Validation of Accuracy of Enzyme-Linked Immunosorbent Assay in

that ELISA is both overreporting (false positives) and underreporting (false negatives) compared to the KinExA system. The large number of hybridoma c...
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Validation of Accuracy of Enzyme-Linked Immunosorbent Assay in Hybridoma Screening and Proposal of an Improved Screening Method Kazuhiro Sasaki,* Thomas R. Glass, and Naoya Ohmura

Biotechnology Sector, Central Research Institute of Electric Power Industry, 1646 Abiko, Abiko City, Chiba, Japan 270-1194

The 96-well plate format of enzyme-linked immunosorbent assay (ELISA) is the de facto standard in screening hybridomas for active antibody. Despite its widespread use, there have been few or no systematic attempts to validate its accuracy and answer the fundamental question, is it finding all the positives? We report here on a comparison between ELISA and a semiautomated flowbased kinetic exclusion assay (KinExA), both used in screening the same hybridoma cell line. Our finding is that ELISA is both overreporting (false positives) and underreporting (false negatives) compared to the KinExA system. The large number of hybridoma cells (e.g., cultured in six 96-well plates) that must be checked is daunting in considering any method other than ELISA for routine screening. To overcome this, we devised a sampling strategy in which wells are combined in a specified pattern, allowing a significant reduction in the total number of measurements required.

The practical application of antibodies continues to expand dramatically. From the diagnostic testing of humans for which they were first applied they have moved to tests for animals,1,2 fish,3,4 plant disease,5 contamination of foods by bacteria,6 envi* To whom correspondence should be addressed. Phone: +81+471-82-8211. Fax: +81-471-83-3347. E-mail: [email protected]. (1) Pan, I. C.; Huang, T. S.; Hess, W. R. J. Clin. Microbiol. 1982, 16, 650-655. (2) Meyer, R. K.; Oesch, B.; Fatzer, R.; Zurbriggen, A.; Vandevelde, M. J. Virol. 1999, 73, 9386-9392. (3) Mourton, C.; Romestand, B.; de Kinkelin, P.; Jeffroy, J.; Le Gouvello, R.; Pau, B. J. Clin. Microbiol. 1992, 30, 2338-2345. (4) Biosca, E. G.; Marco-Noales, E.; Amaro, C.; Alcaide, E. Appl. Environ. Microbiol. 1997, 63, 537-542. (5) Holzem, A.; Nahring, J. M.; Fischer, R. J. Gen. Virol. 2001, 82, 9-15. (6) Barna-Vetro, I.; Gyongyosi, A.; Solti, L. Appl. Environ. Microbiol. 1994, 60, 729-731. 10.1021/ac048823k CCC: $30.25 Published on Web 03/01/2005

© 2005 American Chemical Society

ronmental monitoring for PCB,7-9 pesticides,10 hormones,11-13 and so on. Besides testing, antibodies are currently in use as human therapeutics and there are many significant efforts underway to develop new antibody-based drugs.14 Antibodies are produced not only in animals but in immortalized monoclonal mammalian cell culture (hybridoma),15 and phage displayed antibody libraries constructed by genetic engineering.16,17 Regardless of application, every technique using an antibody requires selection of the particular antibody to be used. The selection complexity varies with the production technique used. In the case of polyclonal antibodies produced in animals, selection of one antisera from a small number of antisera from individual animals is the simplest case. On the other extreme, phage display libraries can easily have millions of separate antibodies to choose from. Hybridoma technology represents a middle ground in which hundreds to thousands of potential antibody clones must be considered. Selection starts with a screening assay in which potential antibodies are screened for binding to the target molecule. ELISA is the de facto standard for antibody screening (7) Chiu, Y. W.; Carlson, R. E.; Marcus, K. L.; Karu, A. E. Anal. Chem. 1995, 67, 3829-3839. (8) Baker, D. B.; Bushway, R. J.; Adams, S. A.; Macomber, C. Environ. Sci. Technol. 1993, 27, 562-564. (9) Schuetz, A. J.; Weller, M. G.; Niessner, R. J. Anal. Chem. 1999, 363, 777782. (10) Dankwardt, A. In Encyclopedia of Analytical Chemistry; Meyers, R. A., Ed.; John Wiley & Sons Ltd.: Chichester, U.K., 2000. (11) Ohmura, N.; Lackie, S. J.; Saiki, H. Anal. Chem. 2001, 73, 3392-3399. (12) Glass, T. R.; Saiki, H.; Blake, D. A.; Blake, R. C., II; Lackie, S. J. Anal. Chem. 2004, 76, 767-772. (13) Snyder, S. A.; Villeneuve, D. L.; Snyder, E. M.; Giesy, J. P. Environ. Sci. Technol. 2001, 35, 3620-3625. (14) Brekke, O. H.; Sandlie, I. Nat. Rev. Drug Discovery 2003, 2, 52-62. (15) Kohler, G.; Milstein, C. Nature 1975, 256, 495-497. (16) Cai, X.; Garen, A. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 9261-9266. (17) O’Brien, P. M.; Aitken, R.; O’Neil, B. W.; Campo, M. S. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 640-645.

