Clostridium botulinum: Detection of anti-botulinum toxin antibodies in serum or plasma, using neutralization reference test in mice.
The accepted reference method to detect and quantify antibodies against botulinum toxin is the Mouse Neutralization Assay, in which a solution of botulinum toxin, quantified at 50% Lethal Dose for the mouse (LD50), is mixed with various dilutions in base 2 or base 4 of the test serum/plasma, and after incubation, batches of mice are inoculated intraperitoneally. The highest dilution of the test serum that reduces toxicity is the antibody titer against the corresponding botulinum toxin in the serum. This dilution, compared to an international standard, allows results to be obtained in international units (IU/mL) (1 IU is defined as the amount of antibodies that neutralizes 10,000 LD50 of toxins A or B, or 1,000 LD50 of type E). The amount of toxin used in the tests is that which is neutralized by 0.02; 0.005 and 0.0125 IU/mL of antitoxin for types A, B and E, respectively. Sera that do not protect the mouse at the titer of 1:4 are reported as <0.02 IU/mL for type A, or <0.005 IU/mL for type B. This test is laborious, expensive, and long-term. implementation, so alternatives have been sought based on enzyme immunoassay (ELISA) methods using microplates coated with botulinum toxin. However, the values obtained by ELISA sometimes do not completely correlate with the mouse neutralization test.
As a prior step to the neutralization test, it is necessary to calculate the minimum lethal dose (m.l.d.) of toxin. The calculation of the minimum lethal dose is carried out by diluting a culture filtrate in base of 10, diluting it by half with physiological saline solution to obtain the same dilution as the toxin mixed with patient serum or plasma, and inoculating with each dilution mixture to some laboratory animals. In this way, the maximum dilution (the highest) that causes the death of the inoculated animals is calculated. The inoculated volume that caused the death of the animals will contain a minimum lethal dose. The animals are inoculated with the minimum lethal dose (m.l.d.) of diluted toxin together with the patient´s serum/plasma (v:v) pure and with base 10 dilutions.
The report indicates the presence or absence of specific antibodies by neutralization test against the minimum lethal dose in mice of the toxin used.