Vox Sang. 33: 335-345 (1977)
Development of Hemagglutination Assays 11. Enhancement in the Sensitivity of an RPHA Test for HBsAg
Bernard Horowitz, Martin Stryker, Johannes Vandersande, Arnold Lippin and Kenneth R . Woods New York Blood Center and Department of Biochemistry, Cornell University Medical College, New York. N.Y.
Abstract. Studies were undertaken to enhance the sensitivity of a previously developed RPHA test for HBsAg. A net increase in sensitivity of approximately 3-fold was achieved by modifying the elution procedure used to purify chimpanzee anti-HBs by affinity chromatography. A further 3- or 4-fold sensitivity increase was achieved by increasing the volume of specimen tested. A concomitant increase in nonspecific agglutination usually observed with increased specimen size was avoided by incubating the reaction mixture at 37 or 45°C. Evaluation of the test in detection of HBsAg in blood obtained from volunteer donors indicates that the materials produced at the New York Blood Center using the modified test protocol compare favorably with a commercial RPHA test. Modifications which did not contribute toward enhancing sensitivity are also reported.
Introduction The close association between type B hepatitis and HBsAg' (Australia antigen) [20, 231 is generally accepted. Screening tests to detect HBsAg in donor blood have been developed to reduce the risk of posttransfusion hepatitis. RPHA and RIA techniques are the most sensitive now routinely employed for this purpose, being 10- to 100-fold more sensitive than CEP and 100to 1,000-fold more sensitive than AGD [I, 3,15,19,29,31]. The detection rate of HBsAg amongst blood donors in the United States has varied between 0.1 and 3%, depending on the sex, age, ethnic back-
ground, and residence of the donor population [7, 8, 14,281. It is impossible to accurately quantitate the effect on posttransfusion hepatitis of screening all donor blood for HBsAg because of the rapid trend to eliminate paid blood donors and the elimination of HBsAg-positive donors from the donor population. Early studies using AGD and CEP to screen donor blood indicated that HBsAg-positive blood accounted for about 25% of the cases of posttransfusion hepatitis [ l l , 121, estimated at 30,000150,000 cases per year in the USA [21]. HBsAg has since been demonstrated by RIA and RPHA in CEP-negative units of blood administered to patients who subse-
3 36
Horowitz/Stryker/Vandersande/Lippin/Woods
quently developed HBsAg-positive hepatitis [lo, 15, 18,301. Despite routine screening of all collected blood by RIA or RPHA, a substantial number of cases of posttransfusion hepatitis still occurs, though it now appears likely that only a small proportion of these cases are caused by the hepatitis B virus [2, 9, 11, 2-51. To further explore the involvement of hepatitis B virus in posttransfusion hepatitis and to reduce its occurrence, more sensitive assays for this virus are needed. The sensitive detection of antigen with RPHA techniques can be considered a priori to depend upon the (1) purity and avidity of the antibody used in coating the stabilized cells, (2) composition of the diluent in which the test is run, (3) surface properties of the stabilized erythrocytes, (4) surface properties of the hemagglutination plate, ( 5 ) volume of specimen tested, (6) temperature at which the test is run, and (7) contact time between antigen and antibody-coated RBCs. Exploration of these parameters has given rise to modifications of a previously reported RPHA test for HBsAg [26] resulting in increased sensitivity. Numerous approaches which did not enhance sensitivity are also presented with the hope that they will prove beneficial in the development of RPHA test procedures for the assay of other biologically important molecules as well as in hepatitis testing.
Materials Human type 0-negative erythrocytes, human sera from volunteer donors, and human serum albumin, y-globulin, and fibrinogen, were prepared at The New York Blood Center. Chimpanzee sera containing anti-HBs and HBsAg-Sepharose were generously provided by Dr. A . M . Prince.
Hemagglutination plates were obtained from Ames Co., Linbro Chemical Co., Inc., and Cooke Laboratory Products. Iz6I-NaI carrier-free, was purchased from New England Nuclear. Siliclad wcs obtained from Clay Adams, Polybrene from Aldrich Chemical Co., Inc., and Auscell from Abbott Laboratories. Neuraminidase, isolated from CI. perfringens, was obtained from Worthington Biochemical Corp.; and the beads used in the detection of anti-HBs in solution were 0.25 inch diameter, ball grade TI, polystyrene, natural, supplied by the Harvey Westbury Corp., Westbury, N.Y. All other reagents were of the highest listed purity available commercially in the USA.
Methods Procedures f o r RPHA The methods used throughout this paper essential to the RPHA technique were those previously reported [26] except where noted. RBCs were coated with anti-HBs as previously modified [16]. The concentration of EDTA in SD was increased to 1 0 m M when larger specimen volumes were used. Quanfitation of Anti-HBs Attached t o Red Blood Cells The quantity of active anti-HBs attached to the cell surface was determined as previously described [16]. Quantitation of Anti-HBs in Solution Preparation of beads. Affinity chromatographypurified anti-HBs (10 pg!ml) was attached to polystyrene beads as described by Cart irnd Trcyyur [5]. HBsAg was then combined with the bound anti-HBs by incubating the beads for 2 h at 45 ' C in a solution containing a level of HBsAg barely detectable by CEP. The beads were washed with distilled water and stored desiccated over CaCI,. Preparation of IW-anti-HBs. Irsl-anti-HBs was prepared by the method of Greenwood et ctl. (131. Assay f o r anti-HBs. TO 160 !(I of leaI-anti-HBs (0.28pCi, 5.5,uCilmol) in a solution of 50% fetal calf serum, 0.5% human serum albumin, 2% nor-
Improved RPHA Test for HBsAg
337
Table 1. Effect of changes in RPHA test conditions
Change
Effect
1 Increase purity of antibody attached to RBCs
Enhances sensitivity
2 Increase specimen size
Enhances sensitivity; must overcome resultant nonspecific agglutination
3 Alter composition of cell suspension diluent Vary gelatin concentration from 0.001 to 1% Replace gelatin with bovine serum albumin, PEG 4000, dextran T20 or dextran T80 Add bovine serum albumin, PEG4000, dextran, or dextran T80 to SD as previously constituted Vary ionic strength from 0.025 to 0.5
No change in sensitivity; size of positives are increased at lower concentrations of gelatin
Vary pH from 6.0 to 8.0 Vary EDTA concentration from 1 to 10 mM
4 Alter surface properties of RBCs a Treat with neuraminidase b Attach human serum albumin, IgG, or fibrinogen to antibody-coated RBCs c Subject antibody-coated RBCs to 1-10 cycles of freezing and thawing d Prepare RBCs from other donors of type 0-negative
5 Alter surface properties of hemagglutination plate a Treat plate with Polybrene or Siliclad b Use plates of other manufacture c Coat plate with anti-HBs
6 Alter physical conditions during the incubation period a Increase temperature to 37 or 45 "C.
b Continuously agitate cell-specimen mixture during a preincubation period
Causes spontaneous agglutination
No change in sensitivity
No change in sensitivity; minimum ionic strength needed to overcome spontaneous agglutination is 0.1 No change in sensitivity No change in sensitivity; higher concentrations of EDTA are needed with larger specimen volumes
Reduces attachment of antibody to RBCs Causes spontaneous agglutination No change in sensitivity
No change in sensitivity
No change in sensitivity No change in sensitivity No change in sensitivity
Little if any change in sensitivity; elevated temperature allows the reaction to proceed faster and reduces false-positive rate No change in sensitivity
Horowitz/StrykerlVandersandeilippin/Woods
338
Table 11. Purification of anti-HBs by affinity chromatography Step
Volume Protein ml concentration mg/ml
Ethanol-fractionated y-globulin Affinity chromatography Procedure 1 p H 10.85 Procedure 2 p H 9.6 pH 10.85
f
2 $:
6
12 7.2 4.5
20
Total protein mg
Quantity required for 50% inhibition 1, pg
Recovery, %
Fold purification
120
15.0
100
1 .o
22.7
0.25
3.0
0.66 2 0.25 2
57
0.25
1.8 0.95
1.23k0.582 0.32 k 0.09 3
19
12.2
37
46.9
0.21
Determined by competitive radioimmune assay as described in text. Average of four purifications. Average of seven purifications.