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although other methods have been tried for special purposes.18,19 Recently, we described a flow-based immunoassay system (KinExA system) capable of theoretical (antibody limited) sensitivity in analysis of small analytes.11 Others have reported that the same system is 10-1000 times more sensitive than an enzyme-linked immunosorbent assay (ELISA) and other assay formats evaluated.11,20 It occurred to us to evaluate the KinExA as a screening platform and compare its performance to ELISA, the industry standard. In our laboratory, hybridomas are grown in six to eight 96well microculture plates. ELISA starts with a large advantage over KinExA because of its high throughput and ready use of the microplate assay format. In contrast, KinExA does not allow multiple measurements at the same time. The advantage of the KinExA is its sensitivity; some hybridomas grow slowly and produce little antibody at the time of screening, requiring high sensitivity for detection. Therefore, ELISA might miss some antibodies that KinExA’s superior sensitivity could detect. Another potential advantage of KinExA is that occurrence of false positives might be reduced because antibodies contact the solid-phase immobilizing antigen for a much shorter time, which may reduce nonspecific binding to the solid phase. For the present study, we screened the same cell line using both ELISA and KinExA for comparative purposes. We produced antibodies against chelated (EDTA) lead using hybridoma technology and screened the resultant hybridomas three times, once using ELISA, a second time using KinExA to measure every well, and a third time using KinExA in conjunction with a sampling strategy to reduce the overall time for screening. As results, we present evidence that screening with KinExA does find more positives and report fewer false positives than ELISA. In addition, under certain circumstances, the time and labor for KinExA can actually be less than for ELISA. MATERIALS AND METHODS Materials. BALB/cA Jcl inbred mice were purchased from Clea Japan, Inc. (Tokyo, Japan). 1-(4-Isothiocyanobenzyl)ethylenediamine-N,N,N′,N′-tetraacetic acid (isothiocyanobenzyl-EDTA) was obtained from Dojindo (Kumamoto, Japan). Keyhole limpet hemocyanin (KLH) and ovalbumin (OVA) were obtained from Sigma-Aldrich (A2512 and H7017; St. Louis, MO). Cy-5 conjugated F(ab′)2 fragment of goat anti mouse IgG 286402 was obtained from Jackson ImmunoResearch (West Grove, PA). Myeloma cell (NS0) was purchased from The Institute of Physical and Chemical Research Cell Bank (Tsukuba, Japan). Preparation of Protein-Chelate Conjugates. Preparation of protein-chelate complexes was based on Chakrabarti’s report.21 Briefly, 2 mg of protein (KLH or OVA) was dissolved in 3 mL of 100 mM boric acid (pH 9.0) and then mixed with 1 mg of isothiocyanobenzyl-EDTA overnight. During this time, isothiocyanobenzyl-EDTA bound with the amino group of the protein. Before coordinating lead to conjugated EDTA, the pH of the solution was adjusted to 7.0 using a desalting column (10DG; Bio(18) Canziani, G. A.; Klakamp, S.; Myszka, D. G. Anal. Biochem. 2004, 325, 301-307. (19) Zilka, N.; Vechterova, L.; Kontsekova, E.; Novak, M. J. Immunol. Methods 2003, 272, 49. (20) Blake, D. A.; Jones, R. M.; Blake, R. C., II; Pavlov, A. R.; Darwish, I. A.; Yu, H. Biosens. Bioelectron. 2001, 16, 799-809.

