British Journal of Urology (1975), 41,489494 0

The Immunological Integrity of Matrix Substance A and its Possible Detection and Quantitation in Urine SHEILA MOORE and G. GOWLAND

University of Leeds, Department of Immunology, The General Infirmary. Leeds.

Boyce, King and Fielden (1962) described “Matrix Substance A” (Matrix A) as “a component peculiar to renal calculous matrix and calculous patient urine.” It was identified as a muco- protein, immunologically distinct from any component of normal urine, and said to be present in all calcigerous calculi and excreted in the urine of patients with renal calculous disease.

Boyce and King (1963) found a material, immunologically identical with Matrix A in the renal parenchyma of all patients known to have formed calculi. Subsequently Matrix A was also found in the urine of patients with diseases involving renal damage and repair (Keutel and King, 1 964).

Patients and Methods

Patients 24-hour urine specimens were collected from 25 patients who were either attending the Stone Clinic or were admitted to the ward with renal colic: 1 : 1,OOO phenylmercuric nitrate was added to the 24-hour urine samples as a preservative, as described by King, Boyce, Little and Artom (1958). All the colic patients were shown to have a renal calculus.

Normal Controls Urines were collected from 20 healthy members of staff with no history of renal disease.

Preparation of Renal Stone Antigen 3 separate antigenic extracts were prepared from batches of renal stones of varied chemical com- position. The method of preparation was basically that of Boyce et al. (1962). The stones were ground prior to decalcification in EDTA pH 7-7. It was 2 to 4 weeks before decalcification ap- peared complete. The preparations were then dialysed against running tap-water and freeze-dried.

Separate preparations were made from the following groups of stones : A. Carbonate stones. B. Stones with oxalate, calcium, potassium and magnesium. C. Uric acid stones. D. High oxalate and calcium stones. E. High nitrate stones.

Total Non-dialysable Solids from Urine (TNDS) This substance was prepared as described by Boyce et al. (1962).

Antisera to Renal Stone Antigen Antisera were prepared in New Zealand White rabbits, a total of 8 animals being used. The first rabbits were treated as suggested by Anderson, Lepper and Winzler (1960). The renal stone anti- gen was suspended in sterile normal saline (20 mg/ml) and 1 ml of this suspension injected at

47/5-~ 489

490 BRITISH JOURNAL OF UROLOGY

weekly intervals for 5 weeks. Each inoculum was divided into equal portions and given intra- muscularly into the animals' forequarters. As the resultant antibody produced only weak pre- cipitin lines when tested by agar diffusion, 2 intravenous doses were subsequently given.

Later, Freunds adjuvant was used with the first 2 doses, followed by a mixture of intravenous and intramuscular doses over a period of 4 months.

The first 4 rabbits received injections from only 1 batch of renal stone antigen, the others were injected with material from all 3 batches of renal stone antigen.

Serological Tests for the Presence andlor Quantitation of Renal Stone Antigen in Urine

I . Immunodiflusion Ouchterlony's method (1958) of double diffusion in agar was used.

2. Tanned Cell Agglutination Test It was proposed to treat sheep red blood cells with tannic acid and sensitise them with either renal stone antigen or specific antibody.

It was found that TNDS caused agglutination of sheep red cells and renal stone antigen lysis. Red cells as a passive carrier in a test using either of these reagents was therefore unsuitable.

3. Latex Agglutination Inhibition Test It was found possible to coat latex particles with renal stone antigen and do a latex agglutination inhibition test for the detection of renal stone antigen. It was necessary to include 0.025 % bovine serum albumin as a stabilising agent after the particles had been coated, and in the test diluent.

Dilutions of the material to be tested were made. A constant amount of antibody of known titre was added and allowed to react. Latex particles, coated with renal stone antigen were then added. The agglutination of the latex particles was noted, it being absent where the test material contained sufficient renal stone antigen to react with all the antibody present.

It was possible with this test to detect 25 mg/l of renal stone antigen present in urine. This test appeared to be insufficiently sensitive when compared with other tests used to detect the amount of substance present in patients' urines. Details of this method can be found in Moore (1974).

4. Radioimmune Assay This was derived from the method of Sarsfield and Gowland (1973). Antibody was coupled to cyanogen bromide-activated cellulose. This was added to dilutions of the substance being tested for renal stone antigen. After time had been allowed for the antigen-antibody reaction to take place, any excess antigen was removed by washing the cellulose. Antibody, labelled with "Iodine was first absorbed with washed cells from human peripheral blood and then added and given time to attach to the antigen-antibody complexes on the cellulose. Free radioactive material was removed by washing and the residual radioactivity measured in a Packard Auto-gamma Spectro- photometer. The radioactivity present, in excess of that present in negative controls, was taken as a measure of the concentration of renal stone antigen present. This method was capable of de- tecting 2 mg/l renal stone antigen. For details of the method see Moore (1974).

