Double antibody immunoassays of enzymes such as prostatic acid phosphatase

Claim 3

The method of claim 1 wherein after step (e) and in or before step (g) a non-interfering, water soluble detergent is added to further enhance the solubility of constituents present at any time in step (g).

Claim 4

The method of claim 1 wherein said reaction medium is a kinetic reaction medium and said enzyme is assayed kinetically.

Claim 5

The method of claim 1 wherein after step (e) and in or before step (g) a non-interfering, water soluble detergent is added to further enhance the solubility of constituents present at any time in step (g), said reaction medium is a kinetic reaction medium, and said enzyme is assayed kinetically.

Claim 6

The method of claim 1 wherein steps (a) through (d) are conducted simultaneously and said Ab 1 is selected from the group consisting of monoclonal and polyclonal antibodies and said Ab 2 is selected from the group consisting of monoclonal and polyclonal antibodies.

Claim 7

The method of claim 5 wherein after step (e) and in or before step (g) a non-interfering, water soluble detergent is added to further enhance the solubility of constituents present at any time in step (g).

Claim 8

The method of claim 5 wherein said reaction medium is a kinetic reaction medium and said enzyme is assayed kinetically.

Claim 9

The method of claim 5 wherein after step (e) and in or before step (g) a non-interfering, water soluble detergent is added to further enhance the solubility of constituents present at any time in step (g), said reaction medium is a kinetic reaction medium, and said enzyme is assayed kinetically.

Claim 10

A kinetic method for assaying prostate acid phosphatase (PAP) in a sample comprising: (a) contacting said sample with a first antibody (Ab 1 ) against said PA, wherein the amount of Ab 1 employed is at least sufficient to bind substantially all of said PAP in said sample; (b) incubating a first medium containing said Ab 1 and said PAP for a time at least sufficient to enable said Ab 1 to bind to substantially all of said PAP to form Ab 1 ˜PAP complexes; (c) contacting said Ab 1 ˜PAP complexes with a second antibody (Ab 2 ) capable of recognizing Ab 1 , wherein the amount of Ab 2 employed is at least sufficient to bind substantially all of said Ab 1 ˜PAP complexes and form a precipitate with substantially all of said complexes; (d) incubating a second medium containing said Ab 2 and said Ab 1 ˜PAP complexes for a time at least sufficient to enable (i) said Ab 2 to bind to substantially all of said Ab 1 ˜PAP complexes to form Ab 2 ˜Ab 1 ˜PAP complexes and (ii) substantially all of said Ab 2 ˜Ab 1 ˜PAP complexes to precipitate out of said second medium; (e) separating said precipitated Ab 2 ˜Ab 1 ˜PAP complexes from said second medium; (f) contacting said separated, precipitated Ab 2 ˜Ab 1 ˜PAP complexes with a solution (i) having a pH≦about 3 and (ii) comprising γ-globulin in an amount at least sufficient to prevent substantially any precipitation of Ab 2 ˜Ab 1 ˜PAP complexes at a pH suitable for formation of said precipitate, thereby forming a resulting solution; (g) contacting said resulting solution with a non-interfering, water soluble detergent capable of further enhancing the solubility of constituents present at any time in step (i); (h) contacting said detergent containing resulting solution with a water soluble n-alcohol having at least one terminal hydroxyl group and capable of participating in a transphosphorylation reaction, said alcohol being employed in an amount such that it is not a rate limiting factor in a reaction between a substrate and said alcohol in the presence of said PAP; (i) contacting said alcohol and detergent containing resulting solution with a buffered reaction medium comprising said substrate, a reagent capable of reacting with a product of said alcohol, substrate, PAP reaction to produce a detectable product, and a buffer having a pH compatible with an assay of PAP activity; and (j) kinetically measuring said detectable product of step (i).