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Rad Laboratories; Hercules, CA) to prevent metal precipitation. To make Pb-EDTA-protein conjugate, 80 µL of 10 µM PbCl2 solution was added to 4 mL of 1 mg/ml EDTA-protein solution. Immunization of Mice and Hybridoma Production. Three mice were injected intraperitoneally at 2-week intervals with PbEDTA KLH conjugate emulsified in adjuvant (TiterMax Gold; CytRx; Norcross, GA). Fourth injections were given 7 days after the third injection. Four days after the fourth injection, spleen cells were fused with myeloma cells using poly(ethylene glycol) 1500 (783641; Roche Diagnostics GmbH; Penzberg, Germany). The fused cells were cultured in six 96-well flat-bottomed microculture plates for 14 days. Solid-Phase Preparation. Antigen-coated Sepharose beads were prepared as follows. One milliliter of NHS-activated Sepharose (17-0906; Amersham Biosciences; Uppsla, Sweden) suspension was washed with 0.1N HCl and then neutralized with phosphatebuffered saline (PBS) buffer (pH 7.4). Metal-EDTA-OVA conjugate solution (1 mg of protein/mL) was added to Sepharose beads and mixed gently overnight. Finally, Sepharose beads were blocked against nonspecific binding with BSA solution (1 mg/ mL) by mixing over 2 h. Immunoassay Procedure for KinExA. Antibody assays were performed with KinExA 3000 (Sapidyne Instruments; Boise, ID). The KinExA 3000 consists of a single flow cell located at the focal point of a filter fluorometer. Solid-phase material (OVA-PbEDTA Sepharose particles in the present case) is suspended and flowed into the flow cell where it is trapped against a screen. Basic operation for KinExA was shown in Figure 1. A single sample (hybridoma supernatant) measurement began with flow of PBS at 0.25 mL/min for 30 s to establish a baseline. Next, hybridoma supernatant (diluted or mixed as described in Results and Discussion) flowed through the solid phase for 120 s at a rate of 0.25 mL/min. During this period, specific antibody present in the sample (if any) accumulated on the solid-phase Pb-EDTA. The solid phase was then washed in PBS for 60 s, followed by flowing Cy-5-anti mouse IgG for 120 s, followed by an additional 30 s of PBS, all at 0.25 mL/min. Finally, residual bulk fluorescence (i.e., not attached to the solid phase) was removed by a 60-s flow of PBS at 1.0 mL/min. The signal difference between the final wash and the initial baseline was calculated and used as a measure of antibody in the sample. Thirteen sample lines are available, and a customized computer interface allows unattended sequential measurement of each, including automatic replacement of the solid phase. Fluorescence readings are recorded automatically once per second and are available for later analysis. Immunoassay Procedure for ELISA. To immobilize EDTAOVA conjugate to polystyrene microplates (Falcon 353911; Becton, Dickinson and Co.; Franklin Lakes, NJ), 50 µL of the conjugate solution (0.01 mg/ml) was added to each well and kept overnight at 4 °C. After the plate was washed with water, 50 µL of 1 mM PbCl2 solution was added to make the Pb-EDTA complex on the solid phase. After washing with PBS containing 0.1% (w/v) Tween20 (T-PBS buffer), 200 µL of T-PBS with 10% fetal bovine serum (FBS) was added to block for 1 h at room temperature. The blocking solution was discarded, and then each well was washed with T-PBS. Each hybridoma supernatant (25 µL) was allowed to bind Pb-EDTA-OVA on the plate for 30 min. After discarding the supernatant and washing with T-PBS for three times, the

