- Email: [email protected]
[10] Quantification of complement fixation by the C1 fixation and C1 fixation and transfer tests
[10]
QUANTIFICATION
OF C O M P L E M E N T
FIXATION
165
the steps before adding complement are done in EDTA buffer to prevent the interaction of serum C with the labeled cells. In that case the cellsantibody-antigen mixtures should be washed 1 × with VBS before the addition of C. In experiments where the antibody to the label is of the non-C-fixing type, the cells-antibody-antigen mixtures are washed 1 x with VBS and are than exposed to an anti-antibody that is C fixing (e.g., rabbit anti-goat) for 1 hr at 37° before adding C. The amount of the second antibody must be excess, i.e., addition of more second antibody does not increase lysis. Because some antisera and guinea pig C contain natural antibody to the sheep cells and possibly to the label, it is necessary to absorb the antisera with sheep cells and the C with labeled sheep cells. In some instances one or more of the reagents may contain colored substances that may interfere with the spectrophotometric determination of hemoglobin. In such instances washing 1 x before addition of C will eliminate this problem. There are several advantages of the PHI test. The most outstanding of them is that its performance requires equipment that is found in most laboratories, i.e., a centrifuge and a spectrophotometer. Sheep cells and guinea pig serum are easily obtained in most countries and if not available commercially can be procured from domestic sources. The accuracy and precision of the PHI test approaches that of RIA without the use of radioactive materials while its sensitivity compares favorably with RIA.
[10] Q u a n t i f i c a t i o n o f C o m p l e m e n t F i x a t i o n b y t h e C1 Fixation and C 1 Fixation and Transfer Tests
By TmOR BORSOS The complement fixation (CF) tests is a widely used diagnostic and investigative tool for the detection of antigen-antibody complexes. The principle of the CF test is based on the ability of certain classes of immunoglobulins (Igs) when in aggregated form or as antibodies (Abs) in combination with antigens (Ags) to interact with and bind ("fix") serum complement (C). The fixation of C by Ag-Ab complexes results in reduction of the concentration of free C; the residual C activity is measured by its hemolytic activity, i.e., the ability of C to lyse red cells sensitized with Ab. The CF test can be a sensitive (nanogram amounts of Ab or Ag) and precise test; however, the results cannot usually be interpreted on a mo-
METHODS IN ENZYMOLOGY, VOL. 74
Copyright ~) 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181974-4
166
IMMUNOASSAYMETHODS
[10]
lecular basis, i.e., there is no direct relationship between the number of CF complexes and the quantity of C fixed by them. The development of a sound theory for the hemolytic activity of C and its individual components, coupled with great advances in devising molecular measurements for their hemolytic activity permitted the development of CF tests that are based on the measurement of the interaction with Ag-Ab complexes with a single component of C. The advantages of CF tests based on the fixation of a single component of C as opposed to a CF test based on fixation of " w h o l e " C activity has been discussed by Rapp. 1 One of the widely used tests of this nature is based on the fixation of the first component of C (C1) by Ag-Ab complexes. 2 There are two types of CF tests using C 1 as the indicator molecule: in the C1 fixation and transfer test, a particulate Ag-Ab complex interacted with purified C1 (the active form of C1) is washed, and the bound C1 is then transferred to indicator cells, which, under appropriate conditions, will lyse. From the degree of lysis, the number of C1 molecules present in the original mixture can be calculated. In the second type of C 1F test, the Ag-Ab complex is first exposed to carefully determined, limiting amounts of C 1 followed by measurement of hemolytic activity of the residual, unbound C1 in the mixture. Because the hemolytic test for C1 activity enumerates hemolytically active C1 molecules on a molecular basis, both of these CF tests yield estimates of minimal numbers of CF complexes on a molecular basis. The C1FT test is performed in three stages and can be summarized as follows: 1. Fixation stage: AgAb + excess C1 wash AbAgC'I + C1 2. Transfer stage: AgAbC1 + EAC4 ~ EAC14 + AgAb 3. Lysis stage: EAC14 + C2 + C3-9--~ lysis The first stage is performed at low ionic strength (/x = 0.065) to favor binding of C1 to Ag-Ab complexes, whereas the second stage is performed at an ionic strength (tz = 0.15) that permits transfer of C1 to the indicator cells. In case the Ig in the Ag-Ab complex is non-C-fixing, the Ag-Ab complex is exposed to an anti-Ig antibody where the anti-Ig Ab is of the CF class. After washing, excess C1 is added and the test is performed as indicated in the sequence. The CIFT test is designed to measure particulate Ag-Ab complexes. A C1 fixation test was therefore designed to permit the measurement of Cl H. J. Rapp, in "Immunological Methods" (J. F. Ackroyd, ed.), p. 1. Blackwell, Oxford, 1964. 2 T. Borsos and H. J. Rapp, J. Immunol. 95, 559 (1965).