Claim 11

A kinetic method for assaying prostate acid phosphatase (PAP) in a sample comprising: (a) contacting said sample with a first antibody (Ab 1 ) against said PA, wherein the amount of Ab 1 employed is at least sufficient to bind substantially all of said PAP in said sample; (b) incubating a first medium containing said Ab 1 and said PAP for a time at least sufficient to enable said Ab 1 to bind to substantially all of said PAP to form Ab 1 ˜PAP complexes; (c) contacting said Ab 1 ˜PAP complexes with a second antibody (Ab 2 ) capable of recognizing Ab 1 , wherein the amount of Ab 2 employed is at least sufficient to bind substantially all of said Ab 1 ˜PAP complexes and form a precipitate with substantially all of said complexes; (d) incubating a second medium containing said Ab 2 and said Ab 1 ˜PAP complexes for a time at least sufficient to enable (i) said Ab 2 to bind to substantially all of said Ab 1 ˜PAP complexes to form Ab 2 ˜Ab 1 ˜PAP complexes and (ii) substantially all of said Ab 2 ˜Ab 1 ˜PAP complexes to precipitate out of said second medium; (e) separating said precipitated Ab 2 ˜Ab 1 ˜PAP complexes from said second medium; (f) contacting said separated, precipitated Ab 2 ˜Ab 1 ˜PAP complexes with a solution (i) having a pH≦about 3 and (ii) comprising γ-globulin in an amount at least sufficient to prevent substantially any precipitation of Ab 2 ˜Ab 1 ˜PAP complexes at a pH suitable for formation of said precipitate, thereby forming a resulting solution; (g) contacting said resulting solution with a non-interfering, water soluble detergent capable of further enhancing the solubility of constituents present at any time in step (i); (h) contacting said detergent containing resulting solution with a water soluble n-alcohol having at least one terminal hydroxyl group and capable of participating in a transphosphorylation reaction, said alcohol being employed in an amount such that it is not a rate limiting factor in a reaction between a substrate and said alcohol in the presence of said PAP; (i) contacting said alcohol and detergent containing resulting solution with a buffered reaction medium comprising said substrate, a reagent capable of reacting with a product of said alcohol, substrate, PAP reaction to produce a detectable product, and a buffer having a pH compatible with an assay of PAP activity; and (j) kinetically measuring said detectable product of step (i).

Claim 12

The method of claim 9 wherein said Ab 1 is rabbit Ab 1 ; said Ab 2 is goat anti-rabbit IgG; said solution has a pH of from about 2 to about 2.8 and is selected from the group consisting of phosphoric acid, citric acid, formic acid, and succinic acid solutions containing said γ-globulin, wherein said γ-globulin is rabbit γ-globulin; said detergent is selected from the group consisting of Brij-35 and Triton X-100; said water soluble n-alcohol is an alkyl diol having 2 to 5 carbon atoms; said buffered reaction medium has a pH of from about 5 to about 5.5 and is buffered by a buffer selected from the group consisting of citrate, succinate and acetate buffers; and said substrate is selected from the group consisting of glycerol phosphate, α-naphthyl phosphate, and β-naphthyl phosphate.

Claim 13

The method of claim 10 wherein said solution has a pH of from about 2.5 to about 2.7 and is a citric acid solution containing said rabbit γ-globulin, said alkyl diol contains from 3 to 5 carbon atoms; said buffered reaction medium has a pH of from about 5.3 to about 5.4; said substrate solution is selected from said group consisting of α-naphthyl phosphate and β-naphthyl phosphate; and said reagent comprises a diazonium salt.

Claim 14

The method of claim 11 wherein said alkyl diol is selected from the group consisting of 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, and 1,5-pentanediol; and wherein said diazonium salt is selected from the group consisting of fast garnet GBC salt, fast blue B salt, naphthanyl diazo red RC, diazotized p-nitroaniline, fast red ITR salt, fast red TR salt, and hexazotised p-rosaniline salt.

