System for identification of cells by electrophoresis

• US 4,707,237 A
• Filed: 09/09/1982
• Issued: 11/17/1987
• Est. Priority Date: 09/09/1982
• Status: Expired due to Fees

• Current Assignee: CIBA Vision Corporation (Bayer AG)
• Original Assignee: CIBA Vision Corporation (Bayer AG)
• Inventors: Lepp, Cyrus A., Mussatto, James J., Ziegenmeyer, James M.
• Primary Examiner(s): Niebling, John F.
• Application Number:
  US06/416,461
• Time in Patent Office: 1,895 Days
• Field of Search: 204/180 G, 204/299 R, 204/182.8
• US Class Current: 204/466
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: G01N 27/447 using electrophoresis

Abstract

Cells are identified electrophoretically through isoenzyme analysis. The use of standard and control cells allows semi-skilled workers to achieve reproducible results. A coding sequence is assigned to the cels to be identified using a grid to interpret the electrophoretic separations observed on a series of electrophoretic films, each film being developed for a particular isoenzyme. Comparison of the coding sequence with a compendium of coding sequences for known cells identifies the cells to be identified.

Claims

1. An electrophoretic method for identifying cells comprising the steps of:

(a) simultaneously performing on an electrophoretic film, multiple electrophoretic separations on a sample from a control, a standard population of cells, and at least one sample from an unknown population of cells each of the samples containing a least some electrophoretically separable substances which are characteristic of all the cells;

(b) developing the electrophoretic separations of control, standard, and unknown cells with regard to an electrophoretically separable, characteristic substance;

(c) comparing the developed separations for the control and standard samples to determine the range of separation for the characteristic substance under the conditions used; and

(d) based on the range of separation so determined, comparing the separations for the unknown and standard samples to identify the unknown cells.

2. The method of claim 1 wherein the electrophoretically separable substances are proteins.

3. The method of claim 2 wherein the electrophoretically separable substances are isoenzymes.

4. The method of claim 3 wherein the isoenzymes are purine nucleoside phosphorylase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, mannose phosphate isomerase, peptidase B, aspartate aminotransferase and lactate dehydrogenase.

5. The method of claims 1, 2, 3 or 4 wherein the control population of cells is a population of mouse L cells.

6. The method of claim 5 wherein the standard population of cells is a population of HeLa cells.

7. The electrophoretic method of claim 1 comprising:

(a) performing electrophoretic separations on a series of samples from the unknown population of cells;

(b) developing the electrophoretic separations of the unknown samples with regard to a series of different, preselected characteristic substances;

(c) determining from the developed samples the electrophoretic mobility exhibited by the unknown cells to be identified for each of the preselected substances;

(d) assigning to the electrophoretic mobility for each substance so determined a symbolic representation;

(e) arranging the symbolic representations in a predetermined order to form a coding sequence for the unknown population of cells; and

(f) comparing the coding sequence of the unknown population of cells with a compendium of predetermined coding sequences for known cells to identify the unknown cells.

8. The method of claim 7 wherein the electrophoretic mobility is determined using a grid having a plurality of spaced lines by which the symbolic representations are assigned.

9. The method of claims 7 or 8 wherein the symbolic representations are letters of the alphabet.

10. The method of claims 7 or 8 wherein the electrophoretically separable substances are proteins.

11. The method of claims 7 or 8 wherein the electrophoretically separable substances are isoenzymes.

12. The method of claims 7 or 8 wherein the series of electrophoretically separable substances consists of purine nucleoside phosphorylase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, mannose phosphate isomerase, peptidase B, aspartate aminotransferase and lactate dehydrogenase.

13. The method of claims 7 or 8 wherein the electrophoretically separable substances are developed to produce a group of similar colors.

14. The method of claim 13 wherein the electrophoretically separable substances are developed to form an insoluble formazan which is colored purple-blue or purple-red.

