The present invention relates to a method and a reagent for detectin bacterial and ungal infections with a high sensitivity.

Various methods for rapidly detecting bacterial infections throug Gram-negative bacteria with a relatively high sensitivity are based upon th reaction of a lysate of amebocytes or "blood cells" from horseshoe crabs. Th lysate reacts to bacterial endotoxins, which are lipopoiysaccharides, such that gel is formed. This gel phenomenon, which is a defense reaction of the horseshoe crab, is elicited by the lipopoiysaccharides activating a serine protease, which in turn converts coagulogen into coagulin. The previous determination methods are based, on one hand, on the gel reaction itself, such as measurement of the gel formation rate, turbidity changes, etc., and, on the other hand, upon the activated serine protease. The last mentioned method, which is the definitely most sensitive one and permits detection of conceήtra-

-6 -9 tions as low as 10 - 10 mg/ml, utilizes the capability of the serine protease to enzymatically hydrolyze certain synthetically prepared polypeptides, such that specific end groups are cleaved off and thereby form compounds which may be detected colorimetrically or fluorometrically. This method is described in, for example, the German Offenlegungsschrift 2,740,323. A commercial reagent, marketed by Kabi Peptide Research Ltd., Mδlndal, Sweden, contains a synthetic peptide having a terminal p-nitroanilide group and a lysate from the American horseshoe crab or Limulus polyphemus. On addition of bacterial lipopoiy¬ saccharides the serine protease of the lysate is activated and the yellow colour of the p-nitroaniline released thereby may be read colorimetrically.

While the above described reaction applies to blood cell lysates from all horseshoe crab species, there is only a small number of living species of this early, almost fossile marine animal. Among those the above horseshoe crab; Limulus polyphemus, which exists along the American Atlantic coast, and the Japanese horseshoe crab, Tachypleus tridentatus, may be mentioned. These animals, which thus are relatively rare in the oceans, are also becoming depleted, and this is particularly the case for Limulus polyphemus which is the one that has so far been most utilized commercially for the above mentioned test method. Since the horseshoe crab cannot be cultured in an aquaculture, a shortage of such lysate may be expected in the future.

Till now there has, however, been no substitute for the blood cell lysate of the horseshoe crab, the horseshoe crab being regarded as an old and primitive

odd branch of the articulates or arthropods with a unique blood system which in several respects differs substantially from those of other arthropods. The above mentioned endotoxin reaction has therefore been believed to be limited to this specific animal species. Thus, for example, the horseshoe crab only has one type of blood cells, while the other arthropods have three to nine distinct blood cell types depending on class and species. A more important difference in this connection is, however, that the coagulogen of the horseshoe crab is in the blood cells, while in other arthropods it has been shown to be included in the plasma, where the main coagulation reaction also takes place. Both plasma and blood cells would therefore be required for a coagulation activation in the latter case, which thus would make the preparation of a fairly stable activable preparation like that from the horseshoe crab impossible.

According to the invention it has, however, been found, that coagulogen and at least one serine protease are included in the blood cells of the arthropods crustaceans and insects and are involved in the activation of a coagulation-like reaction in a similar way as in the horseshoe crab, and that therefore both bacterial and fungal infections may be determined qualitatively as well as quantitatively using a blood cell or hemocyte lysate from these animals. A lysate from these arthropods is activated quantitatively by lipopoiysaccharides, or LPS, i.e. bacterial endotoxin, as well as by another type of carbohydrates, viz. β-l,3-glucans, which are part of the cell walls of essentially all fungi. While the biochemical mechanism is not completely known, the 3-1,3-glucans very specifically activate a serine protease which, in addition to converting coagulogen into coagulin, converts prophenoloxidase into the active enzyme phenoloxidase.

