 of (%)
THREE
SERA
283
TABLE III DAY-TO-DAY NOASSAY
PRECISION
STUDIES
ON THREE SERA
ASSAYED
BY THE FLUORESCENT
IMMU-
FOR C3
Figures represent means of 40 separate determinations
performed
by two different operators over a
2-month period. Samples
Low normal Middle normal High normal
Mean (mg/dI)
59.4 129.8 226.4
S.D. (mg/dI)
Coefficient of variation (o/o)
5.1 10.9 21.6
8.6 8.4 9.5
[ 111. The serum samples were also assayed for C3 by two commercial RID kits; kits purchased from Behring Diagnostics, Somerville, N.J., which used the “end point” method of Mancini et al. [l] and kits purchased from Meloy Laboratories, Springfield, Va., which used the “early read out” method of Fahey and McKelvey [2]. There was good agreement between the C3 value obtained by the FIA method and the C3 value determined by both of the RID methods (Table V). The correlation coefficient for the FIA values and the “end point” method RID values was somewhat greater (r = 0.91) than the correlation coefficient for the FIA values and the “early read out” method RID values (r = 0.88). It is interesting to note that the FIA values correlated with values from either RID methods better than the values from the two RID methods correlated with each other (r = 0.84). C3 values as determined by the Behring kits (mean 77.8 mg/dl + 13.4) and the FIA method were consistently lower than the C3 values determined by the Meloy kits (mean 165.4 mg/dl + 37.1). The fact that these two manufacturers use different internal standards could account for the difference and suggests the need for an international reference preparation for C3 similar to that available for the immunoglobulins [ 121. Another possible explanation could be differences in the antibodies used in these systems. The Behring kits are said to contain antibodies directed against only P-1-A globulin while the Meloy kits and the FIA used antibodies directed against both P-1-A and p-1-C globulins. In conclusion, the FIA method described is very reproducible over a relatively large operating range. The FIA method gives values of C3 which agree with values for C3 obtained by at least one established method, RID. The method is rapid, requiring only 2 h to perform. The FIA method has the
TABLE IV EFFECT OF EXTRANEOUS IMMUNOASSAY
PROTEIN ON THE DETERMINATION
OF C3 BY THE FLUORESCENT
Figures represent means of 10 separate determinations. Additions
No addition 200 ~1 rabbit serum
Mean
S.D.
(mg/dI)
(meldI)
Coefficient of variation (W)
123.8 123.6
6.2 6.4
5.0 5.2
284
TABLE
V
COMPARISON RADIAL
OF
C3
VALUES
OBTAINED
IMMUNODIFFUSION
BY
THE
FLUORESCENT
IMMUNOASSAY
Method
Correlation
Equation
coefficient
(r)
regression
line
Y
FIA
RID
*
0.9 1
y = 0.89x
+
FIA
RID
**
0.86
y = 2.37.x
-
24.0
RID
**
0.84
y = 2.33.x
-
15.6
*
* RID * * RID
by
the method
by the
method
AND
of the
x
RID
(FIA)
(RID)
of
Mancini
of
Fahey
6.6
et al. Ill. and
McKelvey
[21.
sensitivity to measure as little as 125 ng/ml. Thus, the sensitivity of the FIA method should allow easy measurement of C3 in other biological fluids which cannot be assayed by other currently available methods without prior concentration. The digital read out of a fluorometer and the linear standard curve obtained by the FIA method allows for easier interpretation of data. References Mancini. Fahey,
G..
Carbonara,
J.L.
and
A.O.
McKelvey.
LaureII,
C.-B.
(1966)
Anal.
Ritchie.
R.F.
(1967)
J. Lab.
Bloemmen.
F.J..
J. Immunol.
Methods
6
Cap&
P.J.A.
7
Knapp,
W.,
8
Grabar,
P. and
9
Laurell,
C.-B.
10
Rinderknecht,
11
KohIer,
12
Rowe,
P.F. D.S.,
Radl,
(1974) and
10.