[10]
QUANTIFICATION OF COMPLEMENT FIXATION
167
fixation by any CF complexes. The sequence of reaction of the C1F test can be summarized as follows: 1. Fixation stage: AgAb + C1 (measured quantity)---, AgAb-C1 ÷ reduced free C1 2. Lysis stage: (AgAbC1) + free C1 + EAC4 + C2 + C3-9 ~ lysis Both stages are performed a t / z = 0.065 or below. The test is essentially a competitive inhibition test; 'because it is performed at ionic strength below 0.15, there is little or no transfer of AgAb bound C1 to EAC4. Materials The following reagents are needed to perform the various tests based on the fixation of C1. These descriptions are adapted from Rapp and Borsos :3 with permission of the publisher. Veronal-Buffered Saline (VBS). For stock solution: dissolve 83.0 g of NaCI and 10.19 g of sodium 5,5-diethylbarbiturate in about 1.5 liters of distilled or deionized water (flee of endotoxin and pyrogens). Adjust pH of solution to 7.35 with 2 N HCI and add water to exactly 2.00 liters. One liter isotonic buffer is prepared by mixing 200 ml of stock solution with 1.0 ml of a solution containing 1.00 M MgC12 and 0.150 M C aC12 and by adding water to exactly 1.00 liter. Gelatin is added to achieve a final concentration of 1 g/liter. The ionic strength of this buffer is 0.150. Verona#Buffered (VB)Sucrose. For stock solution, dissolve 486.1 g of reagent grade sucrose and 5.095 g of sodium 5,5-diethylbarbiturate in about 500 ml of distilled or deionized water. Adjust the pH of the solution to 7.35 with 2 N HCI and add water to exactly 1.00 liter. Isotonic veronalbuffered sucrose is prepared by mixing 200 ml of stock solution with 1.0 ml of the MgCI2-CaC12 solution and by adding water to exactly 1.00 liter. Gelatin is added to achieve a final concentration of 1 g/liter. The ionic strength of this buffer is 0.009. To obtain a VBS-sucrose buffer of ionic strength of 0.065, mix 2 parts of VBS with 3 parts of VB sucrose. EDTA Solution. For stock EDTA (0.1 M), dissolve 37.2 g of disodium ethylenediaminetetraacetic acid (Na2H~EDTA) in about 800 ml of distilled or deionized water. Adjust the pH to 7.65 with freshly prepared 2 N NaOH. (Caution: addition of NaOH must be slow and with vigorous stirring.) Bring the volume to 1.00 liter with water. The stock solution (stored at 4°) must not be used more than 3 weeks after it was prepared. a H. J. Rapp and T. Borsos, "'MolecularBasis of Complement Action," Chapter 7. Appleton, New York, 1970.
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IMMUNOASSAYMETHODS
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Isotonic VBS-EDTA buffer (0.01 M EDTA) is prepared by mixing 9 parts of isotonic VBS (containing gelatin but not added MgCl~ and CaCI~) with 1 part of the 0.1 M EDTA solution. Complement Components and Cellular Intermediates
First Component of C (C1). The preferred method for obtaining guinea pig C1 in partially purified form is described herein; in case it is not feasible to prepare one's own C1, quite satisfactory preparations of C1 may be purchased from Cordis Laboratories, Miami, Florida. Twenty milliliters of cold fresh (frozen or fresh frozen) guinea pig serum are adjusted to pH 5.6 by carefully adding 0.15 M HCI delivered under the meniscus with constant stirring. The mixture is dialyzed for 2 hr with constant mixing against about 2 liters of ice-cold water. The dialyzed serum is centrifuged at 900g. The precipitate is washed 3 x (at 900g) with ice-cold 0.015 M NaCl; after the last centrifugation, the precipitate is resuspended in 2.0 ml of VBS-sucrose buffer (/.t = 0.065) (without gelatin) and extracted at room temperature for about 15 min. The tube is centrifuged at 900 g for 20-30 min and the (sometimes opalescent) supernatant is applied in two equal parts to the top of a sucrose gradient (8-30% sucrose in VBS, # -- 0.065). The tubes are centrifuged at about 100,000g for 16-18 hr at 10-15 °. All C1 activity is found near the bottom of the tube. The C1 prepared by this method is stable for many months at - 20° to - 40° and is free of interfering substances. Second Component of C (C2). Suitable guinea pig C2 can be purchased from Cordis Laboratories, Miami, Florida. Alternatively, C2 that is of sufficient activity, purity, and stability can be prepared by DEAE- and CM-cellulose chromatography. For the processing of 20 ml of guinea pig serum, 27 g of DEAE-(Brown Co.) and 20 g of CM-cellulose are used. It is important that both the DEAE- and CM-cellulose are washed according to these instructions; other methods of washing have been known to affect the binding properties of these celluloses in such a way that C2 activity could not be recovered during the separation procedures. The celluloses are washed first with 0.5 M NaCl (the use of several liters is recommended), followed by resuspension of the DEAE in about 1 liter of 0.015 M NaCI and the CM in about 1 liter of 0.03 M NaC1. The slurry of DEAE is stirred vigorously and the pH is adjusted to 7.4 with 1-2 N HCI. Equilibration may take 15-20 min. The slurry of CM is vigorously stirred and its pH is adjusted to 4.7 with freshly made 1-2 N NaOH. It is important that the pH not be below 4.7 once equilibration occurs (C2 is inactivated rapidly at pH 4.5 or below). Three glass columns of about 60-cm length are set up (ID of Nos. 1 and 2 about 5 cm and No. 3 about 3 cm).
