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Year : 1997  |  Volume : 63  |  Issue : 6  |  Page : 361-365

"Immunoperoxidase" an essential modality of immunohistochemistry

Correspondence Address:
P D Gupta

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Source of Support: None, Conflict of Interest: None

PMID: 20944379

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The immunoperoxide (IP) technique can be used for the detection of immune deposits in various tissue pathologies. This article focusses on the standardization of the reagents anti IgG, anti IgA and anti C3C which are used to target these immune deposits. Lymph node tissue was used as substrate for standardization of anti IgG and anti IgM. For anti IgA, intestine was used while for anti C3C kidney tissue rapidly proliferating glomerulo nephritis - RPGN was utilized. Sensitivity of the technique was found to be high. Also the results on paraffin section were excellent and could be interpreted using simple light microscope. It is concluded that once standardization is achieved, IP can be used as an essential modality in the detection of immune deposits in a wide variety of immunologically mediated dermatologic disorders.

Keywords: Immunoperoxidase (IP), Immune deposits, Standardization, Optimum dilution

How to cite this article:
Gupta P D, Rapose A. "Immunoperoxidase" an essential modality of immunohistochemistry. Indian J Dermatol Venereol Leprol 1997;63:361-5

How to cite this URL:
Gupta P D, Rapose A. "Immunoperoxidase" an essential modality of immunohistochemistry. Indian J Dermatol Venereol Leprol [serial online] 1997 [cited 2020 Jun 4];63:361-5. Available from: http://www.ijdvl.com/text.asp?1997/63/6/361/4618

Immunohistochemistry [1] is widely used as a histopathologic tool for confirmatory evi­dence in diagnosis of a wide variety of tissue pathologies. In Dermatology, the fluorescent technique [2] (immunofluorescence-IF) has largely contributed to the understanding of the pathogenesis and in the classification of various immunological disorders - especially vesiculo-bullous and collagen vascular disorders. But disadvantages inherent to the IF tech­niques [3] include lack of permanence of the preparations, naturally occurring autofluorescence and trouble and expense of employing dark field ultraviolet micros­copy. The massive cost of setting up an IF laboratory is the main reason why this tech­nique is available in only a few centres in India.

We therefore took upon ourselves, the chal­lenge to set up an equivalent alternative tech­nique which will suit the Indian economy, yet at the same time giving equipotent re­sults. [4] This inspired us to set up the Immunoperoxidase laboratory at Seth G.S. Medical college and K.E.M. hospital, Mumbai.

  Materials and Methods Top

Sections 4-5 μ of formalin fixed, paraffin embedded biopsy specimens were used for the test. The reagents used were: primary an­tibody - anti IgG (Dako A 0423), anti IgM (Dako A 0425), anti IgA (Dako A 0262), anti C3C (Dako A 0062), (These are the most commonly detected immunoglobulins in immunologically mediated skin diseases), sec­ondary antibody swine anti rabbit serum (Dako Z 0196), peroxidase antiperoxidase (PAP) complex [5],[6] (Dako Z 0113), swine se­rum (Dako X 0901). Diamino benzidine 7 (DAB) (Sigma D 5637) was used as colouring agent and hematoxylin was used as counter­stain.

To establish any new diagnostic tool, stan­dardization is the first and the most essential step. Standardization implies ideal dilution of the reagents, so that the optimal staining is obtained with minimal background. In the IP techniques, standardization is required for primary and secondary antibodies and the PAP complex.

Step No.1 : Standardization of secondary antibody (IIo Ab) and PAP

This is the first step and is done using a known primary antibody as constant. We used anti S100 protein as constant. The tar­get organ was nerve tissue of salivary gland.

Dilutions attempted were as follows:

Step No.2 : Standardization of anti IgG and anti IgM

Target antigen was IgG and IgM present in lymph node tissue. Dilutions attempted were 1:25, 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:2000, 1:4000.

Step No. 3 : Standardization of anti Ig A The target antigen was Ig A and the tissue used was the intestinal mucosa. Dilutions at­tempted were 1:20, 1:40, 1:80, 1:160, 1:300, 1:500, 1:1000.

Step No. 4 : Standardization of anti C 3 C The target antigen was C3C, present in kid­ ney (RPGN). Dilutions attempted were 1:25, 1:50, 1:100, 1:200, 1:400. All the dilutions were carried out using phos­phate buffered saline. Around 50-100ml of reagent was layered on the sections and the slides were incubated at 37°C in a humid chamber. After each step of the procedure, the sections were gently rinsed in buffer.

Mounting was done using DPX and viewed under a simple light microscope.

Tests for sensitivity (controls)

This included the omission of primary anti­body or use of inappropriate antibody (we used chorio embryonic antigen).

  Results Top

The following tissues were used successfully as substrate in order to achieve optimum di­lution of the reagents

Positivity was detectable as brown deposit (due to DAB) against a blue background (due to hematoxylin counterstain.

