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This application is a continuation-in-part of copending United States patent application serial number 442,309, filed 11-28-89, for New In vitro Test for Fecal Leukocyte, the subject matter of which is incorporated herein as though recited in full.
l. Field of the Invention
The present invention relates to diagnosis of inflammatory processes by examination of body fluids or tissues for leukocyte or white blood cells (WBC) .
2. Prior Art
A need exists for a simple, reliable in vitro test for the diagnoses of inflammatory process, such as fecal leukocytes (eve after they are morphologically disrupted) , to indicate that a su set of patients with the common problem of diarrhea that is in-flammatory and thus requires specific, more costly diagnostic an therapeutic attention.
Diarrheal illnesses are extremely common (causing 2 to 12 o more illnesses per person per year) throughout the world, and of ten pose diagnostic and therapeutic questions for the physician. Fortunately, important diagnostic clues can be obtained by con-sidering whether the diarrhea is a noninflammatory process aris-ing typically from the upper small bowel, or whether it is an in flammatory diarrhea arising from an invasive process in the ileu or colon. Although the majority of cases are noninflammatory and will often respond to simple oral rehydration therapy, it is im- portant to distinguish the invasive, inflammatory diarrheas, which are usually caused by Shigella, Salmonella, Campylobacte or Clostridium bacteria, that may be, more severe and should b the focus of more expensive culturing for these invasive pathogens. The invasive, inflammatory diarrheas may also requi specific antibiotic treatment. A particularly helpful diagnost clue to distinguishing inflammatory from noninflammatory diar- rheas has been the examination for large numbers of leukocytes (white blood cells or "pus cells") in the diarrheal fecal specimens themselves. However, this requires that the physician or promptly examine mucus from a cup fecal specimen under a mic scope, stained for clearly distinguishable leukocytes in the fe cal debris. This requires the immediate availability of a skill person with a microscope to stain and examine fresh fecal specimens in the clinic or emergency area where the patient is seen. Despite extensive efforts, this is difficult to accomplis especially with this extremely common problem in a busy clinic setting.
There are many potential markers for leukocytes in the primary and secondary granules. Leukocyte esterase was explored as a potential marker for fecal leukocytes, since an analogous test exists for leukocytes in urine. However, it was discovered that leukocyte esterase was non-specifically positive for all stool samples, both those with and without leukocytes.

The diagnosis of inflammatory processes is best made by examina tion of body fluids, such as sputum, vaginal or cervical secre-tions or tissue biopsies, for leukocytes. While this traditiona method requires a skilled microscopist examining stained materi on slides using a microscope, the WBCs may be destroyed by pathogenic microorganisms or toxins or on transport or storage, or absorbed onto swabs, or may be present in some areas, but no other areas of the specimen. Further, the test is highly releva to the detection to dangerously low white blood cell counts, pa ticularly neutropenia. Such low white blood cell counts severel limits the less costly widespread use of emerging new drugs suc as clozapine (Clozaril) for schizophrenia or AZT for AIDS.
A simple, in vitro test for a leukocyte marker was develop that is sensitive to the numbers of fecal leukocytes typically found in inflammatory diarrheal specimens and that can be quick and easily done with a minimum of training, either in the clini later (after transportation or storage) in the laboratory.
Surprisingly, the test has been found to provide a simple sensi tive screen for as few as 200 WBCs/ul.
The marker found most specific for leukocytes in fecal specimens is based on lactoferrin, an iron-binding glycoprotein found concentrated in secondary granules in leukocytes
(Hetherington et al. "An Enzyme-Linked Immunoassay (ELISA) for Measurement of Lactoferrin", J IMMUN METH, 65:183-190 (U.S.A.) 1983) . Standard immunoassay methods of radial immunodiffusion, latex agglutination and enzyme-linked immunosorbent assays (ELISA) that employ antibodies against lactoferrin have proved successful in detecting specimens. However, the latex agglutina- tion method seems the simplest and most applicable in vitro test These and further and other objects and features of the in- vention are apparent in the disclosure, which includes the above and ongoing written specification, including the claims and the drawings.
Fig. 1 graphically illustrates results of 22 fecal specimen tested for C. difficile cytotoxin tested with the leukocyte lac- toferrin latex agglutination assay showing that with increasing cytotoxin titer, an increasing percent (to 100%) of lactoferrin, evidence of an inflammatory process.