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QUANTIFICATION OF COMPLEMENT FIXATION
169
The bottom of the column may not be of sintered glass. The columns should be constructed so that air pressure can be applied at the top so that liquids can be passed through rapidly due to the increased pressure. Column No. 1 receives an amount of DEAE slurry to contain 20 g of DEAE; No. 3 the remainder (about 7 g); column No. 2 receives all the CMcellulose. Once the celluloses have settled (take care that the cellulose column never runs dry 9, a thin layer of glass wool is laid on top of each (approximately ½inch); to hold the glass wool in place a 1-inch layer of glass beads are added. Column No. 1 is then washed with about 500 ml of 0.03 M NaC1 and No. 3 with 200-300 ml of 0.015 M NaCI (there should be several liters of each of these solutions prepared). The columns are ready for use when the liquid levels entered the glass wool layer. Twenty milliliters of guinea pig serum are treated as indicated for the preparation of C1. The supernatant fluid of the precipitated serum is removed and its pH is adjusted to 7.4 with 0.15 M NaOH and its ionic strength to 0.08 with 3.0 M NaCI. The adjusted supernatant is poured into column No. 1 and permitted to enter the cellulose; add about 600 ml of 0.08 M NaCI. After discarding the first 100 ml of effluent, collect the next 300 ml and mix with 500 ml of ice-cold water; apply to and pass through column No. 2. Collect 1 liter of effluent in a beaker containing 1 liter of ice-cold water. The 2 liters of effluent are well mixed and passed through column No. 3. After all of the 2 liters of effluent has passed through (and been discarded), wash the column with 200-300 ml of 0.015 M NaCI, then adjust the fluid level to the top of the cellulose column. Add 200 ml of 0.25 M NaC1 and start the pass through. Discard the first 30 ml and collect the next 100 ml. This should contain C2. C-EDTA. Usually it is prepared by diluting fresh (frozen) guinea pig serum 1/40 in 0.01 M EDTA buffer. This reagent can be kept (cold) for several weeks. Sheep Cells (E). Sheep cells can be obtained from several commercial sources. Cells should not be used until 1 week after collection. Ten to twenty milliliters of the blood (containing an anticoagulant) are mixed with 0.01 M EDTA buffer and are incubated for a few minutes at 37°. The cells are washed 2x with 0.01 M EDTA buffer and 3× with VBS (+ gel + Ca'-'+Mg'+). One billion cells when lysed in 25 ml of water have an OD of 0.420 at 541 nm (1 cm light path). Anti-Forssman Antibody (A). Antibody may be procured commercially. It should be obtained from rabbits immunized with boiled stromata of sheep red cells. Sera should be isolated with high IgM antibody concentration. Optimal antibody levels are determined in the following manner (buffer, VBS): In a series of test tubes place 0.1 ml o r E (1.0 x 109/ml) and 0.1
170
IMMUNOASSAY METHODS
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ml of dilutions of anti-Forssman antiserum; after incubation for 10 min at 37°, add 1.0 ml of a 1/300 dilution of fresh guinea pig serum as a source of C. (The appropriate dilution of C may have to be determined by trial and error.) After 1 hr of incubation the cells are removed by centrifugation and the amount of lysis determined by measuring hemoglobin at a wavelength of 541 nm. A plot of percentage lysis versus relative concentration of antiserum should yield a curve that is concave to the abscissa. Where percentage lysis becomes independent of concentration at high levels of A (cf. Ref. 3), i.e., a plateau is reached say at 1/100 dilution of A, then a 1/50 dilution should be appropriate for preparing EAC4. EAC4. Mix 5 ml of E (in VBS-sucrose buffer, /~ = 0.065, 1.0 × 109 E/ml) with the appropriate dilution of A. Incubate for 10 min. Wash 1×. Add 1 ml of C1 (about 200 effective molecules of C1 per cell; see following section for titration of C1), cool to 0°. Add by quick mixing, 50 ml of ice-cold guinea pig (or human) serum diluted 1/10 in 0.01 M EDTA buffer. Mix rapidly and well. Incubate for 20-30 min. Wash by centrifugation 2× with 0.01M EDTA and 3× with VBS. Dilute to contain 1.5 × 108 cell/ml. Store at 4° . This reagent should be stable and useful for 1-2 weeks. Titration ofC-[. 4 Place in a series of test tubes (13 × 100 mm is ideal) 0.1 ml of EAC4 (1.5 × 108 E/ml) (buffer throughout this part of the test is VBS-sucrose, /z -- 0.065). Mix with the cells 0.1 ml of a dilution of C1. Incubate 15 min at 30°. Add 0.1 ml of C2 (at least 50 effective molecules per cell). Incubate 10 min at 30°. Add 1.0 ml of C-EDTA and incubate 1 hr at 37°. Centrifuge and measure amount of hemoglobin in supernatant at a wavelength of 412 nm. Calculate fraction of cells lysed (y). Plot on log-log paper the - I n ( 1 - y ) against relative concentration of C1. At -ln(1 - y) = 1, the cells carry an average number of one CI molecule per cell, i.e., the dilution of C-1 yielding - l n ( l - y) -- 1 contains 1.5 × 10~ effective C1 molecules per milliliter. Table I shows the protocol for titrating CI. Test for A g - A b Based on the Fixation and on the Transfer of CiIn the introduction I have described the principles of the various C-fixing tests utilizing the fixation and measurement of C1 on a molecular basis. I shall now describe in detail the various forms of the tests and show an example for each. Direct C I F T Test. In the fixation stage (VBS-sucrose buffer; 9 /z = 0.065) mix portions of the particulate Ag with various dilutions of Ab. Wash in VBS-sucrose buffer,/z = 0.065, 2 ×. Add 0.1 ml of C1 to contain about 6 × 101° C1 molecules per milliliter. Controls should include Ag solo, Ab solo, tube solo. Incubate 20 min at 30°. Wash by centrifugation
[lO]
171
QUANTIFICATION OF COMPLEMENT FIXATION TABLE I PROTOCOL FOR TITRATION OF C [ Tube n u m b e r
Reagent E A C 4 (1.5 x 10 s E/ml) C 1 dilution (0.1 ml) VBS-sucrose ~/x = 0.065)
Unit
ml
1
2
3
4
5
0.1
0.1
0.1
0.1
0.1
1000
2000
4000
8000
1000 0.1
ml
6
7
8
9
0.1
0.1
0.1
0.1
0.2
0.1
0.2
0.1
0.1
