Quantization of lymphocyte mediated sheep red blood cell hemolysis, using an ELISA reader

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Journal of Immunological Methods, 142 (1991) 137-139 © 1991 Elsevier Science Publishers B.V. 0022-1759/91/$03.50 ADONIS 00221759 JIM 06053

L e t t e r to the editors

Quantitation of lymphocyte mediated sheep red blood cell hemolysis, using an ELISA reader T.B. Poduval Molecular Biology and Agriculture Division, Bhabha Atomic Research Centre, Bombay-400 085, India (Received 4 March 1991, revised received 23 May 1991, accepted 24 May 1991)

Dear Editors, Sheep red blood cells (SRBC) are widely used as a test antigen, to assess the immune status of experimental animals. A variety of methods have been developed for enumerating antigen-induced antibody-secreting cells. The localized hemolysis in gel (LHG) technique which, with its modifications, is one of the more widely used methods to enumerate antibody secreting cells (Jerne et al., 1963; Cunningham, 1968). One major limitation of L H G assay is that the total number of plaque forming cells (PFC) does not truly reflect the antibody producing capabilities of the individual lymphocytes in a given lymphoid organ. Larger plaques may contain cells secreting relatively large amounts of antibody. Methods have been evolved to measure the plaque size, in addition to the plaque number, which are either extremely tedious or not accurate (Dresser, 1990). It has been shown that assay conditions must be rigidly controlled for valid size comparisons to be made (Jerne, 1974; Dresser, 1990). In Cunningham PFC assay in liquid medium, Brownian movements or slight convection current can lead to occlusion of small plaques in a few hours. Air bubble in the chamber, uneven surface of the glass slides lead to movement in the liquid medium with consequent loss of definition of small plaques.

Correspondence to: T.B. Poduval, Molecular Biologyand Agriculture Division, Bhabha Atomic Research Centre, Trombay, Bombay-400085, India.

A spectrophotometric determination of lymphocyte mediated SRBC hemolysis in vitro has been developed (Simpson and Gozzo, 1978) to quantitate the amount of anti-SRBC antibody secreted by immune spleen cells. This method may more accurately reflect the antibody production. This technique has been successfully used in our laboratory to monitor the antibody response to SRBC in mice, which are chronically fed with ethanol (Poduval et al., 1990). In the present report, an ELISA reader has been used, to measure the extent of SRBC hemolysis, which reflects the presence of antibody producing cells. SRBC drawn from a single sheep, stored at 5 °C in Alsever's solution for at least 7 days and not later than 25 days were washed three times with 5-10 vols. of isotonic phosphate-buffered saline containing calcium and magnesium, pH 7.2 (PBS). The last wash was done by centrifuging at 800 x g for 10 min. 1 vol. of packed, washed cells were evenly suspended in 19 vols. of PBS to make 5% SRBC suspension. Swiss male mice, aged 8-10 weeks, were immunized i.p. with 0.2 ml of 5% SRBC. On day +5 and day + 8 of SRBC immunization, mice were killed and their splenic cell suspensions were prepared in PBS. A part of the splenic suspension was stored at 5°C. Standardized SRBC suspension for the assay was prepared as follows. In brief, 5% SRBC suspension, prepared as above, was diluted in PBS to make 0.3% SRBC. To 1 vol. of 0.3% SRBC, 2 vols. of distilled water were added arid 100 ~1 of the clear lysate obtained after centrifugation at 500 x g for 10 min, was transferred to ELISA microwell

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modules (Nunc, Denmark) and optical density (OD) measured using a 410 nm narrow bandpass filter, in an ELISA plate reader (Mini Reader II Dynatech Laboratories). SRBC concentration was adjusted according to the following relation:

Vf

~

0"8

h

h

r

r

0.7 0.6 ~ 0.4 ~

~XOD 0.9

3

Day+5 ~..4bOhr

O

3hr

0.3 0.2

where OD represents the optical density of the lysate of 0.3% SRBC suspension, Vi is an accurately measured aliquot of the 0.3% SRBC suspension and Vf is the final volume to which it should be adjusted by the addition of PBS. This standardized SRBC suspension was lysed by the addition of twice the volume of distilled water and OD of 100 tzl of this lysate measured as above. Standardized SRBC suspension giving an OD between 0.9 and 0.95 by this method was used in the assay. For the assay, 50/zl of spleen cell suspension (2.5-40 × 106 cells/ml) and 50/zl of standardized SRBC suspension were mixed in ELISA microwell modules and 50 ~I 1:50 fresh guinea pig serum (complement) absorbed with SRBC, was added. A high control, which contains SRBC and distilled water, a low control containing SRBC and PBS and a complement control containing SRBC and complement were also included in the assay. A second set of experiments were performed in test tubes according to the method reported earlier (Simpson and Gozzo, 1978). The ELISA microwell modules and the test tubes were incubated in a humidified chamber at 37°C for 1 h and the contents were cen-

