Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B06—GENERATING OR TRANSMITTING MECHANICAL VIBRATIONS IN GENERAL
  • B06B—METHODS OR APPARATUS FOR GENERATING OR TRANSMITTING MECHANICAL VIBRATIONS OF INFRASONIC, SONIC, OR ULTRASONIC FREQUENCY, e.g. FOR PERFORMING MECHANICAL WORK IN GENERAL
  • B06B1/00—Methods or apparatus for generating mechanical vibrations of infrasonic, sonic, or ultrasonic frequency
  • B06B1/18—Methods or apparatus for generating mechanical vibrations of infrasonic, sonic, or ultrasonic frequency wherein the vibrator is actuated by pressure fluid
  • B06B1/183—Methods or apparatus for generating mechanical vibrations of infrasonic, sonic, or ultrasonic frequency wherein the vibrator is actuated by pressure fluid operating with reciprocating masses
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2337/00—N-linked chromogens for determinations of peptidases and proteinases
  • C12Q2337/10—Anilides
  • C12Q2337/12—Para-Nitroanilides p-NA
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/81—Protease inhibitors
  • G01N2333/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
  • G01N2333/811—Serine protease (E.C. 3.4.21) inhibitors
  • G01N2333/8121—Serpins
  • G01N2333/8125—Alpha-1-antitrypsin
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
  • G01N2333/976—Trypsin; Chymotrypsin

Definitions

• the degree of clinical manifestation in al -antitrypsin deficiency is highly variable, ranging from complete absence of clinical symptoms to an early onset of severe emphysema with pronounced disability. But the homozygotes run greater risk of lung disease'than the heterozygotes. The high risk of developing emphysema is increased with the cigarette smokers in this population.
• Methods currently available for estimating antitrypsin levels in serum include enzymologic procedures involving the inhibition of digestion of protein substrates such as casein, fibrinogen and hemoglobin, and inhibition of hydrolysis of synthetic substrates such as benzoyl-L-arginine p-nitroanalide and p-toluene sulfonyl-L-arginine methyl ester.
• protein substrates such as casein, fibrinogen and hemoglobin
• synthetic substrates such as benzoyl-L-arginine p-nitroanalide and p-toluene sulfonyl-L-arginine methyl ester.
• a rapid and simple procedure is needed for the semi-quantitative measurement of antitryptic activity in human sera, a procedure which would make possible the screening of largepopulations for the genetically controlled u antitrypsin deficiency that is associated with an unusual familial variety of pulmonary emphysema.
• Such inform ation would be a valuable guide for premarital testing and counseling that could prevent the unfortunate births of homozygotes with a high probability of future crippling emphysema. Such information would also serve as a precautionary screening in employment examinations in industries with high exposure to irritating fumes, dusts or excessive smog. A background would be available for medical advice on the dangers of cigarette smoking.
• the object of this invention is to provide a rapid semi-quantitative procedure for determining antitrypsin levels in human sera in which one set of control sera serves as a reference for the mass screening of many individual human sera.
• This invention is concerned with a diagnostic test package for use in an improved semi-quantitative method for estimating antitrypsin levels in human blood.
• the diagnostic test package contains a strip of unexposed and developed color film, sample test vials (each containing a constant amount of freeze-dried trypsin for individual serum sample determinations when properly diluted with serum), and three reference vials (each containing a different amount of freezedried trypsin which serve as reference control sera when properly diluted with buffer).
• Antitrypsin levels in human sera are determined by the color changes resulting from the hydrolysis of the gelatin substrate of color film by the serum trypsin reagent with side by side comparisons of the color changes produced by simulated normal, heterozygous and homozygous sera.
• test method of this invention utilizes the depth of hydrolysis (with resultant color changes) into the trilayered/tri-colored gelatin sandwich of an unexposed and developed color film strip as the semi-quantitative measure of the trypsin inhibitory capacity of human sera. Side by side comparisons are made with simulated normal, heterozygous and homozygous antitrypsin sera.
• test sera (0.2 ml.) are each added to individual vials containing 0.30 mg. of freeze-dried trypsin and mixed until all the trypsin has dissolved. After about 5 minutes incubation at room temperature, a drop (0.0l0.03 ml.) of each mixture is placed on the gelatin side of an unexposed and developed color film strip (any positive color film may be used, preferably Agfachrome). After approximately 5 minutes, the film strip is immersed in a pH 3 water rinse bath which stops the gelatin hydrolysis reaction. The film strip is gently rubbed or squeegeed until colored particles no longer are leached from the spotted area (approximately 30 seconds).
• the film strip is then removed from the bath, dried and the colored spots compared with those obtained from the five minute hydrolysis action of a drop (0.01-0.03 ml.) of each of the simulated normal, heterozygous and homozygous sera (prepared inan identical manner as the test sera, i.e., 0.2 ml. of 0.05-0.10 M tris, 0005-002 M) CaCl,, pH 8 buffer solution added to each of three individual vials containing respectively 0.06 mg, 0.18 mg. and 0.26
• a light aqua-blue colored spot is obtained with homozygous oi -antitrypsin human sera and the corresponding simulated homozygouscontrol.
• a blue-violet spot results from the action of heterozygous (l -antitrypsin sera and the corresponding heterozygous control.
• Normal a -antitrypsin sera and the simulated normal control react very little on the gelatin substrate of the color film under the conditions of the test, and the color of the spot on the film is black (film unchanged) to black-- violet.
• the test sera are reported as normal,
• the trypsin reagent vials (containing 0.30 mg.
• freeze-dried trypsin are prepared by making up a 3.0
• tyrpsin inhibitory capacity (I.l.().), expressed as mg. of trypsin capable of being inhibited by antitrypsin, taken from tho litm'atnrv for nornnil. hvtcrozygous and homozygous ai-antitrypsin deficiency.
• the trypsin reagent has purposely been made more concentrated than the actual trypsin reagent concentration necessary to titratc exactly the mean normal I.l.0. of 1.2 lug/ml. This use of a slighly higher trypsin reagent concentration is used to reduce the possibility of false negatives.

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• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Analytical Chemistry (AREA)
• Mechanical Engineering (AREA)
• Immunology (AREA)
• Microbiology (AREA)
• Molecular Biology (AREA)
• Biophysics (AREA)
• Physics & Mathematics (AREA)
• Biotechnology (AREA)
• Biochemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

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Cited By (8)

• 1970
  • 1970-12-14 US US00098213A patent/US3730843A/en not_active Expired - Lifetime
• 1971
  • 1971-04-19 GB GB1297609D patent/GB1297609A/en not_active Expired
  • 1971-10-28 CA CA126,337A patent/CA956869A/en not_active Expired
  • 1971-10-28 SE SE7113740A patent/SE392648B/xx unknown
  • 1971-11-11 DE DE19712156040 patent/DE2156040A1/de active Pending

Non-Patent Citations (4)

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