Cross-Reactions between Mycobacteria

Cross-Reactions between Mycobacteria

I. Crossed Immunoelectrophoresis of Soluble Antigens of Mycohacferium Zeprnemurium and Comparison with BCG

0. CLOSS, M. HARBOE & ANNE M. WASSUM Institute for Experimental Medical Research, University of Oslo, Ullevll Hospital, Oslo. Norway

Closs, O., Harboe, M. & Wassum, Anne M. Cross-Reactions between Myco- bacteria 1. Crossed Immunoelectrophoresis of Soluble Antigens of Myco- bacterium lepraemurium and Comparison with BCG. Scand. J. Immunol. 4 ,

Crossed irnmunoelectrophoresis (CIE) has been used to characterize the soluble antigens of Mycobacterium Icpraemuriuni (Douglas strain) (MLM). The anti- gen was prepared by sonification of intact bacilli, purified as described. A pool of immunoglobulin isolated from hyperimmune rabbit antisera was used as antibody. By this procedure 42 antigens from MLM could be defined in a highly reproducible manner. An analysis of crossed line immunoelectropho- resis (CLIE) and CIE with intermediate gel as methods for studying antigenic cross-reactivity are presented. Based on this analysis both BCG antigen and BCG antibody were added separately to the intermediate gel of the MLM reference system to study the cross-reactivity between this mycobacterium and MLM. It was concluded that 23 out of the 42 MLM antigens defined in our reference system showed various degrees of cross-reactivity. However, most of these cross-reactions were weak. The method appears to be well suited for studying cross-reactions between mycobacteria, but similar studies using other mycobacteria are needed before it can be decided how closely MLM is related to BCG.

0. Closs, M . D., Institute for Experimental Medical Research, Ullc,vtil Hos- pital, Oslo, Norway

SUPPI. 2, 173~-185, 1975.

In recent years efforts to develop an effective immunoprophylaxis against leprosy have added new aspects to the study of the an'tigenic com- ponents of mycobacteria. The source of anti- gen for immunization against leprosy may be either the non-cultivable Myc~ihac te r ium lep- rue itself o r another, antigenically related, mycobacterium.

Studies on soluble antigens indicate that there is considerable antigenic similarity be- tween the various mycobacterial species. Ac- cording to Stanford (12) some antigens are common to all mycobacteria; others are shared

by slow-growing species; some are shared by fast-growing species; finally, some antigens are specific for each species.

Like M . leprae, Mycobacteriurn lepraernuri- urn (MLM), the causative organism in murine leprosy, is non-cultivable, extremely slow- growing, and a n obligate intracellular parasite. As pointed out previously (4) the immuno- pathology of murine and human leprosy shows important similarities which make murine lep- rosy particularly interesting as an experimen- tal model for its human counterpart. It there- fore seems important to learn more about the

174 0. Clo.ss. M . Harboc & Anne M . Wassurn

antigenic composition of MLM, and its rela- tion to other mycobacterial species. including M. leprae.

Several workers have demonstrated a certain antigenic relationship between M L M and M . avium (5. 13). and a similar relationship has also been established between M . leprae and M . avium (8). However, these observations are bajed on immunodiffusion techniques of rel- atively low sensitivity which only allow a limited number of antigens 'to be dearly defin- ed. While standard immunodiffusion techniques seem quite satisfactory for taxonomic purposes (1 2), more sensitive and quantitative techniques are required to assess the degree and structur- al basis of the antigenic cross-reactivity be- tween the various species of mycobacteria.

Rexnt ly . crossed immunoelectrophoresis (CIE) has been introduced in the analysis of mycobacterial antigens (3, 1 1 , 15) and anti- bodies (3). The technique seems to be definite- ly superior to immunodiffusion ad modum Ouchterlony in that i t allows a far larger num- ber of antigens o r antibodies to be identified and provides more quantitative information.

An immunological cross-reaction takes place when antibodies produced by immunization with one antigen react with another antigen. I f it is not due to contamination of the im- munogen by the cross-reacting substance, a cross-reaction shows that structural similarity exists between the two antigens. Such structur- al similarity may be analysed at three different levels:

At the level of one antigenic determinant. Here structural relatedness may be un- ambiguously expressed as either identity or partial identity. At the level of one antigen molecule. Here it is not possible to distinguish between partial identity due to a few determinants being identical and partial identi'ty due to many determinants being partially identi- cal. At the level of one complex structural unit, such as an animal or bacterial cell, which contains many different kinds of antigen molecules. Here structural relationship may

also be expressed as the proportion of an- tigen molecules exhibiting cross-reactivity.