Results

Properties of Antisera prepared against Renal Stone Antigen All the antisera were tested by double diffusion in agar against TNDS from normal urine, normal human serum, renal stone antigen and extracts of renal stones of differing chemical composition. Antisera were also tested against these reagents after absorption with normal human serum ; normal TNDS; normal human serum and normal TNDS; and washed cells from human peri- pheral blood.

IMMUNOLOGICAL INTEGRITY OF MATRIX SUBSTANCE A 49 I

Fig. 1. Antistone serum reacted against TNDS, normal human serum and mixed renal stone antigen. Fig. 2. Antistone serum absorbed with TNDS and normal human serum, reacted against TNDS, normal human serum and mixed stone antigen. Fig. 3. Antistone serum, absorbed with human blood cells, reacted against TNDS, normal human serum and mixed stone antigen. Fig. 4. Antistone serum, absorbed with human blood cells, reacted against extracts of renal stones of differing chemical composition. Types of stones: Group A, Carbonate stones; Group B, Stones with oxalate, calcium, potassium and magnesium; Group C, Uric acid stones; Group D, High oxalate and calcium stones; Group E, High nitrate stones.

Reactions similar to those obtained by Boyce et al. (1962) with their whole antiserum were obtained using the antistone sera produced in this laboratory. These antisera reacted with renal stone antigen, normal human serum and normal TNDS (Fig. 1).

When antigens prepared from various chemical types of renal stones were reacted with the antisera, all preparations gave reactions except the uric acid stones.

However, with absorbed antisera, results did not correspond with those obtained by Boyce er al. (1962). These authors claimed that absorption with normal human serum and TNDS left them with a single, strongly reacting antigen-antibody system. In our hands, after such absorption, 3 and sometimes 4 separate antigen-antibody systems were apparent which appeared to be specific for components in the renal stone antigen (Fig. 2). Absorption with washed human blood cells

492 BRITISH JOURNAL OF UROLOGY

Table I Tests for the Presence of Stone Specific Antigens in the Urine or TNDS of Normal Individuals and a Variety of Patients

Immunodiffusion Radio-immune Assay A A

I > ( \

Category NO. + - % NO. + - % Tested Positive Tested Positive

Normal 20 0 20 0 20 0 20 0 Stone formers 16 11 5 69 12 5 7 42 Stone formers with urinary infection 9 4 5 4 4 8 4 4 50 All stone formers 25 15 10 60 20 9 1 1 45

alone produced a similar result (Fig. 3). I t can be clearly seen from the figures that the residual systems after absorption are only weakly reactive-some appearing too weakly to show on photo- graphs.

Boyce et al. (1962) found that all renal stones, except uric acid or cystine calculi, gave a single precipitin line with their absorbed antiserum. Although the unabsorbed antisera used in the pre- sent study reacted with assorted groups of stones, the absorbed antisera only reacted with the mixed stone antigen (which contained stones of many descriptions) and the groups of stones con- taining carbonate (Fig. 4).

Since the specific precipitin line which Boyce et ul. (1962) designated Matrix Substance A could not be obtained, the systems detectable with absorbed antisera, and not reactive with TNDS or human serum are henceforth referred to as “stone specific antigens” (SSA) As cell absorption proved to be so easy and efficient, this method was used in any further experiments where ab- sorbed serum was used.

Diflusion Reactions of Cell Absorbed Antisera with Urine Non-dialysable products from urine were tested at a concentration of 10 x that in the original urine sample.

20 normal control urines were tested in this way. No precipitin lines were seen. Urines from 25 patients were tested. 10 of the specimens showed no reaction with the antibody.

5 gave 1 precipitin line, 8 gave 2 lines and 2 specimens gave 3 precipitin lines (see also Table 1).

Results of Radioimmune Assay ( R A) All the 20 urines from normal individuals with no history of renal infection gave negative results, i.e.<2 mg/l.

Of the tests on the urine from 20 proven stone formers, 9 gave positive results, the amount of SSA detected varying from 2.2 to > 10 mg/l. 42% of stone formers without urinary infection and 50

Table I1 compares the results from 20 stone formers tested by both immunodiffusion and radio- immune assay. It is clear that these 2 tests do not correlate. Since our antisera obviously detect more than 1 stone specific antigen, the most likely explanations would be that:

with urinary infection were positive (see Table I).

(u) different individuals excrete different stone specific antigens ; (6) the 2 tests have different efficiencies with respect to different antigenic systems.

It is also obvious that immunodiffusion will detect antigens which are not detected by radio- immune assay and vice versa.

IMMUNOLOGICAL INTEGRITY OF MATRIX SUBSTANCE A

Table 11

Results from 20 Urines from Proven Stone Formers tested by both Immunodiffusion (ID) and Radioimmune Assay (RA) for the Presence of Stone Specific Antigens

493

Positive by Negative by Positive ID Negative ID Both Methods Both Methods Negative RA Positive RA

5 2 9 4

Table III Occurrence of Matrix Substance A or Stone Specific Antigens

% Positive Detected by

Boyre et al., 1962 All stone formers

Keutel and King, 1964 All stone formers Recurrent stone formers Other conditions All patients examined

Present experiments All stone formers Recurrent stone formers All stone formers Recurrent stone fomers All stone formers

68 Immunodiffusion

28.6 Immunodiffusion 39.6 54.5 36.5

60 Immunodiffusion 65 45 Radioimmune assay 45 85 Immunodiffusion and

radioimmune assay

Despite the non-correlation of the 2 tests (only 5/20 positive by both methods), if both radio- immune assay and immunodiffusion are performed in parallel, 85 % (17/20) of stone formers give a positive result showing that they excrete one or more SSAs in their urine.