second antibody (goat anti-mouse IgG-alkaline phosphatase conjugate; 170-6520; Bio-Rad Laboratories) was allowed to bind for 30 min. Binding of the second (alkaline phosphatase-labeled) antibody was detected using an alkaline phosphatase substrate kit (172-1063; Bio-Rad Laboratories). Alkaline phosphatase activity was measured using p-nitrophenyl phosphate as the substrate and monitoring absorbance at 410 nm with a plate reader (EL808; BioTek instruments; Highland Park, IL).

Figure 1. KinExA principle and representative data. Panel A shows the principle of operation for the screening measurement. In A 1, sample flows over antigen-coated beads and specific antibody is captured. In A 2, the captured antibody is labeled with a Cy5 antispecies antibody, and in A 3, the unbound label is washed out. Arrows from panel A indicate corresponding portions of a representative signal trace in panel B. Panel C shows the principle for a confirmation assay; it is the same as panel A except excess PbEDTA is added in the first step (see panel B 1) preventing binding of Pb-EDTA-specific antibody to the solid phase.

RESULTS AND DISCUSSION Comparison of KinExA and ELISA Measurements for Every Well. Hybridomas were prepared from a mouse immunized with Pb-EDTA-KLH (to prepare anti-Pb-EDTA antibody) in the manner described in the Materials and Methods section. A total of six 96-well plates (576 individual wells) were used for hybridoma culture. As a first check of KinExA’s validity for screening in comparison with ELISA, every well was measured twice, once with ELISA and once with KinExA. To make equitable conditions, we used the same volume of culture medium (25 µL) and the same antigen conjugate (Pb-EDTA-OVA) for both systems. The two systems are analogous in that cell culture supernatant is exposed to the solid phase to allow any Pb-EDTA-specific antibody present to bind to the antigen conjugate on the solid phase. In both systems, a labeled antispecies antibody is applied to quantify the captured primary antibody. Signal from KinExA is determined as the difference of fluorescence before applying secondary antibody and after washing free secondary antibody (Figure 1), while the ELISA signal is an absorbance measurement. The major difference is that the KinExA system is flow based so each increment of the sample contacts the solid phase for a very short time during its passage through the flow cell. It should be noted that the 25 µL of supernatant was applied full strength in the ELISA well but was diluted to a volume of 1 mL in PBS-BSA before applying it to KinExA system. The reason for this is that the KinExA is a flow-based system and the sample lines must be charged with sample prior to use, resulting in a loss of ∼150 µL of sample. Because the sample flows over the solid phase, captured antibody accumulates on the solid phase and diluting a sample in the manner done here does not normally reduce sensitivity.11 Signals from the 576 culture wells are summarized in histograms in Figure 2. To allow easy comparison, signals in Figure 2 were normalized relative to the assay range, from the minimum detected signal (0%) to the maximum detected signal (100%). Normal distribution curves are overlaid in Figure 2 for comparison, and both sets of data clearly include more large values than would be expected from random fluctuations in a normal distribution. At least some of the large signals are presumed due to positive antibody cultures while the large groupings evident in the lowsignal regions of both measurement methods are presumed to represent nonspecific binding from negative samples containing no specific antibody. To determine positive samples, temporary cutoff points were selected to reject the large low-signal groupings of both methods. The value used for the cutoffs is somewhat arbitrary. As discussed later, a lower cutoff will identify more positives, but a larger proportion of these will be false positives. The cutoffs used, indicated as “screening cutoffs” in Figure 2, are 2.3% for KinExA, and 8.0% for ELISA. Using these cutoff points, 30 samples from KinExA and 44 samples from ELISA were Analytical Chemistry, Vol. 77, No. 7, April 1, 2005

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Figure 2. Histogram comparison of KinExA and ELISA response. Signals are scaled to run from zero (the minimum signal observed) to 100% (the maximum signal observed). Curved lines indicate normal frequency distributions.

