Archivee of

Arch. Dermatol. Res. 265, 189-194 (1979)

Deermatological search

O Springer-Verlag 1979

A Semiquantitative Measurement of Anti-Nuclear Antibody Using Immuno-Microfluorimetry* Hiroaki Ueki ~, Itaru Yoskii 2, Akira Ikeda 2, and Nozomi Nohara 3 1 Departent of Dermatology, Kawasaki Medical School, Kurashiki, Japan 2 Department of Anatomy, Kawasaki Medical School 3 Department of Dermatology, Okayama University Medical School, Okayama, Japan

Summary. 1. Using microfluorimetry, the strength of fluorescence was measured on fluoresceinated anti-nuclear antibody of SLE sera. The indirect "Sandwich" method was applied using human peripheral lymphocytes as substrates tissue. 2. The results of using three FITC-labelled anti-human IgG conjugates of different types were compared with each other. 3. More specific and more consistent results were obtained with conjugates with a lower F/P ratio and lower antibody and protein concentration than those in the case of conjugates with a higher F/P molar ratio and higher antibody and protein concentration. 4. The immuno-microfluorimetric technique is very useful for the objective determination of the results of fluorescence microscopy and for the comparison of conjugates of different qualities.

Key words: Immuno-microfluorimetry- Microfluorimetry - A N F -

F/P

molar ratios

Zusammenfassung. 1. Mit Hilfe der Immun-Mikrofluorimetrie wurde die Fluorescenz-Stfirke der antinuklefiren Antik6rper bei SLE-Seren gemessen. Es wurde die indirekte Immun-Fluorescenz-Methode angewandt, und menschliche periphere Lymphocyten als Antigene benutzt. 2. Es werden die Resultate aus der Anwendung von drei verschiedenen FITC-markierten anti-human IgG-Konjugaten miteinander verglichen. 3. Die spezifischeren und bestfindigeren Resultate wurden mit Konjugaten der niedrigeren F/P mol-Relation und der niedrigeren Antik6rper- und EiweigKonzentration erlangt, nicht mit den Konjugaten der h6heren F/P molRelation und der h6heren Antik6rper- und Eiweil3-Konzentration. * This study was supported by Kawasaki Medical Grant No. 51206 for project research Offprint request to: Prof. Dr. Hiroaki Ueki (address see above)

0340-3696/79/0265/0189/$1.20

190

H. Ueki et al. 4. Diese i m m u n f l u o r i m e t r i s c h e T e c h n i k ist geeignet, die U n t e r s u c h u n g s e r gebnisse, o b j e k t i v u n t e r d e m F l u o r e s c e n z - M i k r o s k o p zu beurteilen u n d K o n j u gate unterschiedlicher Qualit~it m i t e i n a n d e r zu vergleichen. Sehliisselwiirter: I m m u n - M i k r o f l u o r i m e t r i e F/P molare Relationen

Mikrofluorimetrie - ANF -

I m m u n o f l u o r e s c e n c e technique is n o w a d a y s widely a p p l i e d to biological, medical, a n d l a b o r a t o r y studies, being used in a p a r t for r o u t i n e p u r p o s e s in hospitals as, for example, in detecting F T A ( f u o r e s c e n t t r e p o n e m a l a n t i b o d y ) , A N F (anti-nuclear a n t i b o d y ) , a n d o t h e r a u t o a n t i b o d i e s or i m m u n e deposits in h u m a n tissues. The increasing d e m a n d s for the i m m u n o f l u o r e s c e n t test led us to c a r r y o u t trials on the s t a n d a r d i z a t i o n o f the technique, as the results f r o m different l a b o r a t o r i e s are n o t always in a g r e e m e n t owing to different optics, conjugates, materials, a n d p r o c e dures. T h e r e are a l r e a d y r e p o r t s o f a t t e m p t s to s t a n d a r d i z e the i m m u n o f l u o r e s c e n t technique. K a w a m u r a et al. [4, 5] have d e v e l o p e d a new m e t h o d using a superwide d a r k field c o n d e n s o r ( T i y o d a ) a n d a f l u o r i m e t r i c a p p a r a t u s a p p l i e d to the s t u d y o f the F T A test. O t h e r trials were p e r f o r m e d b y G o l d m a n [2]. I n the field o f d e r m a t o l o g y , G i a n n e t t i a n d C o r m a n e [1] have t r i e d to m e a s u r e the specific fluorescence o f p e m p h i g o i d a u t o a n t i b o d i e s , using different conjugates u n d e r the fluorescence microscope. Recently, I k e d a [3], using an O l y m p u s fluorescence m i c r o s c o p e , r e p o r t e d o n an i m m u n o - f l u o r i m e t r i c analysis o f a specific fluorescent a n t i b o d y a g a i n s t m o u s e lens crystallins. In the p r e s e n t s t u d y the fluorimetric trials were carried o u t on A N F , using an O l y m p u s f l u o r i m e t r i c a p p a r a t u s with a single b e a m a n d a s p o t exciting system. One o f the m o s t i m p o r t a n t a d v a n t a g e s o f this i n s t r u m e n t is t h a t a c o n s t a n t light value can be o b t a i n e d b y D C electric current.

