Chromogenic Test Research Articles

Sensitive amplification immunoassay for human IgG and anti-human IgG by using an enzyme cascade system

A sensitive heterogeneous immunoassay for human IgG and anti-human IgG was developed using an enzyme cascade system in limulus amoebocyte as a signal amplification system. Lipopolysaccharide (LPS) was conjugated to human IgG and anti-human IgG was adsorbed on polystyrene beads. The LPS-labelled human IgG mixed with unlabelled human IgG was allowed to react in a competitive manner with the immobilized anti-IgG on the polystyrene bead surface. After B/F separation, the LPS activity in the supernatant (free) and LPS activity on the bead (bound) were measured by using the chromogenic limulus test. IgG could be measured in the range 10 −7-10 −11 g ml −1. LPS-labelled anti-IgG and IgG absorbed on polystyrene beads were prepared, and LPS-labeled anti-IgG mixed with unlabelled anti-IgG was allowed to react again in a competitive manner with solid-phase IgG. The LPS activity specifically bound to the bead was then measured. Anti-IgG could be measured in the range 10 −7-10 −11 g ml −1.

Laboratory and therapeutic control in the Thrombosis Centre Rotterdam using chromogenic prothrombin time tests

Growing interest is observed in chromogenic substrate assays, because of their better precision, performance and possibilities for automation. Applied to a Cobas Bio centrifugal analyzer, we compared Nycotest-Chrom (N-test) and Thromboquant-PT (Tbq) with Thrombotest (TT). Precision tests were performed with samples from 4 plasma pools: normal (45 sec), low (100 sec), middle (150 sec) and high (200 sec) segments of the therapeutic range of TT. N-test had the best precision profile in both intraassay and interassay determinations compared with Tbq. Both chromogenic substrate assays were better than TT in this respect. By orthogonal regression a provisional therapeutic range was calculated from 312 determinations and later adjusted in a confirmation experiment with natural logarithm regression in 946 determinations. Compared with a TT range of 105-180 sec, the therapeutic ranges were 71-120 sec for N-test and 63-103 sec for Tbq. In a clinical therapeutic control phase, 110 patients were randomized in equal proportions to two groups A and B. Every 2 weeks for a period of 16 weeks, blood samples were tested for N-test, Tbq and TT. In group A, dose adjustment was based on N-test, and in group B on Tbq. The monitoring physician was blinded for Tbq and TT in group A and for N-test and TT in group B. No differences were found between the groups for mean TT, N-test, Tbq or mean dosage, nor differences were found in complication rate. A definite therapeutic range was complied using sensitivity/specificity curves together with their 95% confidence limits. Given TT 105-180 sec, the therapeutic range of N-test is from 80 (95% confidence limits: 77-82) to 110 (95% confidence limits: 108-115) sec, and of Tbq from 68 (95% confidence limits: 66-71) to 95 (95% confidence limits: 91-98) sec. It was concluded that the two chromogenic substrate assays performed equally safe in the monitoring of patients on oral anticoagulant therapy, despite differences in diagnostic correspondence with the reference TT.

Endotoxemia in Patients on Hemodialysis

We examined endotoxins and limulus amebocyte lysate-reactive material (LAL-RM) in serum from 87 patients on hemodialysis, using the conventional chromogenic limulus test (CCLT) and a new specific endotoxin assay with factor G-free LAL (endotoxin-specific test: EST). All patients with regenerated cellulose dialyzers had increased CCLT values, whereas the EST values were not higher than in controls. This discrepancy can be explained by the LAL-RM which is cellulose-derived and cross-reacts with LAL via factor G. Using EST for measurements of endotoxin, 6 patients out of 87 had pathological endotoxemia and all of these patients were associated with either cirrhosis, infection, or malignancy. Some patients who had no complications showed fever before or during dialysis, but they did not have high endotoxin levels. EST is useful for the diagnosis of endotoxemia in patients on hemodialysis because of the presence of LAL-RM in serum. Endotoxemia is rare in patients on hemodialysis, if they are not associated with infection, cirrhosis, or other complications.