(1965)
15,
Med.
Immunochemistry
94,
C.A.
(1953)
Biochem.
J.J.
Andersen,
5,
den
Berg,
P.,
Schuit,
H.R.E.
165-178
Biochim.
Experientia
MulIer-Eberhard,
Methods Biophys.
5,259-273 Acta
10,
193
358-361
16,430 H.J.
(1967)
S.G.
and
Hijmans,
512-517 van
J. Immunol. 10,
254
45-52 70,
Methods
(1974)
2, 235-
84-90
337-355
Anal.
B. and
J.F.
J. Immunol.
Haaijman,
J.S.
H. (1960) Grab,
Clin.
J. Immunol.
Williams,
and
Heremans. (1965)
Biochem.
J.,
Ploem,
(1965)
and
E.M.
(1972)
J. Immunol. Bull.
WHO
99, 46.
1211-1216 67-69
W.
(1976)
Clinica Chimica Acta, 78 (1977) 271-284 @ ElsevierlNorth-Holland Biomedical Press
CGA 8623
A SOLID PHASE FLUORESCENT IMMUNOASSAY FOR THE QUANTITATION OF THE C3 COMPONENT OF HUMAN COMPLEMENT
MICHAEL
W. BURGETT
Bio-Rad Laboratories, (Received
January
*, SANDRA
Richmond,
18th,
J. FAIRFIELD
Calif. 94804
and JAMES
F. MONTHONY
(U.S.A.)
1977)
Summary A non-competitive method for the determination of the C3 component of human complement in serum is described. This procedure involves use of a specific antibody covalently attached to derivatized polyacrylamide beads and a fluorescently labeled specific antibody. Reproducible results were achieved for C3 in serum in the range of 20 mg/dl to 195 mg/dl within 2 h. C3 levels as low as 125 ng/ml can also be measured. Fluorescent immunoassay and radial immunodiffusion were used to determine C3 levels in healthy adults. Good agreement was found between the two methods.
Introduction The C3 component of complement in human serum currently can be immunologically assayed by three commercially available methods. These methods, radial immunodiffu,.on (RID) [ 1,2], electroimmunoassay (EIA) [ 31 and nephlometry [4] all suffer from one or more drawbacks. RID lacks sensitivity, requires extended incubation time and is often difficult to interpret. EIA also is lacking in sensitivity and is difficult to interpret. Nephlometry requires the filtration of all reagents and some samples and an expensive apparatus. The purpose of this paper is to describe a solid phase fluorescent immunoassay (FIA) for human C3 which is rapid, sensitive, relatively simple and can be performed with ordinary laboratory equipment and an inexpensive filter fluorometer. In the method described, an excess of a commercially available solid phase antibody is reacted with a diluted serum sample. The antigen solid phase antibody complex thus formed is reacted with fluorescently labeled antibody. * Correspondence should be addressed to: Michael W. Burg&t. Ph.D., Senior Research Chemical Division, Bio-Rad Laboratories. 32nd Griffin Avenue, Richmond, CA 94804,
Biochemist, U.S.A.