ma1 human serum, 5 m M Tris-HC1 (pH 7.2), and 0.1% sodium azide was added 40,uI of appropriately diluted test specimen containing anti-HBs followed by a bead coated with HBsAg. After incubation of the mixture for 1 h at 45 O C , the bead was washed 10 times with distilled water and the bound radioactivity determined with a gamma counter. A standard solution of anti-HBs was used to calibrate each run, and the amount of specimen needed to inhibit the binding of 1251-anti-HBs by 50% was determined. The curves in figure 1 illustrate the inhibition observed when varying concentrations of either alcohol-precipitated chimpanzee y-globulin (from animals immunized with HBsAg) or affinity chromatography-purified antiHBs were added to this system. The affinity chromatography-purified preparation used was 25 times more effective in inhibiting the binding of radioactive antibody than the ethanol-precipitated y-globulin solution.
Method of Protein Determination Protein concentration was determined spectrophotometrically assuming an extinction coefficient 1% of E 280nm= 13.5.
Results Table I lists the approaches which were explored in the attempt to improve the sensitivity of a previously reported RPHA test for HBsAg [26]. Two proved successful and are reported in detail below.
Modification in RPHA Test Purity of anti-HBs. Previously, the antibody used in coating cells was purified by 1 Abbreviations: HBsAg = hepatitis B surface antigen; anti-HBs = chimpanzee antibody directed against HBsAg; RPHA = reversed passive hemagglutination; RIA = radioimmunoassay; AGD = agar gel diffusion; CEP =counter electrophoresis; SD = cell suspension diluent, consisting of 0.1 M sodium phosphate buffer, pH 7.2, 0.1 % gelatin, 1 % normal human type AB serum, 1 mM EDTA, and 0.02% sodium azide; EDTA = ethylenediamine tetraacetic acid; RBCs = human erythrocytes stabilized by treatment with pyruvic aldehyde and formaldehyde.
Improved RPHA Test for HBsAg
3 39
15 C
c n i 50 E
c
a c
Y
8 25
Protein, pg
Fig. 1. Radioimmune assay for anti-HBs. Alcohol fractionated ( 0 )or affinity chromatography purified (0) anti-HBs was added in the amounts indicated along with 125I-anti-HBs to HBsAgcoated beads as described under Methods. The amount of radioactivity bound was determined.
application of a solution of alcohol-precipitated hyperimmune y-globulin to a column of insolubilized HBsAg at neutral pH and, after a neutral wash, elution at pH 10.85 [26]. This process produced a 20- to 25-fold increase in the purity of anti-HBs (table IJ). It was found that inclusion of an intermediate wash with pH 9.6 buffer resulted in a 2-fold increase in purity of anti-HBs eluting at pH 10.85 (table 11). RBCs coated with this more highly purified antibody bound, on the average, 1.7-fold more anti-HBs and proved 2.9-fold more sensitive in detecting antigen (table 111). The optimum concentration of protein for coating cells was determined for each lot of antibody. Anti-HBs
Table 111. Performance of cells coated with anti-HBs prepared by two different methods ~~
Purification procedure
~~
~
Antibody lot No.
~
Uptake of 125I-HBsAg RPHA titer-' CPm
x
Spontaneous agglutination
1 Eluted directly with
with pH 10.85 buffer
29-32 33 38
39 40 42 t 44
3,125 2,493 3,414 3,491 3,378 3,559
X = 3,243 f396
1.25 1.25-5.00 2.50 0.62 2.50 2.50
none none none none none slight
Tt=2.10+0.90
2 Eluted with p H 10.85 buffer after pH 9.6
wash.
45 41 52 53 54 55 56
7,450 4,742 4,793 5,218 4,955 5,594 5,302
X = 5,436 f937
5.00-10.0 10.0 2.50 1.25 1.25-10.0 5.00 1.00
x = 6.00 f3.00
none none none none none none none
Horowitz/Stry ker/Vandersande/Li ppin/Woods
340
~-~
~
Table 1V. Effect of temperature on use of increased specimen volumes ~
Volume cell suspension, pl
Number of specimens tested
Temperature
A Original protocol (cells settle at 1 g ) 50 60 ambient 50 60 ambient 100 118 ambient 100 118 ambient 100 118 ambient 100 118 ambient
Number showing nonspecific agglutination
2 5 2 5 7.5 10
0 6 0 8 11 14
0.0 6.8 9.3 11.9
28 1 4 5 2
13.9 1.7 13.3 17.2 I .o
B Modified protocol (cells settle with centrifugation) too 202 ambient 10 100 100 100 100
59 30 29 193
31 O C 37 oc 31 O C 45 o c
eluted at p H 9.6 was least effective in the preparation of cells for use in detecting HBsAg (data not shown). Other procedures for increasing purity were also tried in an attempt either to enhance the sensitivity of the cells or to overcome the spontaneous agglutination which occurs when excess antibody protein binds to RBCs. As none resulted in higher purity antibody nor did they improve the present test, they will only be listed here: repurification of affinity chromatography purified antiHBs on a second, similar affinity column; QAE-Sephadex chromatography of either alcohol precipitated y-globulin or affinity chromatography-purified anti-HBs; and Sepharose 4-B chromatography of affinity chromatography-purified anti-HBs. In addition, it was suggested that the spontaneous agglutination which occurs when too much antibody is attached to cells might result from aggregates of IgG present in the solu-
~
Volume of specimen tested, pl
10 15 20 10
Falsepositive rate, 95
0.0 10.0
tion of anti-HBs. However, when the solution of anti-HBs was analyzed in the analytical ultracentrifuge, aggregates were not observed. Volume of specimen tested. Only a limited volume of specimen can be used in an RPHA procedure without causing nonspecific agglutination. The previously described protocol [26] used 1 0 p l of specimen first diluted 6-fold (or 1.67 pl). The results in table IVA indicate that 2 p l of specimen could be used without encountering an undue false-positive rate. 5 pl of specimen substantially increased the false-positive rate even when the volume of cell suspension was increased from 50 to 100p1. The specimen concentration with the original protocol was therefore limited to 3.8-4.8%. It is interesting to note that the same nonspecific agglutination occurred when increased specimen volumes were added to uncoated RBCs. The specimen concentration could be in-
Improved RPHA Test for HBsAg
341
Table V. Enhanced sensitivity rcsulting from larger specimen volume
Specimen
Subtype
Highest dilution positive previous protocol *
modified protocol ~
ad ad ad (purified) aY ay (purified)
16 2 16,000 16 1,600
Sensitivity increase (fold)
~
~~
64 4 128,000 32 3,200 Average 3.6
1
10 ,MI of specimen first diluted 1:5 was added to 50 ,dcell suspension as originally described [26]. 10 ul of specimen was added directly to 100 p l cell suspension as in figure 2.