According to the invention a fungal or bacterial infection may thus rapidly and easily be detected by, in a per se known manner, contacting a sample to be examined with, on one hand, a blood cell lysate from crustaceans or insects, or an activable serine protease or proteases isolated therefrom, and, on the other hand, a detector substance which through the enzymatic action by such a blood cell lysate activated by a bacterial or fungal extract may form a physically or chemically detectable compound, and determining the latter. A corresponding reagent or reagent kit for detecting or determining fungal or bacterial infections therefore comprises, on one hand, a blood cell lysate from crustaceans or insects and, on the other hand, such a detector substance.

Although generally blood cell lysates from all crustaceans and insects could be used according to the invention, among the crustaceans (Crustacea) the

ten-footed crustaceans or the so-called decapods are preferred. Fresh-wate decapods as well as marine decapods may be used. As examples of fresh-wate crayfish may be mentioned Astacus astacus (river crayfish), Pacifastacu leniusculus (signal crayfish), Astacus pallipes, Orconectes limosus, Astacu leptodactylus, Cambarus affinis (North-American crayfish), Procambarus ciarkii Among suitable marine decapods Cancer pagurus (common or edible crab) Carcinus maenas (shore crab), Homarus vulgaris (lobster), Palinurus vulgari (spiny lobster), Nephrops norvegicus (Norwegian lobster), Cailinectes sapidu may be mentioned. Among these crustaceans several are directly suited fo culturing in an aquaculture, e.g. river crayfish and signal crayfish, but als lobster and crab, and these are therefore preferred for the purposes of th invention.

Among insects particularly those of Orthoptera and Lepidopter

(butterflies) may be mentioned. Examples of the first mentioned order ar Schistocerca gregaria (desert locust) and Locusta migratoria (common locust).

Among butterflies Galleria melonella (wax-moth), Hyalphora cecropia and

Bo byx mori (silk-moth) may be mentioned. Although insects probably would not be of the same interest for lysate production as crustaceans, the culture of, for example, silk-moth for this purpose might very well be contemplated. Due to the fact that blood cell lysates from, e.g., the above mentioned crustaceans may be used, a simple and continuous source of blood cell lysates having a very uniform quality is assured, since hemolymph from, e.g., crayfish may be collected during the major part of the year. The hemolymph of the horseshoe crab, on the other hand, may only be collected during a very limited period of time.

As mentioned above the coagulation process for hemolymph from crustaceans and insects involves activation of at least one serine protease. Due to the presence of such an active serine protease essentially the same type of peptide compounds, which have been used or suggested or the previously known Limulus lysate process, may be used. Thus, the invention comprises a method and a reagent, wherein the detector substance, i.e. the substance to be affected by the activated lysate, is a peptide compound of the formula

R X-Arg-R ₂ wherein R . is a peptide moiety protected at the N-terminal and having at least tw a ino acid units, X is Gly or Ala, preferably Gly, and R- is a residue attached to the C-terminal of the arginine residue represented by Arg through an acide amide and/or ester linkage, and which in the presence of the blood cell lysate

activated by bacteria or ungi may be hydrolyzed enzymatically into and/or a mineral acid salt thereof. The residue R ₂ is further such that th compound R-H formed may be detected physically or chemically, e.g colorimetrically or spectrophotometrically, and is preferably a fluorogenic or chromogenic compound. Preferably, the group R ₂ is derived from p-nitroanilide 5-nitro-α-naphtylamide, β-naphtylamide, α-naphtyl ester, β-naphtyl ester indoxyl ester, N-methylindoxyl ester, (4-methyl)umbelliferyl ester and/o resorfin ester, the corresponding compounds R^H being p-nitroaniline, 5-nitro α-naphtylamine, β-naphtylamine, α-naphtol, β-naphtol, indoxyl, N-methyl indoxyl, 4-methyI-umbeliiferon and resorfin. Of these the first two compound are chromogenic, while the other are fluorescent compounds.

Suitable protecting groups for R, are, for example, benzoyl, acetyl carbobensoxy, tert-butoxycarbonyl and p-toluenesulf onyl.