15. A method for identifying cells comprising the steps of:

(a) electrophoretically separating extracts from standard cells, control cells and the unknown cells to be identified simultaneously on separate tracks of an electrophoretic film;

(b) developing the electrophoretic film with regard to a first preselected electrophoretically separable characteristic substance using a reagent specific for that substance to produce a pattern of one or more bands of an insoluble product in each track for which the substance is present in the extract, the pattern, where present, being indicative of the extent of electrophoretic migration of the first preselected substance for the standard, control, and unknown cells;

(c) interpreting the developed electrophoretic film by reference to a grid having a plurality of spaced lines thereon and having a sequence of symbols associated with the lines, the interpretation occurring by aligning the film with the grid by reference to the developed pattern in the track for the standard cells and determining by reference to the grid first and second symbols corresponding to the developed patterns in the tracks for the extracts from the control cells and the unknown cells, respectively;

(d) recording the first and second symbols so determined at first and second locations, respectively, on a data sheet;

(e) repeating steps a, b, c, and d for a predetermined number of additional electrophoretically separable characteristic substances, the symbols determined for these substances being recorded on the data sheet at predetermined locations located relative to the first and second locations to form a first coding sequence for the control cells and a second coding sequence for the unknown cells;

(f) comparing the coding sequence for the the control cells with a reference coding sequence for those cells to determine whether the electrophoretic separations have been performed properly; and

(g) for properly performed separations, comparing the coding sequence for the unknown cells with a compendium of predetermined coding sequences for known cells to identify the unknown cells.

16. The method of claim 15 wherein the electrophoretically separable substances are proteins.

17. The method of claim 15 wherein the electrophoretically separable substances are isoenzymes.

18. The method of claim 17 including the additional step of determining the total isoenzyme activity of the cells to be identified for one or more of the isoenzymes for which an electrophoretic separation has been developed.

19. The method of claim 18 including the additional step of determining the isoenzyme activity represented by each band of a developed pattern having more than one band by apportioning the total isoenzyme activity among the bands based on the relative optical densities of the bands.

20. The method of claim 15 wherein the electrophoretic films, the reagents and the data sheet are color-coded with respect to the electrophoretically separable substances, one color being used to identify each substance.

21. The method of claim 15 wherein the symbols are letters of the alphabet.

22. The method of claims 17, 18 or 19 wherein the isoenzymes are purine nucleoside phosphorylase, gluocose-6-phosphate dehydrogenase, malate dehydrogenase, mannose phosphate isomerase, peptidase B, aspartate aminotransferase and lactate dehydrogenase.

23. The method of claims 15, 16, 17, 18 or 19 wherein the same insoluble product is produced during development for each of the electrophoretically separable substances.

24. The method of claim 23 wherein the insoluble product is a formazan.

25. A system for identifying cells electrophoretically comprising:

(a) extracts from a standard and a control cells;

(b) an extract from an unknown cells;

(c) a set of reagents for developing characteristic substances in electrophoretic separations of the standard, control, and unknown cells, each reagent being identified by a code distinct from the codes used for each other reagent, the number of reagents being chosen to permit characterization of the unknown cells;

(d) a set of electrophoretic films having means for receiving samples from the extracts of the standard, control, and unknown cells, the films being identified by the codes used for the reagents, one code for each film;

(e) an electrophoretic device; and

(f) means for interpreting the electrophoretic films including means for compiling a sequence of symbols indicative of the separation achieved for the developed characteristic substances of the unknown cells.

26. The system of claim 25 wherein the symbols are associated with a grid of spaced lines.

27. The system of claim 25 wherein the compiling means is coded with the codes for the reagents.

28. The system of claim 25 wherein the reagents develop the electrophoretic separations for the presence of proteins.

29. The system of claim 25 wherein the reagents develop the electrophoretic separations for the presence of isoenzymes.

30. The system of claim 29 further including a second set of reagents identified by the same code as the first set of reagents for determining total isoenzyme activity of the cells to be identified.

31. The system of claims 25, 28, 29 or 30 wherein the reagents are premeasured.

32. The system of claims 29 and 30 wherein the isoenzymes are purine nucleoside phosphorylase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, mannose phosphate isomerase, peptidase B, aspartate aminotransferase and lactate dehydrogenase.

33. The system of claims 25, 28, 29 or 30 wherein the reagents develop the electrophoretic separations to a group of similar colors.

34. The system of claim 32 wherein each of the reagents includes a soluble tetrazolium dye which is reduced to an insoluble formazan which is colored purple-blue or purple-red.

35. The system of claims 25, 28, 29 or 30 wherein the standard cells are mouse L cells and the control cells are HeLa cells.

36. The system of claim 25 further including means for decoding the sequence of symbols to determine the identity of the cells to be identified.

37. The system of claim 36 wherein the decoding means is a compendium of known sequences of symbols for known cells.

38. The system of claims 25, 29 or 30 wherein the code is a color code.