Improved microtechnique for endotoxin assay by the Limulus amebocyte lysate test

By crossed immunoelectrophoresis 36 different anode-migrating antigens were demonstrated in sonicated antigen preparations of Psetidomonas aeruiginosa. We numbered these antigens to establish a reference precipitin pattern. Antigen no. 31 was identified as the lipopolysaccharide (LPS) antigen, because it was found to be responsible for the 0-group specificity and because it reacted with anti-LPS monoclonal antibodies and with Limlllus amoebocyte lysate. Purified outer membrane proteins F (porin), H,, and I used as antigens formed precipitins with the reference antibodies, thus establishing their antigenicity. LPS that copurified with protein F and slightly contaminated protein H2 was detectable as an extra precipitin (antigen no. 31). The use of monoclonal antibodies specific for smooth LPS and rough LPS revealed different antigenic determinants in the LPS molecule and suggested that antigen no. 5 could be the core region of the LPS which is equivalent to the rough LPS. Antibodies against these outer membrane antigens were detected in patients with chronic P. aeruiginosa pneumonia and in patients with acute P. aertiginosa bacteremia. Antibodies with the same specificity were also found in rats chronically infected with P. aeriuginosa 7 days postinfection. This demonstrates the surface accessibility and antigenic reactivity of outer membrane antigens.

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“…Thus far, the most sensitive method for the detection or quantitation of endotoxin has been the Limulus amoebocyte lysate test (14,29).…”

Section: Discussionmentioning

confidence: 99%

Immunogenicity of Pseudomonas aeruginosa outer membrane antigens examined by crossed immunoelectrophoresis

Lam¹,

Mutharia²,

Hancock³

et al. 1983

Infect Immun

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“…The blood path was then rinsed with 200 ml sterile water for injection by recirculation at room temperature for 2 h at a flow rate of 250 d m i n . LAL tests were performed according to the instructions from the suppliers of the test kits, and L A L micro-tests were performed according to Gardi and Arpagaus (4).…”

Section: Methodsmentioning

confidence: 99%

Hollow‐Fiber Dialyzers and Their Pyrogenicity Testing by Limulus Amebocyte Lysate

Henne¹,

Schulze²,

Pelger³

et al. 1984

Artificial Organs

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Aqueous extracts of cellulose hollow fibers (CHF) exhibit positive reactions in some Limulus amebocyte lysate (LAL) tests. However, in spite of LAL activity, the extracts produce no fever reaction in rabbits. A comparison of lysates from different suppliers shows pronounced activity differences when extracts of cuprammonium-derived CHF are tested. One of the lysates, which is fully reactive against standard endotoxin, shows no reaction with such extracts, nor do CHF extracts diminish its sensitivity to standard endotoxin. Investigations of the cuprammonium process have shown that endotoxins introduced by the linters are degraded and washed out. Other endotoxin introduction, particularly by the process water, has been excluded. Oxidative or acidic degradation of cellulose does not result in the formation of LAL-reactive material (LAL-RM). On the other hand, sterile cotton wool shows LAL reactivity, and cellulose acetate regains LAL reactivity when it is saponified. Thus, it appears likely that the LAL-RM found in CHF is of purely cellulosic origin and crossreacts with a number of commercially available lysates.

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“…A firm gel clot is one that remains solid in the bottom of the reaction tube when the tube is inverted. Methods to enhance the visualization of clot formation in microtiter volumes have been described (113,167,299). With all gel clotbased techniques, a semiquantitative result can be obtained through serial dilution of samples and standards.…”

Section: The Lal Assaymentioning

confidence: 99%

Endotoxemia: methods of detection and clinical correlates.

Hurley¹

1995

Clinical Microbiology Reviews

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Improved microtechnique for endotoxin assay by the Limulus amebocyte lysate test

Cited by 28 publications

References 6 publications

Immunogenicity of Pseudomonas aeruginosa outer membrane antigens examined by crossed immunoelectrophoresis

Immunogenicity of Pseudomonas aeruginosa outer membrane antigens examined by crossed immunoelectrophoresis

Hollow‐Fiber Dialyzers and Their Pyrogenicity Testing by Limulus Amebocyte Lysate

Endotoxemia: methods of detection and clinical correlates.

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