278
The solid phase antibody antigen fluorescently labeled antibody sandwich is then separated from unreacted labeled antibody. The fluorescence of the solid phase antibody complex is determined and is directly proportional to the amount of C3 present. This method differs from previously described solid phase fluorescent immunoassays [5,6,7] in that the total fluorescence of a bead suspension is measured rather than the fluorescence of individual beads. Materials and methods Immobilized antibody The immobilized antibody used in this study, Immunobeadt” C3 Reagent (Catalog No. 170-5178, Control Nos. 15088 and 20972), was obtained from Bio-Rad Laboratories, Richmond, Calif. This reagent is a derivatized polyacrylamide bead support to which a highly purified rabbit anti-human C3 (P-l-A, p-1-C globulin) is covalently bound. The small size (l-8 pm diameter) and hydrophilic nature of the bead support allows them to form suspensions which are stable for up to 2 h. The reagent was supplied in a lyophilized form. When rehydrated with phosphate buffered saline (PBS, 0.01 M potassium phosphate buffer, pH 7.2, 0.15 M NaCl, 0.01% NaN,) the reagent was found to be stable for at least 30 days at 4°C. Fluorescent labeling of antibody Rabbit immunoglobulins against human C3 (P-l-A, p-1-C globulin) also were obtained from Bio-Rad Laboratories. The preparations used were shown to be monospecific by immunoelectrophoresis [8] and cross rocket immunoelectrophoresis [9]. These immunoglobulins were labeled with fluorescein isothiocyanate (FITC) by a modification of the method of Rinderknecht [lo]. A 1% immunoglobulin solution was dialyzed extensively against 0.05 M sodium bicarbonate, pH 8.5, containing 0.075 M NaCl. Approximately 1.4 mg of FITC on Celite (Calbiochem, La Jolla, Calif.) was added for each mg of immunoglobulins and the mixture was stirred slowly for 1.5 h. Celite was removed by centrifugation at 1400 X g for 20 min. Unbound FITC was separated from the immunoglobulin by dialysis against PBS. The FITC labeled immunoglobulin could be used without further purification if the fluorescein to protein ratio of the material was between 2 and 4. The FITC labeled immunoglobulins were stable for more than 2 months at 4°C and could be lyophilized for longer storage without loss of activity. Determination of the optimal amount of immobilized antibody Our assay required that the immobilized antibody be maintained in excess so that all the C3 present in the samples to be tested would be bound. To determine the optimal amount of immobilized antibody to be used in the assay, increasing amounts of the immobilized antibody (0.25 mg to 3.5 mg) were added to a dilution of human serum which contained 538 ng of C3. Blank samples containing the immobilized antibody, but no C3, were run concurrently. The final volume of each reaction tube was brought to 1.0 ml with PBS. After 2.5 h at 37°C an additional 2.0 ml of PBS was added to each reaction tube. The samples were mixed and centrifuged at 1000 X g for 8 min.
279
The supernatant fluid was discarded from each sample and the pellet was resuspended in 1 ml of PBS. A large excess of FITC labeled antibody (108 pg) was then added to each sample; they were then mixed and incubated 1.5 h at 37°C. The immobilized antibody complex was washed twice by centrifugation and resuspended in PBS to remove unreacted FITC labeled antibody. Borosilicate disposable culture tubes (12 X 75 mm) were used both as reaction tubes and as cuvettes for fluorescence determination. The washed, immobilized antibody complex was resuspended in 2.25 ml PBS and the fluorescence of the sample determined with a Perkin-Elmer Model 204 spectrofluorometer (excitation wavelength 485 nm, emission wavelength 525 nm). A typical experiment is shown in Fig, 1. An estimation of the C3 binding capacity of the immobilized antibody can be made by extrapolating the linear portion of the curve to 100% maximal fluorescence. In the example shown 1.8 mg of immobilized antibody was required to bind the 538 ng C3 added. Therefore, the capacity was approximately 300 ng C3 per mg of immobilized antibody. The two lots of Immunobeadst” C3 reagent used in this study had capacities of approximately 300 ng and 260 ng C3 per mg of immobilized antibody respectively. Determination
of the optimal amount
of FITC
labeled antibody
As with the immobilized antibody, it was necessary that excess labeled antibody be present in the assay system in order to react with all the C3 present in the sample to be tested. To determine the optimal amount of FITC labeled antibody, assay tubes were set up containing 2.0 mg of immobilized antibodies and 538 ng human C3 in a final volume of 1 ml PBS. Blank tubes containing only the immobilized antibody in 1 ml PBS were run concurrently. After incubation at 37°C for 2.5 h, 2 ml of PBS was added to each sample; they were then mixed and centrifuged at 1000 X g for 8 min. Supernatants were .
