GB2270315A

GB2270315A - Intrinsic factor-horseradish peroxidase conjugates

Info

Publication number: GB2270315A
Application number: GB9317356A
Authority: GB
Inventors: Dean William Schroer
Original assignee: Becton Dickinson and Co
Current assignee: Becton Dickinson and Co
Priority date: 1992-09-04
Filing date: 1993-08-20
Publication date: 1994-03-09
Also published as: FR2695406B1; DE4329530A1; JPH06205673A; CA2104413A1; CA2104413C; AU662235B2; FR2695406A1; AU4476493A; GB9317356D0

Abstract

This invention preferably presents a non-isotopic competitive assay for Vitamin B12 (B12), utilizing intrinsic factor (IF) labelled with horse radish peroxidase (HRP). The conjugation is accomplished by oxidizing surface carbohydrates on the HRP with NalO4, and reacting this with IF.

Description

2270315 INTRINSIC FACTOR - HORSE RADISH PEROXIDASE CONJUGATES

BACKGROUND OF INVENTION

Assays for the serum level of Vitamin B12 have proven to be extremely challenging to develop. This is due primarily to the high sensitivity required, on the level of picograms, as well as the fact that normal serum contains endogenous B12 binders. These binders must be treated to release the B12 prior to the assay; such treatment is quite harsh and generally requires a separate step to accomplish it. The treatment involves the use of either heating to elevated temperatures (100OC), commonly termed "boil" assays, or the use of strong chemical agents, "no-boil" assays. one example of a no boil assay is presented in U.S. Patent No.. 4,300,907 to Mansbach et al.

Because of these requirements, until quite recently virtually all commercially available assays for B12 have been radioassays which utilize a radioactive isotopic labeled binding protein for detection. A number of other formats have been discussed in the literature including the use of chemi luminescent (Clin. Chem., Vol. 35, No. 6, p. 1194, Abst. #609 (1989)), fluorescent (Clin. Chem., Vol. 37, No. 6, p. 978 Abst. #326 (199M, and color-labelled B12 (available from Ciba Corning) detectors. These assays utilize microbeads coated with B12 binding protein for detection, and, as such, may not be compatible with many automated detection methods.

Additionally, enzyme linked assays utilizing alkaline phosphatase (e.g. Clin. Chem., Vol. 38, No. 6, P. 1089, P-2173 Abst. #0662 (1992), Clin. Chem., Vol. 38, No. 6, p. 1095 Abst. #0691 (1992), Clin. Chem., Vol. 37, No. 6, p. 978, Abst. #320 (1991) and B galactosidase (Clin. Chem., Vol.

33, No. 6, p. 963, Abst. #403, (1987)) have been reported; however, both of these enzymes are quite large. Since the time required for competitive immunoassays is heavily dependent on the diffusion rate, and since the diffusion rate is approximately inversely proportional to the cube root of the molecular weight, the utility of these formats in such assays limited.

SUMMARY OF INVENTION

This invention presents a non-isotopic competitive assay for Vitamin B12 (B12). Briefly, the assay utilizes intrinsic factor (IF) labelled with horse radish peroxi dase (HRP). The HRP can be conjugated to the IF by oxidizing surface carbohydrates on the HRP.

This method, it has been found, permits formation of the IF/HRP conjugate without deleteriously affecting the B12 binding sites on the IF. Thus the conjugates can be used in assays for B12. Further, because the IF is labelled.with HRP, the assays can be run on automated equipment adapted to utilize the signal generated by the HRP/substrate reaction. Additionally, because the method permits relatively large amounts of HRP to be conjugated to IF (e.g. a high HRP/W ratio), the signal generated will also be high, increasing the assay sensitivity.