Incubate 15 rain, 30 ° C2"
ml
0.1
0.1
0.1
0.1
C-EDTA E D T A buffer H20
ml b ml ml
1.0
1.0
1.0
1.0
0.1
Incubate 15 min, 30 ° 1.0
1.0 1.0
1.0 1.0
Incubate 60 rain, 37°; centrifuge OD4~2 of supernatant OD412 of control y" ~' z x dilution C 1/EAC4/ml
1.02
0.756
0.485
0.315
0.950 0.864 1.99 1990
0.686 0.624 0.977 1954 1965 e
0.415 0.377 0.474 1896
0.245 0.223 0.252 2020
0.010
0.070
0.010
1.11
0.60
1.10
" At least 50 C2 molecules per EAC4. b Ice-cold. " Fraction of cells lysed. ' N u m b e r o f effective C / molecules per cell. " To calculate the absolute n u m b e r of C 1 molecules, multiply by the n u m b e r of cells/ml: 1.97 x Iff~ C1/cell/ml x 1.5 x 10s EAC4/ml = 3.0 x 10~ C[/ml,
1 x ; transfer to a fresh set of tubes and wash 4 × . R e s u s p e n d in VBS (0.15) and m a k e dilutions. Incubate 0.1 ml of each dilution with 0.1 ml o f E A C 4 (1.5 x 10 ~ in VBS,/~ -- 0.15). Incubate for 20 min at 30 °. Add 0.1 ml of C2 in VB sucrose /x = 0.009) containing about 50 C2 molecules per cell. I n c u b a t e 15 min at 30 °. Add 1.0 ml of ice-cold C-EDTA. Incubate 1 hr at 37 °. Centrifuge and determine fraction of cells lysed and the n u m b e r of C1 molecules as shown for C1 titration. Because the dilution of the original A g - A b c o m p l e x to yield - l n ( 1 - y) = 1 is now known, we can calculate the n u m b e r of C1 fixing sites in the original A g - A b mixture. The data should be corrected for the appropriate blanks. Caution: Precentrifugation of C1 and antiserum (Ab) at several thousand g is essential for success-
172
IMMUNOASSAY
METHODS
[10]
e-
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~',,-
¢.q O0
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[10]
QUANTIFICATION
OF
COMPLEMENT
FIXATION
t~ O~
O
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t.~
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,6 L) X
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173
174
IMMUNOASSAY
METHODS
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175
Q U A N T I F I C A T I O N OF C O M P L E M E N T F I X A T I O N
IgG, direct Ci FT IgM
(U
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v,-
IgG, indirect C'IFT qU
10
"3 IU O
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5 d
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64
Relative antibody concentration Fi6. 1. Dose-response curves of rabbit IgM and IgG anti-Forssman antibodies as determined by the C 1FT test. In the indirect test the second antibody (used in excess) was a rabbit anti-allotype antibody. (Supplied by Dr. R. Mage.) Relative concentration of 1 for lgM, 10 ng; IgG direct, 250 ~g; IgG indirect, 2.5 /zg.
fully carrying out this test. An example of a protocol for a C1FT test is shown in Table II. The Indirect CIFT Test. This test is performed exactly as the direct C1FT except before adding C1, an anti-Ig of the appropriate specificity is added. Caution: Anti-Ig must be precentrifuged before use and may have to be absorbed with Ag. This test can be used (1) to quantitate the Ig to the Ag. For this purpose the anti-Ig must be used in excess, i.e., an increase in the amount of anti-Ig will not increase fixation of C1; (2) to quantitate anti-Ig. The dose-response curve of CI fixation is then the function of the anti-Ig concentration. Caution: Carry controls for antigen and anti-Ig; anti-Ig may have to be absorbed with Ag to remove CF natural Ab to the
176
IMMUNOASSAY METHODS
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Ag. Examples of dose-response curves for the direct and indirect C1FT are shown in Fig. 1. CIF Test. Various amounts of C-fixing complexes in 0.1 ml are incubated with 0.1 ml of limiting amounts of C1 at 30° in VBS-sucrose buffer (/.t = 0.065). Ideally about (1.5-3.0) × 108 C1 molecules per milliliter should be used; however, the actual amount will be dictated by the C1 binding activity shown by the controls (e.g., Ab solo, Ag solo). After incubation of the mixture for 20--30 min, 0.1 ml of EAC4 (1.5 x 108/ml) in VBS-sucrose buffer (/z = 0.065) are added and the mixture is incubated for 20 min at 30°. C2 and C-EDTA are then added as in the C-1 titration. 4 It should be evident that this test is essentially a C-1 titration where C1 is "deviated" by the Ag-Ab complex before EAC4 is added. An example of a protocol for C-iF test with aggregated IgG is shown in Table III. Discussion The development of the direct 2 and indirect CIFT 5 and the C1F 6 tests permitted us to obtain quantitative C fixation data on a molecular basis. The tests have been successfully applied to the quantitation of soluble complexes and of cell surface Igs of any c l a s s . 2"3'5-14 The test has many obvious advantages, among which are sensitivity, precision, flexibility, adaptability, and rapid performance. Under ideal conditions, nanogram quantities of Ag or Ab can be detected on a molecular basis. Among the drawbacks are a need for high-quality reagents and oversensitivity to interfering substances such as enzymes, endotoxins, and chelating agents. While the test was never intended to replace conventional CF tests, it has proved itself a valuable research tool capable of yielding information on the molecular nature of C fixation and on the nature of Ag-Ab interaction.
4 T. Borsos and H. Ji Rapp, J. lmmunol. 91,851 (1963). 5 T. Borsos, H. R. Colten, J. S. Spalter, N. Rogentine, and H. J. Rapp,J. Immunol. 101,392
(1968). T. Ishizaka, K. Ishizaka, T. Borsos, and H. Rapp, J. lmmunol. 97, 716 (1966). 7 T. Ishizaka, K. Ishizaka, S. Salmon, and H. Fudenberg, J. lmmunol. 99, 82 (1967). B. J. Mills and N. R. Cooper, J. lmmunol. 121, 1549 (1978). 9 W. Augener, H. M. Grey, N. R. Cooper, and H. J. Miiller-Eberhard, Immunochemistry 8, 1011 (1971). io G. Fiist, G. A. Medgyesi, E. RajnaviSlgyi, M. Csecsi-Nagy, K. Czikora, and J. Gergely, Immunology 35, 873 (1978). 11 T. Ishizaka, T. Tada, and K. Ishizaka, J. Immunol. 100, 1145 (1968). ~2 W. D. Linscott, J. Immunol. 11)2, 1322 (1969). 13 W. D. Linscott, J. lmmunol. 104, 1307 (1970). J4 j. p. Atkinson and M. M. Frank, J. Clin. Invest. 54, 339 (1974).
