ENGLISH
IVD
CF-Fungal Antigens and Positive Controls
INTENDED USE:
The fungal antigens and positive controls are used to detect antibodies in patient serum by the complement
fixation (CF) procedure to aid in the diagnosis of four specific fungal diseases: Histoplasmosis, Blastomycosis,
Coccidioidomycosis, and Aspergillosis.
EXPLANATION:
Patient sera should be tested with each of the antigens, since there is some overlap in antigenicity between the
various fungi and the symptoms of the diseases are very similar. Higher CF titers are usually observed on
patient sera when they are tested against the same antigen as the etiologic agent of their infection.
BIOLOGICAL PRINCIPLES:
The test is based on the Laboratory Branch Complement Fixation (LBCF) Test Procedure (as published by the
Centers for Disease Control, Atlanta, GA)5. The principal of the CF test is that antibodies present in patient sera,
when mixed with their appropriate antigens will "fix" or take-up complement (a component of fresh serum). This
"fixation" of complement is determined by adding an assay system consisting of sheep red blood cells (SRBC)
sensitized with anti-SRBC (hemolysin) and measuring the percentage of lysis of the SRBC (complement is
required for lysis). If complement has been "fixed" the indicator SRBC's will not be lysed.
The CF test involves two basic principles:
1.) Complement (C') is irreversibly reacted (fixed) by certain classes of antibody-antigen complexes (certain
classes of antibodies do not fix complement). The degree of fixation is governed by the relative concentration
of antibody or antigen.
2) The lysis of SRBC that have been sensitized with hemolysin is dependent upon complement being present.
The CF test (Serum + Antigen + C' — incubate — + sensitized SRBC) is interpreted as follows:
Antibody present = NO HEMOLYSIS
Antibody absent = HEMOLYSIS
MATERIALS AVAILABLE:
A. Optimally Diluted Antigens: These antigens are prediluted to their optimal concentration for use in the
LBCF procedure and are ready for use as supplied. The optimum dilutions for these antigens have been
determined by "box-titration" against sera from proven cases of the mycoses with known titers. The optimally
diluted antigens are available as follows:
DESCRIPTION REF#SIZE
Histoplasma Mycelial CF Antigen Dilute
H101505 ml
Histoplasma Yeast CF Antigen Dilute
H301505 ml
Blastomyces CF Antigen Dilute
B101505 ml
Coccidioides CF Antigen Dilute
C101505 ml
Aspergillus CF Antigen Dilute
A101505 ml
B. Concentrate Antigens: The concentrate antigens must be box-titered against known patient sera (the
positive control sera are from hyperimmunized animals and may NOT be used for box-titering) to determine the
optimum dilution of each antigen for use in the LBCF test. The optimum titer of each lot number is available on
request. Generally, the antigens will have optimum titers between 1:16 and 1:64 depending upon the particular
antigen. The concentrate antigens available include:
DESCRIPTION
REF#SIZE
Histoplasma Mycelial CF Antigen Concentrate
H70150 5 ml
Histoplasma Yeast CF Antigen Concentrate
H90150 5 ml
Blastomyces CF Antigen Concentrate
B50150 5 ml
Coccidioides CF Antigen Concentrate
C90150 5 ml
Aspergillus CF Antigen Concentrate
A50150 5 ml
C. Antibody Controls: The Positive Controls are from hyperimmunized goats. Each Positive Control should
give a 1:32 titer (+/- 1 dilution) with its optimally diluted homologous antigen. The Negative Control should be
negative in the CF test with all antigens.
DESCRIPTION
REF. #
SIZE
Histoplasma Mycelial CF Positive Control
H20110 1 ml
Histoplasma Yeast CF Positive Control
H40110 1 ml
Blastomyces CF Positive Control
B20110 1 ml
Coccidioides CF Positive Control
C20110 1 ml
Aspergillus CF Positive Control
A20110 1 ml
Negative Control
N80110 1 ml
MATERIALS NOT PROVIDED: (some vendors are suggested)
1. Guinea pig complement (Cambrex Bio Science, Cat.# 30-956J)
2. Hemolysin Rabbit Anti-Sheep Erythrocyte Stroma (Cambrex Bio Science, Cat.# 55-402J)
3. Sheep Blood Alsevers (SRBC) (Colorado Serum Co., Cat.# CS1112 #7833 [recommend using blood
from neutered sheep])
4. Veronal Buffered Diluent (VBD, 5X concentrate) (Cambrex Bio Science, Cat.# 12-624E)
5. Gelatin (Sigma, Cat. # G2500)
6. 5% phenol solution in saline
7. pH meter
8. 15 ml Centrifuge tubes
9. Centrifuge
10. Refrigerator (2-8ºC)
11. Pipettes (1, 5, & 10 ml)
12. Glass serological tubes (15 X 125 mm & 12 X 75 mm)
13. Incubator, 37°C
14. Water baths or heat block (37°C & 56°C)
15. Microtiter equipment:
a. 25-50 µl multichannel micropipetter
b. "U"-bottom polystyrene microtiter plates
c. reading mirror (concave, 1.5 X mag.)