Table VI. Performance trials of modified RPHA test for HBsAg
Procedure
Number tested
Modified protocol Auscell
5,173
Positive on initial screen
Positive on repeat screen
Confirmed positive
74 (1.4%) 75 (1.4%)
47 (0.91 %) 47 (0.91%)
16 (0.31%) 16 (0.31%)
creased without an undue increase in the false-positive rate if the RPHA reaction mixture was heated at 37 "C for 2 h or 45 "C for 1 h (table IVB). A maximum specimen volume of between 10 and 15 ,~1(9.1-13.0%) proved satisfactory. In order to avoid convection problems when working at these elevated temperatures a previously described centrifugation procedure [24,29, 32) was used. This procedure consisted of centrifugation of the entire plate for 2min at 1,000 rpm using IEC rotor 259 and the plate holders supplied by Ames, followed by tilting the plate at an angle of 60" from the horizontal. Unagglutinated cells streamed downward forming a short solid line with even
edges. Agglutinated cells remained as a tight button, sometimes remaining in the center of the well and other times falling intact to the edge of the well (fig. 2). A weak positive reaction was characterized by the occurrence of distinct granularity within the solid pattern caused by the streaming cells. The rate of nonspecific agglutination with increased specimen size could also be reduced by heating the specimen alone at 56 O C for 30min followed by RPHA testing at ambient temperature. For example, none of the 28 samples causing agglutination when tested at ambient temperature (table IVB, line 1) caused agglutination if they were first heated at 56°C for 30 min.
342
Horowitz/Stry ker/Vandersande/Lippin/Woods
Fig.2. Modified RPHA assay for HBsAg. Sera (10 pl), positive or negative for HBsAg, were added to 100 ti1 of anti-HBs-coated RBCs at a
conccntration of 0.0575% in SD. After incubation at 45 "C for 1 h the samples were ccntrifuged and held at a 60" angle for 15 min.
Effect on Sensitivity of the New Test Protocol The new test procedure consists of addition of 10 ,ill of specimen to 100 pl of cells suspended at a concentration of 0.0375% (v/vj in SD, incubation at 45 "C for 1 h, and centrifugation fcr 2 min at 1,000 rpm followed by tilting the plate at an angle ot 60". Sensitivity was determined by testing 5 HBsAg-positive specimens which were serially diluted in tubes and randomized. Three of the specimens were of subtype ad and 2 of subtype ay. The cells used for both protocols were coated with anti-HBs which was eluted from the affinity column after washing with pH 9.6 buffer. These results, therefore, do not take into account the 2.9-fold
increase in sensitivity caused by using more highly purified anti-HBs. The results are shown in table V. The increase in sensitivity ranged from 2- to 8-fold, averaging 3.6fold. Performance Trials of Modified RPHA Il4aterials and Protocol The performance of the newly modified test under routine serum screening conditions is summarized in table VI. Specimens from 5,173 volunteer donors were tested in parallel with Auscell at the New York Blood Center. The results obtained with the two tests were virtually identical: 74-75 (1.4%) of the specimens were positive on initial screening, 47 (0.91%) of these were repeat-
Improved RPHA Test for HBsAg
ably positive on subsequent screening, and 16 (0.31%) of these were established as HBsAg-positive by both tests. The false-positive rate for both tests was 0.60%.
Discussion RPHA is currently used throughout the world in testing for hepatitis-associated antigens. The detection of other antigens such as rubella, herpes, carcinoembryonic antigen, u-fetoprotein, or certain drugs by RPHA may prove exceedingly valuable as our knowledge of this technique increases. Inherently, the technique is capable of detecting antigen at a concentration in the specimen of approximately 10-lG M. Current RPHA techniques are at least several orders of magnitude less sensitive than this, yet they have proven to be among the most sensitive analytical techniques available. The principal factors believed to influence sensitivity are the binding constant of antibody to antigen, antibody density on the cell surface, availability of bound antibody to participate in intercellular bridging, the number of cells which must aggregate before a positive agglutination reaction is observable, the specimen size, the length of time allowed for the agglutination reaction to occur, and the temperature at which the reaction takes place. Wider application of RPHA testing depends somewhat on the realization of its sensitivity potential. While a significant literature dealing with the forces which affect the agglutination of unfixed erythrocytes has developed [4, 6, 17, 22, 271, little attention has been given to evaluating factors affecting the sensitivity of stabilized cells. Manufacturer and user alike prefer the use of stabilized cells for
343
their convenience, reproducibility, and reliability over an extended time period. Application of the findings reported herc to the previously described test for HBsAg [26] resulted in a net increase in sensitivity of approximately 10-fold. Two independent improvements were responsible for this increase. First, the purity of the anti-HBs used in coating the cell was enhanced by a refinement in the method used to elute anti-HBs from a column of insolubilized HBsAg. A 2-fold increase in purity of the antibody resulted in a 3-fold increase in sensitivity. Seccond, sensitivity was increased through the use of a larger specimen volume. The previous protocol tested 1.67 pl of specimen at a final concentration of 2.8%. Higher specimen concentrations were not used to avoid spontaneous agglutination (a high false-positive rate). With the modified protocol in which the specimen was heated, either separately or after addition to the cells, 10 pl of specimen could be tested without encountering a high rate of spontaneous agglutination. A comparison of the two procedures showed that the modified protocol increased the sensitivity 3.6-fold, on the average. The explanation of why heating reduces nonspecific agglutination remains speculative. It cannot even be currently concluded that heating the specimen separately at 56 OC achieves its effect in the same manner as heating the specimen plus cells at 37 or 45 "C. However, it should be emphasized that the observed nonspecific agglutination results from the interaction between specimen and uncoated RBCs as well; that is, the observed nonspecific agglutination did not depend upon an interaction between the bound y-globulin and an element in serum. Nonspecific agglutination may result from
344
the aldehyde treatment itself since specimen concentrations as high as 85% are occasionally but routinely used with unfixed erythrocytes in clinical laboratories. Both of the above-mentioned improvements rely on increasing the likelihood of immune complex formation by increasing the concentration of the reactants (antibody and antigen). In contrast, methods which are believed to enhance sensitivity with fresh erythrocytes by modulating their zeta potential [ 2 2 ] do not appear to increase sensitivity of RPHA reactions using aldehyde-stabilized erythrocytes. Future increases in sensitivity would seem most likely to come about from (1) methods which allow the attachment of higher quantities of antibody without inducing spontaneous agglutination, (2) methods which allow the use of greater specimen concentration, (3) use of higher affinity antibodies, (4) alternate methods of agglutinate visualization, and ( 5 ) methods which optimize cellcell interaction by influencing the shape of the cells before stabilization. Each of these areas is worthy of study in order to realize the full potential of RPHA techniques. Acknowledgements We are grateful for the continued involvement of Dr. A . M . Prince and for the technical assistance provided by Mr. Jay Maracic.