Fig. 2 graphically illustrates the increasing titer of cytotoxin which is associated with increasing titers of lactofer-rin, but decreasing appearance of leukocytes, which demonstrates the destruction of leukocytes by the cytotoxin analogous to that seen in vitro
This in vitro test for a leukocyte marker must be sensitive to the numbers of fecal leukocytes typically found in inflam- atory diarrheal specimens. Typically, at least 5,000 to 10,000 polymorphonuclear neutrophils per mm3 are found in inflammatory diarrheal specimens.
(a) Radial Immunodiffusion Assay Studies were conducted using the commercially available L Partigen radial immunodiffusion plates (Calbiochem) for lactof rin. The leukocytes were obtained from fresh, whole blood processed with a neutrophil isolation kit. The fecal suspensio with and without leukocytes were initially mixed with an equal amount of 0.1% Triton-X to lyse the leukocytes (an addition th was subsequently found to be unnecessary) . Forty microliters o this suspension was placed in the wells and the plates were checked daily for visible rings around the wells. Within three days, measurable rings were visible around the wells which had stool samples with fecal leukocytes added and were absent in samples without fecal leukocytes. The ring size with fecal leukocytes at 10,000/mm3 was 9,05 mm. The ring size with fecal samples that included 7,700 leukocytes/mm3 was 7.05 mm and no ring was seen in fecal samples with no leukocytes. Similar results were obtained after 10 days' refrigeration of the feca leukocyte suspension as well.
Because the Calbiochem LC-Partigen kits became unavailabl continuing work necessitated preparation of plates with purcha anti-lactoferrin antibody. The preparation of plates for radia immunodiffusion assay was made by adding 6 ml of 1% agarose co taining 1:1000 rabbit anti-lactoferrin antibody (30 ul per 6 m agarose per standard 2 x 3" slide) . The rabbit anti-human lac-toferrin was obtained from Sigma Company, St., Louis, Missouri (Catalog # L-3262) . The agarose was prepared using 1% low gel perature agarose (Sea Plaque from FM Corporation) , 3%
polyethylene glycol-6000 from Fisher Scientific and 100 ml pho phate buffered saline. When the agar covered slides were ready for use, 2 mm wells were punched that each readily hold 5 microliters of antigen preparation each.
Using these methods for radial immunodiffusion, dose response curves with purified lactoferrin (Sigma Chemical Co - pany, St, Louis, Missouri) showed it was detectable at 0.02- 0. mg/ml (ug/mm3) with a ring size of 4.1 - 4.3 mm respectively. Repeated studies with human PMN's (poly orphonuclear neutrophil revealed optimal sensitivity of approximately 2.08 X 103 MN/mm3, giving a zone diameter of 4.7 mm. This corresponds to a calculated lactoferrin concentration of 0.0104 ug/ul based on the ap proximate concentration of 1 ng lactoferrin per 200 PMN's
(Hetherington et al. "An Enzyme-Linked Immunoassay (ELISA) for Measurement of Lactoferrin", J IMMUN METH, 65:183-190 (U.S.A.) 1983). This result using 0.3% cetyl trimethyl ammonium bromide (CTAB, a detergent used to lyse neutrophils for their release of lactoferrin) was slightly better than that seen in the absence o CTAB, and was in the same general range of radial immunodiffusio assay results with purified lactoferrin noted above. Thus the se sitivity of radial immunodiffusion appeared to be approximately 2000 PMN's/mm3, a number far lower than the expected concentra-tion of leukocytes (PMN's) in inflammatory fecal specimens, judg ing by microscopy with numerous leukocytes per high power field. Natural inflammatory diarrheal specimens revealed two patients with documented Salmonella gastroenteritis showing distinct ring ranging from 4 to 13 mm in size, as well as two patients with C. difficile cytotoxin giving rings of 7.0 to 8.4 mm. In addition, the normal stool specimen was repeatedly negative on three dif- ferent occasions. CTAB and Triton detergents did not seem to ad any sensitivity to naturally inflammatory fecal specimens.
