. J. Immunol., Vol. 5, 1976.

Prozone Effects in Indirect Immunofluorescence E. UNDER & A. MIETTINEN Department of Serology and Bacteriology, University of Helsinki, Helsinki. Finland

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Linder, E. & Miettinen. A. Prozone Effects in Indirect Scand. J. Immunol. 5, 513-519, 1976.

Immunofluorescence.

A marked prozone effect was observed in indirect immunofluorescence (IrL) with rabbit antisera against rat renal proximal tubular epithelial brush bord' r (BB) antigens: anti-BB antibodies were not detectable if used in high concentrations but were readily demonstrated if sufficiently diluted. The prozone effect occurred in spite nf demonstrated binding of anti-BB antibodies to target antigens. No prozone was seen when direct IFL was used, and 'cross-reacting' antigens in small-intestinal epithelial brush border were detectable hy indirect [FL without pro2One. It was concluded that the anti-immunoglobulin conjugates were unable to reach antigenic determinants on tightly clustered immunoglobulin molecules. Thus prozone effects seem to depend primarily on the density ot' antigen determinants in the target tis.sues. Etvert Linder, M.D,, Department of Serolo^y and Badcriology, Helsinki, Haartmaninkatu j . SP-00290 Helsinki 29. Finland

As a rule, indirect immunofluorescence (IFL) is more sensitive than the direct method. This is the case when the number of available antigenic sites in the target tissue is limited, but it is also true when there is a limited number of specific antibodies capable of reacting with these antigens (7, 15). The reason for the higher sensitivity of the indirect method is that more than one labeled anti-immunoglobulin molecule may react with each immunoglobulin molecule bound to the target antigen in the first layer (15). Our previous studies on renal tubular epithelial brush border (BB) antigens indicated that indirect IFL may fail in cases where the direct method demonstrates bright specific fluorescence. Antibodies raised in rabbits against a membrane fraction prepared from rat kidney stained proximal tubular epithelia, giving a distinct apical fluorescence when the direct IFL method was used. By indirect IFL, however, only diffuse or faint staining was observed in these structures (M). The present investigation was undertaken in order to ex-

University of

plain the observed discrepancy between staining patterns observed using direct and indirect IFL staining. The failure to demonstrate discrete staining by indirect TPL in spite of strong specific staining by thc direct method was observed only if specific anti-BB antibodies were used in high concentrations. This prozone phenomenon occurred despite binding of specific anti-BB antibodies and appeared to be due to differences in the target antigens. MATERIALS AND METHODS Antisera. Rabbits were immunized with a rat kidney proximal epithelial brush border fraction isolated by sucrose density gradient centrifugation as described previously ( H ) . Two sheep antisera against rabbit IgG were prepared as described (12). Immunologically pure antibodies were prepared using glutaraldehyde-polymerized tissue homogenates ( i ) and rabbit IgG coupled to cyanogenbromide-activated Sepharose beads (2).

P-. Linder & A. Miettinen Taiile I. Direct and indirect immunofluorescence (IFL) by two rabbit anti-rat kidney brush border (BB) whole sera and purified immunoglobulin fractions* Antiserum no.