Claim 15

The method of claim 12 wherein said solution has a pH of about 2.53; said detergent is Brij-35; said alkyl diol is 1,5-pentanediol; said buffered reaction medium has a pH of about 5.35 and said buffer is citrate buffer; said substrate is α-naphthyl phosphate; and said diazonium salt is fast red TR.

Claim 16

The method of claim 9 wherein: (I) said Ab 1 is present in a solution comprising about 5.4±15% mmol/L citric acid, about 21.7±15% mmol/L sodium citrate, and about 2±20% mg/ml rabbit antiserum, Ab 1 ; (II) said Ab 1 and said PAP are incubated for at least about 10 minutes; (III) said Ab 2 is present in a solution comprising about 7.8±15% mmol/L citric acid, about 20.5±15% mmol/L sodium citrate, and about 10±20% mg/ml goat anti-rabbit IgG antiserum; (IV) said Ab 2 and said Ab 1 ˜PAP complexes are incubated for at least about 10 minutes; (V) said solution comprises about 20±15% mM citric acid and about 0.625±15% mg/ml rabbit γ-globulin and has a pH of about 2.53±15%; (VI) said detergent comprises about 4±15% mM Brij-35; (VII) said alcohol comprises about 150±15% mM 1,5-pentanediol; (VIII) said buffered reaction medium comprises about 5.8±15% mM α-naphthyl phosphate, about 3.6±15% mM fast red TR, about 75±15% mM citrate, and has a pH of about 5.35±15%; and (IX) said kinetic measurement is made at about 10±10% minutes after commencement of step (i).

Claim 17

The method of claim 14 wherein: (I) said Ab 1 solution comprises about 5.4±10% mmol/L citric acid, about 21.7±10% mmol/L sodium citrate, and about 2±15% mg/L rabbit antiserum, Ab 1 ; (II) said Ab 1 and said PAP are incubated for about 30±33% minutes; (III) said Ab 2 solution comprises about 7.8±10% mmol/L citric acid, about 20.5±10% mmol/L sodium citrate, and about 10±15% mg/ml goat anti-rabbit IgG antiserum; (IV) said Ab 2 and said Ab 1 ˜PAP complexes are incubated for about 30±33% minutes; (V) said solution comprises about 20±10% mM citric acid and about 0.625±10% mg/ml rabbit γ-globulin and has a pH of about 2.53±10%; (VI) said detergent comprises about 4±10% mM Brij-35; (VII) said alcohol comprises about 150±10% mM 1,5-pentanediol; (VIII) said buffered reaction medium comprises about 5.8±10% mM α-naphthyl phosphate, about 3.6±10% mM fast red TR, about 75±10% mM citrate, and has a pH of about 5.35±10%; and (IX) said kinetic measurement is made at about 10±25% minutes after commencement of step (i).

Claim 21

The method of claim 15 wherein: (I) said Ab 1 solution comprises about 5.4±5% mmol/L citric acid, about 21.7±5% mmol/L sodium citrate, about 2±5% mg/l rabbit antiserum, Ab 1 , about 10 mg/ml±15% mannitol, and from about 0.005 to about 0.1 mg/ml thimerosal; (II) said Ab 1 and said PAP are incubated for about 30±15% minutes; (III) said Ab 2 solution comprises about 7.8±5% mmol/L citric acid, about 20.5±5% mmol/L sodium citrate, about 10±5% mg/ml goat anti-rabbit IgG antiserum; about 10 mg/ml±100% mannitol, and from about 0.005 to about 0.1 mg/ml thimerosal; (IV) said Ab 2 and said Ab 1 ˜PAP complexes are incubated for about 30±15% minutes; (V) said solution comprises about 20±5% mM citric acid and about 0.625±5% mg/ml rabbit γ-globulin and has a pH of about 2.53±10%; (VI) said detergent comprises about 4±5% mM Brij-35; (VII) said alcohol comprises about 150±5% mM 1,5-pentanediol; (VIII) said buffered reactin medium comprises about 5.8±5% mM α-naphthyl phosphate, about 3.6±5% mM fast red TR, about 75±5% mM citrate, and has a pH of about 5.35±5%; and (IX) said kinetic measurement is made at about 10±25% minutes after commencement of step (i).

