Process for preparing Limulus lysate

• US 4,322,217 A
• Filed: 04/14/1981
• Issued: 03/30/1982
• Est. Priority Date: 06/27/1980
• Status: Expired due to Term

• Current Assignee: Biowhittaker Technologies, Inc. (Lonza Group AG)
• Original Assignee: Mallinckrodt Incorporated (Mallinckrodt plc)
• Inventors: Dikeman, Roxane N.
• Primary Examiner(s): Warden, Robert J.
• Application Number:
  US06/254,233
• Time in Patent Office: 350 Days
• Field of Search: 23/230 B, 252/408, 435/4, 435/7, 435/29
• US Class Current: 435/7.21
• CPC Class Codes: G01N 33/50 Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q
• CPC Class Codes: G01N 33/579 involving limulus lysate

Abstract

This invention relates to a process for treating Limulus Lysate having improved sensitivity to endotoxin, to lysate reagents utilizing such lysate, and to the use of such lysate reagents.

Claims

1. A process for treating under lysate treating conditions Limulus amebocyte lysate having decreased sensitivity to endotoxin due to the presence of an endogenous lysate inhibitor prior to any subsequent reaction with endotoxin with an enhancing amount of a lysate sensitivity enhancing agent having lysate sensitivity enhancing characteristics to neutralize or partially neutralize said lysate inhibitor thereby increasing the lysate sensitivity to endotoxin, said lysate enhancing agent being selected from the group consisting of

(I) amphoteric surfactants having the following formulae:

##STR8## wherein R₁ is an alkylene radical having from 1 to 4 carbon atoms;

Y and Y'"'"' are each (1) hydrogen, (2) lower alkyl or (3) hydroxy lower alkyl;

R₂ and R₃ are each (1) lower alkyl or (2) hydroxy lower alkyl;

n is 0 or 1, when n is 0, R₄ is alkyl containing from about 8 to about 18 carbon atoms;

when n is 1, R₄ is an alkylene radical having from 1 to about 6 carbon atoms;

R₅ is an alkyl containing from about 8 to about 18 carbon atoms;

M is hydrogen, sodium, potassium or ammonium;

(II) anionic surfactants have the following formulae;

space="preserve" listing-type="equation">(R.sub.6).sub.n.sbsb.1.(Y)Ar.(SO.sub.3 M).sub.n.sbsb.2 (E)

space="preserve" listing-type="equation">R.sub.5 OSO.sub.3 M (F)

wherein

R₅, Y and M have the same meaning as set forth above

R₆ is an alkyl from 8 to 24 carbon atoms

n₁ is an integer from 1 to 3

n₂ is 1 or 2

Ar is phenyl or naphthyl;

(III) cationic surfactants having the following formula;

##STR9## wherein R₅, Y and Y'"'"' have the same meaning as set forth above; and

(IV) nonionic surfactants having the following formula;

space="preserve" listing-type="equation">R.sub.5 R.sub.7 R.sub.8 N→

O (H)

wherein

R₅ has the same meaning as set forth above

R₇ and R₈ are each methyl or ethyl; and

those nonionic surfactants selected from the group consisting of the condensation product of about 10 to 30 moles of ethylene oxide with the monoester of a hexahydric alcohol containing 6 carbon atoms with the ester group containing 10 to 20 carbon atoms.

2. A process according to claim 1, wherein the enhancing agent is selected from the group of amphoteric surfactants represented by formula (A) of Claim 1.

3. A process according to claim 2, wherein the enhancing agent is present in an amount from about 0.01 to about 0.05% (w/v).

4. A process according to claim 3, wherein n is 0, R₄ is tetradecyl, R₂ and R₃ are each methyl, and RY₁ is trimethylene.

5. A process according to claim 3, wherein n is 1, R₄ is trimethylene, R₂ and R₃ are each methyl and ##STR10##

6. A process according to claim 1, wherein the enhancing agent is selected from the group of amphoteric surfactants represented by formula (D) of claim 1.

7. A process according to claim 6, wherein the enhancing agent is present in an amount of from about 0.01 to about 0.05% (w/v).

8. A process according to claim 7, wherein n is 1, R₄ is propylene, R₂ and R₃ are each methyl and ##STR11## is methylene.

