ANALYTICAL

BIOCHEMISTRY

(1990)

188,5-B

lmmunochemical Detection of Glycosphingolipids on Thin-Layer Chromatograms Bernhard

Kniep

GBF-Institute

Received

and Peter

for Biotechnological

December

F. Miihlradt Research, D-3300 Braunschweig,

Federal

Republic of Germany

27,1989

A sensitive immunochemical method was developed for the detection of glycosphingolipids on thin-layer chromatograms. The procedure involves oxidation of diol groups of glycosphingolipids with sodium periodate, derivatization of the formed aldehyde groups with digoxigenin-hydrazide, and reaction of the bound digoxigenin with an alkaline phosphatase-labeled polyclonal anti-digoxigenin antibody. The latter is detected by an insoluble indigo-like dye as a result of dephosphorylation of 5-bromo-4-chloro-3-indolyl phosphate. The detectability of all glycosphingolipid species was improved over that of the orcinol and resorcinol staining methods. Two nanograms of the standard gangliosides GM 1, GD 1 A, and GT 1 was detected, whereas the detection limit for short-chain neutral glycosphingolipids was in the range of 20-50 ng. Long-chain glycosphingolipids were detectable with a particularly high sensitivity. Selective staining of the gangliosides could be achieved by the use of low periodate concentrations. Q 1990 Academic Press, Inc.

The chemical detection of glycosphingolipids (GSLs)’ on thin-layer chromatograms using either the orcinol (specific for carbohydrates) or the resorcinol (specific for sialic acids) spray reagents shows a poor sensitivity. The detection limit for GSLs is about 100-200 ng with each of these reagents. In contrast, immunostaining methods permit GSL detection in the nanogram range, provided i Abbreviations used: The designations for gangliosides are GMi, Gal~l-3GalNAc~l-4(NeuAcol2-3)Gal~l-4Glcl-1Cer; GDIA, NeuAccu23Gal~l-3-GalNAc@l-4(NeuAca2-3)Gal~1-4Glc~l-1Cer; GDIB, Gal/313GalNAc/314(NeuAccu2 - 8NeuAccu2 - 3)Gal@l4Glcl1Cer; Gria, NeuAca2-3Galfll3GalNAcjYl4(NeuAccu2-8NeuAco2-3)Gal/314Glcl1Cer. Other abbreviations used are: BCIP, 5-bromo-4-chloro-3-indolyl phosphate; CD, cluster of differentiation; DIG, digoxigenin-succinylt-aminocaproic acid hydrazide; GSLs, glycosphingolipids; HPTLC, high-performance thin-layer chromatography; mAb, monoclonal antibody. 0003~2697/90 $3.00 Copyright 0 1990 by Academic Press, All rights of reproduction in any form

that antibodies of the desired specificity are available. There have been some trials to develop sensitive GSL detection methods. Introduction of 3H label and subsequent autoradiography improve the detectability of GSLs (1) but require a cumbersome purification of the radioactive GSLs prior to thin-layer chromatography. Another method was based on periodate treatment of GSLs, reaction of the formed aldehyde groups with a hydrazine-alkaline phosphatase conjugate, and finally detection of the bound alkaline phosphatase by a chromogenic substrate (2). However, the hydrazine-alkaline phosphatase conjugate is not commercially available and, when we tried this method, we had problems with nonspecific staining of the background. For staining GSLs on thin-layer plates, we adapted a staining procedure originally developed for the detection of glycoproteins on nitrocellulose or other carriers (3). The method described here is also based on periodate oxidation of the carbohydrate portion of GSLs. Digoxigenin-x-hydrazide (X = spacer) reacts with the aldehyde groups, and the digoxigenin is recognized by an alkaline phosphatase - labeled polyclonal anti - digoxigenin antibody. Phosphatase activity is finally detected by dephosphorylation of 5-bromo-4-chloro-3-indolyl phosphate, leading to an insoluble indigo-like blue dye. This method, called DIG staining, combines the general applicability of the orcinol stain with the sensitivity of an immunostain. The DIG stain method detected about 20 times lower amounts of gangliosides than neutral GSLs, because the former compounds are richer in cleavable vicinal OH groups. MATERIALS

AND

METHODS

Glycosphingolipids and substrates. Lactosylcer amide, monosialogangliosides, and polar neutral GSLs were obtained from neutrophils and purified in our laboratory by Folch partition (4) and DEAE-Sepharose chromatography (5). Lactosylceramide was finally purified by HPLC (6) and its concentration determined 5

Inc. reserved.

