Antibodies without animals

Gold HK, Garabedian HD, Dinsmore RE, et al. Restoration of coronary flow in myocardial infarction by intravenous chimeric 7E3 antibody without exogenous plasminogen activators. Observations in animals and humans. Circulation 1997 95(7) 1755-1759. 

Many different types of lesions have been observed (very often at autopsy) in animals suffering from severe pantothenic acid deficiency. These may involve the skin, the adrenals, the entire gastrointestinal tract, nerves, and spinal cord. Functionally, in chickens fertility may be reduced by pantothenic acid deficiency to practically zero64 without any outward signs being shown by the fowls. Recently, pantothenic acid deficiency has been found to produce duodenal ulcers in about 60 per cent of the rats tested.65 It is required for bone development66 and is implicated in antibody responses.67... 

The use of specific antibodies labeled with a fluorescent dye to localize substances in tissues was first devised by A. H. Coons and his associates. At first, the specific antibody itself was labeled and applied to the tissue section to identify the antigenic sites (direct method) (1). Later, the more sensitive and versatile indirect method (2) was introduced. The primary, unlabeled, antibody is applied to the tissue section, and the excess is washed off with buffer. A second, labeled antibody from another species, raised against the IgG of the animal donating the first antibody, is then applied. The primary antigenic site is thus revealed. A major advantage of the indirect method is the enhanced sensitivity. In addition, a labeled secondary antibody can be used to locate any number of primary antibodies raised in the same animal species without the necessity of labeling each primary antibody. 

Selectivity in lAC depends on the specificity of the immobilized antibody and, thus, monoclonal antibodies are preferentially used. In that case, a large amount of sample can be subjected to immunoaffinity cleanup without any retention of matrix components. This opens the possibility to determine very low concentrations of drug residues in edible animal products. For example, 20 ng chloramphenicol in 1 L milk can be determined with a recovery of 99% when 1 L of defatted milk is submitted to immunoaffinity cleanup. The chromatograms obtained after LC analysis were as clean as those obtained when 10 ml milk containing the same amount of chloramphenicol was also submitted to immunoaffinity cleanup (170). 

The mixture is injected once a week for 3 weeks, and then the animal is maintained for 3 weeks without additional injections. Approximately 25-40 ml of blood is removed from the animal s ear vein to test for antibodies. One week after the first bleed the rabbit is boosted with half the antigen amount used earlier along with incomplete adjuvant incomplete adjuvant does not contain the mycobacteria. Serum is again removed 2 weeks after this injection and tested for antibody response. The enzyme-linked immunosorbent assay is used to determine if the titer (or antibody concentration) of the serum is sufficiently high to establish antibody binding to the antigen. This 3-week cycle is repeated as long as necessary to obtain the antibodies desired. 

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