Materials and Methods

Optical System and Apparatus An Olympus single beam microfluorimeter was used in our present study. Its optical system is shown briefly in Table 1, and elsewhere in the literature [3]. The lighting value was kept constant (the fluctuation rate was less than 1% in an hour) by applying a DC electric current and a special type electric plate. The size of the pin hole was adjusted to such a scale that each single fluorescent nucleus of the lymphocytes could be observed just inside of the hole. For the microfluorimetry, the fluorescent magnified image was formed in the plane of the image diaphragm and only this light flux was measured by the photomultiplier.

Immunofluorescent Proceduresfor ANF Smears of human peripheral leukocytes were air-dried on plass slides, fixed in 95 ~ ethanol for 5 min, and washed for 15 min in PBS (phosphate buffered saline; pH 7.2; 0.005 M). Diluted sera of SLE patients were dropped on the fixed smears and incubated for 30 min in a moist chamber at 20~C. After washing three times in PBS for 15 min, the smears were incubated in the FITC-tabelledanti-human IgG antibody solutions of different types for 30 rain, in a moist chamber at 20~ and washed again in PBS. The treated smears were mounted in PBS-Elbanol. The FITC-labeled conjugates were twice absorbed with acetone powder of mouse liver before use. Three kinds of different conjugates were used and their characteristics are shown in Table 2.

Immuno-Mirofluorimetry of ANF

191

Table l. Optical system applied to immuno-microfluorimetryOlympus fluorescencelight microscope with an Olympus single beam microfluorimeter and transmitting illumination system Light source: High pressure mercury vapor bulb of 200 W (Wacom, BMO-200 D) Exciting filter: BG 12 blue filter Barrier filter: Wratten 2 B Condenser: Light field condenser Spot exciting system Pin hole: Diaphragm: Sensitivity: Observation:

5 6 1023 x 400

Table 2. Characteristics of FITC-labeled anti-human IgG antibody solutions used in this study A" F/P (fluorescein/protein) weight ratio F/P molar ratio Ab P/P (specific antibody protein/ protein) molar ratio Ab P/F (specific antibody protein/ fluorescein) molar ratio Protein-bound FITC concentration Total protein concentration Specific antibody concentration Dilution used

Bb

C~

6.7 mcg/mg 2.8

6.7 mcg/mg 2.8

4.4 mcg/mt 1.78

0.041 0.015

0.041 0.015

0.047 0.026

289.0 mg/ml 43.4 mg/ml 1.8 mg/ml 1:10

289.0 mcg/mt 43.4 mg/ml 1.8 mg/mt 1:50

17.5 mcg/ml 4.0 mg/ml 188 mcg/ml 1:5

a Conjugate A b Conjugate B c Conjugate C

The blocking tests were performed applying the unlabeled anti-human IgG antibody. Each fluorescence strength (FS) from fluorescentlymphocyteswas measured as relative electric values from 0 to 100.

Results Specific fluorescence (FS) was measured using the fluorimeter. T e n fluorescent nuclei were m e a s u r e d at r a n d o m in each sample a n d s h o w n as closed circles, with the b a c k g r o u n d fluorescence as_ opened cirlces. Figure 1 shows the results of A N F o b t a i n e d in the study using the conjugate A. FS of A N F showed higher values in c o n c e n t r a t e d sera o f patients especially in u n d i l u t e d a n d 10-fold diluted sera o f SLE. I n over 10-fold dilutions, FS revealed c o n s t a n t values between 20 a n d 50, while in the more c o n c e n t r a t e d sera, FS ranged in value between 50 a n d 100, a n d were n o t completely blocked by the earlier applications of u n l a b e l e d a n t i - h u m a n IgG, or a b s o r b e d with the h u m a n IgG.

H. Ueki et al.

192 FITC-labeled anti-human IgG F/P molar ratio 2.8 Specific Ab content 180 mcg/ml

F] u o r e s c e n c e

Strength

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Fig. 1. Fluorescence strength of A N F is shown as closed circles, with the background fluorescence as open circles. Results were obtained using the conjugate A. High non-specific fluorescence was observed both in the undiluted and the 10-fold diluted sera of SLE patients, while the relatively lower specific fluorescence was measured in the more diluted sera

Dilution of $era

FITC-labeled anti-human IgG F/P molar ratio 2.8 Specific Ab. content 36 mcg/ml

Fluorescence Strength

Pin hole 6 Diaphragm 5 Sensitivity 103 760 V

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Fig. 2. Results obtained using the conjugate B. The specific fluorescence was measured constantly in the diluted sera, although high non-specific fluorescence was observed in the concentrated sera

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Results using the conjugate B are shown in Fig. 2. The FS values were lower than those of Fig. 1. The sera of patients of over 20-fold dilutions showed constant FS values between 10 and 20, while FS in undiluted sera ranged between 20 and 30, FS in 10-fold diluted sera were between 25 and 45, respectively. The most constant results were obtained in the study using the conjugate C, as shown in Fig. 3. The specific FS values ranged between 10 and 20, which were completely absorbed with the human IgG and blocked with the unlabeled antihuman IgG. The FS values were shown to be constant both in the concentrated and the diluted sera. The background fluorescence strength ranged between 3 and 15, shown to be lower in the study using conjugate C, while the study using conjugate A revealed relatively higher values. The relationship between the specific FS and sensitivities of the microfluorimeter are demonstrated in Fig. 4. The FS values and the sensitivities of the fluorimeter paralleled each other well.