Discussion

Boyce et al. (1962) described their absorbed antiserum as showing 1 single precipitin line when reacted with stone antigen or with urines from stone-forming patients. After absorption, the antisera prepared during this investigation still gave 3 or 4 lines of reaction with the stone antigen and 1 to 3 lines of reaction with urines from stone-forming patients. However, the results obtained using the absorbed antiserum for immunodiffusion do indicate that the antiserum was detecting substances absent in the urine of healthy people but often present in urine from stone-forming patients. We have termed these substances stone specific antigens (SSA).

Radioimmune assay is generally very sensitive and might be expected to detect substances in lower concentration than gel diffusion. When urines were tested for stone specific antigens, the number positive by the immunodiffusion method was greater than the number positive by radio- immune assay. Factors which might contribute to this result might be that the 2 tests are detecting different antigenic systems; that attachment of antibody to cellulose masks too many antigen

494 BRITISH JOURNAL OF UROLOGY

combining sites; or that the antibody shows a degree of non-avidity in a fluid system to one or more of the antigens (SSA). However this assay will detect SSA in certain urine samples which are negative when tested by immunodiffusion (Table 11).

Table I11 compares the results of Boyce et al. (1962), Keutel and King (1964) and the present experiments. It is obvious that the results obtained by different workers using the same technique vary; and that different techniques may not give comparable results.

These facts could probably best be explained on the basis that “Matrix Substance A ” is not a single antigenic entity or that renal stone antigen (or “Matrix Substance A”) preparations ob- tained in different laboratories or from different sources contain a variable number of antigenic components. This would make it uncertain as to whether different techniques would detect the same antigen and also raises the possibility that not all the antigenic components need be excreted by any 1 patient or at any one time by the same patient.

However, despite the fact that several different SSAs obviously exist, using a combination of radio-immune assay and immunodiffusion we have been able to detect some form of SSA in the urine of 85 of renal stone formers, but not in the urine of healthy individuals.

Summary

There would seem to be no doubt that most stone-forming patients at some time during the course of their disorder excrete substances not present in normal urine. However, considerable doubt must now exist that “Matrix Substance A” is a single antigenic entity or that its presence is con- fined to the active formation of renal calculi.

We wish to thank Dr C. K. Anderson, Consultant Renal Pathologist, for bringing Matrix A to our attention and Mr R. E. D. Williams, Consultant Urologist. for arranging specimens from patients. We are indebted to the staff of the M.R.C. Mineral Metabolism Unit, University of Leeds and Mr Bryden of St Paul’s Hospital, London, for supplying specimens of renal stones.

References

ANDERSON, A. J., LEPPER, M. H. and WINZLER, R. J. (1960). Studies on urine colloids. 11. Production of an antibody to the insoluble organic matrix of renal calculi and a determination of a soluble antigen in the urine of patients with poliomyelitis. American Journal of the Medical Sciences, 240, 31 1-318.

BOYCE, W. H. and KING, J. S. (1963). Present concepts concerning the origin of matrix and stones. Annals of the New York Academy of Science, 104,563-578.

BOYCE, W. H., KING, J. S. and FIELDEN, M. L. (1962). Total nondialysable solids (TNDS) in human urine. XIII. Immunological detection of a component peculiar to renal calculous matrix and to urine of calculous patients. Journal of Clinical Investigation, 41, 1180-1189.

KEUTEL, H. J. and KING, J. S. (1964). Further studies of matrix substance A. Investigative Urology, 2, 115-122. KING, J. S., BOYCE, W. H.. LITTLE, J. M. and ARTOM, C. (1958). Total non-dialyzable solids (T.N.D.S.) in human

urine. I. The amount and composition of T.N.D.S. from normal subjects. Journal of Clinical Investigation,

MOORE, S . (1974). “Matrix Substance A”. Serological properties and prospective imrnuno-assay. Thesis for Fellow-

OUCHTERLONY, 0. (1958). Diffusion-in-gel methods for immunological analysis. Progress in Allergy, 5 , 1-78. SARSFIELD, J. K. and GOWLAND, G. (1973). A modified radioallergosorbent test for the in vi/ro detection of allergen

37, 315-321.

ship of the Institute of Medical Laboratory Technology.

antibodies. Clinical and Experimental Immunology, 13, 619-624.

The Authors

Sheila Moore, FIMLT. Chief Laboratory Technician. G. Gowland, MRCPath, Professor of Immunology.

Requests for reprints to G. Gowland.