An Enzyme Linked Immunosorbent Assay (ELISA) for Detecting IgG Sensitized Erythrocytes K. W. BRUNER, JR. AND C. W. KISSLING From the St. Francis Hospital of Wichita, Wichita, Kansas

Thls paper describes an Enzyme Linked Immunosorbent Assay (ELISA) for detecting IgC sensitized erythrocytes utilizing a commercially available mtihuman 1%; conjugated with alkaline phosphatase. Erythrocyte hemoiysis in the assay was minimized by dissolving the pnitrophenyl phosphate substrate in a arbonate- bkarbooate buffer. Nonspecific absorption of the enzyme conjugate to erythrocytes and glassware was reduced by adding 1% bovine serum albumin to wash solutions. Assay sensitivity wm increased with greater concentrations of enzyme conjugate and erythrocytes in the incubation stage. The sensitivity of the described ELISA procedure is approximately equal to that of the standard antiglobulin test. Some possible future applications of ELISA in the blood bank are discussed.

develop an uncomplicated ELISA procedure for the detection of IgG sensitized erythrocytes and to compare the sensitivity of this new procedure with that of the standard antiglobulin technique. Materials and Methods Substrate

We evaluated pnitrophenyl phosphate (pNPP)* dissolved in three different buffers: Glycine buffer,* 2-amino-2-methyl-1-propanol buffer (221 buffer),* and a carbonate-bicarbonate buffer. Concentrations tested were from 5 mg p-NPP per ml buffer to 0.625 mg p-NPP per ml buffer. To enhance enzyme activity, 0.001 M. MgClz was added to all buffers5

ENZYME-LINKED immunosorbent assay (ELISA) is a relatively new labeled antibody t e c h n i q ~ e . in ~ , which ~ the label is an Enzyme-Conjugate enzyme. The antibody and enzyme are Goat anti-human IgG (heavy and light chain) conjugated in such a manner that both the conjugated to alkaline phosphatase (ALPase)t immunologic and enzymatic activities are was used. Dilutions of conjugate from 1:lOO to retained.2 Recent years have seen the 1: 1600 were tested to determine optimal reactivity development of many ELISA procedures in our test system. The dilutions were made with buffered normal saline containing 1% in the fields of microbiology and t o x i c o l ~ g y . ~ ~phosphate ~~ bovine serum albumin. Many of these tests are extremely sensitive, but are not as precise as radioimmunoassay Antisera (RIA) procedure^.^ The disadvantage of Patient sera containing IgG alloantibodies RIA is that it uses unstable reagents and anti-Fy", anti-K, anti-D; selected commercial requires expensive and sophisticated equipantisera anti-B (Spectra), anti-e (Ortho), and an ment. ELISA uses stable reagents and is IgM anti-P, (Pfizer);and an eluate from cord cells with a positive direct antiglobulin test due to within the capability of any laboratory immune anti-A were used to sensitize erythrocytes possessing a spectroph~tometer.~~'*~ possessing corresponding antigens. Antisera To date, ELISA applications in the blood were diluted with normal saline to obtain weakly bank have been minimal. RIA has been sensitized cells. shown to be useful in detecting erythrocyte antigens and antibodies.6 Therefore, we Cell Suspensions wanted to utilize ELISA as a substitute Human erythrocytes were obtained in either CPD or EDTA anticoagulant. The cells were for RIA in such studies. We attempted to

* Sigma Chemical Co., St. Louis, MO.

Received for publication October 2, 1978; accepted November 18. 1978.

t Miles Laboratories, Inc., Elkhart, Indiana.

0041-1132/79/1100/0773 $00.75 6 J. B. Lippincott Co. Transfusion November-December 1979

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six times with normal saline containing 1% bovine serum albumin (NS-1% BSA). Cell suspensions from 3 to 50 per cent in NS-1% BSA were tested to find the most sensitive concentration for ELISA.

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FIG.1. Effect of changing the enzyme conjugate and erythrocyte concentrations on ELISA sensitivity. (A Absorbance is the absorbance produced by IgG sensitized test cells minus the absorbance produced by unsensitized control cells). washed at least three times with normal saline before sensitization. After sensitization for 30 minutes at 37 C, the red blood cells were washed 1.0,

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ELISA Procedure Two drops of an erythrocyte suspension were transferred to a test tube containing two drops of diluted ALPase conjugate. The cells and conjugate were incubated together for 30 minutes at 37 C, then washed six times with NS-1% BSA. Following the final wash and decanting of the supernatant fluid, the cells were allowed to resuspend in a residual wash solution remaining in the tube. The entire cell volume in each tube was then transferred to test tubes containing one milliliter of buffered substrate. The mixture was incubated at 37 C for 30 minutes. After centrifugation, 0.9 ml aliquots of the supernatant fluid were transferred to tubes containing 2 ml of 0.05 N NaOH for absorbance readings on a spectrophotometer. The blank consisted of 0.9 ml buffered substrate added to 2 ml of 0.05 N NaOH. The product of enzymatic conversion, p-nitrophenol, forms a yellow enolate ion above pH 7.6 with maximum absorption at 410 nm. For our purposes, an E L S A test was considered positive if the absorbance of the sensitized erythrocytes exceeded the absorbance of all four negative controls. 1.a

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FIG. 2. Comparison of ELISA and antiglobulin test in detecting erythrocytes sensitized with anti-K. Dilutions of sensitizing antiserum arc given in parenthesis beneath the strength of antiglobulin reactions.