References Aach, R. D.; Grisham, J . W., and Parker, C. W.: Detection of Australia antigen by radioimmunoassay. Proc. natn Acad. Sci. USA 68: 10561060 (1971). Alter, H. J.; Holland, P. V.; Purcell, R. H.; Lander, J . 3.; Feinstone, S. M., and Morrow, A. G.:
Horowitz/Stryker/Vandersande/Lippin/Woods
Posttransfusion hepatitis after exclusion of commercial and hepatitis-B antigen-positive donors. Ann. intern. Med. 77: 691-699 (1972). 3 Barker, L.F.; Geretz, R. J.; Hoofnagle, J . H., and Nortman, D. F.: Viral hepatitis B. Detcction and prophylaxis; in Greenwalt and Jamieson Transmissible disease and blood transfusion, pp. 81-111 (Grune & Stratton, New York 1975). 4 Brooks, D. E. and Seaman, G . V. F.: Electroviscous effect in dextran erythrocyte wspensions. Nature new Biol. 238: 251-253 (1972). 5 Catt, K. and Tregear, G. W.: Solid phase radioimmune assay in antibody-coated tubes. Science 158: 1570-1572 (1967). 6 Chien, S.: Symposium on ultrastructural and electrochemical aspects of red cell aggregation. 7th European Conference on Microcirculation. Biblthca anat, vol. 11, pp. 244-309 (Karger, Basel 1973). 7 Derrick, J. D.; Young, A. M., and Pert, J. H.: Large-scale blood-screening program for hepatitis B. N.Y. St. J. Med. 75: 1693-1698 (1975). 8 Dodd, R.Y.; Ni, L. Y.; Mallin, W.S., and Greenwalt, T. J.: Hepatitis B (surface) antigen testing by radioimmunoassay. Am. J . din. Path. 63: 847-853 (1975). 9 Feinstone, S. M.; Kapikian, A. Z.; Purcell, R. H.; Alter, H. J., and Holland, P. V.: Transfusion-associated hepatitis not due to viral hepatitis type A or B. New Engl. J. Med. 292: 767-770 (1975). 10 Giorgini, G. L., jr.; Hollinger, F. B.; Leduc, L.; Issarescu, S.; George, J.; Blackman, A,, and Thayer, W. R., jr.: Radioimmunoassay detection of hepatitis type B antigen. A prospective study in blood donors and recipients. J. Am. med. Ass. 222: 1514-1518 (1972). 11 Gocke, D. J.: A prospective study of posttransfusion hepatitis. The role of Australia antigen. J. Am. med. Ass. 219: 1165-1170 (1972). 12 Goldfield, M.: Some epidemiological studies of transfusion-associated hepatitis; in GreenWalt and Jamieson Transmissible disease and blood transfusion, pp. 141-151 (Grune & Stratton, New York 1975). 13 Greenwood, F. C.; Hunter, W. M., and Glover, J. S.: The preparation of 1slI-labelled human growth hormone of high specific radioactivity. Biochem. J . 89: 114-123 (1963).
Improved RPHA Test for HBsAg
14 Hollinger, F. B.; Aach, R. D.; Gitnick, G. L.; Roche, J. K., and Melnick, J. L.: Limitations of solid-phase radioimmunoassay for HBAg in reducing frequency of post-transfusion hepatitis, New Engl. J. Med. 289: 385-391 (1973). I5 Hopkins, R.; Robertson, M.; Ross, D.; Turnbull, W. M., and Das, P. C.: Detection of hepatitis B surface antigen among Scottish blood donors: Evaluation of sensitive tanned-cell haemagglutination-inhibition test. Br. med. J. iic 409-41 1 ( I 975). I6 Horowitz, B.: Development of hemagglutination assays. I. Attachment of anti-HBs antibody to stabilized erythrocytes. Vox Sang. 33: 324-334 (1977). 17 Jandl, J. H. and Simmons, R. L.: The agglutination and sensitization of red cells by metallic cations: interactions between multivalent metals and the red cell membrane. Br. J. Haemat. 3: 19-38 (1957). I8 Xoretz, R. L. and Gitnick, G. L.: Prevention of post-transfusion hepatitis. Role of sensitive hepatitis B antigen screening tests, source of blood and volume of transfusion. Am. J. Med. $9: 754-760 (1975). 19 Ling, C. M . and Overby, L. R.: Prevalence of hepatitis B virus antigen as revealed by direct radioimmune assay with IW-antibody. J. Immun. 109: 834-841 (1972). 20 London, W. T.; Sutnick, A. I., and Blumenberg, B. S.: Australia antigen and acute viral hepatitis. Ann. intern. Med. 70: 55-59 (1969). 21 NHLI Blood Resource Study: Supply and use of the nation’s blood resource. National Heart and Lung Institute, NIH, vol. 1, Appendix C, June 30 (1972). 22 Pollack, W.; Hager, H. J.; Reckel, R.; Toren, D. A., and Singher, H. 0.: A study of the forces involved in the second stage of hemagglutination. Transfusion 5: 158-183 (1965). 23 Prince, A. M.: An antigen detected in the blood during the incubation period of serum hepatitis. Proc. natn. Acad. Sci. USA 60: 814821 (1968). 24 Prince, A. M.; Brotman, B., and Ikram, H.: Hemagglutination assay: Subtyping by hemagglutination inhibition, an ultra-sensitive identity test for HB antigen; in Vyas, Perkins and
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25
26
27
28
29
30
31
32
Schmid Hepatitis and blood transfusion, pp. 147-154 (Grune & Stratton, New York 1972). Prince, A. M.; Brotman, B.; Grady, G. F.: Kuhns, W. J.; Hazzi, C.; Levine, R. W., and Millian, S. J.: Long-incubation post-transfusion hepatitis without serological evidence of exposure to hepatitis-B virus. Lancet ii: 241-246 (1974). Prince, A.M.; Ikram, H.; Chicot, D.; Wright, R.; Vnek, J.; Neurath, R.; Lippin, A., and Swiss, S.: A new reversed passive hemagglutination test for detection of HBsAg. Vox Sang. 29: 319-329 (1975). Schnebli, H. P. and Bachi, T.: Reaction of lectins with human erythrocytes. I. Factors governing the agglutination reaction. Expl Cell Res. 91: 175-183 (1975). Szmuness, W.; Hirsch, R. L.; Prince, A. M.; Levine, R. W.; Harley, E. J., and Ikram, H.: Hepatitis B surface antigen in blood donors: further observations. J. infect. Dis. 131: 111118 (1975). Vyas, G . N . and Shulman, N. R.: Hemagglutination assay for antigen and antibody associated with viral hepatitis. Science 170: 332-333 (197d). Wallace, J.; Ban, A., and Milne, G. R.: Which techniques should be used to screen blood donations for hepatitis B surface antigen. Br. med. J. ii: 412-414 (1975). Walsh, J. H.; Yalow, R., and Berson, S. A,: Detection of Australia antigen and antibody by means of radioimmunoassay techniques. J. infect. Dis. 121: 550-554 (1970). Wegmann, T . G . and Smithies, 0.:A simple hemagglutination system requiring small amounts of red cells and antibodies. Transfusion 6: 67-73 (1966).