Claim 22

A kit for assaying an enzyme in a sample comprising: (a) a first component comprising a first antibody (Ab 1 ) against said enzyme, wherein the amount of Ab 1 therein is at least sufficient to bind substantially all of said enzyme in said ample to form Ab 1 ˜enzyme complexes; (b) a second component comprising a second antibody (Ab 2 ) capable of recognizing Ab 1 , wherein the amount of Ab 2 therein is at least sufficient to bind substantially all of said Ab 1 ˜enzyme complexes to form Ab 2 ˜Ab 1 ˜enzyme complexes and form a precipitate with substantially all of said complexes; (c) a third component comprising a solution (i) having a pH at least sufficient to solubilize precipitated Ab 2 ˜Ab 1 ˜enzyme complexes and (ii) comprising γ-globulin in an amount at least sufficient to prevent substantially any precipitation of Ab 2 ˜Ab 1 ˜enzyme complexes at a pH suitable for formation of said precipitate; and (d) a fourth component comprising a reaction medium capable of being used to assay said enzyme.

Claim 23

A kit for assaying an enzyme in a sample comprising: (a) a first component comprising a first antibody (Ab 1 ) against said enzyme, wherein the amount of Ab 1 therein is at least sufficient to bind substantially all of said enzyme in said ample to form Ab 1 ˜enzyme complexes; (b) a second component comprising a second antibody (Ab 2 ) capable of recognizing Ab 1 , wherein the amount of Ab 2 therein is at least sufficient to bind substantially all of said Ab 1 ˜enzyme complexes to form Ab 2 ˜Ab 1 ˜enzyme complexes and form a precipitate with substantially all of said complexes; (c) a third component comprising a solution (i) having a pH at least sufficient to solubilize precipitated Ab 2 ˜Ab 1 ˜enzyme complexes and (ii) comprising γ-globulin in an amount at least sufficient to prevent substantially any precipitation of Ab 2 ˜Ab 1 ˜enzyme complexes at a pH suitable for formation of said precipitate; and (d) a fourth component comprising a reaction medium capable of being used to assay said enzyme.

Claim 24

The kit of claim 20 further comprising a non-interfering, water soluble detergent.

Claim 25

The kit of claim 20 wherein said reaction medium is a kinetic reaction medium.

Claim 26

The kit of claim 20 further comprising a non-interfering, water soluble detergent and wherein said reaction medium is a kinetic reaction medium.

Claim 27

The kit of claim 20 wherein the first and second components are combined into a single component and wherein Ab 1 is selected from the group consisting of monoclonal and polyclonal antibodies and Ab 2 is selected from the group consisting of monoclonal and polyclonal antibodies.

Claim 28

The kit of claim 24 further comprising a non-interfering, water soluble detergent.

Claim 29

The kit of claim 24 wherein said reaction medium is a kinetic reaction medium.

Claim 30

The kit of claim 24 further comprising a non-interfering, water soluble detergent and wherein said reaction medium is a kinetic reaction medium.

Claim 31

A kit for kinetically assaying prostate acid phosphatase (PAP) in a sample comprising: (a) a first component comprising a first antibody (Ab 1 ) against said PAP, wherein the amount of Ab 1 therein is at least sufficient to bind substantially all of said PAP in said sample to form Ab 1 ˜PAP complexes; (b) a second component comprising a second antibody (Ab 2 ) capable of recognizing Ab 1 wherein the amount of Ab 2 therein is at least sufficient to bind substantially all of said Ab 1 ˜PAP complexes to form Ab 2 ˜Ab 1 ˜PAP complexes and form a precipitate with substantially all of said complexes; (c) a third component comprising a solution (i) having a pH≦about 3 and (ii) comprising γ-globulin in an amount at least sufficient to prevent substantially any precipitation of Ab 2 ˜Ab 1 ˜PAP complexes at a pH suitable for formation of said precipitate; (d) a fourth component comprising a non-interfering, water soluble detergent; (e) a fifth component comprising a water soluble n-alcohol having at least one terminal hydroxyl group and capable of participating in a transphosphorylation reaction, said alcohol therein being in an amount such that it is not a rate limiting factor in a reaction between a substrate and said alcohol in the presence of said PAP; (f) a sixth component comprising a buffered reaction medium comprising said substrate, a reagent capable of reacting with a product of said alcohol, substrate, PAP reaction to produce a detectable product, and a buffer having a pH compatible with an assay of PAP activity.

