QUANTITATIVE IMMUNOFLUORESCENCE STUDIES C . E. D. Taylor and G . V. Heimer

Public Health Laboratory Department of Microbiology Central Middlesex Hospital Park Royal, London NWlO 7NS, England

In 1891, the physicist William Thomson, later Lord Kelvin,' made a statement that has an important bearing o n immunofluorescence techniques in their present stage of development. ". . . when you can measure what you are speaking about and express it in numbers you know something about it; but when you cannot measure it, when you cannot express it in numbers, your knowledge is of a meagre and unsatisfactory kind: it may be the beginning of knowledge, but you have scarcely, in your thoughts, advanced t o the stage of science, whatever the matter may be." For several years, this concept has been reflected in t h e need expressed by many workersZp7 for a n objective means of assessing the brightness of fluorescence emission in microscopic preparations. As a result, several microphotometers for measuring fluorescence emission are now commercially available, but their use generally has been restricted because of their high cost and complexity. Earlier this year, we published a paper8 that describes and evaluates a relatively simple and inexpensive means of measuring immunofluorescence emission from single cells. Our equipment, which incorporates fiber optics, may be used in conjunction with any fluorescence microscope. Furthermore, it has been calibrated, so that measurements of fluorescence emission from any selected small area may be expressed in standard international physical units, which thus offers the possibility of comparing results obtained in different laboratories or even in t h e same laboratory on different occasions. First, I should like t o describe briefly our apparatus and the method of using it before reporting o n its application in the quantitative assessment of fluorescent conjugates. Our apparatus includes a modified inverted microscope that incorporates a tungsten halogen lamp, a Tiyoda dark-field condenser, and a primary alldielectric interference filter, together with a matching secondary yellow glass filter. The interference filter is specifically designed for use with microscopic preparations treated with fluorescein isothiocyanate-labeled antibodies. It transmits a certain amount of red light, the intensity of which is reduced by placing a blue glass filter in the excitation light path. To prevent microscopic preparations from fading while the equipment is being adjusted and an object is being selected for measurement of its fluorescence emission, an additional orange glass filter is inserted temporarily in t h e excitation light path. A variable diaphragm on the xl00 objective has been set in a fixed position so as t o ensure a constant numerical aperture that matches the dark-field condenser. The photometric equipment consists of the following components:

a x 1 0 microscope eyepiece, in t h e center of which is a fiber optic probe that collects light from a known area o n the microscope slide. The size of this area depends o n the magnifying power of the objective lens. Probes of

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different sizes are available and may be used in combination with objective lenses of different magnifying power. Routinely, we use a probe with a circular tip 150 p m in diameter and that collects light from a circular area on the microscope slide of 3.75 p m in diameter with a x40 objective lens and 1.5 p m in diameter with a xl00 objective lens. a flexible fiber optic, which connects the fiber optic probe in the microscope eyepiece to a side-looking photomultiplier tube, which has a n integral electrostatic shield and S4 photosurface and which is in a housing equipped with a photopic correction filter f o r the S4 photosurface and a variable shutter. The photomultiplier tube is connected t o a battery-operated log linear photometer, the output of which may be connected to a chart recorder. The apparatus was calibrated initially in July 1971 at t h e Aeronautical Quality Assurance Directorate, Ministry of Defence, Harefield, Middlesex, England, by comparing fluorescence emission from a piece of uranium glass, used as a transfer standard, with emission from a standard light source calibrated in t h e National Physical Laboratory, Teddington, Middlesex, England. The uranium glass has emission characteristics similar t o those of fluorescein. With our light source supplied with a standard voltage and the optics adjusted t o give a maximum reading on the photometer, the apparatus was caolibrated in candellas per square meter (cd m-2) a t an ambient temperature of 21 C. Recalibration has been performed o n two occasions in a similar manner by the same authority. Readings obtained from the uranium glass are within the limits of experimental error (+lo%) for the calibration procedure, which therefore indicates acceptable stability. T o measure fluorescence emission from a single cell in a microscopic preparation, t h e orange glass protection filter is first inserted in the excitation light path before bringing the image of the object to be measured beneath the tip of t h e probe. The orange glass protection filter is then removed, and the maximum response o n t h e chart recorder is regarded as representing the fluorescence emission from that particular object. As a model for our work, we have used one of the well-known pathogenic serotypes of Escherichia coli, namely, serotype 026, treated with homologous antibody conjugated with fluorescein. Routinely, we take a single reading from each of 10 bacteria, one in each of 10 microscopic fields at least five fields distant from one another. TABLE 1 shows replicate readings of fluorescence emission obtained in a typical experiment. F o r each dilution of the conjugate are shown 10 readings and their means, standard deviations, and coefficients of variation. In TABLE 2 may be seen the summarized results of five such experiments (based on a total of 50 replicate readings for each dilution of the conjugate). There are many possible uses f o r equipment such as this, for example determining the working dilution of a conjugate o r determining endpoints in routine titrations of the many different antibodies demonstrable by immunofluorescence in human and animal sera. The possibility exists, also, of incorporating fiber optics in an immunofluorescence system for the rapid and automated detection of pathogenic microbes in clinical specimens. First, however, we thought it important t o perform more basic work and t o consider how this means of measuring fluorescence emission might be used for evaluating fluorescent conjugates. After all, these are, so to speak, the basic tools of the trade. Without good conjugates, all efforts are wasted.

