Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1983-01-28
1986-06-03
Marantz, Sidney
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 29, 435 34, 436149, 436519, 436810, 436827, G01N 3350
Patent
active
045929947
DESCRIPTION:
BRIEF SUMMARY
The present invention concerns a method for the determination of the content and species of microorganisms in a sample. There are already several different procedures to determine species and amount, but a common drawback with all these is that they require a long time, often several days to make one determination. To date workers have used a procedure involving plating, which means spreading, on a plate or glass surface, a layer of substrate to which is added the microorganisms whose number and species. At first nothing is seen in the medium, but later are to be determined on the microorganisms divide and eventually form a number of colonies that are observable by the naked eye. Based on the assumption that one cell creates one colony, the number of microorganisms are allowed to divide, the species of the microorganism can be determined. Another procedure to determine amount and kind of microorganism is to use specific sorbents containing antibodies. Such sorbents immobilize a predetermined species of microorganism against which the antibodies are directed. When the cells have been captured in this way they can be incubated in a growth medium, and when the microorganisms, through division, have given rise to a larger group of cells that is observable by the naked eye, the amount of microorganisms can be determined by colony counting. The choice of substrate to which the adsorbent with the microorganisms are added gives an indication of the kind of microorganisms that has been adsorbed, since only certain microorganisms are able to grow in a certain substrate, while others need a different substrate for their growth.
The present invention is aimed at creating a system of procedures by which it is possible to determine biochemical data concerning microorganisms within a short period of time, in many cases within two hours.
The procedure is based upon the use of an adsorbent for a preselected microorganism, where the sample from which the microorganism is bound together with the sorbent, is washed after the adsorption the in order to eliminate all non-specifically bound cells. The washed microorganisms bound to the sorbent are exposed to a substrate so that the cell metabolism is started. For this metabolism there is a need for certain substances concomittant with generation of metabolic products.
The quantity of generated products is directly related to the number of adsorbed cells. One or more products may be utilized either directly or indirectly to determine the amount of microorganisms. The species of the microorganisms is determined by the adsorbent due to its specific binding properties.
The adsorbent is as a rule a carrier provided with antibodies directed against the microorganisms that is to be adsorbed. If it is desirable to adsorb all microorganisms, then as an alternative either mixtures of antibodies or other binding structures with a broader specificity, eg. lectins are used.
The above mentioned substrate may be or may contain oxygen and the products may be eg. those generated by consumption of oxygen due to metabolic activity. The amount of oxygen consumed can be determined with established measuring equipment.
Other parameters may be the pH of the substrate-medium. The pH-value can be determined by using a pH-indicator and fotometric equipment.
Still another parameter is reduction equivalents formed during the metabolism. These reduction equivalents are uncoupled from the ordinary metabolism by eg. artificial electron acceptors, the degree of reduction of which can be determined by established technique.
Still another parameter may be the amount of carbon-dioxide generated during the metabolism, as this amount can be determined by using established analytical instruments.
Moreover, for certain types of cells specific metabolites may be excreted, the concentration of which in the medium can be used to determine the number of cells bound to the sorbent. Analysis in such a case is performed with a dedicated specific analysis developed for the individual metabolites.
Still another physical prope
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Alfa-Laval AB
Marantz Sidney
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