PRECAUTIONS:
A. All reagents are intended for in vitro diagnostic use only!
B. Specific standardization is necessary to produce our high quality reagents and materials. IMMY cannot
guarantee the performance of its products when used with materials purchased from other manufacturers.
C. Do not use reagents containing foreign matter, particulates or aggregates, which indicate contamination or
improper storage or handling. Note: Histoplasma Yeast Antigens (REF H30150 & H90150) contain a
suspension of yeast cells and should be cloudy.
D. Specimens must not contain bacteria, visible lipids, or other obvious signs of contamination.
E. Do not store specimens in a frost-free type freezer. Repeated freezing and thawing of the specimens can
affect test results.
F. When handling patient specimens, adequate measures should be taken to prevent exposure to etiologic
agents potentially present in the specimen.
G. All reagents are preserved with sodium azide [0.095% (w/w)]; it is therefore recommended that excess
reagents simply be discarded in an appropriate waste receptacle.
STABILITY AND STORAGE:
Fungal antigens should be stored at 2-8°C and are stable until the expiration date as long as titers with the
positive controls are 1:32 ± 1 dilution. The positive controls may continue to be used as long as titers with their
homologous antigen is 1:32 ± 1 dilution. The positive control is stable until the expiration date when stored at 2-
8°C PRIOR to rehydration. The rehydrated positive control will remain stable for up to one month if stored at 2-
8°C. For storage periods longer than one month, the rehydrated positive control should be aliquotted and frozen
(NOT in a frost-free freezer) where it will remain stable until the expiration date. Repeated freezing and thawing
should be avoided. When the positive controls are in use, the period at room temperature should be kept as
short as possible.
REAGENT PREPARATION:
Positive Control: The Positive Controls are rehydrated by adding 1 ml of distilled or DI water to the vial and
incubating at room temperature until completely dissolved (2-3 minutes), followed by gentle mixing.
1X VBD Preparation:
1. Combine 100 ml DI water and 0.5 g gelatin to a 1 L flask and bring to a boil
2. Remove from heat and allow to cool to room temperature
3. Add 300 ml DI water (Gel-Water Solution)
4. Add 100 ml 5X VBD and mix by inverting (1X VBD)
Hemolysin Titration:
Hemolysin should be titrated each time new lot numbers of sheep cells or hemolysin are used. Upon receiving a
new lot number of hemolysin, a stock 1:100 solution should be prepared. The procedure for the preparation of
this stock solution is shown below.
Preparation of Stock 1: 100 Hemolysin Dilution
1. Add 4.0 ml of 5% phenol solution to a 125 ml Erlenmeyer flask
2. Add 94 ml of cold VBD to the flask and mix by swirling.
3. Add 2ml of glycerinized hemolysin and mix by swirling. (Note: hemolysin is already diluted 1:2 with
glycerine as received).
4. Store the 1:100 stock hemolysin at 2-8ºC for up to 1 year.
Hemolysin Titration Procedure
1. Label a 15 X 125 mm tube for the 1:1000 hemolysin dilution.
2. Add 9 ml of cold VBD to the tube
3. Add 1 ml of 1:100 stock hemolysin dilution and mix by pipetting.
4. Label six 15 X 125 mm tubes with the final hemolysin dilutions shown in Table 1.
5. Pipette the volumes of VBD shown in Table 1 into the appropriate tubes.
Table 1:
Preparation of Further Hemolysin Dilutions
Final Hemolysin
Dilution
VBD
ml
1:1000 Hemolysin
Dilution, ml
1:1500
0.5
1.0
1:2000
1.0
1.0
1:2500
1.5
1.0
1:3000
2.0
1.0
1:4000
3.0
1.0
1:8000
7.0
1.0
6. Pipette 1 ml of 1:1000 hemolysin dilution into each tube and mix
7. Prepare appropriate dilutions of complement using cold VBD. See Table 2 in the Complement
Titration section for appropriate volumes of VBD and complement.
Note: Preparing multiple dilutions of complement [i.e. 1:300, 1:350, 1:400, etc.] and running titrations with
each simultaneously will frequently save time since some dilutions may not plot satisfactorily.
8. Incubate the complement dilutions at 2-8° C for 20 minutes, but use within 2 hours.
9. Label 7 tubes, one for each hemolysin dilution shown in Table 1.
10. Sensitize the cells by adding 1 ml of standardized 2.8% SRBC to each tube. (See Reagent
Preparation Section for preparation of 2.8% SRBC suspension.)
11. Slowly, and with constant swirling, add 1.0 ml of the appropriate hemolysin dilution to each of the
tubes.
12. Shake the rack to mix, and incubate in a water bath at 37° C for 15 minutes.
13. For each dilution of complement, label 7 tubes, one for each hemolysin dilution shown in Table 1,
with the appropriate hemolysin and complement dilution.