QUANTIFICATION
OF C O M P L E M E N T
FIXATION
165
the steps before adding complement are done in EDTA buffer to prevent the interaction of serum C with the labeled cells. In that case the cellsantibody-antigen mixtures should be washed 1 × with VBS before the addition of C. In experiments where the antibody to the label is of the non-C-fixing type, the cells-antibody-antigen mixtures are washed 1 x with VBS and are than exposed to an anti-antibody that is C fixing (e.g., rabbit anti-goat) for 1 hr at 37° before adding C. The amount of the second antibody must be excess, i.e., addition of more second antibody does not increase lysis. Because some antisera and guinea pig C contain natural antibody to the sheep cells and possibly to the label, it is necessary to absorb the antisera with sheep cells and the C with labeled sheep cells. In some instances one or more of the reagents may contain colored substances that may interfere with the spectrophotometric determination of hemoglobin. In such instances washing 1 x before addition of C will eliminate this problem. There are several advantages of the PHI test. The most outstanding of them is that its performance requires equipment that is found in most laboratories, i.e., a centrifuge and a spectrophotometer. Sheep cells and guinea pig serum are easily obtained in most countries and if not available commercially can be procured from domestic sources. The accuracy and precision of the PHI test approaches that of RIA without the use of radioactive materials while its sensitivity compares favorably with RIA.
[10] Q u a n t i f i c a t i o n o f C o m p l e m e n t F i x a t i o n b y t h e C1 Fixation and C 1 Fixation and Transfer Tests
By TmOR BORSOS The complement fixation (CF) tests is a widely used diagnostic and investigative tool for the detection of antigen-antibody complexes. The principle of the CF test is based on the ability of certain classes of immunoglobulins (Igs) when in aggregated form or as antibodies (Abs) in combination with antigens (Ags) to interact with and bind ("fix") serum complement (C). The fixation of C by Ag-Ab complexes results in reduction of the concentration of free C; the residual C activity is measured by its hemolytic activity, i.e., the ability of C to lyse red cells sensitized with Ab. The CF test can be a sensitive (nanogram amounts of Ab or Ag) and precise test; however, the results cannot usually be interpreted on a mo-
METHODS IN ENZYMOLOGY, VOL. 74
Copyright ~) 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181974-4
166
IMMUNOASSAYMETHODS
[10]
lecular basis, i.e., there is no direct relationship between the number of CF complexes and the quantity of C fixed by them. The development of a sound theory for the hemolytic activity of C and its individual components, coupled with great advances in devising molecular measurements for their hemolytic activity permitted the development of CF tests that are based on the measurement of the interaction with Ag-Ab complexes with a single component of C. The advantages of CF tests based on the fixation of a single component of C as opposed to a CF test based on fixation of " w h o l e " C activity has been discussed by Rapp. 1 One of the widely used tests of this nature is based on the fixation of the first component of C (C1) by Ag-Ab complexes. 2 There are two types of CF tests using C 1 as the indicator molecule: in the C1 fixation and transfer test, a particulate Ag-Ab complex interacted with purified C1 (the active form of C1) is washed, and the bound C1 is then transferred to indicator cells, which, under appropriate conditions, will lyse. From the degree of lysis, the number of C1 molecules present in the original mixture can be calculated. In the second type of C 1F test, the Ag-Ab complex is first exposed to carefully determined, limiting amounts of C 1 followed by measurement of hemolytic activity of the residual, unbound C1 in the mixture. Because the hemolytic test for C1 activity enumerates hemolytically active C1 molecules on a molecular basis, both of these CF tests yield estimates of minimal numbers of CF complexes on a molecular basis. The C1FT test is performed in three stages and can be summarized as follows: 1. Fixation stage: AgAb + excess C1 wash AbAgC'I + C1 2. Transfer stage: AgAbC1 + EAC4 ~ EAC14 + AgAb 3. Lysis stage: EAC14 + C2 + C3-9--~ lysis The first stage is performed at low ionic strength (/x = 0.065) to favor binding of C1 to Ag-Ab complexes, whereas the second stage is performed at an ionic strength (tz = 0.15) that permits transfer of C1 to the indicator cells. In case the Ig in the Ag-Ab complex is non-C-fixing, the Ag-Ab complex is exposed to an anti-Ig antibody where the anti-Ig Ab is of the CF class. After washing, excess C1 is added and the test is performed as indicated in the sequence. The CIFT test is designed to measure particulate Ag-Ab complexes. A C1 fixation test was therefore designed to permit the measurement of Cl H. J. Rapp, in "Immunological Methods" (J. F. Ackroyd, ed.), p. 1. Blackwell, Oxford, 1964. 2 T. Borsos and H. J. Rapp, J. Immunol. 95, 559 (1965).
