Method of measuring total protein of sample with the aid of electrophoretic image

• US 4,666,578 A
• Filed: 02/24/1986
• Issued: 05/19/1987
• Est. Priority Date: 02/27/1985
• Status: Expired due to Fees

• Current Assignee: Olympus Optical Company Ltd. (Olympus Corporation)
• Original Assignee: Olympus Optical Company Ltd. (Olympus Corporation)
• Inventors: Yamamoto, Hidehiko
• Primary Examiner(s): NOT, DEFINED
• Application Number:
  US06/832,221
• Time in Patent Office: 449 Days
• Field of Search: 204/183.3, 204/180.1, 204/299 R, 204/300 R
• US Class Current: 204/549
• CPC Class Codes: G01N 27/44721 by optical means

Abstract

A method of measuring an amount of total protein TP_i of a test sample from an electrophoretic image of the test sample including a step of forming an electrophoretic image of a standard sample having a known amount of total protein TP₁, a step of integrating absorbance values of the electrophoretic image of the standard sample to derive an integrated value of concentration D₁ of the electrophoretic image, a step of integrating absorption values of the electrophoretic image of the test sample to derive an integration value of concentration D_i of the electrophoretic image of the test sample, and a step of deriving the amount of total protein TP_i of the test sample in accordance with an equation of ##EQU1## .

Claims

1. a method of measuring an amount of total protein contained in a sample comprising the steps of

forming electrophoretic images of a standard sample having a known amount of total protein and of a test sample having an unknown amount of total protein;

deriving an integrated value of concentration of the electrophoretic image of the standard sample;

deriving an integrated amount of concentration of the electrophoretic image of the test sample; and

calculating the unknown amount of protein of the test sample in accordance with the known amount of total protein of the standard sample and the integrated values of concentration of the electrophoretic images of the standard sample and test sample.

2. A method according to claim 1, wherein said electrophoretic images of the standard and test samples are formed by applying the standard and test samples on a single substrate and by subjecting the substrate to electrophoresis.

3. A method according to claim 1, wherein an integrated value of concentration of an electrophoretic image of a sample is derived by scanning the electrophoretic image photoelectrically to derive a first electric signal representing a transmittivity of the electrophoretic image, supplying the first electric signal ot a log-amplifier to derive a second electric signal representing an absorption of the electrophoretic image and integrating the second electric signal to derive the integrated value of concentration of the electrophoretic image.

4. A method according to claim 3, wherein said electrophoretic image is scanned two-dimensionally and said second electric signal is integrated over whole the electrophoretic image.

5. A method according to claim 3, wherein said electrophoretic image is scanned linearly along a center line of the electrophoretic image and said second electric signal is integrated along the center line of the electrophoretic image.

6. A method according to claim 1, further comprising a step of correcting an integrated value of concentration of electrophoretic image of a sample.

7. A method according to claim 6, wherein said correcting step comprises deriving correction coefficients k_Alb, k.sub.α

.sbsb.1_-G, k.sub.α

.sbsb.2_-G, k.sub.β

-G and k.sub.γ

-G for correcting difference in dyeing degree for respective protein components, deriving integrated values of concentration d_Alb, d.sub.α

.sbsb.1_-G, d.sub.α

.sbsb.2_-G, d.sub.β

-G and d.sub.γ

-G for fraction images of respective protein components, and deriving an integrated value of concentration D of the electrophoretic image as a sum of products of the integrated values of concentration of fraction images and corresponding correction coefficients;

space="preserve" listing-type="equation">D=k.sub.Alb ·

d.sub.Alb +k.sub.α

.sbsb.1.sub.-G ·

d.sub.α

.sbsb.1.sub.-G +k.sub.α

.sbsb.2.sub.-G ·

space="preserve" listing-type="equation"> d.sub.α

.sbsb.2.sub.-G +k.sub.β

-G ·

d.sub.β

-G +k.sub.γ

-G ·

d.sub.γ

-G.

8. A method according to claim 7, wherein said correction coefficients are determined in accordance with a wavelength of light by means of which the electrophoretic image is optically scanned.

9. A method according to claim 2, wherein an electrophoretic image of the standard sample and a plurality of electrophoretic images of a plurality of test samples are formed on the same substrate and the method further comprises a step of correcting variation in amounts of samples applied on the substrate.

10. A method according to claim 9, wherein said correcting step comprises

forming a plurality of electrophoretic images of the standard sample on a substrate,

deriving integrated values of concentration d₁, d₂ . . . d_N of said plurality of electrophoretic images of the standard sample;

deriving an average value d_R of said integrated values of concentration d₁, d₂ . . . d_N ;

deriving correction coefficients K₁, K₂ . . . K_N in accordance with said integrated values of concentration and the average value; and

deriving corrected integration values of concentration by dividing integrated values D₁, D₂ . . . D_N of electrophoretic images of test samples by said correction coefficients K₁, K₂ . . . K_N, respectively.

11. A method according to claim 10, wherein said steps of forming the electrophoretic images of the standard sample and deriving the integrated values of concentration of electrophoretic images of the standard sample are performed plural times by using a plurality of substrates, and said average value of integration value is calculated from average values of a plurality of integrated values of concentration.

12. A method according to claim 3, wherein said electrophoretic images of samples are moved in a stepwise manner in a direction perpendicular to an electrophoretic direction and are scanned by means of a linear image sensor array aligned in the electrophoretic direction.

13. A method according to claim 1, further comprising a step of calculating amounts of respective protein components of a sample by multiplying fraction percentages of respective protein components by an amount of total protein of the relevant sample.

14. A method according to claim 1, further comprising a step of detecting condition of electrophoretic images of samples, and deleting a deriving operation of an amount of total protein of a sample when the condition of electrophoretic image of the relevant sample is detected to be abnormal.