14. Add 0.4 ml of cold VBD to each tube
15. Add 0.4 ml of the appropriate complement dilution to each tube and shake the rack to mix.
16. Add 0.2 ml of sensitized SRBC to each of the appropriately labeled tubes, and shake the rack to
mix.
17. Incubate tubes in a 37° C water bath for 1 hour, shaking after 30 minutes.
18. Centrifuge tubes at 600g for 5 minutes.
19. Prepare color standards and record the percent lysis for each tube. (See Color Standards )
20. Plot the percent (%) Hemolysis on the y-axis of standard (linear-linear) graph paper versus the
appropriate hemolysin dilution on the x-axis.
21. Examine the graph for a plateau. (i.e. the region where increasing the concentration of hemolysin
produces no marked increase in lysis.)
22. The second dilution on the plateau is the optimum hemolysin dilution to be used to sensitize cells
for the complement titrations, antigen titrations, and serum tests. This dilution is termed the 100%
hemolytic unit of hemolysin (H'
50
).
Figure 1
Complement Titration:
Complement must be titrated each time new lot numbers of hemolysin, complement or SRBC's are used.
Freshly rehydrated complement should be aliquoted into sealed vials or tubes and stored at -60°C or colder.
A thawed aliquot should be used for all titrations.
Keep Complement, and all dilutions, cold (e.g. refrigerate or ice bath) during and after thawing (2 -8ºC).
Procedure:
1. Prepare sensitized, standardized SRBC's as indicated in the "Reagent Preparation" section.
Note: 3 or 4 different complement dilutions should be titered simultaneously to increase the probability of
obtaining values that will give a valid titration curve.
2. For each C' dilution that is to be titered, label two sets of four 12 X 75 mm tubes with the appropriate
complement dilution and the numbers 1 through 4. (Each titration is performed in duplicate.)
3. Prepare C' dilutions according to Table 2.
Table 2:
Complement Dilutions
Complement
Dilution
ml
Complement
ml
VBD
1:200
0.25
49.75
1:225
0.25
56.00
1:250
0.25
62.25
1:275
0.25
68.50
1:300
0.25
74.75
1:325
0.25
81.00
1:350
0.25
87.25
1:375
0.25
93.55
1:400
0.25
99.75
4. For each C' dilution that is to be tittered, add reagents to the duplicate sets of tubes in the order shown
in Table 3.
Table 3:
Complement Titration
Reagent Tube# 1 2 3 4
VBD (ml)
0.60
0.55
0.50
0.40
C' Dilution (ml)
0.20
0.25
0.30
0.40
Sensitized SRBC's (ml)
0.20
0.20
0.20
0.20
5. Shake the tubes and place in a 37°C water bath for 30 minutes. Shake after 15 min. of incubation.
6. Remove the tubes from the water bath, and centrifuge to pack the cells.
7. Determine the percent hemolysis in each tube by comparing with the color standards, and interpolating
to the nearest 5% when a tube does not exactly match one of the standards. (See Reagent Preparation
-Color Standards)
8. Average the percent lysis for each pair of replicate tubes with the same volume and dilution of C'
Table 4:
Lysed to Unlysed Ratio
Y
Y
Y
Y
(100-Y)
Y
(100-Y)
Y
(100-Y)
10 0.111 40 0.67 70 2.33
15 0.176 45 0.82 75 3.00
20 0.250 50 1.00 80 4.00
25 0.330 55 1.22 85 5.70
30 0.430 60 1.50 90 9.00
35 0.540 65 1.86
Do not graph when Y exceeds 90% or when Y is less than 10%
9. Using Table 4, determine the value of the ratio of lysed to unlysed cells Y/(1 00 - Y) for the average
percent lysis of tubes 1-4 for each C' dilution.
For example, if the average of the percent lysis for a pair of tubes was 35%, the corresponding value of
the ratio Y/(100-Y) would be 0.540.
10. Using log-log graph paper, plot the averages for each of the four points for a single C' dilution using the
volume of C' plotted against the corresponding Y/(100-Y) value (see Figure 2.)
Note : To plot satisfactorily, two of the Y/(1 00-Y) values must be less than 1, and two must be greater
than 1. In the example depicted in Figure 2, the 1:375 C' dilution plotted satisfactorily.
11. Join the two points plotted for tubes 1 & 2, and using a ruler, find the midpoint of this line.
12. Repeat this process for the points plotted for tubes 3 & 4
13. Draw a line connecting the two midpoints for the lines determined in steps 11 & 12 above.
14. Determine the slope of the midpoint line plotted in step 13. To determine the slope, use a millimeter
ruler to measure vertically from the point where the midpoint line intersects a vertical line down to the
same level where the midpoint line intersects a horizontal line. This length is the "rise". Next, measure
horizontally from the point where the midpoint line intersects a horizontal line across the same level
where the midpoint line intersects a vertical line. This length is the "run". The slope is calculated by
dividing the "rise" by the "run". The slope must equal 0.2 +/- 0.02 for a satisfactory titration.