[10]
QUANTIFICATION OF COMPLEMENT FIXATION
167
fixation by any CF complexes. The sequence of reaction of the C1F test can be summarized as follows: 1. Fixation stage: AgAb + C1 (measured quantity)---, AgAb-C1 ÷ reduced free C1 2. Lysis stage: (AgAbC1) + free C1 + EAC4 + C2 + C3-9 ~ lysis Both stages are performed a t / z = 0.065 or below. The test is essentially a competitive inhibition test; 'because it is performed at ionic strength below 0.15, there is little or no transfer of AgAb bound C1 to EAC4. Materials The following reagents are needed to perform the various tests based on the fixation of C1. These descriptions are adapted from Rapp and Borsos :3 with permission of the publisher. Veronal-Buffered Saline (VBS). For stock solution: dissolve 83.0 g of NaCI and 10.19 g of sodium 5,5-diethylbarbiturate in about 1.5 liters of distilled or deionized water (flee of endotoxin and pyrogens). Adjust pH of solution to 7.35 with 2 N HCI and add water to exactly 2.00 liters. One liter isotonic buffer is prepared by mixing 200 ml of stock solution with 1.0 ml of a solution containing 1.00 M MgC12 and 0.150 M C aC12 and by adding water to exactly 1.00 liter. Gelatin is added to achieve a final concentration of 1 g/liter. The ionic strength of this buffer is 0.150. Verona#Buffered (VB)Sucrose. For stock solution, dissolve 486.1 g of reagent grade sucrose and 5.095 g of sodium 5,5-diethylbarbiturate in about 500 ml of distilled or deionized water. Adjust the pH of the solution to 7.35 with 2 N HCI and add water to exactly 1.00 liter. Isotonic veronalbuffered sucrose is prepared by mixing 200 ml of stock solution with 1.0 ml of the MgCI2-CaC12 solution and by adding water to exactly 1.00 liter. Gelatin is added to achieve a final concentration of 1 g/liter. The ionic strength of this buffer is 0.009. To obtain a VBS-sucrose buffer of ionic strength of 0.065, mix 2 parts of VBS with 3 parts of VB sucrose. EDTA Solution. For stock EDTA (0.1 M), dissolve 37.2 g of disodium ethylenediaminetetraacetic acid (Na2H~EDTA) in about 800 ml of distilled or deionized water. Adjust the pH to 7.65 with freshly prepared 2 N NaOH. (Caution: addition of NaOH must be slow and with vigorous stirring.) Bring the volume to 1.00 liter with water. The stock solution (stored at 4°) must not be used more than 3 weeks after it was prepared. a H. J. Rapp and T. Borsos, "'MolecularBasis of Complement Action," Chapter 7. Appleton, New York, 1970.
168
IMMUNOASSAYMETHODS
[10]
Isotonic VBS-EDTA buffer (0.01 M EDTA) is prepared by mixing 9 parts of isotonic VBS (containing gelatin but not added MgCl~ and CaCI~) with 1 part of the 0.1 M EDTA solution. Complement Components and Cellular Intermediates
First Component of C (C1). The preferred method for obtaining guinea pig C1 in partially purified form is described herein; in case it is not feasible to prepare one's own C1, quite satisfactory preparations of C1 may be purchased from Cordis Laboratories, Miami, Florida. Twenty milliliters of cold fresh (frozen or fresh frozen) guinea pig serum are adjusted to pH 5.6 by carefully adding 0.15 M HCI delivered under the meniscus with constant stirring. The mixture is dialyzed for 2 hr with constant mixing against about 2 liters of ice-cold water. The dialyzed serum is centrifuged at 900g. The precipitate is washed 3 x (at 900g) with ice-cold 0.015 M NaCl; after the last centrifugation, the precipitate is resuspended in 2.0 ml of VBS-sucrose buffer (/.t = 0.065) (without gelatin) and extracted at room temperature for about 15 min. The tube is centrifuged at 900 g for 20-30 min and the (sometimes opalescent) supernatant is applied in two equal parts to the top of a sucrose gradient (8-30% sucrose in VBS, # -- 0.065). The tubes are centrifuged at about 100,000g for 16-18 hr at 10-15 °. All C1 activity is found near the bottom of the tube. The C1 prepared by this method is stable for many months at - 20° to - 40° and is free of interfering substances. Second Component of C (C2). Suitable guinea pig C2 can be purchased from Cordis Laboratories, Miami, Florida. Alternatively, C2 that is of sufficient activity, purity, and stability can be prepared by DEAE- and CM-cellulose chromatography. For the processing of 20 ml of guinea pig serum, 27 g of DEAE-(Brown Co.) and 20 g of CM-cellulose are used. It is important that both the DEAE- and CM-cellulose are washed according to these instructions; other methods of washing have been known to affect the binding properties of these celluloses in such a way that C2 activity could not be recovered during the separation procedures. The celluloses are washed first with 0.5 M NaCl (the use of several liters is recommended), followed by resuspension of the DEAE in about 1 liter of 0.015 M NaCI and the CM in about 1 liter of 0.03 M NaC1. The slurry of DEAE is stirred vigorously and the pH is adjusted to 7.4 with 1-2 N HCI. Equilibration may take 15-20 min. The slurry of CM is vigorously stirred and its pH is adjusted to 4.7 with freshly made 1-2 N NaOH. It is important that the pH not be below 4.7 once equilibration occurs (C2 is inactivated rapidly at pH 4.5 or below). Three glass columns of about 60-cm length are set up (ID of Nos. 1 and 2 about 5 cm and No. 3 about 3 cm).