6

KNIEP

AND

gravimetrically. Globotriaosylceramide, globotetraosylceramide, GMl, GDlA, and GTlB with defined concentrations were obtained from Supelco (Bellefonte, PA). Digoxigenin-succinyl-e-aminocaproic acid hydrazide (DIG), polyclonal sheep anti-digoxigenin Fab fragments (alkaline phosphatase conjugated), and 5-bromo4-chloro-3-indolyl phosphate (BCIP) (components of the “glycan detection kit” Cat. No. 1142372) were kind gifts from Boehringer Mannheim (FRG). Thin-layer chromatography. Precoated silica gel 60 thin-layer chromatography (HPTLC) plates were purchased from Merck (Darmstadt, FRG). The following solvent mixtures were used as mobil phases: Solvent A, chloroform/methanol/water (120/70/17, v/v/v) containing 0.02% CaCl,; Solvent B, chloroform/methanol/ water (50/40/10, v/v/v) containing 0.05% CaClz. Resorcinol and orcinol staining. Both staining procedures were performed as described (78). Fixation of the HPTLC plates. After separation of the GSLs, HPTLC plates were fixed with polyisobutylmethacrylate (Plexigum P 28, Roehm & Haas, Darmstadt, FRG) in n-hexane (HPLC grade, Rathburn Chemicals Ltd., Walkerburn Peeblesshire, Scotland). As previously described (9), the HPTLC plate was chromatographed in this solution until the solvent front reached the top. A Plexigum working solution was prepared from a saturated stock solution. The dilution of the working solution was empirically determined: the concentration of the working solution was adequate when the Plexigum layer covered about 80% of the plate after chromatography. We found this procedure to give a better reproducible coating than the dipping of the plates in fixing solution. Chromatography in fixing solution did not affect the positions of the GSL bands. Antibodies. MAb VIM 2 (10) and mAb VIM 8 (ll), recently classified to the CDw 65 cluster (12), were kind gifts from Dr. W. Knapp (Vienna, Austria). MAb lB2 (13) was kindly donated by Dr. K. Skubitz (Minneapolis, MN). Immunostaining. Immunostaining with the carbohydrate-specific monoclonal antibodies (mAb) VIM 2, VIM 8, and lB2 was performed essentially as described (9) with some modifications (14). In brief, after separation of the GSLs on HPTLC plates and plate fixation, the plates were blocked with PBS/l% BSA and then incubated with the antibody (l/200 diluted ascitis fluid) for 14 h at 4°C. After four washing steps, the HPTLC plate was treated with a l/1000 diluted phosphatase-labeled goat anti-mouse antibody (Dako, Hamburg, FRG) for 90 min at 37°C in the presence of 3% (w/v) polyethylene glycol 6000. The plate was washed four times and finally overlaid with 0.5 mg/ml BCIP in 100 mM glycineNaOH buffer pH 10.4, containing 1 mM MgClz and 1 mM ZnClz . Glycolipid antigens produced a stable indigo-like stain within 30 min.