15. From the point where the midpoint line intersects the vertical "1" line (See Figure 2), draw a horizontal
line to the vertical axis on the left.
16. Read the volume of the C' dilution in ml. This volume contains 150% hemolytic unit of complement (1
C'
50
unit)
17. Determine the volume containing 5 C'
50
units by multiplying the volume containing 1 C'
50
by 5.
1
8. Calculate the dilution of C' necessary to give 5 C
'50
units in 0.4 ml by using the following formula:
Formula:
Example:
Dilution of C' used in titration X*
375 X*
Volume containing 5 C'
50
units = 0.4
0.26 X 5 = 0.4
X*=dilution of C' needed for 5 C'
50
units
X*=115
Sensitized, Standardized Sheep Red Blood Cell Preparation (SRBC):
Preparing Standardized SRBC
1. Determine the volume of sensitized, standardized SRBC needed by multiplying the number of plates by
2.4 then add 1 ml for pipetting
2. To standardize SRBC, wash sheep blood (7 ml blood = ~1 .5 ml packed cells) with VBD by
centrifugation at 600xg for 10 min and remove supernatant by suction - repeat (Only use sheep blood
that is > 5 days and < 28 days old)
3. Transfer SRBC to a graduated centrifuge tube, centrifuge 600xg for 5 minute and record volume of
packed cells. Remove supernatant by suction.
4. Standardize SRBC to 2.8% by using the following table:
Table 5:
Packed Cell VBD Volume Packed Cell VBD Volume
Packed Cell VBD Volume
Volume (ml)
(ml) Volume (ml) (ml)
Volume (ml)
(ml)
0.10 3.40 0.90 30.60 1.70 57.80
0.15 5.10 0.95 32.30 1.75 59.50
0.20 6.80 1.00 34.00 1.80 61.20
0.25 8.50 1.05 35.70 1.85 62.90
0.30 10.20 1.10 37.40 1.90 64.60
0.35 11.90 1.15 39.10 1.95 66.30
0.40 13.60 1.20 40.80 2.00 68.00
0.45 15.30 1.25 42.50 2.05 69.70
0.50 17.00 1.30 44.20 2.10 71.40
0.55 18.70 1.35 45.90 2.15 73.10
0.60 20.40 1.40 47.60 2.20 74.80
0.65 22.10 1.45 49.30 2.25 76.50
0.70 23.80 1.50 51.00 2.30 78.20
0.75 25.50 1.55 52.70 2.35 79.90
0.80 27.20 1.60 54.40 2.40 81.60
0.85 28.90 1.65 56.10
5. Add SRBC to volume of VBD calculated above to a small flask and swirl gently
6. To test the accuracy of the SRBC suspension, add EXACTLY 7 ml of the 2.8% SRBC to a graduated
centrifuge tube (below 1 ml, graduations should be in 0.1 ml increments) and centrifuge 600xg for 10
min.
7. The packed cells should read exactly 0.2 ml. If not, then calculate the CCV using the following formula:
CCV=(PCV/0.2)OCV where CCV=corrected cell volume, PCV=packed cell volume, and OCV=original
cell volume. If CCV > OCV, then calculate the volume of VBD to ADD by CCV-OCV and repeat step 6.
If CCV < OCV, then calculate the volume of VBD to REMOVE by OCV-CCV and repeat step 6 (NOTE:
if VBD is to be added, mix the contents of the centrifuge tube in step 6 and return the contents to the flask
of cells before adding the additional VBD).
Preparing Sensitized, Standardized SRBC
8. To a small flask, add volume of standardized SRBC equal to half the volume of sensitized cells required
9. Prepare an equal volume of optimally diluted hemolysin diluted with VBD and slowly add to cells, while
constant swirling the cells. Make sure to add hemolysin to the cell mixture, not the cell mixture to the
hemolysin.
10. Incubate cell mixture for 15 min in a 37°C water bath
Color Standards
Prepare 0, 30, 50, 70, 90, and 100% Lysis Standards.
1. The Hemoglobin solution is prepared by adding, in order, 1 ml of 2.8% SRBC to a 15x125 mm tube, 7 ml
DI water, mix, and then add 2 ml 5X VBD (this final addition returns the solution to an isotonic state).
2. The 0.28% SRBC suspension is prepared by adding 1 ml 2.8% SRBC to a 15x125 mm tube and 9 ml
1X VBD.
3. Label 6 tubes as follows: 0, 30, 50, 70, 90, and 100.
4. Each color standard is prepared by adding Hemoglobin and 0.28% SRBC as follows:
5. Mix each tube briefly
.