determined to be positive. A total of 18 samples were determined positive by both methods. If the number of positives depended only on the sensitivity of the measurement method, all positives found by the low-sensitivity method should have been included in the positives from the highsensitivity method. The existence of positives specific to the measurement methods suggests that the positives found may depend on the principle of the method rather than on measurement errors. It may be that KinExA’s short contact time (individual antibody molecules have less than 1 s to bind to the solid phase as they flow through) discriminates against slow on-rate antibodies that have plenty of time to bind during the ELISA incubation. On the other hand, antibodies with relatively fast off-rates may dissociate from the ELISA solid phase during the second antibody incubation and be washed away. Such an event seems much less likely in the KinExA system where the total time for such dissociation to occur is only 3.5 min. Of course, these preliminary screening results may still include nonspecific binding antibodies counted as positives. Confirmation of Specific Binding in Screening Results. Confirmation assays were conducted to find and eliminate positives resulting from nonspecific binding. All cell cultures that gave positive results in the first screening test (a total of 56 wells) were retested with and without the addition of 100 µM Pb-EDTA. If the 100 µM Pb-EDTA signal was significantly lower than the zero Pb-EDTA signal, the culture was judged a confirmed positive containing specific antibody. In accordance with the particular assay formats, the procedures used for KinExA and ELISA were slightly different. In KinExA, antigen (Pb-EDTA) was premixed with culture medium and allowed to bind for 20 min before applying to the solid phase. Antibody bound to antigen in solution could not bind to solid-phase antigen, resulting in an inhibited signal (Figure 1), In KinExA, the time the premixed antibody contacts the solid phase is very short, so the equilibrium of premixed antibody and antigen is not changed by the effects of the antigen on solid phase.11,21 On the other hand, in ELISA, a competitive equilibrium is formed among antibody, soluble antigen, and antigen on the solid phase. 1936

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Figure 3. Results of confirmation assay. All antibodies that tested positive in the initial screening were subjected to a confirmation test (see text and Figure 1 for details). Positives indicate antibodies that passed the confirmation, and negatives are those that failed. Some antibodies are included in more than one group.

Confirmation testing results are summarized in Figure 3. In KinExA, 8 out of 30 samples were confirmed as positive, and in ELISA, 3 out of 44 samples were confirmed. False positives, which were from nonspecific binding to solid phase in the first screening, were 22 out of 30 in KinExA and 41 out of 44 in ELISA. (Figure 3) The false positives ratio was 3.8% (22/576) for KinExA and 7.1% (41/576) for ELISA. Confirmed positives and false positives were distributed as shown in Figure 4. In KinExA, four confirmed positives and seven false positives were in signal range from 2.3 (cutoff point for KinExA) to 10%. (Figure 4A) On the other hand, in ELISA, no confirmed positive was in signal range from 8.0 (cutoff point for ELISA) to 70%. (Figure 4B) The temporary cutoff point chosen for KinExA seems reasonable because we got more confirmed positives than ELISA, less false positives than ELISA, and raising it to a higher value results in the loss of confirmed positives. On the other hand, if we set cutoff point for ELISA at 70% instead of 8.0%, the false positives ratio is reduced to 1.9 from (21) Blake, D. A.; Chakrabarti, P.; Khosraviani, M.; Hatcher, F. M.; Westhoff, C. M.; Goebel, P.; Wylie, D. E.; Blake, R. C., II. J. Biol. Chem. 1996, 271, 27677-27685.

Figure 4. Distribution of antibodies detected in the first screening. Positive and negative refer to confirmation test results. Signals are normalized as in Figure 1. The three confirmed positives found by ELISA were all over 70% of the maximum ELISA signal. In contrast, several of the positives found by KinExA were near the cutoff point. Bin size is 5. Panel A, KinExA; panel B, ELISA.