100
1 80 -
mg of Immobilized rabbit anti-C, reagent. Increasing amounts of rabbit anti-C3 Fig. 1. Fixation of C3 by rabbit antiC Immunobeadstm Immunobead Reagents were added to a constant amount (538 ng) of C3. The reaction was completed and the fluorescence determined as described in Materials and methods.
discarded and each pellet was resuspended in 1 ml PBS. Increasing amounts of FITC labeled antibody (3.5 pug-104 pg) were then added to the samples and they were incubated at 37°C for 1.5 h. The immobilized antibody complex was washed twice by resuspension in PBS and centrifugation. The fluorescence of each sample was determined as described previously. A typical experiment is shown in Fig. 2. To insure that the FITC labeled antibody was in excess, 1.5 times the minimal amount required was used in the assay described below. C3 standards A pool of normal human serum was obtained from Pacific Biological Company, Richmond, Calif., for use as a C3 standard. The pool was standardized against a standard human serum purchased from Behring Diagnostics, Somerville, N.J. (Catalog No. 17-592-006, Lot 975H). Four dilutions of this pool were made using 1% bovine serum albumin (Calbiochem, La Jolla, Calif.) in PBS as the diluent and l-ml aliquots of each dilution were lyophilized. The standards were stable for more than six months dry and for one month after rehydration when stored at 4°C. Typically, standard curves were linear over the range of the four dilutions: 25.8 ng to 258 ng (see Fig. 3). When the standard dilution (1 : 151) for serum was used in the assay described below, the four dilutions could be converted to mg/dl levels of 195 mg/dl, 130 mg/dl, 65 mg/dl and 19.5 mg/dl. Fluorescent
immunoassay
of C3 in human serum
The fluorescent immunoassay of C3 in human serum is outlined in Table I. The human serum samples were assayed on the day they were drawn. When aliquots of these samples were stored at 4°C for 24 h or -20°C for seven days, no change in C3 level was found by the FIA. All human serum samples were
I
1
20
I
I
I
40 60 60 !g of FITC labeled rabbit an&C,
1
100
Fig. 2. Titration of FITC labeled rabbit anti-C3. Increasing amounts of FITC labeled rabbit anti-C3 were added to a constant amount of immobilized antibody-C3 complex. The reaction was completed and the fluorescence determined as described in Materials and methods.
281
1
4
200 mgldl (265 ng/aliquot)
$, C, Concentration
Fig. 3. Standard curve for the fluorescent immunoassay performed as described in Materials and methods. The corrected relative fluorescence is the fluorescent reading of the sample minus the fluorescent reading of a blank. The C3 concentration is expressed both as ng per aliquot and mg/dl of a normal dilution. Each point is the mean of 15 determinations with the vertical bars representing r2 SD. of the mean.
diluted 1 : 151 before being assayed, 20 (~1 of serum added to 3.0 ml PBS. A 20-/J aliquot of the diluted serum sample was added to the immobilized antibody which was suspended in 0.5 ml PBS. Preliminary experiments showed that the amount of C3 bound by the immobilized antibody increased very rapidly for the first 30 min of the reaction, but remained relatively constant thereafter. Therefore, a 40-min incubation was used in the assay for the reaction of the immobilized antibody with C3. After 40 min at 37”C, the required amount of FITC labeled antibody was added and the reaction mixture was vortexed. Preliminary experiments showed that the amount of FITC labeled antibody bound by the immobilized antibody-C3 complex increased very rapidly for the first 15 min of the reaction, but remained relatively constant thereafter. Thus a 20-min incubation was used in the assay for the reaction of the FITC labeled antibody with the immobilized antibody-C3 complex. After 20 min at 37”C, the immobilized antibody complex was washed twice by centrifugation at 1000 X g for 8 min and resuspended in 3 ml PBS. A standard TABLE
I
OUTLINE Dilute Sample Add Incubate Add Incubate Wash
Measure
OF THE C3 FLUORESCENT
IMMUNOASSAY
PROCEDURE
Mix 20 ~1 serum sample with 3 ml PBS. Place a 20-~1 allquot of dilute serum sample in an assay tube. 0.5 ml Immunobead C3 reagent. 40 mln at 31°C. FITC labeled antibody. 20 min at 37OC. Add 2 ml PBS, vortex, centrifuge at ca. 1000 X B for 8 min. Decant supernatant. resuspend ln 2.5 ml PBS. Vortex. Centrifuge at ca. 1000 X g for 8 mm. Decant supematant. Resuspend sample in 3 ml PBS. Determine fluorescence.