Table 6:
% Lysis
0
30
50
70
90
100
Hemoglobin 0 0.3 0.5 0.7 0.9 1.0
0.28 % SRBC 1.0 0.7 0.5 0.3 0.1 0
Concentrated Antigen Box Titration:
1. Label one 12 x 75 mm tube for each dilution of known positive sera
2. Heat inactivate sera at 56°C for 30 min
3. Determine amount of each dilution of Ag required (~0.3 ml/row or dilution)
4. Prepare the dilutions of Ag using 1X VBD - mix well
5. Label plates as shown below
Figure 4.
1234567891
01
11
2
A
B
C
D
E
F
G
H
1234567891
01
11
2
A
B
C
D
E
F
G
H
ANTIGEN TITRATION
1:2
1:4
1:8
1:16
1:32
1:128
1:64
None
1:81:161:321:641:1281:2565
2.51.25
A
n
t
i
g
e
n
A
C
C
o
n
t
r
o
l
s
Serum Dilutions
C' Units
Antigen
Dilutions
VBD AC Controls
Serum AC Controls
6. Using the multichannel pipettor, add 25 µl of VBD to all wells in columns 2-9 (NOT in column 1) and
add 50 µl of VBD to columns 10-12, rows A & B (VBD Controls).
7. Add 50 µl of 1:8 diluted serum to all rows in column 1.
8. Using the multichannel pipettor, serially dilute (transfer 25 µl) the 1:8 diluted serum from column 1 to
column 6 (discard 25 µl from column 6).
9. Add 25 µl of 1:2 diluted Antigen to wells 1-9 in row A (i.e. A1-A9). Repeat for each dilution, adding 25 µl
of each antigen dilution to the appropriate row.
10. Using the multichannel pipettor, add 25 µl of 1X VBD to wells H1-H9
11. Mix on shaker for 1 minute
12. Determine the volume of diluted 5 C'H
50
(e.g 550% hemolytic units of complement) required for the test
by multiplying the number of plates by 4.8 (96 wells/plate, 0.050 ml/well) and add 2 ml excess for
pipetting and preparation of other dilutions. At the same time calculate the volume of 2.5 C'H
50
and 1.25
C'H
50
required by multiplying the number of wells for each by 0.050.
13. Calculate the volume of 1X VBD and C' stock required to prepare the 5 C'H
50
.
14. Add the calculated volume of cold 1X VBD to a small flask.
15. Add the calculated volume of C' to the 1X VBD while swirling gently to avoid foaming.
16. Incubate the diluted 5 C'H
50
at 2-8°C or in an ice bath for 20 minutes. The diluted C' must be used
within 2 hours.
17. After the incubation, prepare the calculated amount of 2.5 C'H
50
by diluting the 5 C'H
50
1:2 with cold 1X
VBD. At the same time, prepare 1.25 C'H50 by diluting the 5 C'H
50
1:4 with cold 1X VBD. Incubate the
2.5 and 1.25 C'H
50
at 2-8°C for 20 minutes before use. The diluted C' must be kept cold (2-8°C, or in an
ice bath) and be used within 2 hours.
18. Using the multichannel pipettor, add 50 µl of 5 C
'50
to columns 1-7 (i.e. A1-H7).
19. Add 50 µl of 2.5 C'
50
to column 8 (i.e. A8-H8).
20. Add 50 µl of 1.25 C'
50
to column 9 (i.e. A9-9).
21. Mix on shaker for 1 minute.
22. Stack and cover top plate and incubate overnight (15-18h) at 2-8° C
23. Add 25 µl sensitized, standardized SRBC to all wells of the plates (see Sensitized, Standardized
Sheep Red Blood Cell Preparation)
24. Mix plates on shaker for 2 minutes
25. Cover plates (do not stack) and incubate for 30 minutes in a 37° C incubator - briefly shake plates at 15
minutes
26. See Reading and Recording Results
SPECIMEN PREPARATION:
Serum from patients should be obtained from the clotted blood (anticoagulants should not be used) of patients.
All specimens must be free of visible lipids, white blood cells, platelets, fibrin or other contaminants. Platelets,
white blood cells and fibrin may be removed by centrifugation. Lipids should be removed by filtration.
Sera to be tested within 72 hours of collection may be stored at 2-8°C. For longer storage periods, -20°C or
lower. Avoid repeated freeze-thawing.
Prior to testing, each serum must be heat inactivated at 56°C for 30 minutes to destroy any indigenous
complement.
PROCEDURE:
All testing should be performed as described in the LBCF procedure5. All reagents (hemolysin, complement,
SRBC, and VBD) must be standardized prior to testing the patient specimens or positive control.
PREPARING 1:8 DILUTIONS OF SERA
1. Label one 12 X 75 mm tube for each 1:8 dilution of the following: patient sera, positive control and negative
control.
2. Add 0.7 ml of cold VBD to each tube.
NOTE: For Coccidioides, beginning serum dilutions should be at undiluted (See "Expected Values &
Performance Characteristics, Coccidioidomycosis").