Received: December 24, 1976 Accepted: February 5, 1977
Dr. Bernard Horowitz, Blood Derivatives Program, New York Blood Center, 310 East 67th Street. New York N Y 10021 (USA)
Development of Hemagglutination Assays 11. Enhancement in the Sensitivity of an RPHA Test for HBsAg
Bernard Horowitz, Martin Stryker, Johannes Vandersande, Arnold Lippin and Kenneth R . Woods New York Blood Center and Department of Biochemistry, Cornell University Medical College, New York. N.Y.
Abstract. Studies were undertaken to enhance the sensitivity of a previously developed RPHA test for HBsAg. A net increase in sensitivity of approximately 3-fold was achieved by modifying the elution procedure used to purify chimpanzee anti-HBs by affinity chromatography. A further 3- or 4-fold sensitivity increase was achieved by increasing the volume of specimen tested. A concomitant increase in nonspecific agglutination usually observed with increased specimen size was avoided by incubating the reaction mixture at 37 or 45°C. Evaluation of the test in detection of HBsAg in blood obtained from volunteer donors indicates that the materials produced at the New York Blood Center using the modified test protocol compare favorably with a commercial RPHA test. Modifications which did not contribute toward enhancing sensitivity are also reported.
Introduction The close association between type B hepatitis and HBsAg' (Australia antigen) [20, 231 is generally accepted. Screening tests to detect HBsAg in donor blood have been developed to reduce the risk of posttransfusion hepatitis. RPHA and RIA techniques are the most sensitive now routinely employed for this purpose, being 10- to 100-fold more sensitive than CEP and 100to 1,000-fold more sensitive than AGD [I, 3,15,19,29,31]. The detection rate of HBsAg amongst blood donors in the United States has varied between 0.1 and 3%, depending on the sex, age, ethnic back-
ground, and residence of the donor population [7, 8, 14,281. It is impossible to accurately quantitate the effect on posttransfusion hepatitis of screening all donor blood for HBsAg because of the rapid trend to eliminate paid blood donors and the elimination of HBsAg-positive donors from the donor population. Early studies using AGD and CEP to screen donor blood indicated that HBsAg-positive blood accounted for about 25% of the cases of posttransfusion hepatitis [ l l , 121, estimated at 30,000150,000 cases per year in the USA [21]. HBsAg has since been demonstrated by RIA and RPHA in CEP-negative units of blood administered to patients who subse-
3 36
Horowitz/Stryker/Vandersande/Lippin/Woods
quently developed HBsAg-positive hepatitis [lo, 15, 18,301. Despite routine screening of all collected blood by RIA or RPHA, a substantial number of cases of posttransfusion hepatitis still occurs, though it now appears likely that only a small proportion of these cases are caused by the hepatitis B virus [2, 9, 11, 2-51. To further explore the involvement of hepatitis B virus in posttransfusion hepatitis and to reduce its occurrence, more sensitive assays for this virus are needed. The sensitive detection of antigen with RPHA techniques can be considered a priori to depend upon the (1) purity and avidity of the antibody used in coating the stabilized cells, (2) composition of the diluent in which the test is run, (3) surface properties of the stabilized erythrocytes, (4) surface properties of the hemagglutination plate, ( 5 ) volume of specimen tested, (6) temperature at which the test is run, and (7) contact time between antigen and antibody-coated RBCs. Exploration of these parameters has given rise to modifications of a previously reported RPHA test for HBsAg [26] resulting in increased sensitivity. Numerous approaches which did not enhance sensitivity are also presented with the hope that they will prove beneficial in the development of RPHA test procedures for the assay of other biologically important molecules as well as in hepatitis testing.
Materials Human type 0-negative erythrocytes, human sera from volunteer donors, and human serum albumin, y-globulin, and fibrinogen, were prepared at The New York Blood Center. Chimpanzee sera containing anti-HBs and HBsAg-Sepharose were generously provided by Dr. A . M . Prince.
Hemagglutination plates were obtained from Ames Co., Linbro Chemical Co., Inc., and Cooke Laboratory Products. Iz6I-NaI carrier-free, was purchased from New England Nuclear. Siliclad wcs obtained from Clay Adams, Polybrene from Aldrich Chemical Co., Inc., and Auscell from Abbott Laboratories. Neuraminidase, isolated from CI. perfringens, was obtained from Worthington Biochemical Corp.; and the beads used in the detection of anti-HBs in solution were 0.25 inch diameter, ball grade TI, polystyrene, natural, supplied by the Harvey Westbury Corp., Westbury, N.Y. All other reagents were of the highest listed purity available commercially in the USA.
Methods Procedures f o r RPHA The methods used throughout this paper essential to the RPHA technique were those previously reported [26] except where noted. RBCs were coated with anti-HBs as previously modified [16]. The concentration of EDTA in SD was increased to 1 0 m M when larger specimen volumes were used. Quanfitation of Anti-HBs Attached t o Red Blood Cells The quantity of active anti-HBs attached to the cell surface was determined as previously described [16]. Quantitation of Anti-HBs in Solution Preparation of beads. Affinity chromatographypurified anti-HBs (10 pg!ml) was attached to polystyrene beads as described by Cart irnd Trcyyur [5]. HBsAg was then combined with the bound anti-HBs by incubating the beads for 2 h at 45 ' C in a solution containing a level of HBsAg barely detectable by CEP. The beads were washed with distilled water and stored desiccated over CaCI,. Preparation of IW-anti-HBs. Irsl-anti-HBs was prepared by the method of Greenwood et ctl. (131. Assay f o r anti-HBs. TO 160 !(I of leaI-anti-HBs (0.28pCi, 5.5,uCilmol) in a solution of 50% fetal calf serum, 0.5% human serum albumin, 2% nor-
Improved RPHA Test for HBsAg
337
Table 1. Effect of changes in RPHA test conditions
Change
Effect
1 Increase purity of antibody attached to RBCs
Enhances sensitivity
2 Increase specimen size
Enhances sensitivity; must overcome resultant nonspecific agglutination
3 Alter composition of cell suspension diluent Vary gelatin concentration from 0.001 to 1% Replace gelatin with bovine serum albumin, PEG 4000, dextran T20 or dextran T80 Add bovine serum albumin, PEG4000, dextran, or dextran T80 to SD as previously constituted Vary ionic strength from 0.025 to 0.5
No change in sensitivity; size of positives are increased at lower concentrations of gelatin
Vary pH from 6.0 to 8.0 Vary EDTA concentration from 1 to 10 mM
4 Alter surface properties of RBCs a Treat with neuraminidase b Attach human serum albumin, IgG, or fibrinogen to antibody-coated RBCs c Subject antibody-coated RBCs to 1-10 cycles of freezing and thawing d Prepare RBCs from other donors of type 0-negative
5 Alter surface properties of hemagglutination plate a Treat plate with Polybrene or Siliclad b Use plates of other manufacture c Coat plate with anti-HBs
6 Alter physical conditions during the incubation period a Increase temperature to 37 or 45 "C.
b Continuously agitate cell-specimen mixture during a preincubation period
Causes spontaneous agglutination
No change in sensitivity
No change in sensitivity; minimum ionic strength needed to overcome spontaneous agglutination is 0.1 No change in sensitivity No change in sensitivity; higher concentrations of EDTA are needed with larger specimen volumes
Reduces attachment of antibody to RBCs Causes spontaneous agglutination No change in sensitivity
No change in sensitivity
No change in sensitivity No change in sensitivity No change in sensitivity
Little if any change in sensitivity; elevated temperature allows the reaction to proceed faster and reduces false-positive rate No change in sensitivity
Horowitz/StrykerlVandersandeilippin/Woods
338
Table 11. Purification of anti-HBs by affinity chromatography Step
Volume Protein ml concentration mg/ml
Ethanol-fractionated y-globulin Affinity chromatography Procedure 1 p H 10.85 Procedure 2 p H 9.6 pH 10.85
f
2 $:
6
12 7.2 4.5
20
Total protein mg
Quantity required for 50% inhibition 1, pg
Recovery, %
Fold purification
120
15.0
100
1 .o
22.7
0.25
3.0
0.66 2 0.25 2
57
0.25
1.8 0.95
1.23k0.582 0.32 k 0.09 3
19
12.2
37
46.9
0.21
Determined by competitive radioimmune assay as described in text. Average of four purifications. Average of seven purifications.