(b) Latex Agglutination Assay
For studies using latex agglutination, Baσto-latex 0.81 beads (Code 3102, Difco Laboratories, Detroit, Michigan) were coated with rabbit anti-human lactoferrin (Sigma Chemical Compa Product #L-3262, St. Louis, Missouri) . as follows: 2.5 ml of bea were centrifuged at 3000 rpm for 30 minutes, washed with 5 ml glycine buffer (7.3 g glycine, 10 g NaCl, in 1 liter distilled water adjusted to pH 8.2 - 8.3) and then resuspended in 5 ml of glycine buffer to provide an approximate 1% suspension of beads. To this latex bead suspension was added 0.35 ml rabbit antilac-toferrin antibody and the mixture was incubated at 37 C for 1 hour, after which the antibody-coated beads were spun and resuspended in 5 ml buffer to which 0,005 g azide (0.1%) and 0.0 g bovine serum albumin (1%) were added and the coated bead susp sion was stored at 4 C until used. Studies with titrations or purified Lactoferrin revealed readily apparent agglutination of these latex beads with 0.004-0.0016 mg/ml lactoferrin, at least one log more sensitive than the radial immunodiffusion (RID) as-say mentioned above. This level of greater sensitivity of latex agglutination was also seen with fecal-hypaque separated human PMN's as well with a 1:100 dilution being positive when RID detected only a 1:8 dilution. In addition, leukocytes added to stools as well as the Salmonella and 4 C.difficile cases were positive in the latex agglutination assay. Furthermore, three a ditional control specimens tested on 7 different occasions were all negative. Importantly, these immunoassay results remained clearly positive even after C. difficile cytotoxin totally destroyed the MN morphology over 24 hours in refrigerated specimens.
22 fecal specimens were tested with the leukocyte lactofer rin latex agglutination assay for C. difficile cytotoxin with t results shown in figure 1. These results show that with inσreas ing cytotoxin titers, an increasing percent (to 100%) have Lac- toferrin evidence of an inflammatory process. Remarkably, as shown in Figure 2, the increasing titer of cytotoxin is as- sociated with increasing titers of lactoferrin but decreasing a pearance of leukocytes by microscopy, demonstrating the destruc tion of leukocytes by the cytotoxin analogous to that seen in vitro.
Further data from children with diarrhea in the northeast Brazil have shown that specimens from 16 of 17 children with 1-5 or more fecal leukocytes per high power field on microscopy wit methylene blue stain had lactoferrin latex agglutination titers of 1:50. In contrast, only 3 of 12 methylene blue stained specimens with less than 1 leukocyte per high power field had la toferrin titers of >1:50. Furthermore, despite occasional posi-tives at lower titers, none of 7 specimens from normal control children had lactoferrin titers of >1:50.
(c) Enzyme-Linked Immunosorbent Assay (ELISA) Studies with the development of an ELISA for lactoferrin s gest that it may be even more sensitive than the RID or latex a glutination assays. However, as noted above, the need for in-creased sensitivity may be unnecessary or even inappropriate. F the ELISA testing, wells were coated with varying concentrations of lactoferrin in a sodium bicarbonate buffer at room temperatu for 2-3 hours or overnight at 4 C. Wells and microtiter plates were then washed 3 times with PBS-tween and 1% BSA was added to fill the wells for 30-60 minutes to block nonspecific sites, fol lowed by washing 3 times with PBS-tween. Thereafter 50 ul of ra bit anti-human lactoferrin antibody (at 1:250 dilution, probabl optimal with 1:100 and 1:500 also being effective) was added to each well, followed by 40 minutes incubation at
37 C (or 2 hours at room temperature) , followed by washing 4 times in PBS-tween. Then 50 ul of goat anti-rabbit IgG (Rockland Laboratories, Gilbertsville, Pennsylvania) with peroxidase con-jugation (at 1:1000 dilution) was added for 40 minutes at 37 C (or 2 hours at room temperature) , followed by washing 5 times in PBS-tween. Thereafter, 200 ul of activated peroxidase substrate was added, followed by 45 minutes incubation at room temperature in the dark, after which this was read both visually and spectrophotometrically. The apparent sensitivity was 0,001 ug/uL or less lactoferrin, with conjugate dilution of 1:1000 and primary rabbit antibody dilutions of 1:250, probably representin the optimal conditions for assay. The ELISA technology could also be employed in detection of leukocytes, possibly using a dipstick technology.

While the invention has been described with reference to specific embodiments, modifications and variations of the inven tion may be constructed without departing from the scope of the invention, which is described in the following claims.