9. A process according to claim 7, wherein n is 0, R₂ and R₃ are each methyl and ##STR12## is methylene.

10. A Limulus ambeocyte lysate reagent for determining endotoxin by the formation of an intact clot comprising a buffered aqueous dispersion of Limulus amebocyte lysate present in an endotoxin determining amount having improved sensitivity to endotoxin and an enhancing amount of a lysate sensitivity enhancing agent having lysate sensitivity enhancing characteristics, said lysate enhancing agent being selected from the group consisting of

(I) amphoteric surfactants having the following formulae:

##STR13## wherein R₁ is an alkylene radical having from 1 to 4 carbon atoms;

Y and Y'"'"' are each (1) hydrogen, (2) lower alkyl or (3) hydroxy lower alkyl;

R₂ and R₃ are each (1) lower alkyl or (2) hydroxy lower alkyl;

n is 0 or 1, when n is 0, R₄ is alkyl containing from about 8 to about 18 carbon atoms;

when n is 1, R₄ is an alkylene radical having from 1 to about 6 carbon atoms;

R₅ is an alkyl containing from about 8 to about 18 carbon atoms;

M is hydrogen, sodium, potassium or ammonium;

(II) anionic surfactants have the following formulae;

space="preserve" listing-type="equation">(R.sub.6).sub.n.sbsb.1.(Y)Ar.(SO.sub.3 M).sub.n.sbsb.2 (E)

space="preserve" listing-type="equation">R.sub.5 OSO.sub.3 M (F)

wherein

R₅, Y and M have the same meaning as set forth above

R₆ is an alkyl from 8 to 24 carbon atoms

n₁ is an integer from 1 to 3

n₂ is 1 or 2

Ar is phenyl or naphthyl;

(III) cationic surfactants having the following formula;

##STR14## wherein R₅, Y and Y'"'"' have the same meaning as set forth above; and

(IV) nonionic surfactants having the following formula;

space="preserve" listing-type="equation">R.sub.5 R.sub.7 R.sub.8 N→

O (H)

wherein

R₅ has the same meaning as set forth above

R₇ and R₈ are each methyl or ethyl; and

the nonionic surfactants selected from the group consisting of the condensation product of about 10 to 30 moles of ethylene oxide with the monoester of a hexahydric alcohol containing 6 carbon atoms with the ester group containing 10 to 20 carbon atoms.

11. A reagent according to claim 10, wherein additionally are present the cations, Na⁺, Mn⁺⁺ and Ca⁺⁺ in lysate sensitivity increasing amounts.

12. A reagent according to claim 11, wherein the Lysate is present in an amount to detect from about 0.007 to about 0.050 ng/ml of FDA reference endotoxin EC-2.

13. A reagent according to claim 12, wherein the enhancing agent is selected from the group of amphoteric surfactants represented by formula A of claim 10.

14. A reagent according to claim 13, wherein n is 0, R₄ is tetradecyl, R₂ and R₃ are each methyl and ##STR15## is trimethylene.

15. A reagent according to claim 13, wherein n is 1, R₄ is trimethylene, R₂ and R₃ are each methyl and ##STR16##

16. A reagent according to claim 12, wherein the enhancing agent is selected from the group of amphoteric surfactants represented by formula D of claim 10.

17. A reagent according to claim 16, wherein n is 1, R₄ is propylene, R₂ and R₃ are each methyl and ##STR17## is methylene.

18. A reagent according to claim 16, wherein n is 0, R₂ and R₃ are each methyl and ##STR18## is methylene.

19. A reagent according to claim 10 which is lyophilized.

20. In a method for determining endotoxin under endotoxin determining conditions wherein the endotoxin is reacted with a Limulus amebocyte lysate reagent, the improvement comprising utilizing the Limulus amebocyte lysate reagent of claim 10.

21. A reagent according to claim 10, wherein additionally are present the cations, Na⁺, Sr⁺⁺, Ca⁺⁺ and Mg⁺⁺ in lysate sensitivity increasing amounts.

22. A reagent according to claim 21, wherein the Lysate is present in an amount to detect from about 0.007 to about 0.050 ng/ml of FDA reference endotoxin EC-2.

23. A reagent according to claim 22, wherein the enhancing agent is selected from the group of amphoteric surfactants represented by forumla A of claim 10.

24. A reagent according to claim 23, wherein n is 0, R₄ is tetradecyl, R₂ and R₃ are each methyl and ##STR19## is trimethylene.

25. A reagent according to claim 23, wherein n is 1, R₄ is trimethylene, R₂ and R₃ are each methyl and ##STR20##

26. A reagent according to claim 21, wherein the enhancing agent is selected from the group of amphoteric surfactants represented by formula D of claim 10.

27. A reagent according to claim 26, wherein n is 1, R₄ is propylene, R₂ and R₃ are each methyl and ##STR21## is methylene.

28. A reagent according to claim 26, wherein n is 0, R₂ and R₃ are each methyl and ##STR22## is methylene.