FIG. 3. Comparison of ELISA and antiglobulin test in detecting erythrocytes sensitized with anti-D. Dilutions of sensitizing antiserum are given in parenthesis beneath the strength of antiglobulin reactions.

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Two types of negative controls were tested in duplicate along with the sensitized test cells throughout the ELISA procedure. The antibody control consisted of cells that had been incubated with undiluted antisera to which the cells lacked the corresponding antigen. The conjugate control consisted of cells which had been incubated with only normal saline instead of antisera. Sensitized erythrocytes were tested by the standard antiglobulin method.' All reactions were recorded using a standard grading system.' The sensitized cells were then tested by ELISA, and the results of the two methods compared.

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The p-NPP, in concentrations from 5 mg per ml to 0.625 mg per ml caused red blood cell hemolysis when dissolved in the glycine or 221 buffers. Hemolysis is undesirable as the release of intracellular enzymes causes substrate conversion, resulting in false-positive reactions and decreased assay sensitivity. Optimal results were obtained using a bicarbonate buffer. Hemolysis was minimized by using 5 mg of p-NPP per ml of 0.1 M carbonate-bicarbonate buffer (pH 10.0 to 10.1). Using a pool of cells sensitized with anti-D, it was shown that there was a direct relationship between the amount of ALPase conjugate added to the assay and the amount of red blood cell bound enzyme detected (Fig. 1). For every cell suspension tested from 3 to 50 per cent,

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FIG.5 . Comparison of ELISA and antiglobulintest in detecting erythrocytes sensitized with immune anti-A eluted from cord blood erythrocytes. Dilutions of sensitizing antiserum (eluate) are given in parenthesis beneath the strength of antiglobulin reactions.

greater concentrations of conjugate from 1:1600 to 1:lOO dilutions resulted in a progressively more sensitive assay. Since this conjugate is relatively expensive, dilutions less than 1: 100

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FIG.6. Comparison of ELISA and antiglobulintest in detecting erythrocytes sensitized with anti-Fya. Dilutions of sensitizing antiserum are given in parenthesis beneath the strength of antiglobulin reactions.

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BRUNER AND KISSLING

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not give any positive reactions by the ELISA procedure.

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FIG.7. ELISA reactions of group B erythrocytes sensitized with commercial anti-B typing serum. were not tested and the 1:lOO dilution was chosen for our assay. As may be seen in Figure 1, increasing the number of erythrocytes present in the suspension enhanced the assay sensitivity. At conjugate dilutions of 1:800to 1: 100 there was progressively greater detectable enzyme activity as the cell suspensions were increased from 3 to 50 per cent. Because it becomes more difficult to obtain an adequate specimen and work with higher percentage cell suspensions, the 25 per cent cell suspension was chosen for our assay. With cells coated with anti-K, anti-D, anti-e and IgG anti-A, the ELISA procedure and the antiglobulin test appeared equally sensitive (Figs. 2, 3, 4, and 5). In these tests ELISA could detect a weakly sensitized cell as judged by the antiglobulin method. ELISA could detect a cell which was sensitized 2+ with anti-Fy", but not more weakly sensitized cells (Fig. 6). Figure 7 shows the results of ELISA testing on group B red blood cells which had been incubated with serial dilutions of commercial anti-B. Antiglobulin testing was not done because all antisera dilutions up to 1:32 caused macroscopic agglutination. However, it is noteworthy that the ELISA procedure also easily detected the presence of IgG on group B cells at all the dilutions tested. As would be expected because our conjugate had only anti-IgG activity, PI positive cells sensitized with an IgM anti-PI did