The two techniques used in the present investi- gation, crossed line immunoelectrophoresis (CLIE) and CIE with intermediate gel, allow the analysis to be performed at levels 2 and 3 respectively.

The present paper describes a reference system using CIE to characterize the soluble antigens from MLM. The application of this system to study the cross-reactivity between MLM and M . bovis. strain BCG, is explored as a model for the study of cross-reactions be- tween mycobacteria.

MATERIAL AND METHODS

Preparalion of antigen frtrm M. lepraemurium. Mycohacterium Iepraemurium, Douglas strain. (MLM) were harvested from the liver of mice infected intraperitoneally with a large inoculum 20-25 weeks previously. Each liver was ho- mogenized in 10 ml 0.15 M NaCl (saline) using a Tenbroek all-glass homogenizer (Bellco Glass Inc., Vineland, N.J., U.S.A.). The homog- cnate was centrifuged for 10 min at 200 R, the supernatant collected and spun for 20 min at 10,000 g. AfLer centrifugation the pellet consisted of three layers: T h e top layer, which was heavily contaminated with mouse tissue debris, was partly removed by manipulation with a spatula and washing with saline; the middle layer, which contained most of the bacilli, was carefully collected; the dark bottom layer, which contained red blood cells, was discarded. T h e bacilli were resuspended in 10 ml saline and centrifuged for 10 min at 6,000 g , the supernatant was removed completely, and the pellet was weighed. The pellet was resuspended in saline at a concentration of 60 mg (wet weight) per ml. This suspension was sometimes sonified without further purification to produce soluble antigens for the crossed immunoelectrophoresis (CIE). Before it was used for immunization one additional purifi- cation step was performed: 10 ml of the bacil- lary suspension was thoroughly mixed with mineral oil (Esso White Oil JX, Esso Norske

Soluble Antigens of M. lepraernuriurn 175

A/S, Oslo, Norway) in a Tenbroek homogeniz- er. The mixture was centrifuged for 20 rnin at 20,000 g. Bacilli were collected from the inter- phase after the tube had been frozen to solidi- f y the bottom phase. The thick suspension of bacilli thus obtained was centrifuged at 3,000 g for 30 min and any excess oil removed. The suspension was weighed and 10 ml of saline added per g suspension, followed by sonifiica- tion for 15 min in a Branson Sonifier Model B-12 (Branson Sonic Power Co., Danbury, Conn., U.S.A.) at a measured effect of 80-100 W. The sonicate was centrifuged for 30 rnin at 20,000 g, and the bottom phase containing the water-soluble antigens and the top phase containing bacillary fragments in oil were col- lected separately. The protein concentration in the water phase was determined by the Folin- Ciocalteau method ~(10) using an IgG standard. Some of the antigen solution was stored at 4"C, with 0.02% NaN,, added, for use in CIE. The rest was stored at -20°C together with the insoluble antigen. A mixture of these two preparations, approximately 10 parts soluble + 1 part insoluble antigen, was used for the immunization of rabbits.

Preparation o f antigen from BCG. Four- week-old cultures of BCG (strain Copenha- gen) in Sauton's medium were washed X 1 in saline and centrifuged at 3,000 g for 30 min. The pellet was weighed and resuspended in saline at a concentration of 60 mg per ml. The suspension was then sonified for 15 rnin at 80-100 W. Part of the sonicate was centri- fuged for 20 rnin at 20,000 g, the supernatant collected and stored at 4°C in the presence of 0.02% NaN, for use in the CIE. The rest of the sonicate was stored uncentrifuged at -20°C until it was used for immunization of rabbits.

Production of rabbit antisera. Four rabbits were immunized with each antigen. Before in- jection, the sonicated antigen was thoroughly mixed with an equal volume of Freunds in- complete adjuvant. Each rabbit was given 0.2-0.4 ml of this mixture as multiple intra- cutaneous injections of 2 M O ~1 per site around the neck and in the groins.

The injections were repeated at intervals of 3 4 weeks and the rabbits were bled 8-10

I2

days after the last injection. Some rabbits pro- duced ,a good antiserum after the fourth in- jection, while in others two or three more in- jections were needed. Apart from this, BCG antibody was obtained from Dakopatts, Copen- hagen, Denmark, as X 5.3 concentrated im- munoglobulin. This antibody was produced by immunizing with the same BCG antigen as described above.