Perioperative change of plasma endotoxin levels in early infants

As a preliminary study to elucidate the relationship of endotoxemia to postoperative morbidity, the plasma endotoxin levels in 64 surgical neonates were quantitated by the chromogenic limulus test (Toxicolor test; Seikagaku Kogyo, Tokyo, Japan). The preoperative levels of plasma endotoxin were 64 +/- 59 pg/mL in the group of infants with perforated peritonitis (n = 9), 63 +/- 51 pg/mL in the group of infants with gastroschisis (n = 7), and 15 +/- 16 pg/mL in the group of infants with ileus (n = 28), while the mean level was 6 +/- 5 pg/mL in the remaining 20 surgical neonates who had no signs of ileus or peritonitis. In the serial determination of plasma endotoxin in 28 neonates, the levels on the first postoperative day increased significantly compared with the preoperative levels (16 +/- 18 pg/mL to 46 +/- 25 pg/mL, P less than .01). They decreased gradually to 8 +/- 5 pg/mL within a week in 15 neonates who had no postoperative complications. However, in 13 neonates who had postoperative complications such as wound infection or postoperative ileus, the postoperative levels of plasma endotoxin increased to a much higher level and remained there. In this article the relationship of clinical endotoxemia to postoperative thrombocytopenia and hyperbilirubinemia is analyzed, and the usefulness of evaluating endotoxemia in surgical neonates is discussed.

Effects of different extraction protocols on endotoxin analyses of airborne grain dusts.

The detection of gram-negative bacterial endotoxins in occupational dusts, specifically those from agricultural environments, is of increasing importance in research on occupational lung disease. In this study, the quantitative chromogenic Limulus amebocyte lysate test for the detection of endotoxins in airborne dusts from spring wheat and oats was examined. Different extraction fluids were tested, as were the effects of time on extraction and of repeated freeze-thaw cycles on the extracts. The data suggest that the chromogenic method can be used effectively in the analysis of environmental dusts or their frozen extracts for endotoxin quantitation. Water appears to be the preferable extraction medium, and the length of extraction time may affect the results.

Evaluation of four qualitative methods for detection of beta-lactamase production inStaphylococcus andMicrococcus species

Four qualitative methods for the detection of beta-lactamase production in Staphylococcus and Micrococcus species were evaluated and compared with a quantitative macroiodometric reference method. The disc diffusion test with penicillin G and the cloverleaf method could not separate beta-lactamase-positive from beta-lactamase-negative strains. Two applications of the chromogenic cephalosporin test, using uninduced strains and strains grown on blood agar plates, gave a large number of false negative and false positive results. False negative reactions were most common among uninduced strains, while the false positive reactions were most often recorded for Staphylococcus saprophyticus. A high degree of efficiency was recorded for the nitrocefin spot test, using induced strains grown on antibiotic susceptibility agar, and for the starch-iodine plate method. The starch-iodine plate with methicillin as inducer gave the most reliable results.

False-positive result in Limulus test caused by Limulus amebocyte lysate-reactive material in immunoglobulin products

Limulus amebocyte lysate (LAL)-reactive material other than endotoxin was detected in the plasma and urine of patients after intravenous immunoglobulin therapy. Thirty-seven vials of six different immunoglobulin products were analyzed for the LAL-reactive material by combined use of a conventional chromogenic Limulus test and a chromogenic endotoxin-specific test. The amount of LAL-reactive material in reconstituted immunoglobulin solutions ranged from a mean (standard deviation) of 10.2 (2.1) to 2,448.1 (988.9) pg/ml, and there were statistically significant differences among the six brands. The levels of LAL-reactive material in plasma increased in proportion to the amounts contained in the immunoglobulin products administered. The material accumulated in the blood with repeated administration. Urinary excretion of the material was less than 5% of the total amount administered. Such material seems to be derived from the cellulose-based membranes used during preparation of the blood products. Thus, interpretation of Limulus test results of patients receiving immunoglobulin therapy requires special consideration.

Enterococci highly resistant to penicillin and ampicillin: an emerging clinical problem?

