Clin. exp. Immunol. (1970) 6, 633-637. SHORT COMMUNICATION IMMUNOLOGICAL FINDINGS IN SENSORY CARCINOMATOUS NEUROPATHY. APPLICATION OF PEROXIDASE LABELLED ANTIBODY J. ZEROMSKI Department of Immunology, The Wenner-Gren Institute, Stockholm, Sweden (Received 6 November 1969) SUMMARY An antibody against neurones, which has previously been demonstrated by immunofluorescence, is present in the sera of patients with sensory carcinomatous neuropathy. This antibody was demonstrated by a new technique, namely the use of purified peroxidase-labelled sheep anti-human y-globulin. In cryostat sections of brain treated with a first layer of serum antibody followed by a second layer of peroxidase labelled anti-human y-globulin, heavy coarse brown deposits in nerve cells only from different parts of the central nervous system indicated a positive reaction. The special technical precautions necessary to obtain specific staining of neurones are outlined. INTRODUCTION It has been demonstrated in a series of previous papers (Wilkinson, 1964; Wilkinson & Zeromski, 1965; Zeromski & Wilkinson, 1966) that complement fixing antibodies against the central nervous system are present in sera of patients with the sensory type of carcinomatous neuropathy. The specific activity of these antibodies was found to be directed against intracytoplasmic components of the nerve cells. The reacting antigen was localized by immunofluorescence in the forms of discrete granules distributed around the nucleus of neurons. The reaction was organ specific but not species specific. The reactive antigen was shown to be very labile being destroyed by most fixatives, proteolytic enzymes and organic solvents. Preliminary biochemical studies indicated that this substance was present in the enriched microsomal fraction of brain homogenate and was sensitive to ribonuclease, which suggested a relationship to Nissl substance. Recently Avrameas (1969), Nakane & Pierce (1967) and others have successfully used horseradish peroxidase (HRP) labelled antibody as an immunohistochemical reagent. This Correspondence: Dr Jan Zeromski, Department of Pathological Anatomy, Medical School, 49 Przybys- zewskiego Str., Poznan, Poland. * This work was included in the paper presented at International Conference on Immunity and Tolerance in Oncogenesis-June 1969, Perugia (Italy). N63 N 633
Transcript
Clin. exp. Immunol. (1970) 6, 633-637.
SHORT COMMUNICATION
IMMUNOLOGICAL FINDINGS IN SENSORYCARCINOMATOUS NEUROPATHY. APPLICATION OF
PEROXIDASE LABELLED ANTIBODY
J. ZEROMSKI
Department of Immunology, The Wenner-Gren Institute, Stockholm, Sweden
(Received 6 November 1969)
SUMMARY
An antibody against neurones, which has previously been demonstrated byimmunofluorescence, is present in the sera of patients with sensory carcinomatousneuropathy. This antibody was demonstrated by a new technique, namely theuse of purified peroxidase-labelled sheep anti-human y-globulin. In cryostatsections of brain treated with a first layer of serum antibody followed by a secondlayer of peroxidase labelled anti-human y-globulin, heavy coarse brown depositsin nerve cells only from different parts of the central nervous system indicated apositive reaction. The special technical precautions necessary to obtain specificstaining of neurones are outlined.
INTRODUCTION
It has been demonstrated in a series of previous papers (Wilkinson, 1964; Wilkinson &Zeromski, 1965; Zeromski & Wilkinson, 1966) that complement fixing antibodies against thecentral nervous system are present in sera of patients with the sensory type ofcarcinomatousneuropathy. The specific activity of these antibodies was found to be directed againstintracytoplasmic components of the nerve cells. The reacting antigen was localized byimmunofluorescence in the forms of discrete granules distributed around the nucleus ofneurons. The reaction was organ specific but not species specific. The reactive antigen wasshown to be very labile being destroyed by most fixatives, proteolytic enzymes and organicsolvents. Preliminary biochemical studies indicated that this substance was present in theenriched microsomal fraction of brain homogenate and was sensitive to ribonuclease,which suggested a relationship to Nissl substance.
Recently Avrameas (1969), Nakane & Pierce (1967) and others have successfully usedhorseradish peroxidase (HRP) labelled antibody as an immunohistochemical reagent. This
Correspondence: Dr Jan Zeromski, Department of Pathological Anatomy, Medical School, 49 Przybys-zewskiego Str., Poznan, Poland.
* This work was included in the paper presented at International Conference on Immunity and Tolerancein Oncogenesis-June 1969, Perugia (Italy).
N63N 633
634 J. Zeromski
technique, being at least as sensitive as immunofluorescence can also be used for immuneelectron microscopy. Peroxidase (molecular weight 40,000)-y-globulin complex is stillmuch smaller (molecular weight 200,000) than ferritin alone (molecular weight 850,000),the marker of proteins used for electron microscopy. This might be an important factor forpenetration through biological membranes and for the detection of intracytoplasmicantigens.
In the present communication an attempt was made to demonstrate at the light micro-scope level the site in neurons of the reaction between antigen and neuropathy antiserausing a peroxidase labelled antibody.