Isolation o f immunoglobulins. This was car- ried out as described by Harboe & Ingild (6). Twenty-five grams of (NH,),SO, were added per 100 ml of rabbit antiserum and the mix- ture left on a magnetic stirrer overnight at room temperature. After centrifugation at 3,000 g for 30 min the supernatant was dis- carded and the precipitate washed X 1 with 1.75 M (NH,),SO,. The precipitate was then dissolved in a small volume of distilled water corresponding to %-% of the original serum volume. The protein solution was dialysed 2 X 12 hours against distilled water, 1 X 24 hours against 0.050 M CH,COONa 0.021 M CH,COOH pH 5.0, 2 X 12 hours against dis- tilled water, and 1 X 24 hours against the ace- tate buffer. The precipitated lipoproteins were removed by centrifugation. If needed the supernatant was concentrated by ultrafiltration in a Diaflo cell (Amico Corp., Lexington, Mass., USA) uing a PM 10 membrane. Finally, the immunoglobulin preparation was dialysed against 0.1 M NaCI, 15 mM NaN, and stored at 4°C.

Preparation of the reference pool of anti- MLM immunoglobulins. The sera obtained from different bleedings were all examined by CIE. The weaker ones were pooled separately for each animal and the immunoglobulin was isolated and concentrated corresponding to 3- 5 times the serum concentration. These prep- arations were later mixed with those prepar- ed from the best antisera. It was observed that the serum obtained from one of the rab- bits produced more precipitin lines in CIE than serum obtained from the others, but since the others contained additional precipi- tins against apparently different antigens, all the Ig preparations were pooled. The final composition of the pool was determined after

176 0. Closs, M . Harbor & Anne M . Wassum

mixing the preparations in various proportions in order to produce the maximum number of lines.

Crossed immunoelectrophoresis (CIE). Cross- ed immunoelectrophoresis was carried out in a micro-modification using 5 X 5 cm glass plates as described by Weeke '(14). An electrophore- sis appratus with cooling was used (Dansk Laboratorieudstyr Ltd., Copenhagen, Den- mark). The temperature of the cooling water was 15"C, and the electrophoreses were run in 1 % (W/V) agarose (batch AGS 122, Litex, Glostrup, Denmark) containing barbital/Tris buffer pH 8.6 with ionic strength 0.02. The gel thickness was about 1.5 mm in the first dimen- sion, and 1 mm in the intermediate gel and in the reference gel containing anti-MLM 'anti- body. The areas covered by the gels measured 7.5, 5.0 and 12.5 cm' respectively. Ten pl of MLM antigen was added in the circular an- tigen well and separation in the first dimension was carried out with a potential gradient of 10 V cm-1, after the antigen had been allowed to move out of the well at a lower voltage. Albumin coloured with Evans blue was run in parallel and the first dimension run was ter- minated when it had moved 1.9 cm from the nearest border of the well. The reference gel contained 20 pl per cm2 of the anti-MLM ref- erence pool, either undiluted or diluted 1:2. The effects of various reagents on the MLM reference system were studied by adding them to the intermediate gel. Second dimension elec- trophoresis was performed overnight with a po- tential gradient of 2 V cm-l. Four plates were run simultaneously in the same apparatus. After electrophoresis the plates were pressed under moist filter paper and soft blotting paper (10 min), washed in saline for 4-6 hours, pressed, washed in distilled water for 30 min, pressed again, dried with a hair drier, stained with Coomassie brilliant blue R (Lot 42C- 1530 SIGMA) for 10 min, destained, washed briefly with distilled water and finally dried with a hair drier. For further details see ' (2) .

For characterization of the different peaks, the stained precipitation lines were projected onto a sheet of paper using a Leitz Focomat 1C photographic enlarger (Leitz AG, Wetzlar,

W. Germany) and a copy was drawn. The position of each peak was recorded on milli- metre paper to measure the height in the second dimension and the distance from the centre of the application well in the first di- mension. Fractions of millimetres were mea- sured by interpolation.

Cross-reactivity detected by crossed line im- munoelectrophoresis and crossed immunoelec- trophoresis with intermediate gel