Claim 32

A kit for kinetically assaying prostate acid phosphatase (PAP) in a sample comprising: (a) a first component comprising a first antibody (Ab 1 ) against said PAP, wherein the amount of Ab 1 therein is at least sufficient to bind substantially all of said PAP in said sample to form Ab 1 ˜PAP complexes; (b) a second component comprising a second antibody (Ab 2 ) capable of recognizing Ab 1 wherein the amount of Ab 2 therein is at least sufficient to bind substantially all of said Ab 1 ˜PAP complexes to form Ab 2 ˜Ab 1 ˜PAP complexes and form a precipitate with substantially all of said complexes; (c) a third component comprising a solution (i) having a pH≦about 3 and (ii) comprising γ-globulin in an amount at least sufficient to prevent substantially any precipitation of Ab 2 ˜Ab 1 ˜PAP complexes at a pH suitable for formation of said precipitate; (d) a fourth component comprising a non-interfering, water soluble detergent; (e) a fifth component comprising a water soluble n-alcohol having at least one terminal hydroxyl group and capable of participating in a transphosphorylation reaction, said alcohol therein being in an amount such that it is not a rate limiting factor in a reaction between a substrate and said alcohol in the presence of said PAP; (f) a sixth component comprising a buffered reaction medium comprising said substrate, a reagent capable of reacting with a product of said alcohol, substrate, PAP reaction to produce a detectable product, and a buffer having a pH compatible with an assay of PAP activity.

Claim 33

The kit of claim 25 wherein said Ab 1 is rabbit Ab 1 ; said Ab 2 is goat anti-rabbit IgG; said solution has a pH of from about 2 to about 2.8 and is selected from the group consisting of phosphoric acid, citric acid, formic acid, and succinic acid solutions containing said γ-globulin, wherein said γ-globulin is rabbit γ-globulin; said detergent is selected from the group consisting of Brij-35 and Triton X-100; said water soluble n-alcohol is an alkyl diol having 2 to 5 carbon atoms; said buffered reaction medium has a pH of from about 5 to about 5.5 and is buffered by a buffer selected from the group consisting of citrate, succinate and acetate buffers; and said substrate is selected from the group consisting of glycerol phosphate, α-naphthyl phosphate, and β-naphthyl phosphate.

Claim 34

The kit of claim 29 wherein said solution has a pH of from about 2.5 to about 2.7 and is a citric acid solution containing said rabbit γ-globulin, said alkyl diol contains from 3 to 5 carbon atoms; said buffered reaction medium has a pH of from about 5.3 to about 5.4; said substrate solution is selected from said group consisting of α-naphthyl phosphate and β-naphthyl phosphate; and said reaction comprises a diazonium salt.

Claim 35

The kit of claim 30 wherein said alkyl diol is selected from the group consisting of 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, and 1,5-pentanediol; and wherein said diazonium salt is selected from the group consisting of fast garnet GBC salt, fast blue B salt, naphthanyl diazo red RC, diazotized p-nitroaniline, fast red ITR salt, fast red TR salt, and hexazotised p-rosaniline salt.

Claim 36

The kit of claim 31 wherein said solution has a pH of about 2.53; said detergent is Brij-35; said alkyl diol is 1,5-pentanediol; said buffered reaction medium has a pH of about 5.35 and said buffer is citrate buffer; said substrate is α-naphthyl phosphate; and said diazonium salt is fast red TR.