DETAILED DESCRIPTION OF THE INVENTION

The assays of this invention make use of the well known binding affinity of intrinsic factor (IF) for Vitamin B12 (B12). Briefly, in a preferred competitive assay format, the (liquid) sample (which may contain B12) P-2173 is mixed with IF conjugated to horse radish peroxidase (HRP), and permitted to react. An aliquot of this mixture is then placed in contact with a solid phase containing bound B12. Subsequently, the liquid phase is separated from the solid phase, leaving behind any IF/HRP bound to the solid phase B12; any IF/HRP bound to sample B12 remains in the liquid. The amount of IF/HRP conjugate in the sample can then be measured by addition of HRP substrate and measurement of the reaction product. This is directly related to the quantity of B12 in the sample.

Alternatively, the activity of the bound IF/HRP conjugate would be determined to -ascertain B12 in the sample indirectly.

The substrate utilized is any amenable to HRP action; preferred substrates include:

tetramethylbenzidine, 0-phenylene diamine, luminol/iodophenol, and tyramime Coupling of the HRP to the IF is a critical requirement of this assay. Since many coupling methods require treatment with chemicals which can deleteriously affect the IF and/or HRP, the treatment must be sufficiently mild to permit both of these components to remain unaffected, yet sufficiently strong to permit formation of a conjugate which will not dissociate under storage and/or assay conditions.

One such method utilizes the periodate reaction described by Wilson and Nakane in Immunofluorescence and Related Staining Techniques, Krapp et al (eds.), North Holland Biomedical Press, Amsterdam,- pp. 215-224 (1978), incorporated herein by reference. Briefly, the P-2173 carbohydrate surface of the HRP is oxidized to an aldehyde by addition of NaIO 4 # in a dark reaction chamber at room temperature. The HRP is in aqueous solution, at a concentration of 1-50 gm/1, preferably 5-25 gm/1, more preferably 10-15 gm/1; the NaIO 4 is also in aqueous solution at a concentration of 16-50 mM, preferably 15-40 M more preferably -20-30 mM. The reactants are mixed at a ratio of 10-20, preferably 1216 moles NaIO 4 /mole HRP.

Subsequently, ' the reaction is terminated by the addition of an excess of a glycol, preferably ethylene glycol, and the oxidized HRP is dialyzed against mildly acidic (pH 3-6, preferably 4-5) buffer. About 0.1-3 mg, preferably 0.6-2.4 mg of the purified oxidized HRP is then reacted with about 5-20 ug, preferably 10-15 ug IF, in is aqueous solution at ' a basic pH (8-10, preferably 8.5-9.5). The resultant conjugates are then reduced by an aqueous solution of a metallic or non-metallic hydride, preferably sodium borhydride or lithium aluminum. hydride.

The final product is then exchanged into a neutral salt solution and glycerol is added to facilitate storage.

The above conjugates can be utilized in the competitive assay for Vitamin B12 as described above. The above procedures are particularly suited for making HRP-W conjugates as they minimize the use of expensive IF, by utilizing an excess of HRP to assure all IF is reacted, in addition to leaving the HRP and IF relatively intact and functionally unaffected.

Further, the above assay procedure, using HRP-W conjugates, is particularly suited for use in automated assay instruments, such as the AFFINITY@ analyzer manufactured and marketed by Becton, Dickinson and Company, due to the fact that HRP activity is measured.

P-2173 Since many assays can be formatted to use HRP as the tracer or detector, the versatility of such an assay and, thus, such an instrument is enhanced.

EXAMPLES

The following examples demonstrate certain preferred embodiments of the instant invention, but are not intended to be illustrative of all embodiments.

Example 1 - Preparation of HRP-W Conjugate by Periodate Reaction A 12mg quantity of HRP was dissolved in 1. 0 ml water and shielded from light. A total of 164pl aqueous 25 mM NaIO 4 was added and the system was allowed to react f or minutes at 250C (ambient temperature water bath).

Immediately thereafter, the reaction was terminated by the addition of 200pl- of 1% (V/V) ethylene glycol in water and the system was left undisturbed for 20 minutes at 250C. The resultant solution was buffer exchanged into imM aqueous sodium acetate buffer (pH = 4.4) using a Centricon@ 30 ultrafiltra- tion device to > 99.5% carbonate buffer.

