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Use of SeraSub as a Serum Diluent in the
Manufacturing of ELISA Controls

by: M. Kopnitsky, B. Langer
Zeus Scientific, Inc., Raritan, NJ 08869

ABSTRACT

During the manufacture of ELISA calibrators and controls, it is frequently necessary to dilute antibody positive material to a desired optical density (OD) level. At our manufacturing facility, this was accomplished by diluting the antibody positive sera using antibody (specific antibody) negative sera. This practice maintains the consistency of the solution yet dilutes the positive antibody to a point where the targeted OD is achieved. A study was conducted to explore the possibility of using a commercially available, synthetic serum diluent (SeraSub^TM) in place of the negative sera as a material for diluting positive sera. The potential benefits in doing so included but were not limited to the elimination of potential sera-to-sera adverse interactions, the ability to acquire a large supply of a single lot of serum diluent and the freeing of serum storage space (<-20° C) since the commercial serum diluent may be stored at room temperature. Two different ELISA test systems were used to validate the material. One ELISA test system utilizes an autoimmune (human) IgG antibody as the positive control antibody (SSA IgU ELISA Test System) and the other utilizes a human 1gM antibody as the positive control antibody (EBV-VCA 1gM ELISA Test System). The experimental study involved diluting positive material in both negative serum (control) and in SeraSub at multiple two-fold dilutions starting with the neat positive material and ending with a 1:128 dilution. The bulk materials were aliquotted into screw-capped polypropylene microvials and were stored at multiple temperatures (< -20° C, 2-8° C, room temperature, 37° C and 45° C). Periodically, over a 21 day period, aliquots of each dilution (both test and control diluent) at each storage condition were removed and tested simultaneously on the respective test system. Optical densities (4SOnm) were obtained and maintained in a spreadsheet for analysis following the completion of the testing. At the conclusion of the testing period, scatter plots were generated showing the OD of the sera diluted in negative sera versus the OD of the sera diluted in SeraSub for all dilutions stored at the standard storage conditions of 2-8° C. The anti-SSA antibody scatter plot yielded an r2 value of 0.980 with a slope of 0.991 and a Y intercept of 0.0094. The anti-EBV VCA 1gM antibody scatter plot yielded an r2 value of 0.997 with a slope of 0.967 and a Y intercept of —0.0261. Analysis of the sera stored at elevated temperatures indicated that those serum dilutions prepared using SeraSub were more stable than similar serum dilutions prepared using negative serum. We have concluded that SeraSub may be used as an alternative to negative serum as a control serum diluent.

MATERIALS AND METHODS

Two different types of human serum were used to validate the use of SeraSub. Human serum positive for anti-SSA lgG antibody and another positive for anti-EBV YCA 1gM antibody were used as the antibody positive sera. These types of sera were chosen since they represented the ‘worst case scenarios’ for antibody stability (an autoimmune IgG and an 1gM test system). Two human sera negative for these antibodies were used as the control material for diluting the positive sera, while SeraSub (developed and manufactured by CST Technologies, Inc., Great Neck NY 11021, USA, distributed by CYRX, Raritan, NJ, USA) was used as the experimental serum diluent.

ELISA test systems were used to assess the antibody reactivity of the experimental sera. The anti-SSA IgU antibody specimens were evaluated using the Zeus Scientific, Inc. SSA ELISA test system, and the anti-EBV VCA 1gM antibody specimens were evaluated using the Zeus Scientific, Inc. EBY VCA 1gM test system.
Although the recommended storage conditions for the ELISA control sera are 2-8° C, the study was designed to assess the stability of the experimental sera under stressed storage conditions using both the control and experimental means of diluting the positive sera. The study was set-up as follows: Briefly, the two positive specimens were diluted in both negative serum (control) and in SeraSub (test condition) at multiple two-fold dilutions starting with the neat positive material and ending with a 1:128 dilution. The bulk materials at each dilution were dispensed (O.5mL/aliquot) into screw-capped polypropylene microvials and stored at multiple temperatures (<= ~2O0 C, 2-8° C, room temperature, 37° C and 45° C). Periodically over a 21-day period, aliquots of each dilution at each storage condition were removed and tested simultaneously on the respective test system in triplicate. All assays were performed according to the manufacturer’s instructions. Testing intervals were day 0 (baseline testing), day 3, day 7, day 14 and day 21. Optical densities (4SOnm) were measured and maintained in a Microsoft® Excel® spreadsheet for analysis following the completion of the testing.

RESULTS

The raw data from the various testing intervals appears in Tables 1 through 8 below.

DISCUSSION AND CONCLUSION

It is imperative that manufacturers of ELISA test systems are able to meticulously optimize positive human sera to yield a desired optical density on that respective ELISA test system. Diluting positive sera with negative human serum has been the normal process; however, this practice is difficult in that it is virtually impossible to obtain large quantities of a uniform (nonpooled) serum. Pooling sera overcomes this obstacle, but can introduce problems arising from mixing several human sera together. SeraSub, a synthetic serum diluent, can be purchased in large quantities (greater than 200L per lot), can be stored at room temperature and eliminates the potential for problems surrounding the mixing of different sera.

A cusory review of the data presented in Tables I through 8 above indicates that there is great similarity in the ODs obtained using either negative sera or SeraSub as the diluent for the positive sera. The anti-SSA IgG antibody showed less decline in activity over the 21-day period at stressed temperature
conditions. Although the anti-EBV YCA 1gM antibody deteriorated significantly under stressed conditions of 45° C, it appears that the antibody held-up slightly better when diluted in SeraSub as compared to negative sera. In fact, the SeraSub-diluted 1gM antibody (1:2) was more stable (showed more activity) than the neat, undiluted 1gM positive material.

Scatter plots and regression analyses of the diluted specimens (both methods) stored at 2-8 C indicate a very high degree of correlation between the optical densities obtained. This, coupled with the statistical analyses as recommended by Bland and Altman(1), indicate a very high degree of correlation between the two methods of preparation. The data presented through this investigation indicate that the performance of the antibody positive sera prepared using SeraSub should not differ significantly from the same product prepared using negative sera. In fact, there is data indicating that stability may be improved through the use of SeraSub.
 

REFERENCES

1. Bland, J.M., Altman, D.G., 1986. Statistical Methods for Assessing Agreement Between Two Methods of Clinical Measurement. Lancet. 1(8476), 307-3 10.