The following analysis is based primarily on the description of quantitative immunoelectro- phoretic techniques by Axelsen, Kroll & Wee- ke '(2). It deals with the simplest model pos- sible: a reference system containing one pre- cipitin line (Fig. 1A). due to reaction between the reference antigen AgA and the reference antiserum AbA. By incorporating into the in- termediate gel another antigen (AgX), or anti- bodies against this antigen (AbX), the cross- reactivity between the two sets of reagents can be studied. Crossed line immunoelectro- phoresis (CLIE), in which AgX is added to the intermediate gel, allows direct comparison of this antigen with the reference antigen. CIE with intermediate gel, in which AbX is added to the intermediate gel, cannot be used to define the degree of structural relatedness be- tween AgA and AgX, but is often a more sensitive method than CLIE for demonstrating that a cross-reaction exists. The present anal- ysis compares the expected findings in the two techniques in specific theoretical model situations. It is assumed that AgA and AgX are protein antigens containing a number of different determinants: a , , a,, a:<, . . . a,, and x,, x,, x,, . . . xn, respectively. Based on the nun- ber of determinants which AbA reacts with on AgA, four principally different reference systems may form:

1. AbA i.s directed against many, but not all, determinants on AgA (AhA + a , -t a, + aY . . . + an-n1). Fig. IB shows the pattern of identity formed whenever AbA cannot differentiate between AgA and AgX. The ref- erence peak is moved upwards and fuses completely with the horizontal line formed

Soluble Antigens of M. lepraemurium 177

Fig. 1. Analysis of the effect of adding cross-reacting antigen (AgX) or the corresponding anti- body (AbX) to the intermediate gel in crossed immunoelectropho- resis. Most of the figures are taken from Axelsen, Kr0ll & Weeke (2). I. The reference antibody (AbA) is directed against many deter- minants on the reference antigen (AgA). A. The reference system. B & C. AgA and AgX are im- munologically identical; AbA cannot differentiate between them and AbX contains antibody against AgA in high concentra- tions. D ,& E. Partial identity be- tween AgA and AgX; AbX con- tains antibody against AgA in low concentrations. F & G. Partial identity between AgA and AgX, but only AbX contains antibody against the common determinants. H & I. AgA and AgX have only one determinant in common. 11. The reference antibody is di- rected against only two determi- nants on the reference antigen. A. The reference system. J & K. The patterns of cross-reaction. Symbols: AgA = a, +a,. . . +a, - the reference antigen contains the determinants a,-a,. AbA + a, +a,. . . +an-,,, - the reference antibody is directed against the determinants al-a,-m.

I A g A - a , + a 2 + a 3 . +a,.

AbA- a , + a 2 + a 3 +a,-,,,

AgA -

AbA

H

II A g A - a , + a p . +a,.AbA + a , + a 2

i AbX

178 0. Closs, M . Harbor & Anne M. Wassum

AgA= a,+ a2 ....+ a,. AbA * a, I3l AgA=a ,+a2 +a,.

A''' an+l +an+2 +an+p

A bA

0

Fig. 2. Analysis of the effect of adding cross-reacting antigen (AgX) or the corresponding antibody (AbX) to the intermediate gel in crossed immunoelectrophoresis when the reference antibody (AbA) does not precipitate the reference antigen (AgA). 111. AbA is directed against less than two determi- nants on AgA. A. AbA + a,, AbX -+ a2. B. AbA + 0, AbX + a,+a,. . . +an+ IV. AbA is di- rected against determinants (a,,,,, ani2. . .a,l+,,) which are present in the immunizing antigen and in AgX but not in AgA. Symbols as in Fig. 1.

between AbA and AgX. In this situation AbX may contain high concentrations of antibodies against AgA, which causes the reference peak to move downwards into the intermediate gel (Fig. 1C); if AbX contains antibody against AgA in lower concentrations the effect will be less pronounced (Fig. 1E).

The typical pattern of partial identity is shown in Fig. ID. It proves that at least two determinants are similar on AgA and AgX '(giving rise to the horizontal line) and that at least two determinants are specific for AgA '(giving rise to the 'spurs of the precipitate below the horizontal line). The relative thick- ness of the spurs compared with the horizontal line reflects the degree of structural difference in the two antigens to the extent that the reference antiserum is able to detect it. In this situation, the addition of AbX to the inter- mediate gel usually pulls the peak somewhat downwards and causes its legs to continue down into the intermediate gel (Fig. 1E). The extent of these changes varies with the antibody concentration but is not directly related to the extent of structural similarity between AgX and AgA. Furthermore, partial identity may be present but only AbX reacts with the common determinants. The addition of AgX does not influence the reference peak (Fig. 1F) but the addition of AbX causes depression of the peak and elongation of its legs as shown

in Fig. 1G. The same combination of patterns may arise if AgX is not present in sufficient quantities to cause precipitation. If only AbA reacts with the common determinants the pat- terns obtained when AgX or AbX is added to the reference system are the same as in Figs. 1D and 1A respectively. If the part of AgX which is identical to AgA includes only one determinant, no horizontal line will form when AgX i s added to the intermediate gel, although the reference peak may become ele- vated CFig. IH). AbX may pull the reference peak somewhat downwards and its feet in- wards (Fig. 11). The more determinants AbA reacts with on AgA, the less effect such weak cross-reactivity will have on the position of the reference line.