Sixteen clinical isolates of ampicillin-resistant enterococci (ARE) were recovered from the microbiology laboratory of a 450-bed rehabilitation medical center from January 1981 to September 1987. These isolates were detected when a disk diffusion test using 10 micrograms of ampicillin on a blood agar plate revealed no zones of inhibition. Tube macrodilution tests yielded an MIC of greater than or equal to 16 micrograms of ampicillin per ml. None of the isolates were penicillinase producers by the chromogenic cephalosporin disk test. Ten isolates were Enterococcus faecium, four isolates were E. raffinosus, one isolate was E. gallinarum, and one isolate was not identified (lost). There were 6 male and 10 female patients. The sources of isolates were urine (n = 7), wound (n = 5), ascitic fluid (n = 2), blood (n = 2), peritoneal catheter tip (n = 1), Bartholin's cyst abscess (n = 1), rectal swab (n = 2), and pancreatic abscess (n = 1). The organism was isolated from multiple sites in 4 patients, was a pure culture isolate in 5 patients, and was part of a polymicrobial flora in 11 patients. Six patients were diabetic, and four had liver cirrhosis. All but four patients had received at least one antibiotic within 3 weeks of ARE isolation. The MICs (micrograms per milliliter) for 50 and 90% of isolates tested, respectively, were as follows: ampicillin, 64 and 64; penicillin, 128 and greater than 128; vancomycin, 1 and 2; gentamicin, 4 and 16; ciprofloxacin, 1.6 and 3.2; imipenem, 128 and greater than 128; and daptomycin (LY146032), 1.6 and 6.4. ARE may be an emerging pathogen in the hospitalized patient population.

Endotoxin levels in milk and plasma of mastitis-affected cows measured with a chromogenic limulus test

A chromagenic limulus test (“Toxicolor”) was applied to cow's milk and plasma after treatment with perchloric acid to remove interfering factors. The endotoxin levels in normal cow's milk and plasma were all <10 pg ml −1. In acute mastitis, the milk endotoxin level averaged (1.1 ± 0.7) × 10 3pg ml −1 in the cases where Gram-negative bacteria were isolated, while the plasma endotoxin concentration was normal. The endotoxin levels in the quarters infected with Gram-positive bacteria were all normal, both in milk and plasma. In gangrenous mastitis due to Gram-negative bacteria, the endotoxin concentration was very high in both milk [(9.3 ± 5.3) × 10 6pg ml −1] and plasma (85.2 ± 68.2 pg ml −1). In similar cases due to Gram-positive bacteria, endotoxin levels were all normal, both in milk and plasma, resembling the acute mastitis due to Gram-positive bacteria. The test was considered suitable for the diagnosis of mastitis due to Gram-negative organisms and the levels of endotoxin detected would aid in assessing the prognosis.

Determination of the endotoxin content of egg products using a miniaturized chromogenic Limulus test

A chromogenic Limulus amoebocyte lysate assay was applied to monitor endotoxin concentration in egg products. Analysis of differently contaminated whole egg probes revealed a strong correlation of endotoxin concentration to total bacterial count 6 x 10(4) cfu x ng-1, where cfu = colony-forming unit) as well as to number of Enterobacteriaceae (1 ng/7 x 10(2) cfu). Similar relations were also found for egg white and egg yolk probes. A significant influence of heat pretreatment of egg probes (65 degrees C, 60 min) on endotoxin detection could be excluded. Up to a concentration of 10 mg x ml-1 endotoxin-free whole egg material did not interfere with the test system. A miniaturized version of the chromogenic Limulus test, which can be carried out in microtiter plates, is described.

Reliable five-minute test strip method for identification ofStreptococcus pyogenes

A novel and rapid method (Strep Strip, Lab M) for identification of Streptococcus pyogenes was evaluated. The method combines the established test for pyroglutamyl aminopeptidase (PYR) with a rapid chromogenic test for beta-glucosidase on a paper strip. The test was evaluated with 274 clinical isolates and 237 culture collection isolates of beta-haemolytic streptococci. Streptococcus pyogenes was identified with 100% specificity. Six isolates identified as Lancefield group A and which might therefore be assumed to be Streptococcus pyogenes were shown by the test strip method not to be this species. The beta-glucosidase test on the test strip allowed differentiation between enterococci and Streptococcus pyogenes (both PYR positive) in all cases. The test strip method represents an economical and accurate method for identification of Streptococcus pyogenes in clinical material.

The International Standard for Endotoxin: evaluation in an international collaborative study

An ampouled preparation of bacterial endotoxin, coded 84/650, was evaluated in 35 laboratories in 12 countries for its suitability to serve as the International Standard for Endotoxin. The ampouled preparation was calibrated in terms of the USA National Standard, EC5, in Limulus Amoebocyte Lysate gelation, turbidimetric and chromogenic tests and in rabbit pyrogen tests. On the basis of the results reported here, with the agreement of the participants in the study, and with the authorization of the Expert Committee on Biological Standardization of the World Health Organization, the preparation coded 84/650 was established in 1986 as the International Standard for Endotoxin for Limulus Gelation Tests with an assigned unitage of 14 000 IU of endotoxin per ampoule.