Claim 37

The kit of claim 28 wherein: (I) said Ab 1 is present in a solution comprising about 5.4±15% mmol/L citric acid, about 21.7±15% mmol/L sodium citrate, and about 2±20% mg/ml rabbit antiserum, Ab 1 ; (II) said Ab 2 is present in a solution comprising about 7.8±15% mmol/L citric acid, about 20.5±15% mmol/L sodium citrate, and about 10±20% mg/ml goat anti-rabbit IgG antiserum; (III) said solution comprises about 20±15% mM citric acid and about 0.625±15% mg/ml rabbit γ-globulin and has a pH of about 2.53±15%; (IV) said detergent comprises about 4±15% mM Brij-35; (V) said alcohol comprises about 150±15% mM 1,5-pentanediol; and (VI) said buffered reaction medium comprises about 5.8±15% mM α-naphthyl phosphate, about 3.6±15% mM fast red TR, about 75±15% mM citrate, and has a pH of about 5.35±15%.

Claim 38

The kit of claim 33 wherein: (I) said Ab 1 solution comprises about 5.4±10% mmol/L citric acid, about 21.7±10% mmol/L sodium citrate, and about 2±15% mg/ml rabbit antiserum Ab 1 ; (II) said Ab 2 solution comprises about 7.8±10% mmol/L citric acid, about 20.5±10% mmol/L sodium citrate, and about 10±15% mg/ml goat anti-rabbit IgG antiserum; (III) said solution comprises about 20±10% mM citric acid and about 0.625±10% mg/ml rabbit γ-globulin and has a pH of about 2.53±10%; (IV) said detergent comprises about 4±10% mM Brij-35; (V) said alcohol comprises about 150±10% mM 1,5-pentanediol; and (VI) said buffered reaction medium comprises about 5.8±10% mM α-naphthyl phosphate, about 3.6±10% mM fast red TR, about 75±10% mM citrate, and has a pH of about 5.35±10%.

Claim 39

The kit of any one of claim 33 or 34 wherein said Ab 1 and Ab 2 solutions further comprise at least one composition selected from a group consisting of bulking agents and anti-microbial agents.

Claim 40

The kit of any one of claim 33 or 34 wherein said Ab 1 and Ab 2 solutions further comprise at least one composition selected from the group consisting of bulking agents and anti-microbial agents, wherein said bulking agents are selected from the group consisting of mannitol, sucrose, and lactose, and said anti-microbial agents are selected from the group consisting of thimerosal, sodium azide, and hyamine 3500.

Claim 41

The kit of any one of claim 33 or 34 wherein said Ab 1 and Ab 2 solutions further comprise mannitol and thimerosal.

Claim 42

The kit of claim 34 wherein: (I) said Ab 1 solution comprises about 5.4±5% mmol/L citric acid, about 21.7±5% mmol/L sodium citrate, about 2±5% mg/l rabbit antiserum, Ab 1 , about 10 mg/ml±15% mannitol, and from about 0.005 to about 0.1 mg/ml±15% thimerosal; (II) said Ab 2 solution comprises about 7.8±5% mmol/L citric acid, about 20.5±5% mmol/L sodium citrate, about 10±5% mg/ml goat anti-rabbit IgG antiserum, about 10 mg/ml±15% mannitol, and from about 0.005 to about 0.1 mg/ml±15% thimerosal; (III) said solution comprises about 20±5% mM citric acid and about 0.625±5% mg/ml rabbit γ-globulin and has a pH of about 2.53±10%; (IV) said detergent comprises about 4±5% mM Brij-35; (V) said alcohol comprises about 150±5% mM 1,5-pentanediol; and (VI) said buffered reactin medium comprises about 5.8±5% mM α-naphthyl phosphate, about 3.6±5% mM fast red TR, about 75±5% mM citrate, and has a pH of about 5.35±5%.