3. Add 0.1 ml of each serum to its respective tube and mix.
4. Heat inactivate the diluted sera for 30 minutes in a 56°C water bath.
NOTE: If you are starting your dilutions at any dilution other than 1:8, modify your procedure and labels
accordingly.
PREPARING ANTIGEN DILUTIONS
1. Determine the volume of test antigen required by multiplying the number of patient sera and controls by
0.3 (i.e. 0.025 ml/well, 12 wells/serum) and add 0.5 ml excess for pipetting.
2. Prepare the test antigen at the optimum dilution using cold VBD and mix well.
3. Store antigen at 2-8 °C until use.
Figure 5:
1234567891
01112
A
B
C
D
E
F
G
H
1234567891
01112
A
B
C
D
E
F
G
H
CONTROL SERA TITRATION
VBD
Ag
Pos Cntrl
Neg Cntrl
CS #1
CS #2
CS #3
CS #5
CS #4
CS #6
1:81:161:321:641:1281:2561:81:161:3252.51.25
S
e
r
u
m
A
C
C
o
n
t
r
o
l
W
e
l
l
s
V
B
D
&
A
g
A
C
C
o
n
t
r
o
l
W
e
l
l
s
Serum Only (No Ag)
Serum + Ag
Ag Only (No Serum)
C' Units
REPARING TWO-FOLD DILUTIONS OF SERA
1. After heat inactivation, allow sera to cool to room temperature.
2. Mark plates as shown in the "Control Serum Titration" Figure 5, above for serum dilution wells and
serum Anti-Complementary (AC) Control wells, and label plate with the antigen type, lot number, and,
possibly a plate number.
3. Pipette 25 µl of cold VBD to all wells in columns 2 through 6 and 8 & 9 (not in columns 1 and 7, the
1:8 dilution columns). Pipette 25 µl of VBD into rows A & B, columns 10, 11, & 12 (antigen and VBD
controls).
4. Pipette 50 µl of 1:8 diluted Positive Control to row "A", columns 1 and 7. Repeat for negative control and
each case serum in the appropriate row (e.g. CS#1, etc.).
5. Using a micropipetter*, transfer 25 µl of 1:8 diluted serum from the column 1 well, to the 1:16 (column 2)
well; mix well, and repeat the process for the 1:32 (column 2 to column 3) through the 1:256 dilution for
all sera. Discard 25 µl from the 1:256 column (col. 6).
6. Repeat the process for the serum AC controls for columns 7 through 9 (1:8 through 1:32) and discard
25 µl from col. 9.
NOTE: Using an 8-channel micropipetter, all sera can be serially diluted simultaneously.
7. Pipette 25 µl of optimally diluted antigen to the appropriate wells (columns 1 through 6) for each serum
dilution (NOT Serum AC wells, col. 7, 8, & 9) and the Antigen Control wells (row B, col. 10, 11, & 12).
8. Add 25 µl of cold VBD to the serum AC control wells of the test plate (col. 7, 8, & 9) and to the VBD
control wells (row A, col. 10 through 12).
9. Mix plate on a shaker for 1 minute.
PREPARING DILUTED C'
1. Determine the volume of diluted 5 C'H
50
required for the test by multiplying the number of plates by 4.8
(96 wells/plate, 0.050 ml/well) and add 2 ml excess for pipetting and preparation of other dilutions. At
the same time calculate the volume of 2.5 C'H
50
and 1.25 C'H
50
required by multiplying the number of
wells for each by 0.050.
2. Calculate the volume of VBD and C' stock required to prepare the 5 C'H
50
.
3. Add the calculated volume of cold VBD to a small flask.
4. Add the calculated volume of C' to the VBD while swirling gently to avoid foaming.
5. Incubate the diluted 5 C'H
50
at 2-8°C or in an ice bath for 20 minutes. The diluted C' must be used
within 2 hours.
6. After the incubation, prepare the calculated amount of 2.5 C'H
50
by diluting the 5 C'H
50
1:2 with cold
VBD. At the same time, prepare 1.25 C'H50
by diluting the 5 C'H
50
1:4 with cold VBD. Incubate the 2.5
and 1.25 C'H
50
at 2-8°C for 20 minutes before use. The diluted C' must be kept cold (2-8°C, or in an ice
bath) and be used within 2 hours.
ADDING COMPLEMENT TO THE TEST
1. Pipette 50 µl of 5 C'H
50
to each titration well, control serum well, and each 5 unit control well, all wells
columns 1 through 9 rows A to H and col. 10, rows A & B).
2. Add 50 µl of 2.5 C'H
50
to each 2.5 unit control wells (col. 11, rows A & B).
3. Add 50 µl of 1.25 C'H
50
to each 1.25 unit control well (col. 12, rows A & B).
4. Mix the plates by shaking for one minute.
5. Cover and incubate at 2-8°C for 15 to 18 hours.
PREPARING SENSITIZED SRBC
1. Determine the volume of sensitized SRBC needed by multiplying the number of plates by 2.4, and then
add at least 1.0 ml excess for pipetting.