Comparative Study on a New One-Stage Clotting Assay for Heparin and Its Low Molecular Weight Derivatives

The effects of a normal and a low molecular weight (LMW) heparin fraction were compared by four coagulation methods. Plasma samples of patients were investigated who were treated with normal heparin or with a LMW heparin. The study was undertaken to analyze the interrelationship between the coagulation methods: Heptest, activated partial thromboplastin time, thrombin clotting time, and S 2222 chromogenic anti-factor Xa test. The results showed a high correlation between Heptest and S 2222 anti-factor Xa method for unfractionated and LMW heparin (r = 0.91 and 0.90). Comparing the coagulation times in seconds of Heptest and aPTT, the correlations were r = 0.56 (normal heparin) and 0.15 (LMW heparin). The correlation between the coagulation times of Heptest and thrombin clotting time were r = 0.65 and 0.25, respectively. The correlations between the coagulation methods were higher, when coagulation times rather than transformed values to units per liter of the Heptest and of the S 2222 anti-factor Xa method were employed. Furthermore, the data demonstrate a high sensitivity of the Heptest to conventional and LMW heparin, whereas activated partial thromboplastin time and thrombin clotting time are less sensitive to either heparin. For laboratory control of LMW heparin, Heptest and S 2222 chromogenic method are reliable tests. Therapeutic ranges will have to be established.

リムルステスト及びD-arabinitol測定による深在性真菌症の迅速診断法

Conventional chromogenic limulus test (toxicolor test) reacts with glucan of bakers yeast as well as endotoxin, because toxicolor test contains factor G which is sensitive to glucan and polysaccharide of fungus. A new endotoxin specific limulus test (endospecy test) was developed to improve reaction which responded only to endotoxin. The toxicolor test showed the positive reaction to the culture media of candida albicans 7N strain in RPMI 1640 medium, but the endospecy test did not. D-arabinitol, measured by fluometric enzymatic method, also was positive to culture media. These results suggested that a combination of the toxicolor and the endospecy test and enzymatic measurement of D-arabinitol could be applied to rapid diagnostic methods of fungal infection.

Early diagnosis of invasive candidiasis and rapid evaluation of antifungal therapy by combined use of conventional chromogenic limulus test and a newly developed endotoxin specific assay.

Since beta-1,3-glucan is a common component of fungal cell wall, its detection might be useful in diagnosing invasive candidiasis. Not only endotoxin but beta-1,3-glucan activates proclotting enzyme contained in a conventional chromogenic limulus test (CCLT). Endotoxin activates this enzyme through factor C, while the beta-1,3-glucan activates through factor G. Since endotoxin specific test (EST) contains factor C, endotoxin would be quantified. By subtracting EST value from CCLT value, beta-1,3-glucan would be quantified. We named this value Fungal Index (FI), and examined if it actually reflects the candidal infection. Ninety-two patients were tested for CCLT and EST prospectively. FI increased significantly in candidal infection (p less than 0.05) but remained low in GNR infection. Moreover, FI increased proportionally to the severity of candidal infection. Elevated FI decreased when antifungal therapy was successful. Thus FI was a useful index not only in the diagnosis of invasive candidiasis but also in the evaluation of antifungal therapy.

Characterization of beta-lactam antagonist in Supplement C.

In the course of using Mueller-Hinton agar with 1% Supplement C (SC) (Difco, Detroit, MI) as a susceptibility test medium for Haemophilus influenzae, one lot of SC was encountered whose use was associated with markedly increased ampicillin MICs. Acidimetric and chromogenic cephalosporin filter paper disc tests of SC failed to detect beta-lactamase activity. Macrobroth dilution MIC tests to determine substrate specificity showed SC to antagonize benzylpenicillin and ampicillin but not cephalothin, cefazolin, or cefaclor, with the antagonism being prevented by the addition of clavulanic acid. High pressure liquid chromatographic analysis of reference and reaction solutions of benzylpenicillin with SC showed almost complete degradation of benzylpenicillin to benzylpenicilloic acid after 24 hr at 37 degrees C. For two other lots of SC that had passed MIC quality control testing, similar high pressure liquid chromatographic studies demonstrated slow conversion of small amounts of benzylpenicillin to benzylpenicilloic acid. These findings indicate that the beta-lactam antagonism by SC was due to the presence of a contaminating beta-lactamase directed primarily toward the penicillins.

