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TECHNICAL MANUSCRIPT 494

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. 0 ESTIMATION OF PRECIPITATING ANTIBODY

AUSING A TURBIDIMETRIC TECHNIQUE

William F. Vincent (v0 3 0 (u 0 MfEarl W. Harris u U-E 4

U-U 0 W - 41Sidney Yaverbaum " D4 c-'V)O. a-- ) 0(n ) A-C 1.

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DEPARTENTI (F TiE ARMYFort Detrick

Frederick, Mlaryland 21701

TECHICAL MIOUSCClIPT 494

ISTIMATION OF PRECIPITATING ANTIBODYUSING & TURBIDDMTRIC TECHNIQUE

William. Vincent

Earl W. Harris

Sidney Yaverbaum

Physical Defense DivisionCOIIODITY DEVELOPET & ENGINIEING LABORATORIES

Project 13662706(.071 December 1968

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In conducting the research described in this report, theinvestigators adhered to the 'Guide for Laboratory AnimalFacilities and Care," as promulgated by the Committee onthe Guide for Laboratory Animal Facilities and Care of theInstitute of Laboratory Animal Resources, National Academyof Sciences-NatLonal Research Council.

ABSTRACT

The development of turbidity resulting from the precipi-tatLon between bovine serum albumin (BSA) and anti-BSA serumwas studied in the spectrophotometer. The equivalence ratio

and the titer of precipitating antibody could be determinedby this method. Data obtained with the turbLdimetric methodcorrelated well with those obtained using the classical tubeprecLpi~tn determination. Turbidimetric measurements werealso applied successfully to the measurement of antibodytiter in two other system. Antibody titer can be determinedwith this method in about an hour as compared with the 3 ormore days required by the tube test. In addition, thistechnique requires substantially les reagent than othermethods.

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I. INTRODUCTION

The combination of antibody with a soluble antigen is often accompaniedby viaible turbidity. The extent and rate of turbidity development areusually functions of the relative concentrations of antigen and antibody.Martina and Boyden and iMFalcol were among the first to apply turbidimetricmeasurements to the study of precipitation. The latter investigatorsspeculated that such measurements would be ideally suited to extensiveserologic studies and would eliminate the use of tedious quantitativedeterminations. The use of turbidimetric measurements to study precipi-tation has resulted in the detection of small deviations not observablewith other techniques. Gitlin and Edelhoch,' Junge, Junge, and Krebs,4and Hawkins6 employed turbidimetric measurements for the study of antigen-antibody interactions in precipitation. Little attention, however, hasbeen devoted to the employment of such techniques for the routine deter-mination of precipitating antibody.

This report describes investigations that employ a spectrophotometrictechnique to measure the rate of turbidity development in the precipitinreaction and apply these data to the determination of antibody titer.

11. MATERIALS AND METHDS

A. ANTIGENS

Crystalline bovine serum albumin (BSA) and bovine y-globulin (UGG)were purchased from Pentex Corporation. Periodate-oxidized complexesof these proteins with adenosine-5'-monophosphate (AMP) were preparedaccording to the method of Erlanger and Beiser. 3 For both imunizationand testing, the antigens were suspended in 0.01 M phosphate buffer(pH 6.8) prepared In 0.15 K NaCl. This buffered saline was employed forall dilutions and suspensions.

B. ANTISERA

Antisera against BSA, BOG, and AMP-BSA were prepared in New Zealandwhite rabbits by intramuscular immiunization with 4 to 6 weekly injectionsof 10 mg each of antigen emulsified in Freund's complete adjuvant.

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C. MECIPITIN lIrCTIOEB

Equal volumes (1 ml) of serial dilutions of antigen and a constant con-centration of antiserum were nixed and incubated at 37 C for 2 hours. Afterrefrigeration at 4 C for 48 to 72 hours, the precipitates were collectedby centrifugation, washed twice with 2-ml portions of cold buffered saline,and dissolved in 4 al of 0.1 1 aOH. The protein concentration of thedissolved precipitates was determined using the Folin-Ciocalteu reagent.7

With various modifications, usually in determination of protein ornitrogen, the quantitative tube precipitin test just described is the mostcomonly employed method for determining precipitating antibody titer,

Direct turbidimetric measurements were performed by rapidly mixing equalvolume (0.2 al) of antigen and antibody and transferring the mixture to acuvette with a 1-mm path length. The development of turbidity was read at15-second intervals at 350 q4 on a Beckman DK-2A spectrophotometer. Amixture of buffered saline and antiaere in equal volumes was used in the

reference cuvette to correct for absorbance by serum components such ashemoglobin. The temperature of the cell compartment remained relativelyconstant at about 27 C throughout the experiments.

III. RESULTS

The precipitin curve shown in Figure 1 was obtained by measuring the pro-tein concentration of dissolved BSA:anti-BSA pracipitates using the quanti-tative tube method. The rate of turbidity development using the samedilutions of reactants was measured in the spectrophotometer with resultsshown in Figure 2. The highest rate of turbidity development was observedwith an antigen-antibody ratio corresponding to the equivalence ratio asdetermined by the tube test.

After a relatively short lag period, the rate of turbidity developmentwas essentially constant for about 1 minute after mixing. The rate inthe linear region was measured and was expressed as the /. OD/min. Therate observed with equivalent amounts of antigen and antibody was designatedas the I A CD/mmn. The equivalence ratio, quantity of precipitated protein,and the rate of turbidity development were determined for a number of antigenand antibody dilutions using both the tube precipitin technique and theturbidimetric method, and the results were compared. When the R A OD/minwas plotted as a logarithmetic function against antiserum dilutions, alinear relationship was obtained with the highest concentrations of antibody(Figure 3). A plot of log 9 AOD/min against precipitated protein, asshown in Figure 4, revealed a linear relationship. In contrast, however,with the plot obtained with the antiserum dilutions, linearity in this casewas lost with the higher dilutions of antibody.