ma1 human serum, 5 m M Tris-HC1 (pH 7.2), and 0.1% sodium azide was added 40,uI of appropriately diluted test specimen containing anti-HBs followed by a bead coated with HBsAg. After incubation of the mixture for 1 h at 45 O C , the bead was washed 10 times with distilled water and the bound radioactivity determined with a gamma counter. A standard solution of anti-HBs was used to calibrate each run, and the amount of specimen needed to inhibit the binding of 1251-anti-HBs by 50% was determined. The curves in figure 1 illustrate the inhibition observed when varying concentrations of either alcohol-precipitated chimpanzee y-globulin (from animals immunized with HBsAg) or affinity chromatography-purified antiHBs were added to this system. The affinity chromatography-purified preparation used was 25 times more effective in inhibiting the binding of radioactive antibody than the ethanol-precipitated y-globulin solution.
Method of Protein Determination Protein concentration was determined spectrophotometrically assuming an extinction coefficient 1% of E 280nm= 13.5.
Results Table I lists the approaches which were explored in the attempt to improve the sensitivity of a previously reported RPHA test for HBsAg [26]. Two proved successful and are reported in detail below.
Modification in RPHA Test Purity of anti-HBs. Previously, the antibody used in coating cells was purified by 1 Abbreviations: HBsAg = hepatitis B surface antigen; anti-HBs = chimpanzee antibody directed against HBsAg; RPHA = reversed passive hemagglutination; RIA = radioimmunoassay; AGD = agar gel diffusion; CEP =counter electrophoresis; SD = cell suspension diluent, consisting of 0.1 M sodium phosphate buffer, pH 7.2, 0.1 % gelatin, 1 % normal human type AB serum, 1 mM EDTA, and 0.02% sodium azide; EDTA = ethylenediamine tetraacetic acid; RBCs = human erythrocytes stabilized by treatment with pyruvic aldehyde and formaldehyde.
Improved RPHA Test for HBsAg
3 39
15 C
c n i 50 E
c
a c
Y
8 25
Protein, pg
Fig. 1. Radioimmune assay for anti-HBs. Alcohol fractionated ( 0 )or affinity chromatography purified (0) anti-HBs was added in the amounts indicated along with 125I-anti-HBs to HBsAgcoated beads as described under Methods. The amount of radioactivity bound was determined.
application of a solution of alcohol-precipitated hyperimmune y-globulin to a column of insolubilized HBsAg at neutral pH and, after a neutral wash, elution at pH 10.85 [26]. This process produced a 20- to 25-fold increase in the purity of anti-HBs (table IJ). It was found that inclusion of an intermediate wash with pH 9.6 buffer resulted in a 2-fold increase in purity of anti-HBs eluting at pH 10.85 (table 11). RBCs coated with this more highly purified antibody bound, on the average, 1.7-fold more anti-HBs and proved 2.9-fold more sensitive in detecting antigen (table 111). The optimum concentration of protein for coating cells was determined for each lot of antibody. Anti-HBs
Table 111. Performance of cells coated with anti-HBs prepared by two different methods ~~
Purification procedure
~~
~
Antibody lot No.
~
Uptake of 125I-HBsAg RPHA titer-' CPm
x
Spontaneous agglutination
1 Eluted directly with
with pH 10.85 buffer
29-32 33 38
39 40 42 t 44
3,125 2,493 3,414 3,491 3,378 3,559
X = 3,243 f396
1.25 1.25-5.00 2.50 0.62 2.50 2.50
none none none none none slight
Tt=2.10+0.90
2 Eluted with p H 10.85 buffer after pH 9.6
wash.
45 41 52 53 54 55 56
7,450 4,742 4,793 5,218 4,955 5,594 5,302
X = 5,436 f937
5.00-10.0 10.0 2.50 1.25 1.25-10.0 5.00 1.00
x = 6.00 f3.00
none none none none none none none
Horowitz/Stry ker/Vandersande/Li ppin/Woods
340
~-~
~
Table 1V. Effect of temperature on use of increased specimen volumes ~
Volume cell suspension, pl
Number of specimens tested
Temperature
A Original protocol (cells settle at 1 g ) 50 60 ambient 50 60 ambient 100 118 ambient 100 118 ambient 100 118 ambient 100 118 ambient
Number showing nonspecific agglutination
2 5 2 5 7.5 10
0 6 0 8 11 14
0.0 6.8 9.3 11.9
28 1 4 5 2
13.9 1.7 13.3 17.2 I .o
B Modified protocol (cells settle with centrifugation) too 202 ambient 10 100 100 100 100
59 30 29 193
31 O C 37 oc 31 O C 45 o c
eluted at p H 9.6 was least effective in the preparation of cells for use in detecting HBsAg (data not shown). Other procedures for increasing purity were also tried in an attempt either to enhance the sensitivity of the cells or to overcome the spontaneous agglutination which occurs when excess antibody protein binds to RBCs. As none resulted in higher purity antibody nor did they improve the present test, they will only be listed here: repurification of affinity chromatography purified antiHBs on a second, similar affinity column; QAE-Sephadex chromatography of either alcohol precipitated y-globulin or affinity chromatography-purified anti-HBs; and Sepharose 4-B chromatography of affinity chromatography-purified anti-HBs. In addition, it was suggested that the spontaneous agglutination which occurs when too much antibody is attached to cells might result from aggregates of IgG present in the solu-
~
Volume of specimen tested, pl
10 15 20 10
Falsepositive rate, 95
0.0 10.0
tion of anti-HBs. However, when the solution of anti-HBs was analyzed in the analytical ultracentrifuge, aggregates were not observed. Volume of specimen tested. Only a limited volume of specimen can be used in an RPHA procedure without causing nonspecific agglutination. The previously described protocol [26] used 1 0 p l of specimen first diluted 6-fold (or 1.67 pl). The results in table IVA indicate that 2 p l of specimen could be used without encountering an undue false-positive rate. 5 pl of specimen substantially increased the false-positive rate even when the volume of cell suspension was increased from 50 to 100p1. The specimen concentration with the original protocol was therefore limited to 3.8-4.8%. It is interesting to note that the same nonspecific agglutination occurred when increased specimen volumes were added to uncoated RBCs. The specimen concentration could be in-
Improved RPHA Test for HBsAg
341
Table V. Enhanced sensitivity rcsulting from larger specimen volume
Specimen
Subtype
Highest dilution positive previous protocol *
modified protocol ~
ad ad ad (purified) aY ay (purified)
16 2 16,000 16 1,600
Sensitivity increase (fold)
~
~~
64 4 128,000 32 3,200 Average 3.6
1
10 ,MI of specimen first diluted 1:5 was added to 50 ,dcell suspension as originally described [26]. 10 ul of specimen was added directly to 100 p l cell suspension as in figure 2.