130: ENDOTOXIN LEVELS IN MENINGOCOCCAL INFECTIONS

We studied prospectively plasma endotoxin (lipopoly-saccharides, LPS) levels in forty-five consecutively admitted patients with bacteriologically verified systemic meningococcal disease (SMD). Using a semi-automated, Limulus / chromogenic substrate test (detection limite=25 ng/L, 25 patients (56%) had detectable LPS on admission. LPS>700 ng/L seemed to be critical in developing: 1) Severe, persistent septic shock (15/15 versus 3/30) (p<0.0001). 2) Pathologically elevated serum creatinine (15/15 versus 3/30, p<0.0001), 3) Adult respiratory distress syndrom (5/15 versus 1/30, p=0.01) and 4) Dying (8/15 versus 1/30, p=0,0002, Fisher's exact test). LPS were cleared from the circulation with T/2= 1-3 hours after initiation of antibiotic therapy. Increasing LPS levels were never observed. Blood exchange or plasmapheresis did not significantly increase the LPS clearance. Conclusion: LPS quantitation is of importance as a prognostic marker in meningococcal infections

Measurement of endotoxin in cerebrospinal fluid from neonates and infants using a new endotoxin-specific chromogenic test.

Masumi Inagaki a, Masako Nakajima a, Yukinori Ando a, Sachio Takashima a, Kenzo Takeshita a, Shigenori Tanaka b, Hiroshi Tamura b and Taminori Obayashi ’ a Division of Child Neurology, lnstiiute of Neurological Sciences, Tottori University School of Medicine, Yonago, ’ Tokyo Research Institute, Seikagaku Kogyo Co., Ltd., Higashiyamato, Tokyo and ’ Department of Clinical Pathology, Jichi Medical School, Tochigi-ken (Japan)

Evaluation of a New Two-Hour System for Identification of Urine Isolates

The ability of the RapID SS/U system to accurately identify common isolates from urine was compared with the present conventional 18- to 24-hour system using MicroScan Combo Panels®. The RapID SS/U system uses a combination of conventional tests and single substrate chromogenic tests in a ten-cavity panel that identify select gram-negative bacilli, gram-positive cocci, and yeasts in two hours. A total of 203 clinical urinary isolates were tested as well as quality control organisms in the 12 taxa of the RapID SS U database. The RapID SS/U system correctly identified 194 (95.5%) of 203 isolates to genus level, and 157 (94%) of 167 isolates to species level. The system incorrectly identified nine (4.4%) isolates, one each of Escherichia coli, Citrobacter freundii, and Proteus mirabilis, three yeasts, and three organisms not included in the database. The RapID SS/U system requires minimal hands-on time for inoculation and readout and provides rapid, accurate identification of clinically significant urine isolates.

Induction of Fibrinolysis by Polyanions in Human Plasma

The fibrinolytic potency of several polyanions was comparatively investigated. Fibrinolytic activity was measured in a whole plasma assay using H-D-Val-Leu-Lys-pNA (S-2251) as chromogenic substrate and by a fibrin plate assay using plasminogen rich fibrin plates. In the chromogenic substrate assay potent fibrinolytic polyanions comprised dextran sulfate, GAGPS, pentosan polysulfate, polyanethol sulfate, l-carrageenan and i-carrageenan. Chondroitin sulfates A, B, C, keratan sulfate, ribonucleic acid, k-carrageenan and heparin were weakly fibrinolytic. Hyaluronic acid and lipopolysaccharide from E. coli were inactive. Similar results were obtained when fibrinolytic activity was measured by a fibrin plate assay. All polyanions except lipopolysaccharide produced lysis zones. Induction of fibrinolytic activity in human plasma was shown to be at least partially dependent on Hageman factor. In factor XII deficient plasma no fibrinolysis was induced by any of the polyanions when measured in the fibrin plate assay. In the chromogenic substrate assay corn Hageman factor inhibitor (CHFI) inhibited the activation of S-2251 cleaving enzyme by GAGPS, pentosan polysulfate, polyanethol sulfate, heparin, and ribonucleic acid near completely. The activation by dextran sulfate was inhibited by 45%. Heparin, pentosan polysulfate and GAGPS, three polyanions of therapeutic interest were separately compared. In both assays GAGPS proved the most potent activator, while pentosan polysulfate exhibited 83% and 44% and heparin 32% and 14% of GAGPS fibrinolytic activity in the chromogenic substrate test and the fibrin plate assay, respectively.