METHODS
Sheep anti-human y-globulin serum was treated to obtain pure antibody, using the Avrameas& Ternynck (1969) glutaraldehyde immunoadsorbent technique. Adsorbed antibody waseluted from polymerized human y-globulin by means of 0-1 M glycine-HCl buffer. Thefraction obtained was found to contain immunoelectrophoretically pure sheep y-globulin
Minhn s aAcouln agntThcojgewa furthe puife by prcptainwt
HRP antibody used in carcinomatous neuropathyagar plates revealed that the conjugate contained HRP-active antibody against humany-globulin but not unconjugated antibody or free enzyme.The sera examined included those from neuropathy cases used in the previous work,
known to be positive in immunofluorescent tests and sera of healthy controls. Cryostatsections of freshly collected guinea-pig brain were used as tissue material. The reaction
FIG. 2. Reaction as above. x 504. (a) Occipital lobe; (b) frontal lobe.
procedure was essentially carried out according to Nakane & Pierce (1967) and to Graham& Karnowsky (1966) with one modification, i.e. unfixed sections were first allowed to reactwith the sera tested prior to washing in saline and fixation for 10 min in acetone-alcoholmixture. They were then treated with conjugate followed by the substrate medium for peroxi-dase containing 3,3-diaminobenzidine tetrahydrochloride (Sigma) and 0-01 % H202
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according to Graham & Karnowsky (1966). Control reactions included treatment withnormal human sera, with a conjugate against rabbit y-globulin and a histochemical reactionwithout conjugate.
RESULTS
Guinea-pig brain sections exposed to neuropathy serum and peroxidase labelled antibodyshowed distinct brown deposits within the cytoplasm of the nerve cells. These deposits hada rather heavy, coarse appearance and showed the tendency to coalesce (Figs. 1 and 2).The reaction in neurons from different parts of the central nervous system (frontal, parietal
FIG. 3. Control reaction-section treated with normal human serum followed by HRP con-jugate and HRP substrate medium. No reaction in neurons. x 504.
and occipital lobe) was positive. All neuropathy sera which were positive in an immuno-fluorescence test gave a positive reaction in neurons in tests using peroxidase labelledantibody. Sections treated with normal human sera or with neuropathy sera followed byperoxidase labelled anti-rabbit y-globulin failed to show any peroxidase activity in theneurons (Fig. 3). Similarly, a direct histochemical reaction for peroxidase activity did not
show any brown deposits in the nerve cells.
DISCUSSION
The results in this paper confirm the previous immunofluorescence findings (Wilkinson &Zeromski, 1965) that sera of patients with sensory carcinomatous neuropathy react specifi-cally with cytoplasmic components of the nerve cells. Thus the specific coarse brownishdeposits in neurons seen in this study apparently correspond to the fluorescent granules
HRP antibody used in carcinomatous neuropathy 637observed previously. This observation is of importance because several workers havereported that animal and human cells of the central nervous system exhibit fluorescentgranules, not related to any immunological reaction, when exposed to ultraviolet light(Einarson, 1953; Koenig, 1963; Sarnat, 1968). These granules were however seen whileusing fixed and paraffin embedded material, which was not the case in the sensory neuro-pathy studies, where unfixed cryostat sections were used throughout. Nevertheless, thecurrent paper provides new evidence for specific binding of the antibody with neuronalantigen by means of an entirely different, independent method.To render this reaction specific, it was however necessary to remove unconjugated enzyme
from peroxidase labelled antibody. The presence of unbound HRP resulted in nonspecificreactions within vessels, the meninges and also, to a lesser degree-neurons. It was alsoessential to allow unfixed sections to react first with the serum under test and then afterwashing to fix them in alcohol-acetone mixture before application of subsequent reagents.Such treatment allowed good preservation of the labile neuronal antigen under study beforetreatment with HRP conjugate and HRP substrate medium. There was only negligiblenonspecific staining of myelin and other structures of the central nervous system.The present data suggest that the use of peroxidase labelled antibody will permit future
demonstration of the reaction between the neuropathy antibody and its correspondingneuronal antigen on the ultrastructural level, thus allowing a much closer morphologicallocalization of this antigen than has been possible up till now.
ACKNOWLEDGMENTS
The work reported in this paper was undertaken during the tenure of a Research TrainingFellowship awarded by the International Agency for Research on Cancer.
REFERENCES
AVRAMEAS, S. (1969). Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for thedetection of antigens and antibodies. Immunochemistry, 6, 43.
AVRAMEAS, S. & TERNYNCK, T. (1969) The cross linking of proteins with glutaraldehyde and its use for thepreparation of immunoadsorbents. Immunochemistry, 6, 53.
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GRAHAM, R.C. & KARNOWSKY, M.J. (1966) The early stages of absorption of injected horseradish peroxidasein the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histo-chem. Cytol. 14, 291.
KOENIG, H. (1963) The autofluorescence of lysosomes. Its values for identification of lysosomal constituents.J. Histochem. Cytol. 11, 556.
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