Immuno-Mirofluorimetry of ANF

193 Fluorescence Strength

Ag: Human Leukocytes Ab: SLE Serum (Okase) riTe-labeled anti-human IgG F/P molar ratio 1.78 Specific Ab content c37.6 mco/ml

30 25 20

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Fig. 3. Results obtained using the conjugate C. The specific fluorescence was observed constantly both in the concentrated and the diluted sera

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Fig. 4. The specific fluorescence strength of each of five fluorescent nuclei was measured using the different sensitivities of the fluorimeter. The well paralled results were obtained in the sensitivities of 900 and 970

30. 20, 10, o oo

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700

7~0

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900

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Sensitivity

Discussion

In order to adequately compare immunofluorescent results from different laboratories, it is necessary to standardize materials and technical procedures and reagents including antigens, antibodies, conjugates, fixatives, as well as optical systems. In our present study, conjugates of different types were selected and their relative value was assessed. Detection of A N F is one of the most simple tests for immunofluorescent and microfluorimetric study, because stable nuclear antigens can be obtained constantly from peripheral blood samples and human peripheral lymphocytes show a round shape with no detectable autofluorescence. Each single lymphocyte was observed just within pin hole 6 of the Olympus Fluorescence Microscope System. The fluorimeter always gave a constant value. During the fluorimetric measurements, no loss of fluorescence was observed, and the measurements were carried out in a few seconds. Our present study showed clearly that specific and constant results were obtained using the conjugate C with the F/P molar ratio of 1.78. The higher FS values and the non-specific fluorescence were observed with conjugate A with the

] 94

H. Ueki et at.

F/P molar ratio of 2.8 and the higher antibody and protein concentrations. As stressed repeatedly by Kawamura [4, 6] we also observed non-specific staining of ANF when using conjugate A. When tissue sections were used as antigen substrates in these studies, undesirable non-specific staining was more obvious. If only conjugates with higher F/P molar ratios are available, the conjugate solutions should be used in sufficient dilutions to be blocked with the specific unlabeled antibody solutions. Similarly the sera of patients to be sandwiched should be diluted over 20 times. Gianetti and Cormane [1] have reported similar results using their microfluorimetric test. They showed that non-specific staining decreased more when the conjugate was diluted than the specific staining did. The results in Fig. 4 show that FS values are in good accordance with the sensitivities of the fluorimetric apparatus, when it is used in the sensitivities of 900 or 970. These results certainly can be compared to those obtained from other laboratories using different sensitivities of the fluorimeter. One of the most important advantages of this optical system is that the appropriate-size of pin holes can be selected easily, adapting the size of each fluorescent material, and that only such fluorescence emitted through single cell or tissue can be measured under the microcscope. The measurement is relatively simple and carried out in a few seconds without any loss of fluorescence. With the application of microfluorimetry, immunofluorescent studies can be performed more objectively. Acknowledgements. The technical assistance of Miss T. Hijiya is gratefully acknowledged.

References 1. Gianetti, A., Cormane, R. H. : Quantification of immunofluorescence by microfluorimetry. Arch. Dermatol. Forsch. 246, 249-270 (1973) 2. Goldman, M.: An improved microfluorimeter for measuring brightness of fluorescent antibody reactions. J. Histochem. Cytochem. 15, 3 8 - 4 5 (1967) 3. Ikeda, A. : The immuno-microfluorimetric methods and its practical applications: on the immunofluorescence study of the mouse lens crystallins. Acta Histochem. Cytochem. 7, 265-273 (1974) 4. Kawamura, A., Jr. : Fluorescent antibody techniques and their applications, pp. 38. Tokyo : Univ. of Tokyo and Park Press 1969 5. Kawamura, A., Jr., Mizuoka, K., Matsuhasi, T., Fukuoka, Y. : Methods used in Japan for FTA-ABS tests. In: Immunopathology of the skin. Beutner, E. H., Chorzelski, T. P., Bean, S. F., Jordon, R. E. (eds.), pp. 424. Stroudsburg, PA. U.S.A.: Dowden, Hutchinson & Ross, Inc. 1973 6. Yamamoto, A., Kawamura, A., Jr., Wada, K. : Observation with the fluorescence microscope and standardization of reading results. In: Standardization in immunofluorescence., Holborow, E. J. (ed.), pp. 113. Oxford and Edinburgh: Blackwell Scientific Publications 1970 Received September 4, 1978