Figure 5. Response of antibody 4H8 to Pb-EDTA-OVA and PbEDTA. Using 4H8 antibody, inhibition signals were measured with Pb-EDTA-OVA (open circle) and with Pb-EDTA (closed circle). The antibody Kd is approximately equal to the 50% inhibition point in this figure and is ∼100 times lower (tighter binding) for Pb-EDTA-OVA.

7.1%, and no confirmed positives would be lost. It seems counterintuitive to set the cutoff as high as 70%, and we are doubtful that such a result would stand up to additional testing but that is what the current data set suggests. Another remarkable result in Figure 3 is that all confirmed positives from ELISA are included in the positives from KinExA. KinExA found two positive samples that were missed by ELISA in the first screening. The time when hybridomas start to secrete antibodies can vary widely. These two positives may represent slow-growth hybridomas that KinExA was able to detect because of its superior sensitivity. Perhaps the most interesting feature of Figure 3 is the three samples that were confirmed positive by KinExA and confirmed negative by ELISA. The same antigen was immobilized on both solid phases (beads for KinExA and plates for ELISA), but this was not simple Pb-EDTA. The solid-phase antigen used here consisted of ovalbumin reacted with isothiocyanobenzyl-EDTA chelated with Pb after immobilization. The benzene ring remains after the formation of the EDTA-OVA complex. The protein used on the solid phase (OVA) is different from that injected in the mouse (KLH), but it is still possible that the some produced antibodies could preferentially bind the benzene-Pb-EDTA complex over the Pb-EDTA complex in solution. In the confirmation assay, the Pb-EDTA was supplied at 100 µM, but if the binding affinity for the benzene ring existing between Pb-EDTA

and ovalbumin on the solid phase is high enough, the solid phase may still “win the competition in the ELISA system. In this case, even though the antibody recognizes and binds to Pb-EDTA, it may still end up bound to the Pb-EDTA-OVA at equilibrium. On the other hand, the KinExA assay is not competitive, antibodies that bind Pb-EDTA in solution cannot bind to Pb-EDTA-OVA on the solid phase. Antibody 4H8 was one of the three antibodies that were observed as specific on KinExA but nonspecific on ELISA. The binding affinities of 4H8 to Pb-EDTA and to PbEDTA-OVA were assayed and compared using the KinExA instrument and the affinity measuring methodology previously described.11,21 The results, summarized in Figure 5, show that 4H8 has ∼100 times higher affinity for Pb-EDTA-OVA than for PbEDTA. Evaluation of a Sampling Strategy for KinExA Screening. The results above show KinExA has an advantage over ELISA in that it finds more positive hybridomas. Weighing heavily against the KinExA, however, is the much longer measurement time needed. A single KinExA measurement (for one well) takes ∼10 min, so the total time was 10 min × 96 wells × 6 plates ) 5760 min () 96 h). ELISA screening of the same six plates was accomplished in ∼8 h excluding immobilizing antigen to solid phase. To reduce the measurement number, a “sampling method” was designed and evaluated as follows. In the first round of testing, supernatant from all 96 wells of a single plate was combined into one tube, as diagrammed in Figure 6A,B. As noted above, when testing every well, the supernatant was diluted by a factor of 40 (25 µL of supernatant in 1 mL of PBS/BSA), so the dilution here was only ∼2.5 times greater. The mixed supernatant was applied full strength to the KinExA system, and the fluorescent signal was measured as described in Materials and Methods. If no positive signal was detected from the tube, the entire plate was judged negative and no further testing was performed. For positive plates, a second round of testing, diagrammed in Figure 6C,D, was conducted in which supernatant from each of the 12 wells in a row was mixed into a single tube. Rows were judged either positive or negative and positive rows were subjected to a third round of testing (see Figure 6E,F), in which each well in every positive row was measured. Analytical Chemistry, Vol. 77, No. 7, April 1, 2005

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Figure 6. Example results of the sampling methods. Ninety-six culture mediums from a single plate were mixed and measured on the KinExA 3000; output traces (panel A) and calculated signals (panel B) from six plates are shown. In panels C and D, 12 culture mediums from each row in the positive plates were mixed and 8 samples were measured. Finally, in panels E and F, 12 individual mediums from each positive row were measured.