11. A b A ic. directed against the minimum of determinants sufficient to cause precipitation ( A b A + a, + ad). The reference system and the reaction patterns when AbA cannot differ- entiate between AgA and AgX are as shown in Fig. IA, B, and 'C. When there is partial identity in this system AgX contains less determinants than AgA reacting with AbA and clannot be precipitated; no horizontal line will form, and since AgX will absorb out some of the activity in AbA, this antiserum will no longer precipitate AgA below the level of absorption (Fig. 1 J). If enough AgX is added to the intermediate gel the reference line may

Soluble Antigens of M. lepraemurium 179

disappear completely. AbX will depress the reference peak but cannot pull it down into the intermediate gel (Fig. 1K).

Finally, there are situations when the refer- ence system contains no line but if a cross- reacting antigen or antibody is added to the intermediate gel a new line arises.

111. AbA is directed against less than two determinants on AgA (AbA --$ aJ. The refer- ence system contains no precipitin line. If the antiserum AbX contains antibodies against another determinant on AgA (i.e. AbX-a,) the system may become precipitating and a new line will form in the top gel (Fig. 2A). No line will develop on addition of AgX. If AbX contains antibodies against two or more deter- minants on AgA a new line may develop in the intermediate gel '(Fig. 2B).

IV. A b A contains antibodies against deter- minants present in the immunizing antigen but not in AgA (AbA + a,,, + a,,, . . . + an+&. When working with more complex systems this is a not uncommon situation, e.g. if the immunizing antigen contains insoluble mater- ial. If the determinants in question are present on AgX a straight horizontal line may form when this antigen is added to the intermediate gel (Fig. 2C), but no line develops with AbX.

RESULTS

The M. lepraemurium reference system

Fig. 3A shows the precipitation pattern ob- tained when M . lepraemuriurn (MLM) antigen is run in crossed immunoelectrophoresis (CIE) against the undiluted reference anti-MLM pool. When compared with the drawing of the same pattern in Fig. 3B it is clear that not all of the 42 precipitin lines shown in the drawing are strong and easily recognizable. A few of the lines were in fact weak and could not be identified in every set-up.

The relative electrophoretic mobility of the various peaks in relation to peak no. 13 is given in Table I, together with the relative electro- phoretic mobility of human serum albumin. Peak 13 was selected as reference because of its prominent and consistent appearance. As a

+ f

B ++

Fig. 3. A. Photograph showing the precipitation pattern of M. lepraernuriurn antigen run in cros- sed irnmunoelectrophoresis against a pool of rab- bit anti-Mi. lepraemuriurn antibody. Stained with Coomassie brilliant blue R. B. Drawing of the same pattern as shown in A. Due to lack of space three of the lines have not been numbered: no. 11 (dotted) just beneath no. 12; no. 15 (dotted) above no. 18; and no. 20 just beneath no. 21.

rule the relative electrophoretic mobility of the peaks was found to be highly reproducible, although some peaks showed a less constant position (e.g. nos. 2, 6, 15 and 39). Some of the lines (e.g. nos. 36, 37, 38 and 41) had no distinct peak at all and their position in the first dimension therefore could not be defined.

It was more difficult to obtain the same high degree of reproducibility when measuring the position of individual peaks in the second di- mension, because there was no obvious com- mon baseline from which to make the measure- ments. Neither was the baseline of every peak so well defined that it could serve as a basis for such measurements. The relative position of some of the peaks in the second dimension therefore tended to vary more between dif-

180 0. Closs, M . Harboe C? Anne M . Wassum

Table I. Relative electrophoretic mobility of the antigens of M. Iepraemurium and human serum albumin with antigen no. 13 as reference

Relative electrophoretic No. of

Antigen no. mobility S. D. experiments

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

1.386 1.156 1.127 1.123 1.111 1.085 1.075 1.061 1.057 1.040 1.050 1.028

0.988 0.967 0.980 0.968 0.957 0.933 0.925 0.900 0.902 0.894 0.860 0.833 0.804 0.791 0.792 0.784 0.769 0.710 0.694 0.660 0.618 0.619 N.D. N.D. N.D. 0.534 0.513 N.D. 0.109

-

0.015 7 0.020 7 0.0 10 7 0.005 7 0.01 1 7 0.021 5 0.017 6 0.006 7 0.004 7 0.006 3 0.01 7 5 0.012 6