282
curve was constructed by assaying 20 ~1 of each of the prediluted standards described above. The fluorescence of each sample, each standard, and a blank containing no added serum was determined as described or by using one of the followingfilter fluorometers: Turner Model 111 (excitation filter, Corning 5-60; emission filter, Corning 3-69) or Gilson Spectra/g10 (excitation filter 480-3; emission filter, Corning 3-69). The fluorescence of each sample was corrected by subtracting the fluorescence of the blank sample. The C3 level of each sample was determined from the standard curve. Results and discussion Precision
and sensitivity
The method described was reproducible within the normal lo-fold operating range (19.5 mg/dl to 195 mg/dl). Variation within a single run was less than 5.5% (Table II) while day-to-day variation was less than 9.5% (Table III). The operating range of the FIA method can easily be extended by changing the dilution of serum used. If undiluted serum is assayed, C3 levels as low as 125 ng/ml can be measured. However, an undiluted serum sample would add a much higher concentration of protein and other serum components to the reaction mixture than the normal dilution. To see what effect undiluted serum would have on the assay, a diluted serum sample was asssayed in the presence and absence of a 200+1 aliquot of rabbit serum (Table IV). The addition of 1500 times more rabbit serum protein than normally assayed had no effect on the determination of the C3 level in the human serum sample. This suggests that 200 1.11of undiluted human serum could easily be assayed. Since the low standard contains about 25 ng of C3 it also indicates that as little as 125 ng/ml C3 can be assayed by this method. The level of C3 was determined in four specimens of cerebrospinal fluid (CSF). A 20+1 aliquot of unconcentrated CSF was required to perform the assay. The C3 level as measured by the assay of the four samples varied from 1.6 pg/ml to 5.7 pg/ml. The coefficient of variation for 10 replicates of one of the CSF samples was 3.6%. Comparison
of the values for C3 obtained
Samples of serum from 96 healthy bank. These sera were assayed for C3 from 50.1 mg/dl to 122.3 mg/dl (mean a range for normal subjects reported TABLE
II
SAME
DAY
ASSAYED
PRECISION BY
Samples
Low H&h
THE
STUDIES
FLUORESCENT Mean
normal
Middle
by FIA and RID
adults were obtained from a local blood by the FIA method. The C3 level varied 79.9 mg/dl+ 13.7) which was similar to for C3 by Kohler and Muller-Eberhard
normal normal
(mg/dl)
OF
10
REPETITIVE
IMMUNOASSAY
FOR
DETERMINATIONS
S.D.