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FIGURE L. Quantitative Precipitation Between LSA andAnti-ISA Serum Measured by the Tube Precipitin Technique.The figures by the points correspond to the curves inFigure 2.

Equivalence

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FiGURE 2. Development of Iurbidity vith Various Antigento Antibody Ratios. The figures by each curve correspondto the points in Figure 1.

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flGOU 3. The Z a O014un Values Obtaitned Usion BSA asrhe Antigen with Various Dilutions of Anti-BIS Serum.

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FIGURE 4. Comprison of the Values Obtained with theQuantitative Tube Precipitin Test (Protein Precipitated)vith Those Obtained with the Turbidimetric Technique(Log I L OD/Mn) in the BSA/Anti-BSA System.

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The correlations observed in the BSA system were also observed in aBGG:anti-BGG system. Similar results were also obtained in the immunologitprecipitation between anti-AMP-BSA serum and AMP-BGG as the testing antigen.In this case, an hapten-specific antibody was being measured.

IV. DISCUSSION

HawkinsO noted that, in the equivalence and antigen-excess zones, thereis a considerable lag before the development of turbidity. He noted, asEagle had earlier,2 a significant increase in the velocity of the reactionwhen more concentrated reactants were employed, presumably due to a decrease

in the average inter-particle space and the subsequent increase in thenumber of total collisions. In the experiments reported here, a very shortlag was observed because high concentrations of serum were employed. Inmost cases, the lag period under these conditions was short enough to bedisregarded in calculations.

With the higher concentrations of antibody, the rate of turbidity inthe equivalence zone appeared to follow almost a first-order kineticsreaction rather than the second- or higher-order reactions observed inzones of antigen or antibody excess. With more dilute serum samples, therate of turbidity at equivalence began to deviate from the apparent first-order reaction observed with higher concentrations. As a result, therewas a deviation from linearity when dilute antisera were employed intiter determinations. The opposite relationship was observed when therates were plotted against protein precipitated for the same antiserum,that is, there was a deviation from linearity with the more concentratedserum samples.

The experiments reported here indicate that the measurement of therate of turbidity development can be used effectively to determine theantibody titer of serum when high concentrations of reactants are used.The test can be performed on a large number of serum samples in a varyshort time because there is no necessity for collection and measurementof precipitates. Turbidimetric measurement as a means of titeringantiserum employs far less reagent than the classical tube tests. Thevalues obtained with multiple samples of the same dilution of antiserumor antigen were usually within one standard deviation. This is animportant factor to be considered when the quantity of antiserum availablefor testing is limited.

Excellent correlations were obtained with the three serologic syste_-ndescribed in the current study. The routine application of thisprocedure for precipitating systems should be seriously considered.

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LITERATUR CITED

1. Boyden, A.; DeFalco, R. 1943. leport on the use of the photon-reflectometer in serological comparisons. Physiol. Zool. 16:229-241.

2. Eagle, R. 1932. Specific agglutination and precipitation: II.Velocity of the reactions. J. Immunol. 23:153-186.

3. Erlanger, B.F.; Beiser, S.M. 1964. Antibodies specific for ribo-nucleosides and ribonucleotides and their reaction with DM&. Proc.Nat. Acad. Sci. 52:68-74.

4. Gitlin, D.; Edelhoch, H. 1951. A study of the reaction betweenhuman serum albumin and its homologous equine antibody through themedium of light scattering. J. Immunol. 66:67-77.

5. Hwkins, J.D. 1964. Some studies on the precipitin reaction usinga turbidimetric method. Iuamunology 7:229-238.

6. Junge, J.McB.; Junge, C.O., Jr.; Krebs, B.G. 1955. A study ofantigen-antibody reaction rates by light transmission measurements.Arch. Biochem. Biophys. 55:338-355.

7. Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. 1951.Protein measurement with the Folin phenol reagent. J. Biol. Chem."193:265-275.

8. Martin, D.S. 1942. Use of the photonreflectometer in precipitintitrations of a Loultiple-antigen multiple-antibody mixture by theDean-Webb method. J. Bacteriol. 43:95-96.

9STDU&TION (IF1 UBCIITAUNG A~nTI=D USING A IUE1DIDDMEC ==IGNEI

Dcc er 1968 1

5.Pm~uc m.136627064071 Technical IMauscript 494

IS. MOUDfl@U ffATUTQualified requesters toy obtain copies of this publication from DOG.Foreign announcement and dissemination of this publication by DDC is not authorized.Release or announcement to the public La not authorized.

11. @UUftS"ff0?A0 NOT2 19. PSRPONOW4 MUY*UY "CVITYIDepartment of the AMyIFort Detrick, Frederick, Maryland, 21701

It. AWYAET

The development of turbidity resulting from the precipitation between bovine*arum albumin (BSA) and anti-ISA serum was studied in the spectrophotometer.The equivalence ratio and the titer of precipitating antibody could be determinedby tL-e method. Data obtained with the turbidiustric method correlated well withthose obtained using the classical tube precipitin determtination. Turbidimetricmeasuremenits were also applied successfully to the measurement of antibody titerIn two other systemn. Antibody titer can be determined with this method in aboutan hour as compared with the 3 or more days required by the tube test, In addition,this technique requires substantially less reagent than other methods. ( ...

14. Key WordsTurbidimetric estimationBovine serum a~buainAntibody equivalence ratioAntibody titer determination

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