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TABLE 1 REPLICATE READINGS O F FLUORESCENCE EMISSION (CD M - 2 ) OBTAINED IN A TYPICAL EXPERIMENT Reciprocal Dilutions 4n

Mean Standard deviation Coefficient of variation

320

450 500 500 550 470 490 480 600 400 480

80 370 520 5 20 450 400 470 300 440 350 420

160 290 280 290 350 300 250 300 260 280 250

130 140 180 140 130 160 140 150 160 230

492 53.9 10.95

424 71.36 16.83

285 29.53 10.36

156 30.25 19.39

640 150 90 100 90 60 90 90 80 90 100 94 22.69 24.14

Now, there are two important attributes of a good conjugate. One is its potency and the other its specificity. To begin with, therefore, we have turned our attention to the problem of measuring and expressing the potency of conjugates. One way of doing this might be to express potency in terms of the dilution that corresponds t o an arbitrarily selected fluorescence emission under standardized conditions. Such a method, however, would not take account of the slope of the dose-response line that relates fluorescence emission t o dilution, and this slope may have an important bearing o n the quality of the conjugate. Therefore, we have applied a method of evaluation well established in the field of biologic standardization and have compared test conjugates with a reference conjugate in a quantitative assay in which fluorescence emission is related t o log TABLE 2 REPLICATE READINGS OF FLUORESCENCE EMISSION (CD M-2). OF RESULTS OF FIVE EXPERIMENTS

SUMMARY

Reciprocal Dilutions Experiment 1 mean Experiment 2 mean Experiment 3 mean Experiment 4 mean Experiment 5 mean Grand mean (mean of all 50 values) Standard deviation (of all 50 values) Coefficient of variation = (standard deviation/ grand mean) x 100

40 492 463 455 507 486

80 424 412 429 423 374

160 285 318 274 262 335

320 156 159 190 165 164

640 94 100 107 114 97

480.6

4 12.4

294.8

166.8

102.4

67.23

69.32

43.1

32.28

18.79

13.99

16.81

14.62

19.36

18.35

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dose. Relative potencies were then calculated by standard statistical methods for parallel line assay^.^ FIGURE 1 shows regression lines for three E. coli 026 conjugates (designated A, B, and C). Conjugate A is one with which we have had considerable experience and has proved to give good results in routine work. For purposes of this experiment, therefore, we may regard conjugate A as a reference conjugate. One of the test conjugates (designated B) shows a dose-response line almost parallel t o that of the reference conjugate (designated A), whereas the slope of the dose-response line for the other conjugate (designated C ) is so obviously different from that of t h e reference conjugate that a valid comparison cannot be made. Analysis of the data that relate t o conjugates A and B only confirmed that they were comparable (deviation from parallelism p > 0.05). Accordingly, the potency of conjugate B relative t o conjugate A, the reference preparation, was calculated. If, therefore, the reference conjugate A were t o be allotted an arbitrary potency value of 100 units per unit volume, conjugate B would have a potency value of 50.4 units per unit volume, with fiducial limits of 42.2 and 60.7 at the 95% level of significance. We suggest that such a method of comparing fluorescent conjugates and the principle of allotting potency values to test conjugates in terms of suitable

I 10

20

40

e0

160

320

RECIPROCAL OILUTIONS

FIGURE 1 . Dose-response lines of three E. coli 026 conjugates. A, reference conjugate; B & C, test conjugates.

Taylor & Heimer: Quantitative Studies

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reference preparations should be applied more widely with a view to this becoming a standard procedure. ACKNOWLEDGMENTS We are grateful to Dr. H. G. S. Murray for suggesting a statistical method applicable to parallel line assays.