Coefficient
(mg/dI)
variation
60.0
3.3
5.5
97.6
5.0
5.1
6.5
4.1
157.9
OF
C3 of (%)
THREE
SERA
283
TABLE III DAY-TO-DAY NOASSAY
PRECISION
STUDIES
ON THREE SERA
ASSAYED
BY THE FLUORESCENT
IMMU-
FOR C3
Figures represent means of 40 separate determinations
performed
by two different operators over a
2-month period. Samples
Low normal Middle normal High normal
Mean (mg/dI)
59.4 129.8 226.4
S.D. (mg/dI)
Coefficient of variation (o/o)
5.1 10.9 21.6
8.6 8.4 9.5
[ 111. The serum samples were also assayed for C3 by two commercial RID kits; kits purchased from Behring Diagnostics, Somerville, N.J., which used the “end point” method of Mancini et al. [l] and kits purchased from Meloy Laboratories, Springfield, Va., which used the “early read out” method of Fahey and McKelvey [2]. There was good agreement between the C3 value obtained by the FIA method and the C3 value determined by both of the RID methods (Table V). The correlation coefficient for the FIA values and the “end point” method RID values was somewhat greater (r = 0.91) than the correlation coefficient for the FIA values and the “early read out” method RID values (r = 0.88). It is interesting to note that the FIA values correlated with values from either RID methods better than the values from the two RID methods correlated with each other (r = 0.84). C3 values as determined by the Behring kits (mean 77.8 mg/dl + 13.4) and the FIA method were consistently lower than the C3 values determined by the Meloy kits (mean 165.4 mg/dl + 37.1). The fact that these two manufacturers use different internal standards could account for the difference and suggests the need for an international reference preparation for C3 similar to that available for the immunoglobulins [ 121. Another possible explanation could be differences in the antibodies used in these systems. The Behring kits are said to contain antibodies directed against only P-1-A globulin while the Meloy kits and the FIA used antibodies directed against both P-1-A and p-1-C globulins. In conclusion, the FIA method described is very reproducible over a relatively large operating range. The FIA method gives values of C3 which agree with values for C3 obtained by at least one established method, RID. The method is rapid, requiring only 2 h to perform. The FIA method has the
TABLE IV EFFECT OF EXTRANEOUS IMMUNOASSAY
PROTEIN ON THE DETERMINATION
OF C3 BY THE FLUORESCENT
Figures represent means of 10 separate determinations. Additions
No addition 200 ~1 rabbit serum
Mean
S.D.
(mg/dI)
(meldI)
Coefficient of variation (W)
123.8 123.6
6.2 6.4
5.0 5.2
284
TABLE
V
COMPARISON RADIAL
OF
C3
VALUES
OBTAINED
IMMUNODIFFUSION
BY
THE
FLUORESCENT
IMMUNOASSAY
Method
Correlation
Equation
coefficient
(r)
regression
line
Y
FIA
RID
*
0.9 1
y = 0.89x
+
FIA
RID
**
0.86
y = 2.37.x
-
24.0
RID
**
0.84
y = 2.33.x
-
15.6
*
* RID * * RID
by
the method
by the
method
AND
of the
x
RID
(FIA)
(RID)
of
Mancini
of
Fahey
6.6
et al. Ill. and
McKelvey
[21.
sensitivity to measure as little as 125 ng/ml. Thus, the sensitivity of the FIA method should allow easy measurement of C3 in other biological fluids which cannot be assayed by other currently available methods without prior concentration. The digital read out of a fluorometer and the linear standard curve obtained by the FIA method allows for easier interpretation of data. References Mancini. Fahey,
G..
Carbonara,
J.L.
and
A.O.
McKelvey.
LaureII,
C.-B.
(1966)
Anal.
Ritchie.
R.F.
(1967)
J. Lab.
Bloemmen.
F.J..
J. Immunol.
Methods
6
Cap&
P.J.A.
7
Knapp,
W.,
8
Grabar,
P. and
9
Laurell,
C.-B.
10
Rinderknecht,
11
KohIer,
12
Rowe,
P.F. D.S.,
Radl,
(1974) and
10.
(1965)
15,
Med.
Immunochemistry
94,
C.A.
(1953)
Biochem.
J.J.
Andersen,
5,
den
Berg,
P.,
Schuit,
H.R.E.
165-178
Biochim.
Experientia
MulIer-Eberhard,
Methods Biophys.
5,259-273 Acta
10,
193
358-361
16,430 H.J.
(1967)
S.G.
and
Hijmans,
512-517 van
J. Immunol. 10,
254
45-52 70,
Methods
(1974)
2, 235-
84-90
337-355
Anal.
B. and
J.F.
J. Immunol.
Haaijman,
J.S.
H. (1960) Grab,
Clin.
J. Immunol.
Williams,
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Heremans. (1965)
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