2. Remove the standardized 2.8% SRBC suspension from the refrigerator and swirl gently to suspend cells
(tilt occasionally to detect cells adhering to the bottom).
3. To a small flask, add a volume of 2.8% SRBC equal to half the volume of sensitized SRBC required.
4. Prepare an equal volume of hemolysin (H'), optimally diluted with cold VBD and slowly add the diluted H' to
the SRBC while swirling the SRBC. Never add the cells to the H'
5. Incubate the SRBC/H' mixture for 15 minutes in a 37°C water bath to allow sensitization.
DETECTING FIXATION OF COMPLEMENT
1. Plates should remain at 2-8°C prior to adding the sensitized, standardized SRBC, then pipette 25µl of
sensitized SRBC into all wells of the plates, except rows C through H, columns 10 through 12 (these
are used for color standards).
2. Mix the plates on a shaker for 2 minutes.
3. Cover or seal plates (do not stack) and incubate in a 37°C incubator for 30 minutes, reshake plates at
15 minutes, to resuspend cells and continue incubation.
READING AND RECORDING RESULTS
1. Pipette 0.125 ml of each Color Standard into empty wells on the plates.
2. Centrifuge the plates for 3 minutes at 300 X g.
NOTE: If centrifuge plate carriers are unavailable, incubate plates at 2-8°C for 2 to 3 hours until cells settle.
3. Read and record the controls (antigen and VBD) by comparison to the color standards.
4. Compare the control readings with those in the table below to determine if they are acceptable. If the
controls are not acceptable, the test is invalid; repeat the test.
Table 7:
QUALITY CONTROL:
Positive Controls against each antigen and a Negative Control should be run in each batch to indicate that all
reagents are satisfactory. Internal controls for the CF test include the following:
1. VBD controls (5, 2.5, & 1.25 U): contain VBD, complement (units indicated), and sensitized SRBC; used to
test the complement and hemolytic system (e.g. 1.25 U control should give 40-75% lysis).
2. Serum anti-complementary controls: contains serum dilutions, complement, and sensitized SRBC; used to
detect complement fixing activity of the serum in the absence of added antigen (e.g. serum anti-
complementary activity titer must be at least 4-fold (2 dilutions) lower than the antibody titer for the antibody
titer to be valid. Most common serum AC cause: circulating immune complexes in specimen).
3. Antigen controls (5, 2.5, & 1.25 U): contain optimally diluted antigen, VBD, complement (units indicated)
and sensitized SRBC; used to detect complement binding by the antigen (may indicate antibody present in
the complement; 1.25 U should give 0-75% lysis).
All controls must read correctly for test results to be valid.
TYPE
CONTROL
5
2.5
C' Units
1.25
Antigen AC
100%
85-90% 0-75%
VBD 100% 90-100%40-75%
INTERPRETATION OF RESULTS:
A. Negative Test: Patient sera which fail to "fix" complement in the presence of an antigen should be reported
as "NEGATIVE to named antigen".
B. Positive Test: Patient sera which " fix" complement (have 30% or less lysis with antigen) should be
reported as "POSITIVE to the named antigen" and the titer stated as the highest serum dilution having
30% or less lysis. Serum anticomplementary activity must be at least 4-fold (e.g. 2 dilutions) below the
antibody titer.
C. Anticomplementary (AC) Serum: Patient serum which fixes or destroys complement in the absence of
any added antigen must be reported as "Inconclusive Test Due To Anti-Complementary Activity, Please
Submit Another Specimen". Anti-complementary activity may be due to a number of factors such as
circulating immune complexes, detergent, or microbial contamination in the specimen, and some patient
medications.
LIMITATIONS OF THE PROCEDRUE:
The CF test relies on the property that certain immunoglobulin classes "fix" complement following reaction with
their specific antigen. However, not all immunoglobulin classes produced in a specific immunologic response
have the property of "fixing" complement, most notably IgA and some sub-classes of IgG do not "fix"
complement. For this reason, the CF test may not be positive throughout all stages of an infectious disease.
EXPECTED VALUES & PERFORMANCE
CHARACTERISTICS:
Histoplasmosis: A positive CF test is presumptive evidence of active or recent infection with H. capsulatum. CF
titers of 1:32 or greater or rising titers (a 4-fold or greater increase) with yeast antigens, mycelial antigens, or
both are strong presumptive evidence of histoplasmosis. Titers of 1:8 or 1:16, with either antigen, are generally
considered suggestive of histoplasmosis but are less readily interpreted6. Failure to demonstrate CF antibodies
does not exclude histoplasmosis, especially if only a single specimen is tested.
In "primary pulmonary infection" CF titers to the yeast antigen occur within 10 to 21 days following exposure to
the organism, usually by the time symptoms are present. CF titers with the mycelial antigen are generally
lower and develop later than the yeast antigen titers. However, in certain chronic cases, the mycelial antigen
titers may be higher or be the only antigen positive2.