Table VI. Performance trials of modified RPHA test for HBsAg
Procedure
Number tested
Modified protocol Auscell
5,173
Positive on initial screen
Positive on repeat screen
Confirmed positive
74 (1.4%) 75 (1.4%)
47 (0.91 %) 47 (0.91%)
16 (0.31%) 16 (0.31%)
creased without an undue increase in the false-positive rate if the RPHA reaction mixture was heated at 37 "C for 2 h or 45 "C for 1 h (table IVB). A maximum specimen volume of between 10 and 15 ,~1(9.1-13.0%) proved satisfactory. In order to avoid convection problems when working at these elevated temperatures a previously described centrifugation procedure [24,29, 32) was used. This procedure consisted of centrifugation of the entire plate for 2min at 1,000 rpm using IEC rotor 259 and the plate holders supplied by Ames, followed by tilting the plate at an angle of 60" from the horizontal. Unagglutinated cells streamed downward forming a short solid line with even
edges. Agglutinated cells remained as a tight button, sometimes remaining in the center of the well and other times falling intact to the edge of the well (fig. 2). A weak positive reaction was characterized by the occurrence of distinct granularity within the solid pattern caused by the streaming cells. The rate of nonspecific agglutination with increased specimen size could also be reduced by heating the specimen alone at 56 O C for 30min followed by RPHA testing at ambient temperature. For example, none of the 28 samples causing agglutination when tested at ambient temperature (table IVB, line 1) caused agglutination if they were first heated at 56°C for 30 min.
342
Horowitz/Stry ker/Vandersande/Lippin/Woods
Fig.2. Modified RPHA assay for HBsAg. Sera (10 pl), positive or negative for HBsAg, were added to 100 ti1 of anti-HBs-coated RBCs at a
conccntration of 0.0575% in SD. After incubation at 45 "C for 1 h the samples were ccntrifuged and held at a 60" angle for 15 min.
Effect on Sensitivity of the New Test Protocol The new test procedure consists of addition of 10 ,ill of specimen to 100 pl of cells suspended at a concentration of 0.0375% (v/vj in SD, incubation at 45 "C for 1 h, and centrifugation fcr 2 min at 1,000 rpm followed by tilting the plate at an angle ot 60". Sensitivity was determined by testing 5 HBsAg-positive specimens which were serially diluted in tubes and randomized. Three of the specimens were of subtype ad and 2 of subtype ay. The cells used for both protocols were coated with anti-HBs which was eluted from the affinity column after washing with pH 9.6 buffer. These results, therefore, do not take into account the 2.9-fold
increase in sensitivity caused by using more highly purified anti-HBs. The results are shown in table V. The increase in sensitivity ranged from 2- to 8-fold, averaging 3.6fold. Performance Trials of Modified RPHA Il4aterials and Protocol The performance of the newly modified test under routine serum screening conditions is summarized in table VI. Specimens from 5,173 volunteer donors were tested in parallel with Auscell at the New York Blood Center. The results obtained with the two tests were virtually identical: 74-75 (1.4%) of the specimens were positive on initial screening, 47 (0.91%) of these were repeat-
Improved RPHA Test for HBsAg
ably positive on subsequent screening, and 16 (0.31%) of these were established as HBsAg-positive by both tests. The false-positive rate for both tests was 0.60%.
Discussion RPHA is currently used throughout the world in testing for hepatitis-associated antigens. The detection of other antigens such as rubella, herpes, carcinoembryonic antigen, u-fetoprotein, or certain drugs by RPHA may prove exceedingly valuable as our knowledge of this technique increases. Inherently, the technique is capable of detecting antigen at a concentration in the specimen of approximately 10-lG M. Current RPHA techniques are at least several orders of magnitude less sensitive than this, yet they have proven to be among the most sensitive analytical techniques available. The principal factors believed to influence sensitivity are the binding constant of antibody to antigen, antibody density on the cell surface, availability of bound antibody to participate in intercellular bridging, the number of cells which must aggregate before a positive agglutination reaction is observable, the specimen size, the length of time allowed for the agglutination reaction to occur, and the temperature at which the reaction takes place. Wider application of RPHA testing depends somewhat on the realization of its sensitivity potential. While a significant literature dealing with the forces which affect the agglutination of unfixed erythrocytes has developed [4, 6, 17, 22, 271, little attention has been given to evaluating factors affecting the sensitivity of stabilized cells. Manufacturer and user alike prefer the use of stabilized cells for
343
their convenience, reproducibility, and reliability over an extended time period. Application of the findings reported herc to the previously described test for HBsAg [26] resulted in a net increase in sensitivity of approximately 10-fold. Two independent improvements were responsible for this increase. First, the purity of the anti-HBs used in coating the cell was enhanced by a refinement in the method used to elute anti-HBs from a column of insolubilized HBsAg. A 2-fold increase in purity of the antibody resulted in a 3-fold increase in sensitivity. Seccond, sensitivity was increased through the use of a larger specimen volume. The previous protocol tested 1.67 pl of specimen at a final concentration of 2.8%. Higher specimen concentrations were not used to avoid spontaneous agglutination (a high false-positive rate). With the modified protocol in which the specimen was heated, either separately or after addition to the cells, 10 pl of specimen could be tested without encountering a high rate of spontaneous agglutination. A comparison of the two procedures showed that the modified protocol increased the sensitivity 3.6-fold, on the average. The explanation of why heating reduces nonspecific agglutination remains speculative. It cannot even be currently concluded that heating the specimen separately at 56 OC achieves its effect in the same manner as heating the specimen plus cells at 37 or 45 "C. However, it should be emphasized that the observed nonspecific agglutination results from the interaction between specimen and uncoated RBCs as well; that is, the observed nonspecific agglutination did not depend upon an interaction between the bound y-globulin and an element in serum. Nonspecific agglutination may result from
344
the aldehyde treatment itself since specimen concentrations as high as 85% are occasionally but routinely used with unfixed erythrocytes in clinical laboratories. Both of the above-mentioned improvements rely on increasing the likelihood of immune complex formation by increasing the concentration of the reactants (antibody and antigen). In contrast, methods which are believed to enhance sensitivity with fresh erythrocytes by modulating their zeta potential [ 2 2 ] do not appear to increase sensitivity of RPHA reactions using aldehyde-stabilized erythrocytes. Future increases in sensitivity would seem most likely to come about from (1) methods which allow the attachment of higher quantities of antibody without inducing spontaneous agglutination, (2) methods which allow the use of greater specimen concentration, (3) use of higher affinity antibodies, (4) alternate methods of agglutinate visualization, and ( 5 ) methods which optimize cellcell interaction by influencing the shape of the cells before stabilization. Each of these areas is worthy of study in order to realize the full potential of RPHA techniques. Acknowledgements We are grateful for the continued involvement of Dr. A . M . Prince and for the technical assistance provided by Mr. Jay Maracic.