Particularly suitable peptide derivatives for use in this aspect of th invention are

Bz-He-Glu _τ Gly-Arg-PNA.HCl

Bz-IIe-Glu( -ρiperidyl)-Gly-Arg-PNA.HCl

Bz-Ile-Glu(0-Et)-Gly-Arg-PNA.HC1

Bz-Ile-Glu(θ-i-Pr)-GIy-Arg-PNA.HCl ^'■ Bz-Ile-Ser-Gly-Arg-PNA.HC1

Bz-Ile-Glu(0-Me)-Gly-Arg-PNA.HC1 wherein Bz = benzoyl, Ac = acetyl, Me = methyl, Et = ethyl, i-Pr = isopropyl an

-PNA = p-nitroanilide, and the amino acids are given with the IUPA abbreviations. Some of the peptide compounds are commercially available while th others may be prepared with known methods.

In other respects the process may be effected as is described for th Limulus lysate method in the above mentioned German Offenlegungsschrif 2,740,323. A hemocyte lysate, i.e. a blood cell lysate, from crustaceans and insect according to the invention may be prepared essentially in the same way as th well known Limulus lysate from the horseshoe crab. This method is wel described previously in the literature and need therefore not be described in an detail herein. Thus, a partially purified hemocyte lysate from, for example crustaceans is prepared by first collecting blood from the animal, care bein taken to avoid contamination of the blood. It is to be noted that it is no necessary to kill a crustacean which can be cultured in an aquaculture, such a

crayfish, signal crayfish, etc., when collecting the blood, but blood may b collected from the same animal several times at regular intervals as with human blood donor. This is, of course, a great advantage, since the recovery o blood for hemocyte lysate production according to the invention may then b combined with crayfish culture for food purposes. The hemocytes or blood cell are then isolated through centrifuging and washing in conventional manne After that they are homogenized in a buffer with a high calcium io concentration, centrifuged at about 70,000 g and the supernatant recovere The lysate obtained is more stable than the present commercial lysates fro horseshoe crabs and will remain stable for at least 2 hours. Preferabl however, the supernatant obtained is f reeze-dried to be diluted with water or suitable buffer (pH ~ 8) at the time of use. The solution may optionally b stabilized with 0.5 - 1.5M NaCl, whereby a stability of several days may b achieved. Also a solution of the freeze-dried lysate is stable for at leas 5 hours. To increase the freeze-drying stability, e.g., bovine serum albumi and/or glycine may be added.

A reagent or reagent kit according to the invention for detecting funga and bacterial infections may comprise a blood cell lysate prepared as above an a detector substance according to the previous definition. Preferably, the lysat and the detector substance are in powder form. When using it for testing, fo example, an extract having a suspected fungal or bacterial infection, the tes reagent is dissolved in a suitable buffer (pH ~ 8), and then the extract to b tested is added. The reagent solution is then studied, for example, spectrophoto metrically or colorimetrically depending on the detector substance used. The method and the reagent of the invention are, in comparison wit the horseshoe crab lysate known for endotoxin determination, also extremely sensitive for detecting fungal infections. All fungi containing β-l,3-glucans in the cell walls thereof may be detected, which applies to all existing fungi with few exceptions. To rapidly be able to detect fungal infections in accordance with the invention is of great value. At present, fungal skin infections on humans and animals are cultured, which takes about 2 weeks. Also, mould infections in food, so-called mycotoxins, are becoming a rapidly increasing problem.

The sensitivities hitherto obtained with the use of lysates from crustaceans according to the invention are about 10 -8 - 10 -9 g/ml to endo toxins (bacterial infection) and about 10 ^" g/ml to β-l,3-glucans (fungal infection).

The sensitivity to endotoxins may probably be increased further by eliminating inhibitors of the endotoxin activation which have been shown to be included in

ϋREA f OMPI

the lysate, for example analogously to what is now done with the commercia horseshoe crab lysates.

The blood cell lysates may be rendered endotoxin specific by adding a excess of β-l,3-glucans. In a corresponding manner β-l,3-glucan specificity ma 5 be achieved by adding anti-endotoxin factors, produced in pure form fro crayfish lysate, which will then prevent the endotoxin activation of the crayfis lysate. Alternatively, high contents of endotoxin (LPS) may be added.