In general, higher titers are associated with more severe disease and increasing titers (4-fold or greater rise) on
serial specimens indicates progression of disease. However, persistent low titers may also indicate severe
disease (up to 15% of chronic pulmonary cases have low or negative titers).
Following successful therapy, the CF titers will slowly return to negative, usually within 3 months; however, rare
patients have had persistent titers for up to 6 months following successful therapy.
Sera from patients with other mycoses may cross-react with Histoplasma antigens in the CF test, most notably
blastomycosis and rarely, coccidioidomycosis patients. For more complete serologic testing, immunodiffusion
and latex agglutination tests should be performed in parallel with the CF tests.
Blastomycosis: CF tests with the purified 'A' antigen of B. dermatitidis, may only be positive in
approximately 40% of confirmed cases1. Thus, a negative test has little value and in no way excludes the
possibility of active blastomycosis. Titers of 1:8 or greater are presumptive evidence of active or recent infection.
The antigen frequently gives titers with confirmed cases of histoplasmosis and coccidioidomycosis. High titers
or rising titers are suggestive of blastomycosis and serologic tests for other fungi should be performed
simultaneously. For more complete serologic testing, immunodiffusion tests for blastomycosis should be
performed in parallel with the CF tests.
Coccidioidomycosis: A CF titer of 1:2 or greater may be significant and is presumptive evidence of current or
recent past infection3. Titers greater than 1:16 are usually associated with severe disease. Patients with a limited
number of extrapulmonary lesions, eg. bone, skin, or central nervous system, may have titers below 1:16. Fourfold
or greater decreases in titer on serial specimens usually indicate improvement. In chronic residual
pulmonary disease, a significant number of patients (>30%) may have negative CF tests4.
Antibody demonstrable by the CF test usually develops within 4 to 6 weeks after exposure and reaches a
maximum response within 2 to 3 months. For those patients with positive reactions, the test is of diagnostic and
prognostic value. Low titers are generally associated with mild, localized disease or stable residual lesions,
while high or rising titers are usually associated with progressive or severe disease. A negative test does not
exclude the possibility of active coccidioidomycosis. For more complete serologic testing, immunodiffusion and
latex agglutination tests should be performed in parallel with the CF tests.
Aspergillosis: Less in known about the serological-clinical relationship in aspergillosis than in the previously
discussed mycoses. A CF titer of 1:8 or greater is strong suggestion of Aspergillus infection or allergy. However,
for a definitive diagnosis, the organism must be repeatedly demonstrated by culturing or microscopy.
Approximately 5% of the "normal" population have circulating antibodies against Aspergillus. Insufficient data are
available for a statement regarding cross-reactions between Aspergillus and other fungi. For more complete
serologic testing, immunodiffusion tests should be performed in parallel with the CF tests.
REFERENCES:
1. Campbell, C.C. 1960. The accuracy of serologic methods in diagnosis. Ann. N.Y. Acad. Sci.89:1 63.
2. Beuchner, H.A., et. al. 1973. The current status of serologic, immunologic, and skin tests in the diagnosis of
pulmonary mycoses. Report of the Committee on Fungus Diseases and Sub-Committee on Criteria for
Clinical Diagnosis. Amer. Col. Chest 63:259-270.
3. Chick, E.W., et. al. 1973. The use of skin tests and serologic tests in histoplasmosis, coccidioidomycosis,
and blastomycosis. Amer. Rev. Resp. Dis. 108: 156-159.
4. Smith, C.E., et. al. 1940. Epidemiology of acute coccidioidomycosis with erythema nodosum("San Juoquin"
and "Valley" fever). Amer. J. Publ. Hlth. 30:600-611.
5. U.S. Public Health Service. 1965. Standardized Diagnostic Complement Fixation Method and Adaptation to
Microtest. P.H.S. Pub 1228 (Public Health Monograph no. 74) U.S. Govt. Printing Office, Washington, D.C.
(Note: IMMY has rewritten the LBCF manual and it is available separately, "Diagnostic Micro-CF Procedure
Manual", Cat. # CF1090.)
6. Leland, D.S., et. al. 1991. Variability in Commercial Histoplasma Complement Fixation Antigens. J. Clin.
Microbiol. 29:1723-1724.
International Symbol Usage
In Vitro Diagnostics
V
Expiration Date
H
Sufficient for"#"Tests
X
CE Symbol
C
Catalog Number
h
Manufactured by
N
Lot Number
g
Storage
2-8C
8°C
2°C
l
M Immuno-Mycologics, Inc.
2700 Technology Place
Norman OK 73071 U.S.A.
(405) 360-4669/ (800) 654-3639
Fax: (405) 364-1058
E-mail: info@immy.com
WEB: www.immy.com
Rev.2-12-2007
C
P
MDSS
Schiffgraben 41
30175 Hannover, Germany
pdf