MtiHLRADT

Buffers. Buffer A was 100 mM sodium acetate, pH 5.5. Buffer B was phosphate-buffered saline, pH 7.3, containing 0.05% Tween 21 and 0.02% sodium azide. Buffer C was phosphate-buffered saline, pH 7.3, containing 1% bovine albumin and 0.02% sodium azide. Buffer D was 100 mM glycine-NaOH with 1 mM MgClz and 1 mM ZnCIP, pH 10.4. Periodate oxidation, derivatization with DIG, and staining of GSLs. After separation of the GSLs, the HPTLC plate was dried under vacuum for about 40 min and then fixed as described above. The plate was then dried for 5 min at ambient temperature, placed in an incubation chamber, and soaked with buffer A for 10 min. The HPTLC plate was then treated with a 10 mM sodium periodate solution in buffer A for 30 min at 30°C in the dark, washed four times for 5 min with buffer A in the dark, and treated with a l/1000 dilution of the DIGhydrazide stock solution, as obtained by the supplier, in buffer A for 30 min. After four 5-min washings with buffer B, the plate was treated for 30 min at 37°C with buffer C to block nonspecific binding sites and then incubated overnight with a l/200 dilution of the DIG-specific antibody in buffer C at 4°C. The HPTLC plate was washed three times for 10 min with buffer B and for 10 min with buffer D. The final incubation with 0.5 mg/ml of the phosphatase substrate BCIP in buffer D usually required from 1 to 3 h at 30°C for adequate dye development. The stained plate was then washed with tap water and air-dried. To obtain higher sensitivity, prolonged incubation times may be used. Incubation with the antibody for longer than 48 h is possible without any damage to the silica gel layer. Also, the incubation with the substrate solution can be prolonged, since the background stain was normally very low. RESULTS

The staining of standard gangliosides with DIG is compared to that with resorcinol in Fig. 1A. The detection limit was about 2 ng for each of the standard gangliosides GMl, GDlA, and GTlB. GDlB was present here as an impurity of GTlB. Resorcinol staining gave visible bands only with more than 150 ng of the gangliosides. In Fig. lB, monosialogangliosides from neutrophils were visualized by the DIG stain and for comparison by immunostaining with mAb VIM 2 (specific for monofucosylated long-chain monosialogangliosides of the lactoneo-series (15)) and mAb VIM 8 (specific for monofucosylated monosialogangliosides and neutral GSLs with very long (12 and more) neolacto-type carbohydrate chains (12)). In addition to the bands that were detected by the two specific mAbs, many more GSLs of shorter chain length were visualized by the nonspecific DIG stain. In Fig. 2A, DIG- and orcinol-stained standard neutral GSLs are compared. Again, the neutral GSLs were de-

IMMUNOCHEMICAL

DETECTION

A RESORCINOL

DIG

, I

VIM2 VIM8

f

j

I

--GM3 =2-3sFG 324Sffi 5’-3SN6 3 10 5 12

d

ef

g h I

i

abcdefghi

2

3

FIG. 1. (A) Comparative staining of gangliosides by resorcinol and DIG. On the lanes of both HPTLC plates, 1000 (a), 500 (b), 150 (c), 50 (d), 20 (e), 10 (f), 5 (g), 2 (h), and 1 ng (i) of each ganglioside, GMl, GDlA, and GTl, was applied. GDlB was an impurity of GTlB. After chromatography for 40 min in chloroform/methanol/water (50/40/10, v/v/v) containing 0.05% CaCl,, gangliosides were stained by resorcinol (left) and DIG (right) as described under Materials and Methods. (B) Gangliosides detected by immunostaining and the DIG method. The same mixtures of monosialogangliosides from neutrophils were applied to each HPTLC plate. After chromatography as in (A), plate 1 was stained by the DIG method and plates 2 and 3 were immunostained with mAb VIM 2 and VIM 8, respectively. Abbreviations used in this figure: Gllll, I13NeuAcGg,Cer; GnlA, IV3113 (NeuAc),Gg,Cer; G ma, I13(NeuAc)2Gg&er; GMS, Grl , IV3NeuAc,I13(NeuAc),Gg&er; IPNeuAc-LacCer; 2-3SPG, IV3NeuAcnLc,Cer; 2-6SPG, IVaNeuAcnLc,Cer; 2-3SN6, V13NeuAcnLc,Cer; SlO, VII13NeuAcV3FucnLcsCer; S12, X3NeuAcVII13FucnLc&er.