Table 1. Comparison of Complete Measurement and Sampling Using the KinExA confirmation

complete sampling

screening

positive

negative

30 23

8 6

22 17

Result of Sampling Method and Comparison with Complete Method. Using the sampling method described above, the same hybridoma sample was screened again and the result was compared with that obtained by measuring every well with the KinExA in the first experiment. In the first round of testing, five of the six plates were found to be positive (the data are shown in Figure 6A,C). In the second round, a total of 12 rows were identified as positive leading to 144 individual well measurements for the final step. Finally, a total of 23 positive wells were found from six plates, seven less than were found measuring every well. Of the seven missed by sampling, two were confirmed as specific to Pb-EDTA and five were false positive. Of the 23 positives identified in the screening, 6 were confirmed specific to PbEDTA. The ratio of false positives from the sampling method was 3.0% (17/576) and it was close to the ratio (3.8%) from the complete method. These results are summarized in Table 1. The sampling 1938

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method missed two confirmed positive hybridomas compared with the complete method. The signal values of these two samples were 2.35 and 3.10% (in Figure 2) and so were close to cutoff point (2.3%). As might be expected from the somewhat greater dilution factor, the sampling method tends to miss weak hybridomas. The lowest positive signal from measuring every well was 2.32%. This well was found in the sampling method as well, but only because it happened to lie in the same row as a much stronger positive well. Comparison Sampling Method and ELISA M ethod. As mentioned above, the sampling method found six specific antibodies. (One of these was a very low positive that would probably have been missed if it had not happened to be in the same row as a stronger positive.) This was lower by two (or three without the accident) than the complete sampling method. ELISA found three positive clones, and each clone was contained in the sampling method findings. One out of three clones that were missed by ELISA was the weak clone found by the sampling method by accident; the other two were false positive in ELISA. These results suggest that the screening performance of the sampling method with KinExA detection is somewhat better than ELISA for this data set. The time required for screening using the sampling method, including confirmation, was ∼21 h, which is compared to 16 h for ELISA. The extra time may be considered worthwhile due to the extra positives identified.

Using the sampling method, the number of measurements and time required vary with the number of positive hybridomas and how they are distributed on the plates. In the worst case calculation (every plate is positive), seven or fewer positives can be found more quickly using KinExA than by ELISA. This number goes up somewhat if the positives are found on fewer plates, but the overall positive rate needs to be on the order of a few percent or smaller if KinExA is to be faster than ELISA. Additional Screening Data Using Sampling Method. Using the sampling method, 23 additional screenings (23 mice × 6 plates ) 138 plates) were performed for various metal-chelate complex antigens. In 15 (more than 62%) of the screenings, no positive was found. In these cases, the KinExA screening was considerably more efficient. To conclude that there was no positive using the ELISA method takes a full day versus 2 h using the sampling method and KinExA. On the remaining 8 plates, we found a total of 26 confirmed positive antibodies and 10 false positives. Only two of the eight plates had more than five positives, resulting in screening taking longer than ELISA. CONCLUSIONS For the hybridoma line screened in this work, the KinExA system found over twice as many positive clones (8 vs 3) as the

ELISA system. Using a sampling method to cut the total time needed for screening, the KinExA method still found six positives. All of the positives identified by ELISA were also identified by KinExA. The results also show that clones having a higher affinity for the conjugated form of the antigen (not uncommon) can be misidentified as negatives on the ELISA due to competition effects. In the case of low rates of success in producing specific antibodies (i.e., small numbers of positive clones), the KinExA system offers a considerably more efficient method of screening. If there are no positive clones, this can be determined in 2 h using KinExA versus 8 h for ELISA. Finally, it is the opinion of the technicians in our laboratory that using the KinExA system is easier and less tedious than the ELISA. We anticipate continuing to use the KinExA for routine screening of hybridoma.

Received for review August 10, 2004. Accepted December 3, 2004. AC048823K

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