0.1 2.5

5.0

10 ImmuneSpleenCellsX10 $

20

Fig. ]. O D at 410 nm (ordinate) versus number o f immune spleen cell present in the assay mixture (abscissa): mice were killed on day + 5 or day + 8 of SRBC immunization and splenic cell suspensions were prepared and used immediately (0 h), or after storage at 5°C for 3 h (3 h). Each value represents the m e a n of five replicates.

trifuged at 500 × g for 10 min and 100/xl each of the supernatant was transferred to fresh ELISA modules and the optical density measured. Results are presented in Table I. It is seen that the results obtained by both the methods are very close to each other. Effect of storage of SRBC immune splenic lymphocytes at 5°C on the lysis of SRBC is shown in Fig. 1. The OD values are plotted as a function of the number of SRBC immune splenocytes present in the assay mixture. Results suggest that storage at 5°C for 3 h has no effect on the hemolytic capabilities of the immune splenocytes. The results also indicate an increased lysis of SRBC with an increase in num-

TABLE I I M M U N E S P L E E N C E L L M E D I A T E D S R B C LYSIS: C O M P A R I S O N O F T W O M E T H O D S Splenocyte/ml a (106 )

O D at 410 n m ( m e a n + SE) Day 5

40 20 5.0 2.5

Day 8

Method 1

Method 2

Method 1

Method 2

0.84 + 0.02 0.64 -I-0.02 0.24 + 0.02 0.09 + 0.01

0.85 + 0.63 + 0.22 + 0.08 +

0.43 + 0.01 0.25 + 0.005 0.04 + 0.004 < 0.03

0.41 + 0.02 0.24 + 0.01 0.05 +_0.005 < 0.03

0.03 0.02 0.02 0.01

Refers to the n u m b e r of s p l e n o c y t e s / m l of the original suspension. Mice were killed on day + 5 or day + 8 of SRBC immunization and the assay conducted either by m e t h o d 1 (reported in this paper) or method 2 (Simpson and Gozzo, 1978). Each value represents the m e a n of five replicates.

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ber of splenic lymphocytes. For a given splenocyte number the extent of hemolysis on day + 8 is significantly lower than observed for day +5. Normal lymphocytes at all the concentrations used do not lyse SRBC (data not shown). The method is simple, reliable and very fast. Multichannel pippettes, 96 channel replicator, automatic ELISA reader can greatly increase the speed of the assay. This procedure eliminates subjective error of counting observed in other PFC assays. The method offers scope for the use of multiple replicates. This will help in obtaining a reliable estimate of the true SRBC hemolysis. Results in Table I indicate that the degree of variability within the assay is minimal. The fact that the experiment performed at a later time shows similar results points to the reliability of the assay. The extent of hemolysis may more closely relate to the effective antibody producing capacity of the lymphocytes. A unit change in OD can be more reliably correlated with antibody producing capacity of the host, which no other method may offer. Lymphocyte number ranging from 5 x 104 to 4 x 106 cells can be used in the assay. In the case of PFC assay more than 106 cells interfere with clarity of plaques (Kappler, 1974). Since the number of cells required is small, presence of antibody forming cells in other smaller lymphoid organs can also be assessed. If

the optical surface is not scratched, the ELISA modules can be washed and used again. The method can be adopted to study the in vitro responsiveness of lymphoid cells to other antigens by using antigen coated SRBC. Methods which require large number of replicate cultures can use this method to advantage.

References Cunningham, A.J. and Szenberg, A. (1968) Further improvements in the plaque technique for detecting single antibody-forming cells. Immunology 14, 599. Dresser, D.W. (1990) Fast plaque sizing and some applications of the technique in antibody feedback experiments. J. Immunol. Methods 129, 1. Jerne, N.K., Nordin, A.A. and Henry, C. (1963) The agar plaque technique for recognizing antibody producing cells. In: B. Amos and H. Koprowski (Eds.), Cell-Bound Antibody. Wistar Institute Press, Philadelphia, PA. Jerne, N.K., Henry, C., Nordin, A.A., Fuji, H., Horos, A.M.C. and Lefkovits, I. (1974) Plaque forming cells: methodology and theory. Transplant. Rev. 19, 130. Kappler, J.W. (1974) A micro-technique for hemolytic plaque assays. J. Immunol. 112, 1271. Poduval, T.B., Seshadri, M., Thakur, V.S. and Chauhan, P.S. (1990) Effect of multigeneration alcohol feeding on murine immune system. Ind. J. Exp. Biol. 28, 821. Simpson, M.A. and Gozzo, J.J. (1978) Spectrophotometric determination of lymphocyte mediated sheep red blood cell hemolysis in vitro. J. Immunol. Methods 21, 159.