REFERENCES 1. KELVIN, W. 1891. Electrical units of measurements. I n Popular Lectures and Addresses. 2nd edit. Vol. 1: 80, 81. Macmillan, London, England. 2. CHERRY, W. B. 1970. Fluorescence emission with special reference to standardization in immunofluorescence. In Standardization in Immunofluorescence. E. J. Holborow, Ed.: 127-136. Blackwell Sci. Publ. Oxford and Edinburgh, England. 3. PLOEM, J . S. 1970. Quantitative immunofluorescence. In Standardization in Immunofluorescence. E. J. Holborow, Ed.: 63-68. Blackwell Sci. Publ. Oxford and Edinburgh, England. 4. TAYLOR, C. E. D. 1970. Optical features of immunofluorescence. In Standardization in Immunofluorescence. E. J. Holborow, Ed.: 15-22. Blackwell Sci. Publ. Oxford and Edinburgh, England. 5. TOMLINSON, A. H. 1970. Illumination. In Standardization in Immunofluorescence. E. J. Holborow, Ed.: 5-10. Blackwell Sci. Publ. Oxford and Edinburgh, England. 6. FAGRAEUS, A. 1971. Purpose and scope of the conference. Ann. N.Y. Acad. Sci. 177: 10, 11. 7. ESPMARK, J. A., M. GRANDIEN & N. R. BERGQUIST. 1971. Quantitative evaluation of direct and indirect immunofluorescence tests for viral antigens. Ann. N.Y. Acad. Sci. 177: 98-100. 8. TAYLOR, C. E. D. & G . V. HEIMER. 1974. Measuring immunofluorescence emission in terms of standard international physical units. J . Biol. Stand. 2: 11-20. 9. FINNEY, D. J . 1952. Statistical Methods in Biological Assay. Griffin. London, England.

DISCUSSION DR. F. W. D. ROST: I think that Dr. Taylor has perhaps concealed the amount of time and energy involved in refining his apparatus to its present state. Looking at his abstract, it would appear very easy t o do microfluorometry by just putting a photometer on a microscope. I am sure that Dr. Taylor agrees with me that it is not so simple. Also, the kind of instrumentation is not very important, whether very simple or very complicated, like the machine I will discuss later. As Dr. Taylor said, as long as one is aware of what the apparatus can do and how accurate o r inaccurate it is, the only requirement is t o stay within the limitations of the machine. With respect to standards, I think Dr. Taylor only mentioned that his standard had a similar emission to FITC. I would like t o point out that it is equally important that the standard have the same excitation spectrum, because

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you cannot guarantee that the special distribution of your light, even if i t comes through some filter, may not change. Particularly, for an iodine quartz lamp, the spectrum will change with changes in voltage. So, ideally, one should use a liquid solution of FITC, as with the Thaer-Sernetz standard, or the DASS method. DR. TAYLOR: We appreciate remarks that have been made previously with regard to the limitations of uranium glass. Of course, we are using this material simply as a transfer standard for purposes of calibrating the instrument and not using it for direct comparison of conjugates. We propose that one should make comparisons of like with like, in other words, unknown conjugates with reference conjugates. DR. J. S . PLOEM: I think that we have t o be careful with uranium glass. It is very dependent on the aperture and on the position of the collecting lens and light source. Its only feasible use was as a calibration standard in relation to another reference standard. If one uses uranium glass as a calibration standard, several parts of the microscope may not be touched. As soon as the field diaphragm o r the collecting lens is altered, a different reading is obtained from the uranium glass, The readings, however, were highly reproducible within 1% if one is careful not t o alter the microscopic setup. I think that this cannot be true if more researchers use the same measuring microscope. We talk in terms of standard physical units with our spectrograph, because we want to know the quantum efficiency of fluorochrome. It would be complicated if everyone had to send their microscopes to the National Physics Laboratory for standardization. I think we need only a relative standard. It would, however, be interesting t o have both, because we then could judge the quantum efficiency under certain circumstances. In general, however, it is much simpler t o use a single bead or a single small capillary filled with a fluorochrome as a relative standard. I just want to emphasize that there are different purposes for different standards. DR. TAYLOR: I agree that arbitrary units may be perfectly satisfactory for certain studies and investigations, but if one wants to make permanent records and compare results from one laboratory with those from another, physical units should be used. DR. M. GOLDMAN: I think it is fortunate that Dr. Taylor has introduced the use of fiber optics and that he has put aside difficult centration and mechanical problems involved in the use of diaphragms mounted above the eyepiece, as was and still is the case with some microfluorometers.

Quantitative immunofluorescence studies.

QUANTITATIVE IMMUNOFLUORESCENCE STUDIES C . E. D. Taylor and G . V. Heimer Public Health Laboratory Department of Microbiology Central Middlesex Hosp...
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