Quantitative determinations of endotoxins and β-l,3-glucans respectively, by means of the inventive process may be effected in a manne 10 known per se, e.g. by providing a standard or calibration curve.

The invention will now be described in more detail by means of som specific ^" examples, which, however, are not limiting to the invention in any way.

Example 1 Preparation of a hemocyte lysate 15 Hemocytes from crayfish, Astacus astacus, were collected as describe in Sδderhall, K., Hall, L., Unestam, T., and Nyhlen, L. (1979) J. Invertebr Pathol. 34, 285-294, except that 0.1M sodium citrate was excluded. Th hemocytes were homogenized in 10 mM sodium cacodylate buffer, pH 7.0, wit 100 mM CaCl- and the homogenate was then centrifuged for 20 minutes a 20 70,000 g. The supernatant obtained, containing approximately 2 mg protein/ml may be used either immediately or freeze-dried in 3 ml aliquots. Prior to use i is dissolved in 3 ml of distilled water ^' to a final protein concentration o 2 mg/ml.

Example 2 25 β-l,3-glucan activation of crayfish hemocyte lysate

In order to test the method of the invention with regard to specific β 1,3-gIucan activation of the hemocyte lysate, two volumes of the hemocyt lysate from Example 1 were mixed with one volume of β-l,3-glucans or othe carbohydrates of the concentrations given in the following Table 1 fo 30 30 minutes at 20-22°C. 100 μl of this reaction mixture was added to 600 μl o 0.1M tris-HCl-buffer, pH 8.0, and 100 μl of a 2 mM solution of the syntheti peptide Bz-Ile-Glu(j'-piperidyl)-Gly-Arg-PNA.HCl (obtained from Kabi Peptid Research Ltd., Mδlndal, Sweden). After incubation for 0.5 hour at 37°C 100 μ of 50% acetic acid were added to terminate the reaction, and the released p 35 nitroaniline (PNA) was measured spectrophotometrically at 405 nm.

The β-l,3-glucans used were Zs (the supernatant of a 1% suspension o yeast cell walls, Zymosan, Sigma), Iaminaran M, laminaran G and a linea pentasaccharide composed of β-l,3-D-linked glucosyl residues. Laminaran M an

G were purified from laminaran (Sigma) by using DEAE-moIybdate-Sephade chromatography according to Stark S., J.R. (1976) Carbohydr. Res. 47, 176-17 The linear pentasaccharide was prepared and purified as described in Sδderhal K., and Unestam, T. (1979) Can. J. Microbiol. 25, 406-414.

The results of the enzyme activity determination (serine protease showing the specific β-l,3-glucan activation appears from Table 1 below.

Table 1 β-l,3~glucan activation of serine protease in crayfish hemocyte lysate

Degree of Concentration Enzyme activity polymeri¬ (glucose equiv., min)

Glucan Structure sation μg/ml) (ΔA^/30

None - - 0 0.03

Zymosan (l -→-3)β-D-glucan with varying , _g 60 0.85 supernatant β-l,6-linkages (2 * 10 - 10 ^b )

Laminaran M (l-*-3)β-D-glucan ended 20 300 0.99 with mannitol

Laminaran G (l-*-3)β-D-glucan ended 20 300 0.99 with glucose

Lamioara-pentaose (1^3)β-D-glucan 5 300 0.95

Other carbo¬ hydrates tested - ^' 300-1600 not detected

^x Other carbohydrates tested, were chitin, cellulose, dextran, glucose and mannitol.

Example 3

Freeze-dried hemocyte lysate from Example 1 was dissolved in 3 ml o

0.1 M tris-HCl-buffer of pH 8.0. This solution was then mixed with 1.5 ml o

Zymosan supernatant according to Example 2 and incubated for 30 minutes a 20°C. From this reaction mixture 100 μl were withdrawn and added to 600 μl o

0.1 M tris-HCi (pH 8.0). 100 μl of 2 mM solutions of different synthetic peptide having chromogenic terminal groups (obtained from Kabi Peptide Research Ltd.

