Proteinase-dependent false positive precipitin tests Victoria Wither, Ph.D., and Jerry Dolovich, M.D., F.R.C.P.(C)* Hiandt,on~, Ontario, Canada

A variety of nonimmunologic precipitin-like r‘eartions have been de- scrilwd.l-G In double-diffusion tests in gel, lines rwcmbling precipitin reactions ha\-c been observed between B. szlbfilis alkaline proteinase preparations and the scra of normal individuals with no irltlustrial csposu~ to R. mhtilis products.‘~‘” 0bscrvations on the characteristics of thcsc lines arc dcscrihed here.

In double-diffusion tests, the lines tcntlcd to form mow slowly than usual ant&a-antibody precipitates, and they were generall~~ less distinct in ap- pearxnce. They appeared to 1~ washed from the agar during staining with a.mitlo black in an acetic acid-methanol solxnt. There was no relationship be- twon these lines and the prcscncc or alwncc of scrwn antibody to the B. suhtilis

preparations as determined by ‘9-l aheled antigen-binding methods.11 JVith yariations in cnzpmc i‘anti::cn” eonccntrxtions, lines were elicitctl with

all whole normal hunan sera (X118) and with preparations of the third protein I)cali of’ Scl)hadcx C:-200 gel filtration fracl ions (peak 111) of ~IORXI~ IIIUII:II~ Scra, which contains albumin ant1 a,-ant itq-psin. With the corresponding nraci~oriiolcculc-contairiin~ peak I, thcrc wet2 lines which were less dense ant1 loss constantly demonstrated. No such lines were ohserved with IgG-containing peak 11.

The lines could be elicited with high concentrations of nonpurified R. subtilis

p’otc~inasc preparations and witlt crystalline preparations of the alkaline pro- t,cinasc, subtilopeptidasc A (SPA). 13. subti7i.v alkaline proteinases arc inactivated 1)~ the selective reaction of nonrevcrsiblc inhibitors such as phcnylnrethancs~~l- IhyI fluoride (PXSE’) with the scrinc of the active site of the cr~~yriic.‘~ When pr*otcbirrastl was treated with PBISF, thcw was no apparent loss of antigcnic watt ix-it,y in clouble-cliffnsiori tests with spf‘cific rabbit antisera, but the pry- c*il)it in-like reactions with KITS wcrc eliminated. It is therefore concluded that that i’ormation of the lines is dcpcntlent upon the proteinase activity of the ‘iantigen” preparations.

1 ,ines could be readily produced in I$$ hours by coanterirnnrurroelectrol~lro~(~- sis iC1 HI’) l3 (Fig. I). With this tcchniquc. slcntLcr lines of low density could

T’OZ. 49, No. 1, pp. 59-61

60 Wither and Dolovich 1. ALLERGY CLIN. IMMUNOL. JANUARY 1972

FIG. 1. Nonimmunologic precipitin-like lines demonstrated by counterimmunoelectrophoresis

(CIEP). An illustration of the CIEP pattern is at the left and a corresponding diagram on

the right. t Indicates the anode; - indicates the cathode; E -= enzyme well filled with subtilopeptidase A, 50 mg. per milliliter; S = normal human serum was added; P = peak

III obtained from Sephadex G-200 gel filtration fractionation of normal human serum was

added. Electrophoresis was for 1 l/2 hours. The lines which can be observed between the wells were visible immediately after the electrophoresis.

also be ~)rotlucetl in rtiact.ions ol’ prot,einase with commercial albumin prepara- Gons. (‘IBP was performed with the proteinasc. a cationic protein, in a well anodal to wells filled with serllni or serum fractions. TTniler t.he conditions of clrctrophorcsis. the mobility of gamma globulin was cat.ionic. Thus lines which formed in a location between the wells necessarily inyolvetl reactions of’ pro- Icinasc with non-gamma component,s 0.t’ the strum. Thcsc various observations illustrate that. the lines art’ nonimlnunologi(:.

In an attempt to identify thtl serum components in the lines, numerous linrs were \rashed, remo\-cd with a thin slice of agar, and nsc~l to immunize rabbits. Two rahhit,s WCI~C immunized wit,h lines formed in double-diffusion reactions between a nonpurified R. slthfi/is preparation and KITS. Ont: ra.bbit serum rcactcd in illrnl~~rloelec~tropllorrsis ( TEP) with human n,-antitrypsin ( n,-AT) md cy,-nlacro~lobnlill (+-AI), and thcb n~ond reacted with Q?-M and Tg:Cr. Four rabbits were immunized with lines formed in CIF:P between SPA and ?r;IIS. Two sera reacted with ~Y,-JI ant1 CY-AT, the third reacted with cu,-JI and IgC:, and the fourth with ~11. Two rabbit,s wcrc immunizrd with the lines formed in C.!IEI’ bctwccn Sl’n ant1 peak III 01’ MIS. 111 TEP tests with NTS, both scra reacted only with albumin.

The process 1)~. which the lines were I’o~xwcl has not brew fully definetl. The tlcscribcd findings indiratc that they arc probably not the product, of a process of binding anal aggrtyate I’onnation c0IllpiW:lble to that which product23 ant.igen- antibody prccipitat,es but are more likely produced by products of digestion by- proteinasc. They could not. IJC stained by- the amido black. CIEP experiments with 1251-labclcd cnzynics failed to provide any evidence of a formaCon of zones of aggregated proteins. The presence of antibodies t.o a,-XT and ~-31 in man3 of the antisera resulting from immunization with the nonimmunologic lines suggest.cd that the intrractions of B. subtilis alkaline proteinases with theye strum proteins”. I5 might be the reason for the lines. However, in CIEP tests it was i’owntl that Ibeak III f’rorn a serum with homozygous deficiency of a,-AT was similar to peak II1 from normal human serum. The presence of a,-AT and

VOLUME 49 NUMBER 1

Precipitin tests 61

a,-&1 in the lines could potentially simply reflect a relatively high resistance of these inhibitors to proteolysis.

There is increasing artificial exposure of humans in a variety of settings to proteinases of foreign origins. 7, 8, I6 Such exposures lend themselves to problems which are suitably examined with precipitin tests. In addition, it is usual to use rabbit antisera in precipitin tests in analyses of proteinase preparations. The proteinase-dependent false positive precipitin-like reactions described here illus- t’rate the caution which is required in interpretations of these tests.

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11 Dolovich, J., and Little, D. C.: Correlates of skin test reactions to Badlus subtilis enzyme preparations, J. ALLERGY 49: 43, 1972.

12 Polgar, L., and Bender, M. L.: The reactivity of thiolsubtilisin, an enzyme containing a synthetic functional group, Biochemistry 6: 1344, 11968.

13 Gocke, B. J., and Howe, C.: Rapid detection of Australia antigen by counterimmuno- electrophoresis, J. Immunol. 104: 1031, 1970.

14 \Vicher, V., and Dolovich, J.: Interactions of Bacillus subtilis alkaline proteinases with a,-macroglobulin and or,-antitrypsin, Int. Arch. Allergy Appl. Immunol. 40: 779, 1971.

15 Dolovich, J., and Wither, V.: The binding of Bacillus subtilis alkaline proteinase to a*- macroglobulin, J. Lab. Clin. Med. 77: 95:L, 1971.

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