References Aach, R. D.; Grisham, J . W., and Parker, C. W.: Detection of Australia antigen by radioimmunoassay. Proc. natn Acad. Sci. USA 68: 10561060 (1971). Alter, H. J.; Holland, P. V.; Purcell, R. H.; Lander, J . 3.; Feinstone, S. M., and Morrow, A. G.:
Horowitz/Stryker/Vandersande/Lippin/Woods
Posttransfusion hepatitis after exclusion of commercial and hepatitis-B antigen-positive donors. Ann. intern. Med. 77: 691-699 (1972). 3 Barker, L.F.; Geretz, R. J.; Hoofnagle, J . H., and Nortman, D. F.: Viral hepatitis B. Detcction and prophylaxis; in Greenwalt and Jamieson Transmissible disease and blood transfusion, pp. 81-111 (Grune & Stratton, New York 1975). 4 Brooks, D. E. and Seaman, G . V. F.: Electroviscous effect in dextran erythrocyte wspensions. Nature new Biol. 238: 251-253 (1972). 5 Catt, K. and Tregear, G. W.: Solid phase radioimmune assay in antibody-coated tubes. Science 158: 1570-1572 (1967). 6 Chien, S.: Symposium on ultrastructural and electrochemical aspects of red cell aggregation. 7th European Conference on Microcirculation. Biblthca anat, vol. 11, pp. 244-309 (Karger, Basel 1973). 7 Derrick, J. D.; Young, A. M., and Pert, J. H.: Large-scale blood-screening program for hepatitis B. N.Y. St. J. Med. 75: 1693-1698 (1975). 8 Dodd, R.Y.; Ni, L. Y.; Mallin, W.S., and Greenwalt, T. J.: Hepatitis B (surface) antigen testing by radioimmunoassay. Am. J . din. Path. 63: 847-853 (1975). 9 Feinstone, S. M.; Kapikian, A. Z.; Purcell, R. H.; Alter, H. J., and Holland, P. V.: Transfusion-associated hepatitis not due to viral hepatitis type A or B. New Engl. J. Med. 292: 767-770 (1975). 10 Giorgini, G. L., jr.; Hollinger, F. B.; Leduc, L.; Issarescu, S.; George, J.; Blackman, A,, and Thayer, W. R., jr.: Radioimmunoassay detection of hepatitis type B antigen. A prospective study in blood donors and recipients. J. Am. med. Ass. 222: 1514-1518 (1972). 11 Gocke, D. J.: A prospective study of posttransfusion hepatitis. The role of Australia antigen. J. Am. med. Ass. 219: 1165-1170 (1972). 12 Goldfield, M.: Some epidemiological studies of transfusion-associated hepatitis; in GreenWalt and Jamieson Transmissible disease and blood transfusion, pp. 141-151 (Grune & Stratton, New York 1975). 13 Greenwood, F. C.; Hunter, W. M., and Glover, J. S.: The preparation of 1slI-labelled human growth hormone of high specific radioactivity. Biochem. J . 89: 114-123 (1963).
Improved RPHA Test for HBsAg
14 Hollinger, F. B.; Aach, R. D.; Gitnick, G. L.; Roche, J. K., and Melnick, J. L.: Limitations of solid-phase radioimmunoassay for HBAg in reducing frequency of post-transfusion hepatitis, New Engl. J. Med. 289: 385-391 (1973). I5 Hopkins, R.; Robertson, M.; Ross, D.; Turnbull, W. M., and Das, P. C.: Detection of hepatitis B surface antigen among Scottish blood donors: Evaluation of sensitive tanned-cell haemagglutination-inhibition test. Br. med. J. iic 409-41 1 ( I 975). I6 Horowitz, B.: Development of hemagglutination assays. I. Attachment of anti-HBs antibody to stabilized erythrocytes. Vox Sang. 33: 324-334 (1977). 17 Jandl, J. H. and Simmons, R. L.: The agglutination and sensitization of red cells by metallic cations: interactions between multivalent metals and the red cell membrane. Br. J. Haemat. 3: 19-38 (1957). I8 Xoretz, R. L. and Gitnick, G. L.: Prevention of post-transfusion hepatitis. Role of sensitive hepatitis B antigen screening tests, source of blood and volume of transfusion. Am. J. Med. $9: 754-760 (1975). 19 Ling, C. M . and Overby, L. R.: Prevalence of hepatitis B virus antigen as revealed by direct radioimmune assay with IW-antibody. J. Immun. 109: 834-841 (1972). 20 London, W. T.; Sutnick, A. I., and Blumenberg, B. S.: Australia antigen and acute viral hepatitis. Ann. intern. Med. 70: 55-59 (1969). 21 NHLI Blood Resource Study: Supply and use of the nation’s blood resource. National Heart and Lung Institute, NIH, vol. 1, Appendix C, June 30 (1972). 22 Pollack, W.; Hager, H. J.; Reckel, R.; Toren, D. A., and Singher, H. 0.: A study of the forces involved in the second stage of hemagglutination. Transfusion 5: 158-183 (1965). 23 Prince, A. M.: An antigen detected in the blood during the incubation period of serum hepatitis. Proc. natn. Acad. Sci. USA 60: 814821 (1968). 24 Prince, A. M.; Brotman, B., and Ikram, H.: Hemagglutination assay: Subtyping by hemagglutination inhibition, an ultra-sensitive identity test for HB antigen; in Vyas, Perkins and
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25
26
27
28
29
30
31
32
Schmid Hepatitis and blood transfusion, pp. 147-154 (Grune & Stratton, New York 1972). Prince, A. M.; Brotman, B.; Grady, G. F.: Kuhns, W. J.; Hazzi, C.; Levine, R. W., and Millian, S. J.: Long-incubation post-transfusion hepatitis without serological evidence of exposure to hepatitis-B virus. Lancet ii: 241-246 (1974). Prince, A.M.; Ikram, H.; Chicot, D.; Wright, R.; Vnek, J.; Neurath, R.; Lippin, A., and Swiss, S.: A new reversed passive hemagglutination test for detection of HBsAg. Vox Sang. 29: 319-329 (1975). Schnebli, H. P. and Bachi, T.: Reaction of lectins with human erythrocytes. I. Factors governing the agglutination reaction. Expl Cell Res. 91: 175-183 (1975). Szmuness, W.; Hirsch, R. L.; Prince, A. M.; Levine, R. W.; Harley, E. J., and Ikram, H.: Hepatitis B surface antigen in blood donors: further observations. J. infect. Dis. 131: 111118 (1975). Vyas, G . N . and Shulman, N. R.: Hemagglutination assay for antigen and antibody associated with viral hepatitis. Science 170: 332-333 (197d). Wallace, J.; Ban, A., and Milne, G. R.: Which techniques should be used to screen blood donations for hepatitis B surface antigen. Br. med. J. ii: 412-414 (1975). Walsh, J. H.; Yalow, R., and Berson, S. A,: Detection of Australia antigen and antibody by means of radioimmunoassay techniques. J. infect. Dis. 121: 550-554 (1970). Wegmann, T . G . and Smithies, 0.:A simple hemagglutination system requiring small amounts of red cells and antibodies. Transfusion 6: 67-73 (1966).
Received: December 24, 1976 Accepted: February 5, 1977
Dr. Bernard Horowitz, Blood Derivatives Program, New York Blood Center, 310 East 67th Street. New York N Y 10021 (USA)