The HRP concentration was then determined by absorbance at 403 nm and found to be 9.2 mg/ml. A total of 600pg HRP (66pl) was then admixed with 85gl of 0.5M aqueous sodium carbonate buffer (pH 9.0) and 50pg of IF, and incubated at 250C for 4 hours, protected from light.

Immediately thereafter, the tube was placed in an ice P-2173 bath and 501A of an aqueous solution of NaBH4 (4mg/m1) was added to stop the reaction. The solution was incubated at 40C for 40min. and subsequently buffer exchanged into 1OmM potassium phosphate in normal saline (pH 7.6) using a Centricon@ 30 to > 99.5% phosphate buffer. The conjugate was stored in a 50/50 (V/V) mix of phosphate buffered saline and glycerol.

The conjugate was used in a competitive B12 assay in the AFFINITY@ unit wherein bound B12 (in a coated tube) competes with free B12 for IF-HRP. All IF-HRP reacting with sample (free) B12 is removed from the tube, and the B12 concentration is determined by monitoring HRP activity remaining in the coated tube.

It is apparent that many modifications and variations is of this invention as hereinabove set forth may be made without departing from the spirit and scope hereof. The specific embodiments descried are given by way of example only and the invention is limited only by the terms of the appended claims.

P-2173

Claims

WHAT IS CLAIMED IS:

1. A method for preparing intrinsic factor horseradish peroxidase conjugates comprising:

(i) reacting horseradish peroxidase with NaIO 4 to oxidize surface carbohydrates to form an oxidized horseradish peroxidase; (ii) terminating said reaction and concentrating said oxidized horseradish peroxidase; (iii) reacting said concentrated oxidized horseradish peroxidase with intrinsic factor at a basic pH to form an oxidized conjugate; and (iv) subsequently reducing and concentrating said oxidized conjugates.

2. The method of Claim 1 wherein the ratio of NaIO 4 /horseradish peroxidase (mole/mole) is 10/1 to 20/1.

3. The method of Claim 1 wherein the ratio of concentrated oxidized horseradish peroxidase to intrinsic factor is 100-3000/5-20 (wt/wt).

4. The method of Claim 1 wherein the oxidized conjugates are reduced by the addition of a metallic or non-metallic hydride.

5. An intrinsic factor/horseradish peroxidase conjugate produce by the method comprising (i) reacting horseradish peroxidase with NaIO 4 to oxidize surface carbohydrates to form an oxidized horseradish peroxidase; (ii) terminating said reaction and concentrating said oxid ized horseradish peroxidase; P-2173 (iii) reacting said concentrated oxidized horseradish peroxidase with intrinsic factor at a basic pH to form an oxidized conjugate; and (iv) subsequently reducing and concentrating said oxidized conjugates.

6. The conjugates of Claim 5 wherein the ratio of NaIO 4 /horseradish peroxidase (mole/mole) is 10/1 to 20/1.

7. The conjugates -of Claim 5 wherein the ratio of concentrated oxidized horseradish peroxidase.to intrinsic factor is 100-3000/5-20 (wt/wt).

8. The conjugates of Claim 5 wherein the oxidized conjugates are reduced by the addition of a metallic or non-metallic hydride.

9. An intrinsic factor/horseradish peroxidase conjugate produce by the conjugates comprising (i) reacting horseradish peroxidase with Na104 to oxidize surface carbohydrates to form an oxidized horseradish peroxidase; (ii) terminating said reaction and concentrating said oxidized horseradish peroxidase; (iii) reacting said concentrated oxidized horseradish peroxidase with intrinsic factor at a basic pH to form an oxidized conjugate; and (iv) subsequently reducing and concentrating said oxidized conjugates.

10. In a kit f or the competitive assay of Vitamin B12 utilizing a horseradish peroxidase-labelled intrinsic f actor tracer, the improvement comprising using, as said tracerf the intrinsic factor/horseradish peroxidase conjugate of Claim 5.