tected with a higher sensitivity by the DIG staining method than with the orcinol stain. However, the staining sensitivity was markedly dependent on chain length: globotetraosylceramide, with four carbohydrate residues, had a lower detection limit (about 20 ng) than lac-

A

6

ORCINOL

DIG

DIG 182

d

_

efg

hi

abcdefghi

1

ml.4

0.05

0.1

a2

0.5

1.0

2.0

NoJO&

FIG. 3. DIG staining of neutral GSLs and gangliosides after treatment with different concentrations of periodate. Staining was performed as usual, except that different sodium periodate concentrations were used for each plate as indicated. In each left lane, the gangliosides GMl, GDlA, and GTl (100 ng each of) were applied, and in each right lane the neutral GSLs lactosylceramide, globotriaosylceramide, and globotetraosylceramide (100 ng each of) were applied. Solvent: chloroform/methanol/water (120/70/17, v/v/v) containing 0.02% CaClz. Running time, 30 min. Abbreviations: see Figs. 1 and 2.

tosylceramide, which contains two carbohydrate residues. In another experiment, polar neutral GSLs from neutrophils were stained by the DIG method and compared with the mAb lB2 (specific for Galfil-4GlcNAcPl-R) immunostain. All bands that were recognized by lB2 were also detectable by the DIG staining method. This again shows that the sensitivity of the DIG stain is in the range of an immunostain, particularly when it is applied to the detection of long-chain GSLs. Selective staining of gangliosides. We next considered the possibility of using the DIG method for a detection of gangliosides based on previous findings that sialic acids are oxidized at a lower periodate concentration than the diol groups of other carbohydrates (2). The DIG staining of gangliosides and neutral GSLs using different periodate concentrations during the oxidation step is shown in Fig. 3. As expected, gangliosides were oxidized at lower periodate concentrations than neutral GSLs. According to these data, DIG staining after treatment with 0.1 and 10 mM periodate may provide a first indication of which of the GSLs in a mixture are gangliosides and which are neutral GSLs, the former being selectively stained after 0.1 mM periodate treatment. DISCUSSION

P

^”

7

GLYCOSPHINGOLIPIDS

6 DIG i

bc

OF

2

FIG. 2. (A) Comparative staining of neutral GSLs by orcinol and DIG. From left to right: 1000(a), 500(b), 150(c), 50(d), 20(e), 10(f), 5(g), 2(h), and 1 ng (i) each of the neutral GSLs lactosylceramide, globotriaosylceramide, and globotetraosylceramide were applied and chromatographed in chloroform/methanol/water (120/70/17, v/v/v) containing 0.02% CaCl,. Running time 30 min. (B) Neutral GSLs from neutrophils detected by the DIG method and immunostaining. Polar neutral GSLs from neutrophils were applied to plates 1 and 2 and chromatographed for 50 min in chloroform/methanol/water (50/ 40/10, v/v/v) containing 0.05% CaCl,. Plate 1 was stained by the DIG method and plate 2 was immunostained with mAb lB2. Abbreviations in this figure: Lac, lactosylceramide; Gb3, globotriaosylceramide; Gbl , globotetraosylceramide; nLc,, lactonorhexaosylceramide.

GSLs became of some interest after their identification as target antigens for several cell specific monoclonal antibodies. The antigenic determinants reside mostly on GSLs with very long carbohydrate chains, e.g., the CD 15 (16), CDw 60 (17), and CDw 65 (12) antigens. These antigens are only minor components of the total cellular GSLs and are therefore difficult to characterize. The DIG method described in this paper fulfills the requirements for a sensitive and universal GSL detection method. Its sensitivity for neutral GSLs and particularly for gangliosides reaches the low nanogram range and is thus comparable to the sensitivity obtained by immunostaining procedures. An additional advantage of this

8

KNIEP

AND MtJHLRADT

method is the stability of the fadeless indigo-like dye that is formed by the dephosphorylation of BCIP. ACKNOWLEDGMENTS We thank Reiner technical assistance.

Munder

and Hansjijrg

Thude

for their

excellent

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