0.005 7 0.022 2 0.004 6 0.014 7 0.007 7 0.01 1 7 0.005 7 0.004 5 0.015 4 0.006 7 0.003 5 0.010 5 0.004 7 0.01 1 6 0.012 7

1 0.009 7 0.010 6 0.01 I 6 0.011 6 0.005 7 0.010 7

-

-

- -

-

0.038 2 0.01 1 5

0.005 5 -

Albumin 0.862 3

S.D.=standard deviation. N.D. =not done, usually because the peak was too flat.

ferent runs than their position in the first di- mension. However, this variability was still not very marked and did not create any problems in the identification of the various lines.

I t was noted that different antigen prepara- tions made by sonification of MLM bacilli did not consistently produce the same high num- ber of precipitin lines. Spontaneous precipita- tion of the antigens frequently occurred after freezing and thawing, whereby the number of lines decreased. Storage at 4°C in 15 mM NaN, has so far been found to provide the best conditions for preservation of the soluble antigens from MLM.

Since the MLM antigen is made from bacilli isolated from mouse liver several of the lines observed might be due to contamination with mouse tissue antigens. Therefore, an ultra- sonicated homogenate of normal mouse liver was run in CIE against the anti-MLM refer- ence pool. Only two lines developed; the relative electrophoretic mobility in relation to peak no. 13 in the MLM system was 0.25 and 0.38 respectively. One hundred microlitres of the same mouse liver antigen was also in- corporated into the intermediate gel. None of the lines in the MLM reference system were visibly affected. Most importantly, the lines produced by antigens nos. 39, 40 and 42, which were closest to the two mouse antigens in electrophoretic mobility, remained in their nor- mal position. It was therefore concluded that all the lines included in the M. lepraernuriurn reference system depicted in Fig. 3B were due to antigens present on MLM.

Cross-reactivity between BCG and M. leprae- murium

By incorporating BCG antigen or rabbit anti-BCG antibody into the intermediate gel, cross-reactivity between BCG and MLM was studied with reference to specific antigens. Anti-BCG antibodies from two different sour- ces were tried: whole serum from hyper- immunized rabbits and rabbit anti-BCG im- munoglobulin concentrated X 5.3. Since the changes induced by the latter preparation were more pronounced and #affected a higher pro-

Soluble Antigens of M. lepraemurium 181

Fig. 4. A. Simplified scheme of the precipitin pattern produced in crossed immunoelectrophoresis of M. lepraemurium antigen against pooled rabbit anti-M. lepraemu- rium antibody. Normal rabbit serum has been added to the in- termediate gel. Note the deflec- tion of peak no. 2. B. Same ref- erence system as in A with rabbit anti-BCG immunoglobulin con- centrated X 5 added to the inter- mediate gel. The ,peak marked x is one of two peaks with the mobility of reference peak no. 21 and has not been finally identi- fied.

portion of the antigens, the conclusions were based on results obtained with that prepara- tion. Fig. 4 shows the effect of adding 100 PI anti-BCG immunoglobulin to the intermediate gel, using normal rabbit serum as control. Some of the precipitin lines have been omitted in order to simplify the illustration. By com- paring Figs. 4A and B it is observed that 8 peaks, numbered 3, 14, 25, 26, 27, 32, 34 and 38, have moved down into the intermediate gel. In addition, the legs of three other precipi- tates, numbered 13, 16 and 23, have become extended downwards and their peaks lowered to a varying degree. 'Precipitate no. 30 is somewhat reduced in height and its legs con- tinue into the intermediate gel, although not very far. The line marked 'x' is one of two peaks '(the other not shown) with the position of antigen no. 21 and may represent a new line. Thus, antibodies against 13 out of the 26 MLM antigens shown in Fig. 4A, and possibly against one additional antigen, were present in the anti-BCG immunoglobulin preparation. A

complete list of the MLM antigens reacting with this antibody is given in Table 11.

If Figs. 4A and 5A are compared, it can be seen that precipitate no. 2 looks different when the intermediate gel contains serum in- stead of NaCI. Since normal human serum did not have any effect on position no. 2 this ob- servation indicates that normal rabbits may be pre-immunized against this antigen.