We had hoped to be able to develop a simple, inexpensive ELISA procedure that was more sensitive than the standard antiglobulin method in detecting IgG sensitized erythrocytes. Several formidable problems were encountered in our pursuit. Hemolysis occurred when the red blood cells were suspended in buffered p-NPP substrate. The subsequent release of intracellular enzymes caused marked substrate conversion with unacceptably high levels of background absorbance. Use of a carbonatebicarbonate buffer minimized but did not completely abolish hemolysis. The second major problem was nonspecific absorption of the ALPase conjugate to erythrocytes and glassware. Adding 1% BSA to the saline wash solution substantially reduced but did not completely eliminate nonspecific red blood cell binding. Increased background caused by coating of conjugate to glassware was solved by transfemng the enzyme-coated test cells to a separate substrate reaction test tube. Despite the attempts to decrease nonspecific substrate conversion, the ELISA procedure still lacked sensitivity. We found that by using a larger amount of enzyme conjugate (1 :100 dilution) and an increased red blood cell concentration (25% suspension), the ELISA sensitivity was comparable to but did not exceed that of the standard antiglobulin test. Although increasing the concentrations of conjugate and red blood cells could increase the sensitivity, such a pursuit was inconsistent with our original goal to develop a simple, inexpensive assay. We did find that over 90 per cent of the ALPase conjugate added to the incubation system was not bound to sensitized red blood cells, and only a small part of this could be accounted for by absorption to glassware. Perhaps another enzyme conjugate with more avidity for IgG sensitized erythrocytes would increase the assay sensitivity.

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ELISA ANTIGLOBULIN PROCEDURE

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One thing that makes ELISA attractive for immunohematologic testing is that the end point is a spectrophotometricabsorbance value instead of visual detection and grading of agglutination. As such, ELISA should lend itself readily to automated quantitative measurements for a variety of cellular antigen-antibody systems. For example, this assay would appear to be sensitive enough for adaption to an automated ABO grouping procedure. This study illustrates a potential usefulness for ELISA in the routine blood bank. Acknowledgments The authors wish to thank Dr. E. H. Gerlach of St. Francis Hospital for reviewing this manuscript, and Mr. Rick Reagen of the University of Kansas School of Medicine-Wichita for preparing the illustrations.

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sorbent assay (ELISA). Quantitative assay of ImmunogMuIinG.hmunockmistty&871.1971. 4. -, K. Jonsson, and P. Perlmann: Enzymelinked immunosorbent assay. 11. Quantitative assay of protein antigen, Immunoglobulin G, by means of enzyme-labeled antigen and antibody coated tubes. Biochim. Biophys. Acta 251:

427, 1971. 5. Henry, R. J.,D. C. Cannon, and J.W. Winkelman: Clinical Chemistry Principles and Technics. Hagerstown, Harper & Row, 1974,p. Z5. 6. Jenkins, D. E., W. H. Moore, and A. Hawiger: A method for radioactive antiglobulin testing with '=I labeled anti-IgG. Trimfusion 17:16,1977. 7. Mohr.J.: Is enzyme immunoassay a breakthrough technology? Lab. Manag. June:14. 1977. 8. Van Weeman, B. K., and A. H. W. M. Schuurs: Immunoassay using antigen-enzymeconjugates. FEBS Lett. 15232, 1971. 9. Voller, A., D. E. Bidwell, and A. Bartlett: Enzyme immunoassays in diagnostic medicine. Bull. World Health Organ. 5355. 1976. '10.Wisdom, G. B.: Enzyme-immunoassay. Clin. Chem. 221243. 1976.

References 1. American Association of Blood Banks: Technical Manual of the American Association of Blood Banks, 7th ed. Washington, D.C., 1977. 2. Enpall. E.: Enzyme-linked immunosorbent assay,

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ELISA. I n : Biomedical Applications of Immobilized Enzymes and Proteins, vol. 2. T.MS. Chang, Ed. New York, PlenumPress, 1977,p. 87. ,and P. Perlmann: Enzyme-linked immuno-

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K. WiIliam Bruner. Jr., M.D.. Associate Pathologist and Medical Director, Blood Bank, St. Francis Hospital, 929 N. St. Francis, Wichita, Kansas 67214. Charles W. Kissling, MT (ASCP), Research and Development Technologist, Blood Bank, St. Francis Hospital.

Announcement The International Society of Blood Transfusion will hold a symposium on Diseases Transmitted by Blood with a WHO/ISBT Workshop on Quality Control in Blood Banking, from March 2 to March 8, 1980, in Chandigarh, India. The meeting will be organized by the Indian Society of Blood Transfusion & Immunohaematology, Blood Transfusion Department, PGI, Chandigarh, India. For further information please contact Mrs. Saroop Krishen, Secretary General, Blood Transfusion Department, X I , Chandigarh, India, or Prof. Dr. S. Seidl, Vice President of ISBT, Red Cross Blood Donor Service Hessen, P.B. 730367,P60oO FrankfudMain 73'.

An enzyme linked immunosorbent assay (ELISA) for detecting IgG sensitized erythrocytes.

An Enzyme Linked Immunosorbent Assay (ELISA) for Detecting IgG Sensitized Erythrocytes K. W. BRUNER, JR. AND C. W. KISSLING From the St. Francis Hospi...
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