Mδlndal, Sweden) were added to this mixture, and after incubation for 1 hour a

37°C the reaction was terminated by adding 100 μl of 50% acetic acid. Th released p-nitroaniline was then measured spectrophotometrically at 405 nm

The results for the different chromogenic substrates are listed in Table 2 below

Table 2 Hydrolysis of dif erent chromogenic substrates by preactivated crayfish hemocyte lysate

Chromogenic substrate Enzyme activity

(ΔA^ _Q5 /30 min)

Bz-Ile-Glu-Gly-Arg-PNA.HC1 0.62

Bz-Ile-Glu(y -piperidyl)-Gly-Arg-PNA.HCI 0.84

Bz-Ile-Glu(O-Et)-Gly-Arg-PNA.HCl 0.84 Bz-Ile-Glu(O-i-Pr)-Gly-Arg-PNA.HCl 0.92

Bz-IIe-Ser-Gly-Arg-PNA.HCl 0.90

Bz-Ile-Glu(O-Me)-Gly-Arg-PNA.HCl 0.70

Example 4 A reagent according to the invention consisting of a crayfish hemocyte lysate according to Example 1 in 0.01 M cacodylate buffer (pH 7.0) and an equal amount (about 100 μl) of Bz-Ile-GIu(y-piperidyl)-Gly-Arg-PNA.HCl were mixed with pre-heated (5 minutes, 100 C) extracts of the following fungi: Aphano- myces astaci, Aphanomyces laeγis, Aphanomyces euteiches, Saecharomyces cerevisiae, Aspergillus flavus, Penicillium viridicatum, Candida albicans, Poly- porus annosus, Boletus variegatus. The solutions were then observed colori¬ metrically at 405 nm. For all the ungi a yellow colour was obtained indicating activation of the hemocyte lysate with associated enzymatic hydrolysis of the p-nitroanilide group to p-nitroaniline.

Example 5 A mixture of 100 μl of hemocyte lysate from Example 1, 100 μl of

E. coli endotoxin (Mallinckrodt, Inc., USA) of different concentrations, 100 μl of

" URE

0.1 M tris-HCl, pH 8.0, and 100 μl of a 2 mM solution of Bz-Ile-Glu(^- piperidyl)-Gly-Arg-PNA.HCI (from Kabi Peptide Research Ltd., Mδlndal, Sweden) were incubated for 30 minutes at 37°C. 100 μl of 50% acetic acid were then added to terminate the reaction, and the released p-nitroaniline was measured spectrophotometrically at 405 nm. The absorbance changes obtained appear rom Table 3:

Table 3

Endotoxin concentration Δ ^min / ¹⁰⁰ g/ml hemocyte lysate

1 x 10 ^"6 0.64

1 x 10 ^"7 0.55

1 x 10 ^"8 0.18

1 x 10 ^"9 0.058

Control 0.030

Example 6

In analogy with Example 1 hemocyte lysates were prepared from the following crustaceans:

Pacifastacus leniusculus Astacus pallipes Astacus leptodactilus

Orconectes limosus Cancer pagurus Carcinus maenas Nephrops norvegϊcus The lysates obtained were tested for activation by β-l,3-glucans and endotoxins in analogy with the methods described in Examples 2 and 5, activation (release of p-nitroaniline) being indicated for all the lysates.

Blood cell lysates from the insects Schistocerca gregaria, Galleria melonella, Hyalphora cecropia and Bombyx mori have so far only been tested with regard to activation of phenoloxidase, which, as for the above mentioned crustacean lysates, gave positive results. Because of the great similarities bewteen the blood systems of crustaceans and insects, and since it was ound in connection with the invention that phenoloxidase in turn is activated by a serine protease, also these insect lysates must be expected to be activated in testing as above.