Fig. 5 depicts the various types of changes observed when BCG antigen was added to the intermediate gel. As in Fig. 3, several precipi- tin lines have been omitted in order to make the illustration clearer. A comparison of Figs. 5A and B shows that some of the precipitates, i.e. nos. 16 and 27, have disappeared com- pletely; BCG antigen is thus able to absorb out completely the precipitating activity against these antigens. Precipitates nos. 13 and 34 show attachment to horizontal lines with spur formation. Two other horizontal lines are pres- ent which do not seem to be associated with any of the lines in the reference system. In ad-

Fig. 5. A. Same reference system as in Fig. 4 A but with NaCl in the intermediate gel. B. Same ref- erence system with the soluble fraction of a BCG sonicate added to the intermediate gel. Note that two of the four horizontal pre- cipitin lines shown do not have a clear association with lany of the reference peaks. A B

~ '-

__

182 0. Closs, M . Harboe & Anne M . Wassum

Table 11. Cross-reactivity between BCG and M . Iepraemurium as revealed by crossed line immuno- electrophoresis and crossed immunoelectrophoresis with intermediate gel

Reagent in intermediate gel MLM

antigen Anti-BCG Anti-BCG BCG Con- no. Ig antiserum antigen clusion

1 2 3 4 5 6 7 8 9

10 1 1 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

# M

%

f F

? a f M

? M

%

%

a a a +

# # + % - - a + %

M

M

f ? M

f

m t

M

I

# m #

? %

M

M

a

Arrow pointing upwards: elevation of peak Arrow pointing downwards: reduction of peak - no change observed ? uncertain effect, inconclusive data

$ moderate effect (4) weak effect

$4 strong effect # cross-reactivity not detected = cross-reactivity detected * weak precipitate, no definite change observed.

dition, 7 peaks, numbered 3 . 14, 21. 23, 25, 30 and 38, have increased in height. Altogether, this indicates that in 1 1 out of 27 antigens deter- minants are shared between BCG and MLM. Cross-reactivity in antigens nos. 26 and 32 could not be demonstrated by this technique although these antigens were precipitated by anti-BCG antibody as shown in Fig. 4.

The changes induced by incorporating BCG antigen or antibody into the intermediate gel of the MLM reference system are summarized in Table 11. A total of 23 out of the 42 MLM antigens characterized in this system seemed to share determinants with antigens present in BCG. Three of these cross-reactions were only detected with BCG antibody, in a few instances the reference line was weak and inconsistent, and no definite conclusion could be reached. T h e number of cross-reacting antigens may therefore be even higher than what appears from Table 11. It should be noted, however, that most of the cross-reactions appear to be weak. In no instance was a typical reaction of identity found but the fact that BCG antigen was able to absorb out all precipitating activity against MLM antigens nos. 16 and 27 may in- dicate that they are strongly cross-reacting.

DISCUSS1 ON

Based on different techniques, i.e. gel diffusion (12). immunoelectrophoresis (7) and crossed immunoelectrophoresis (1 I) , efforts have been made by several groups to define a reference system for the study of mycobacterial antigens. Roberts et al. ( 1 1 ) suggested that a single sensi- tive antiserum against M . tuberculosis be used in the analysis of the antigen pattern of all species and strains of mycobacteria in crossed immunoelectrophoresis. While this approach may be well suited for the characterization of closely related species, it will not provide sufficient data concerning species which are only distantly related t o the reference bacteri- um. If the relatives of a mycobacterial species are unknown, the analysis defining its relation- ship to other species should be based on a system homologous for that species. Since

Soluble Antigens of M. lepraemurium 183

little is known of the relationship of M. leprae- murium (MLM) to other mycobacteria, its own antigenic profile should be established as a first step.

The present investigation has demonstrated the existence of 42 different precipitating antigens in MLM, which is the largest number of antigens identified from this bacterium so far. In a gel diffusion system Stanford (13) was able to identify 10 antigens from MLM, while Kwapinski et al. (9), using a somewhat different immunodiffusion system, could iden- tify only 5 antigens. The sensitivity of the pres- ent method could probably be further en- hanced by using more concentrated reagents. However, there seems to be no reason to at- tempt to increase the number of lines further before the possibilities of the system in its present form have been explored. A total num- ber of between 40 and 50 seems to represent the maximum number of lines which it is con- venient to work with.

Since the bacilli have been prepared from mouse liver, there is a chance that some of the peaks that have been identified as MLM antigens are in fact mouse tissue antigens. Stanford l(13) reported that 7 out of 17 lines in his MLM system were also produced by the normal mouse tissue control. Although the presence of mouse antigen in our M,LM prep- aration has not been tested directly with spe- cific antisera, it is unlikely that any of the precipitin lines in the system are due to such antigens. The anti-MLM pool reacted only feebly with a crude extract of mouse liver, and even high concentrations of this antigen did not interfere with any of the reference peaks when added to the intermediate gel. Thus, although roughly similar to the method de- scribed by Stanford (13), our method of puri- fication provides a less contaminated product.