[10]
QUANTIFICATION OF COMPLEMENT FIXATION
169
The bottom of the column may not be of sintered glass. The columns should be constructed so that air pressure can be applied at the top so that liquids can be passed through rapidly due to the increased pressure. Column No. 1 receives an amount of DEAE slurry to contain 20 g of DEAE; No. 3 the remainder (about 7 g); column No. 2 receives all the CMcellulose. Once the celluloses have settled (take care that the cellulose column never runs dry 9, a thin layer of glass wool is laid on top of each (approximately ½inch); to hold the glass wool in place a 1-inch layer of glass beads are added. Column No. 1 is then washed with about 500 ml of 0.03 M NaC1 and No. 3 with 200-300 ml of 0.015 M NaCI (there should be several liters of each of these solutions prepared). The columns are ready for use when the liquid levels entered the glass wool layer. Twenty milliliters of guinea pig serum are treated as indicated for the preparation of C1. The supernatant fluid of the precipitated serum is removed and its pH is adjusted to 7.4 with 0.15 M NaOH and its ionic strength to 0.08 with 3.0 M NaCI. The adjusted supernatant is poured into column No. 1 and permitted to enter the cellulose; add about 600 ml of 0.08 M NaCI. After discarding the first 100 ml of effluent, collect the next 300 ml and mix with 500 ml of ice-cold water; apply to and pass through column No. 2. Collect 1 liter of effluent in a beaker containing 1 liter of ice-cold water. The 2 liters of effluent are well mixed and passed through column No. 3. After all of the 2 liters of effluent has passed through (and been discarded), wash the column with 200-300 ml of 0.015 M NaCI, then adjust the fluid level to the top of the cellulose column. Add 200 ml of 0.25 M NaC1 and start the pass through. Discard the first 30 ml and collect the next 100 ml. This should contain C2. C-EDTA. Usually it is prepared by diluting fresh (frozen) guinea pig serum 1/40 in 0.01 M EDTA buffer. This reagent can be kept (cold) for several weeks. Sheep Cells (E). Sheep cells can be obtained from several commercial sources. Cells should not be used until 1 week after collection. Ten to twenty milliliters of the blood (containing an anticoagulant) are mixed with 0.01 M EDTA buffer and are incubated for a few minutes at 37°. The cells are washed 2x with 0.01 M EDTA buffer and 3× with VBS (+ gel + Ca'-'+Mg'+). One billion cells when lysed in 25 ml of water have an OD of 0.420 at 541 nm (1 cm light path). Anti-Forssman Antibody (A). Antibody may be procured commercially. It should be obtained from rabbits immunized with boiled stromata of sheep red cells. Sera should be isolated with high IgM antibody concentration. Optimal antibody levels are determined in the following manner (buffer, VBS): In a series of test tubes place 0.1 ml o r E (1.0 x 109/ml) and 0.1
170
IMMUNOASSAY METHODS
[10]
ml of dilutions of anti-Forssman antiserum; after incubation for 10 min at 37°, add 1.0 ml of a 1/300 dilution of fresh guinea pig serum as a source of C. (The appropriate dilution of C may have to be determined by trial and error.) After 1 hr of incubation the cells are removed by centrifugation and the amount of lysis determined by measuring hemoglobin at a wavelength of 541 nm. A plot of percentage lysis versus relative concentration of antiserum should yield a curve that is concave to the abscissa. Where percentage lysis becomes independent of concentration at high levels of A (cf. Ref. 3), i.e., a plateau is reached say at 1/100 dilution of A, then a 1/50 dilution should be appropriate for preparing EAC4. EAC4. Mix 5 ml of E (in VBS-sucrose buffer, /~ = 0.065, 1.0 × 109 E/ml) with the appropriate dilution of A. Incubate for 10 min. Wash 1×. Add 1 ml of C1 (about 200 effective molecules of C1 per cell; see following section for titration of C1), cool to 0°. Add by quick mixing, 50 ml of ice-cold guinea pig (or human) serum diluted 1/10 in 0.01 M EDTA buffer. Mix rapidly and well. Incubate for 20-30 min. Wash by centrifugation 2× with 0.01M EDTA and 3× with VBS. Dilute to contain 1.5 × 108 cell/ml. Store at 4° . This reagent should be stable and useful for 1-2 weeks. Titration ofC-[. 4 Place in a series of test tubes (13 × 100 mm is ideal) 0.1 ml of EAC4 (1.5 × 108 E/ml) (buffer throughout this part of the test is VBS-sucrose, /z -- 0.065). Mix with the cells 0.1 ml of a dilution of C1. Incubate 15 min at 30°. Add 0.1 ml of C2 (at least 50 effective molecules per cell). Incubate 10 min at 30°. Add 1.0 ml of C-EDTA and incubate 1 hr at 37°. Centrifuge and measure amount of hemoglobin in supernatant at a wavelength of 412 nm. Calculate fraction of cells lysed (y). Plot on log-log paper the - I n ( 1 - y ) against relative concentration of C1. At -ln(1 - y) = 1, the cells carry an average number of one CI molecule per cell, i.e., the dilution of C-1 yielding - l n ( l - y) -- 1 contains 1.5 × 10~ effective C1 molecules per milliliter. Table I shows the protocol for titrating CI. Test for A g - A b Based on the Fixation and on the Transfer of CiIn the introduction I have described the principles of the various C-fixing tests utilizing the fixation and measurement of C1 on a molecular basis. I shall now describe in detail the various forms of the tests and show an example for each. Direct C I F T Test. In the fixation stage (VBS-sucrose buffer; 9 /z = 0.065) mix portions of the particulate Ag with various dilutions of Ab. Wash in VBS-sucrose buffer,/z = 0.065, 2 ×. Add 0.1 ml of C1 to contain about 6 × 101° C1 molecules per milliliter. Controls should include Ag solo, Ab solo, tube solo. Incubate 20 min at 30°. Wash by centrifugation
[lO]
171
QUANTIFICATION OF COMPLEMENT FIXATION TABLE I PROTOCOL FOR TITRATION OF C [ Tube n u m b e r
Reagent E A C 4 (1.5 x 10 s E/ml) C 1 dilution (0.1 ml) VBS-sucrose ~/x = 0.065)
Unit
ml
1
2
3
4
5
0.1
0.1
0.1
0.1
0.1
1000
2000
4000
8000
1000 0.1
ml
6
7
8
9
0.1
0.1
0.1
0.1
0.2
0.1
0.2
0.1
0.1
Incubate 15 rain, 30 ° C2"
ml
0.1
0.1
0.1
0.1
C-EDTA E D T A buffer H20
ml b ml ml
1.0
1.0
1.0
1.0
0.1
Incubate 15 min, 30 ° 1.0
1.0 1.0
1.0 1.0
Incubate 60 rain, 37°; centrifuge OD4~2 of supernatant OD412 of control y" ~' z x dilution C 1/EAC4/ml
1.02
0.756
0.485
0.315
0.950 0.864 1.99 1990
0.686 0.624 0.977 1954 1965 e
0.415 0.377 0.474 1896
0.245 0.223 0.252 2020
0.010
0.070
0.010
1.11
0.60
1.10
" At least 50 C2 molecules per EAC4. b Ice-cold. " Fraction of cells lysed. ' N u m b e r o f effective C / molecules per cell. " To calculate the absolute n u m b e r of C 1 molecules, multiply by the n u m b e r of cells/ml: 1.97 x Iff~ C1/cell/ml x 1.5 x 10s EAC4/ml = 3.0 x 10~ C[/ml,
1 x ; transfer to a fresh set of tubes and wash 4 × . R e s u s p e n d in VBS (0.15) and m a k e dilutions. Incubate 0.1 ml of each dilution with 0.1 ml o f E A C 4 (1.5 x 10 ~ in VBS,/~ -- 0.15). Incubate for 20 min at 30 °. Add 0.1 ml of C2 in VB sucrose /x = 0.009) containing about 50 C2 molecules per cell. I n c u b a t e 15 min at 30 °. Add 1.0 ml of ice-cold C-EDTA. Incubate 1 hr at 37 °. Centrifuge and determine fraction of cells lysed and the n u m b e r of C1 molecules as shown for C1 titration. Because the dilution of the original A g - A b c o m p l e x to yield - l n ( 1 - y) = 1 is now known, we can calculate the n u m b e r of C1 fixing sites in the original A g - A b mixture. The data should be corrected for the appropriate blanks. Caution: Precentrifugation of C1 and antiserum (Ab) at several thousand g is essential for success-
172
IMMUNOASSAY
METHODS
[10]
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.= "7.