  Discussion Top

The reagents are diluted until only the targetted antibody is detectable and the rest of the antibodies (of unwanted specificity) become undetectable. [9] This is the basis of the checkboard titrations that we used to deter­mine the optimum antibody dilutions. The optimal dilution was determined by antibody concentration that gave the greatest contrast between specific staining and background of all types.

In step No.1 (standardization of Ho Ab and PAP) the known primary antibody used was anti S 100 antibody. S 100 is present in nerve tissue. Salivary gland with nerve elements was used as substrate. Nerve tissue stained brown [Figure - 1] against a blue background.

Once this standard dilution was obtained, the next step involved the standardization of anti IgG and anti IgM. Lymph node was chosen as substrate because lymphocytes are rich sources of IgG and IgM which could be targeted with the antibodies anti IgG and anti IgM respectively. Positivity was detected as brown deposit [Figure - 2].

For standardization of anti IgA, intestine was used as substrate because IgA is secreted onto the lining of intestinal mucosa. IgA was de­tected as a brown lining on the intestinal mucosa.

Kidney tissue diagnosed as rapidly prolifer­ating glomerulonephritis (RPGN) which was positive for C3C by immunofluorescence was chosen as substrate for standardization of anti C3C.

In this process of standardization it was noted that IP staining for immune deposits per­formed on formalin fixed, paraffin embed­ded specimens gave excellent results. These results were concordant with the work of Sinclair et al. [10] This overcomes the problem of the requirement of facilities for frozen section and the expense of immunofluores­cent microscope. This technique has also been shown to be highly sensitive. [11] The IP technique can be used in a wide variety of dermatoses [12] such as vesiculo-bullous disorders, inflammatory disorders, radiodermatitis, collagen vascular disorders and some of the skin tumours. The standardization of the various antibod­ies is no doubt a laborious process. But it is worth the effort because once the standard dilutions are achieved, the procedure be­comes very simple. The fact that IP requires paraffin sections, simple light microscope for interpretation, and the permanency of re­sults, makes this technique a cheap and vi­able alternative to immunofluorescence.

  References Top

1.Marucci AA, Dougherty RM. Use of unlabelled antibody immunohistochemical technique for the detection of human antibody. J Histochem Cytochem 1975; 23: 618-623.  Back to cited text no. 1  [PUBMED]  
2.Beutner EH, Chorzelski TP, Kumar V. Immu­nopathology of the Skin. 3rd Edition, Wiley; New York, 1987.  Back to cited text no. 2    
3.Taylor CR. Immunological techniques. Arch Pathol Lab Med 1978; 102: 113-117.  Back to cited text no. 3  [PUBMED]  
4.Dorling J, Johnson GD, Webb JA, et al. Use of peroxidase conjugated antiglobulin as an alter­native to immunofluorescence for the detection of antinuclear factor in serum. J Clin Pathol 1971; 24: 501-505.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]
5.Slemmon JR, Salvaterra PM, Saito K. Prepara­tion and characterization of peroxidase antiperoxidase Fab complex. J Histochem Cytochem 1980; 28: 10-15.  Back to cited text no. 5  [PUBMED]  
6.Petrali JP, Hinton DM, Moriarty GC, et al. Use of PAP conjugate; J Histochemistry 1974; 22: 782-801.  Back to cited text no. 6  [PUBMED]  
7.Scopse L. Larsson LI. Increased sensitivity in per­oxidase immunocytochemistry; Histochemistry 1986; 84: 221-230.  Back to cited text no. 7    
8.Heyderman E. Immunoperoxidase technique in histopathology : applications, methods and con­trols. J Clin Pathol 1979; 32: 971-978.  Back to cited text no. 8  [PUBMED]  [FULLTEXT]
9.Bigbee JW, Kosek JC, Eng LF. Effects of pri­mary antiserum dilution on staining of "anti­gen rich" tissues with the PAP technique; J Histochem Cytochem 1977; 25: 443-447.  Back to cited text no. 9  [PUBMED]  
10.Sinclair RA, Burns J, Dunnill MS. Immunoperoxidase staining of formalin fixed paraffin embedded human renal biopsies with a comparison of the peroxidase antiperoxidase (PAP) and indirect methods. J Clin Pathol 1981; 34: 859-865.  Back to cited text no. 10  [PUBMED]  [FULLTEXT]
11.Sternberger LA, Sternberger NH. The unlabelled antibody method; J Histochem Cytochem 1986; 34: 599-605.  Back to cited text no. 11  [PUBMED]  
12.Arrese JE, Mosbah BT, Pierard FC, et al. Diag­nostic relevance of the immunoglobulin and complement deposition in cutaneous disease perspectives and new techniques. Med Cut ILA 1993; 21: 130-135.  Back to cited text no. 12    


[Figure - 1], [Figure - 2]


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