The present method seems to be sufficiently sensitive, specific and reproducible to serve as a reference system for the characterization of the antigens of MLM. Wright & Roberts (15) do not recommend the use of their crossed immunoelectrophoretic system for screening or characterizing the antigenic patterns of large numbers of species and strains of myco-

bacteria because of the large expenditure of antiserum and time: 6 ml and 8 days respec- tively per plate. These restrictions apply to a lesser degree to the present method since only 1/24 the amount of antiserum is needed and it takes one quarter of the time.

One major purpose in characterizing the an- tigens of MLM is to define its relationship to other mycobacteria. The present method of using an intermediate gel between the first di- mension gel and the antibody-containing gel as described by Axelsen I( 1) is particularly well suited for this type of analysis. By this proce- dure it is possible to study the effect of either antigen or antiserum on the reference system, and cross-reactivity can be detected even if the structural similarity is too small for a precipi- tate 'to form. The theoretical analysis of va- rious forms of cross-reactivity showed that a variety of situations existed which could each give rise to a distinct pattern of changes in our system (cf. Figs. 1 and 2). In some of these situations, antigen and antibody will be equally effective in locating the cross-reactive antigen, while in other instances only one of the two reagents is able to reveal a relationship. Thus, both the antigen and its corresponding anti- body should be used when searching for cross- reactivity.

In the present investigation, concentrated immunoglobulin from hyperimmunized rabbits was shown to be a very potent reagent for detecting cross-reacting antigens. If the cross- reaction is strong enough to precipitate one of the reference antigens, the extent to which the corresponding peak becomes depressed will only depend on the concentration of antibody added t o the intermediate gel. In this way marked effects may occur even if the cross- reactivity is relatively weak. This method can thus only indicate the number of antigens in- volved in cross-reactions, and does not provide information on the extent of structural simi- larity for each component. Information of the latter type may be obtained by adding the cross-reacting antigen to the intermediate gel.

The various species of mycobacteria have probably developed from a common progeni- tor through a series of mutations (12). During

184 0. Closs, M . Harboe & Anne M . Wassum

this evolutionary process changes in the struc- ture of certain key proteins have been limited by the necessity of preserving their ability to function. Such proteins will probably show some similarity in structure even in widely different species of mycobacteria. Therefore, the number of cross-reacting antigens may not be a sufficient criterion for establishing the relationship between species. The number of antigens which show a high degree of struc- tural similarity is probably more relevant. De- tailed structural studies of isolated antigens from various species appear to be the best method of establishing the relationshiu be- tween various mycobacteria.

Previous studies seem to indicate that MLM is closely related to M . avium (5, 13). Since these investigations are based on an analysis of relatively few antigenic components, the evidence cannot be regarded as conclusive. However, Fukui et al. ( 5 ) have suggested that the a antigen from MLM may be identical with the a antigen from M . avium.

In the present investigation more than half of the 42 antigens detectable in MLM seemed to share determinants with antigens from BCG. It is important to note that in no instance was a reaction of identity observed, but the finding that BCG antigen could absorb out completely the precipitating activity against antigens nos. 16 and 27 indicates that they may be closely related to antigens in BOG. Cross-reactions against antigens nos. 2, 26, and 32 were only detected with BCG antibody and the existence of the corresponding BCG antigens therefore needs to be confirmed before these cross- reactions can be regarded as being definitely established. Until further studies have been carried out, including similar tests with other mycobacteria, these observations do not allow any firm conclusions regarding the relationship between MLM and BCG to be drawn.

Like the antigens from MLM ddected in earlier immunodiffusion studies l(9, 13), most of the 42 antigens presently defined are prob- ably derived from the cytoplasm and may not be liberated unless the bacterium is broken down. Some of them, however, may be located at the surface of the bacterium and may thus

be immunogenic even in in taa bacilli. Further work should be devoted to defining the latter type of antigens, since they may be of pri- mary importance for the early immune re- sponse against the bacterium in infected ani- mals.

ACKNOWLEDGEMENTS

The work was supported by grants from An- ders Jahre’s Fund for the Promotion of Science and the Norwegian Research Council €or Sci- ence and the Humanities. We are grateful to Dakopatts, Copenhagen, Denmark for produc- tion of the anti-BCG immunoglobulin.

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Soluble Antigens of M. lepraemurium 185

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Received 2 December 1974