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O f.. O × oo
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¢.q O0
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[10]
QUANTIFICATION
OF
COMPLEMENT
FIXATION
t~ O~
O
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e4 "O
g~
t.~
X
X
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,6 L) X
t.
g. X O
i.
b
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ZL~ --~.< L~
L~
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173
174
IMMUNOASSAY
METHODS
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O~
o.
( ~ o
t"-I
o
0 0
0
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Z
o"~
4~
o
~'~ ~
t~
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[10]
175
Q U A N T I F I C A T I O N OF C O M P L E M E N T F I X A T I O N
IgG, direct Ci FT IgM
(U
*11
v,-
IgG, indirect C'IFT qU
10
"3 IU O
E
5 d
Z
1
2
4
8
16
32
64
Relative antibody concentration Fi6. 1. Dose-response curves of rabbit IgM and IgG anti-Forssman antibodies as determined by the C 1FT test. In the indirect test the second antibody (used in excess) was a rabbit anti-allotype antibody. (Supplied by Dr. R. Mage.) Relative concentration of 1 for lgM, 10 ng; IgG direct, 250 ~g; IgG indirect, 2.5 /zg.
fully carrying out this test. An example of a protocol for a C1FT test is shown in Table II. The Indirect CIFT Test. This test is performed exactly as the direct C1FT except before adding C1, an anti-Ig of the appropriate specificity is added. Caution: Anti-Ig must be precentrifuged before use and may have to be absorbed with Ag. This test can be used (1) to quantitate the Ig to the Ag. For this purpose the anti-Ig must be used in excess, i.e., an increase in the amount of anti-Ig will not increase fixation of C1; (2) to quantitate anti-Ig. The dose-response curve of CI fixation is then the function of the anti-Ig concentration. Caution: Carry controls for antigen and anti-Ig; anti-Ig may have to be absorbed with Ag to remove CF natural Ab to the
176
IMMUNOASSAY METHODS
[10]
Ag. Examples of dose-response curves for the direct and indirect C1FT are shown in Fig. 1. CIF Test. Various amounts of C-fixing complexes in 0.1 ml are incubated with 0.1 ml of limiting amounts of C1 at 30° in VBS-sucrose buffer (/.t = 0.065). Ideally about (1.5-3.0) × 108 C1 molecules per milliliter should be used; however, the actual amount will be dictated by the C1 binding activity shown by the controls (e.g., Ab solo, Ag solo). After incubation of the mixture for 20--30 min, 0.1 ml of EAC4 (1.5 x 108/ml) in VBS-sucrose buffer (/z = 0.065) are added and the mixture is incubated for 20 min at 30°. C2 and C-EDTA are then added as in the C-1 titration. 4 It should be evident that this test is essentially a C-1 titration where C1 is "deviated" by the Ag-Ab complex before EAC4 is added. An example of a protocol for C-iF test with aggregated IgG is shown in Table III. Discussion The development of the direct 2 and indirect CIFT 5 and the C1F 6 tests permitted us to obtain quantitative C fixation data on a molecular basis. The tests have been successfully applied to the quantitation of soluble complexes and of cell surface Igs of any c l a s s . 2"3'5-14 The test has many obvious advantages, among which are sensitivity, precision, flexibility, adaptability, and rapid performance. Under ideal conditions, nanogram quantities of Ag or Ab can be detected on a molecular basis. Among the drawbacks are a need for high-quality reagents and oversensitivity to interfering substances such as enzymes, endotoxins, and chelating agents. While the test was never intended to replace conventional CF tests, it has proved itself a valuable research tool capable of yielding information on the molecular nature of C fixation and on the nature of Ag-Ab interaction.
4 T. Borsos and H. Ji Rapp, J. lmmunol. 91,851 (1963). 5 T. Borsos, H. R. Colten, J. S. Spalter, N. Rogentine, and H. J. Rapp,J. Immunol. 101,392
(1968). T. Ishizaka, K. Ishizaka, T. Borsos, and H. Rapp, J. lmmunol. 97, 716 (1966). 7 T. Ishizaka, K. Ishizaka, S. Salmon, and H. Fudenberg, J. lmmunol. 99, 82 (1967). B. J. Mills and N. R. Cooper, J. lmmunol. 121, 1549 (1978). 9 W. Augener, H. M. Grey, N. R. Cooper, and H. J. Miiller-Eberhard, Immunochemistry 8, 1011 (1971). io G. Fiist, G. A. Medgyesi, E. RajnaviSlgyi, M. Csecsi-Nagy, K. Czikora, and J. Gergely, Immunology 35, 873 (1978). 11 T. Ishizaka, T. Tada, and K. Ishizaka, J. Immunol. 100, 1145 (1968). ~2 W. D. Linscott, J. Immunol. 11)2, 1322 (1969). 13 W. D. Linscott, J. lmmunol. 104, 1307 (1970). J4 j. p. Atkinson and M. M. Frank, J. Clin. Invest. 54, 339 (1974).