380 386 380/SAS 386/SAS 38a/DEA£ 380/DEAfi 386/DEAE 386/DEAE

I l-f II**' Iff II***

Kidney BB Indirect IFL-h

Direct IFL**

_

+ + •+

-

Intestinal BD Indirect IFLI-

++ +++ ++ ++ +++ +++ ++ ++ ++ -

Direct IFL**

++ ++ ++ ++ -

* Tested on cryostat sections of rat kidney and small intestine. Two anti-BB antisera (nos. 38U and 386) were precipitated at 5095- saturation of ammonium sulpbate (SAS fractions) and subsequently subjected to ion-exhange chromatograpby on DEAE-cellulose. DEAE fraction I w:is eluted with O.OlM phosphate buffer. pH 7.0; DEAE fraction II was eluted with 0.3M phosphate buffer. pH 5.0. t Tested at a 1:5 .serum dilution; serum fractions were adjusted to five times the volume of starting material. A fluorcscein-conjugited sheep anti-rabbit immunoglobulin serum with a fluorescein to protein (F/P) molar ratio of 2.0 and a protein concentratio-i of thc wnrkiny dilution of 35 ^g/ml (0.1 IU/ml) was used in indirect IFL staining. ** Molar F/P ratios of anti-BB conjugates of 1.(13.6 and protein concentrations of working dilutions iif 0.15-0.5 mg/ml. t f Contains mainly IgG. *** Contains IgM, IgA. and traces of IgG.

IgG antibodies were separated from anti-BB anCisera by DEAE-cellulose fractionation (7, 15). Fractions were tested by immunodiffusion using anti-rabbit total serum and anti-rabbit immunoglobulins (Nordic Pharmaceuticals, Tilburg, tbe Netherlands). Jmmnnofhwrescence. Direct and indirect immunofluorescence procedures involved incubations of cryostat sections (unfixed or fixed in a mixture of petroleum spirit and ethanol at room temperature) of rat kidney, liver, and small intestine with antisera as described previously (14); as a general procedure sections were incubated for 30 min and subsequently

washed for 30 min in phosphate-buffered saline, pH 7.2 (PBS), with tbree changes of buffer. For preparation of conjugates, antisera were twice subjected to ammonium sulphate precipitation at 50% saturation, and the protein concentration of the precipitate was adjusted to 10 mg|ml (13) before conjugation with fluorescein isothiocyanate (FITC, Isomer I) (BBL, Cockeysville, Md., USA) (12). The F/P molar ratio of conjugates was 1.0 to 3.6 as calculated from a nomogram (18). The anti-BB antibody conjugates contained 0.150.5 mg protein per ml of working dilution. Two sheep anti-rabbit IgG conjugates were used; their F/P ratios were 2.0 and 3.2. The protein concentration of working dilutions was O.O35-'O.l mg/ml. Tlie conjugate with an F/P ratio of 2.0 was immunologically purifict! 4nd contained 0.1 lU of anti-IgG activity per ml (3) in working dilution. Cross-reacting antiimmunoglobulin antibodies present in tbe conjugate were absorbed with glutaraldehyde-insolubilized rat serum (1). The stained cryostat sections were examined under a Wild fluorescence microscope, using an iodine-quartz 150W lamp as a light source and an FITC interference filter/barrier filter combination (Laboratory of Technical Optics, Lyngby, Denmark). The results were recorded on Kodak Tri X Pan 120 film, using a fixed exposure time.

RESULTS Discrepancy between rest/Its of direct and indirect IPL staining of kidney epithelium Sera from 10 rabbits immunized with the brush border fraction of rat kidney homogenates (anti-BB antisera) reacted by the direct IFL method with renal proximal epithelial brush border. The same antisera tested by the indirect IFL method with two different sheep anti-rabbit IgG conjugates either produced a diffuse granular fluorescence not related to any definite morphological structure or failed to show any staining of kidney tissue (Table I). This result was seen regularly with all antisera tested, regardless of whether the antisera were obtained 1, 3, 9, or 12 months after the start of the immunizations. Antisera were

Prozane in Immunofluorescence 5 ! 5 Table II, Prozones* and end-point titersf of rabbit anti-rat kidney brush border (BB) antisera tested on kidney, liver, and gut by indirect immunofluorescence Target tissue

Anti-BB

Kidney tubules

Bile can:ilicuti

Bnd-point titer

Prozone

titer 6

15

9

19

4 9 6

Prozone

922 924 925 542 921

6 3

17 8

4

927 928

4 4

18 14 15

ftL-r

2

4 4 4

Intestinal vllH

End-point titer

Prozone

12

Prozone effects in indirect immunofluorescence.

J. Immunol., Vol. 5, 1976. Prozone Effects in Indirect Immunofluorescence E. UNDER & A. MIETTINEN Department of Serology and Bacteriology, Universi...
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